Bioactive alkaloids in vertically transmitted fungal endophytes

16 Pages·2014·0.98 MB·English

FunctionalEcology2013 doi:10.1111/1365-2435.12076
DEFENSIVE SYMBIOSIS
Bioactive alkaloids in vertically transmitted fungal
endophytes
Daniel G. Panaccione*,1, Wesley T. Beaulieu2 and Daniel Cook3
1DivisionofPlant&SoilSciences,WestVirginiaUniversity,1090AgriculturalSciencesBuilding, Morgantown,WV
26506-6108USA;2DepartmentofBiology,IndianaUniversity,Bloomington,IN, USA;and3USDAARSPoisonous
PlantResearchLaboratory,Logan,UT, USA
Summary
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 ofbioactive metabolites by the fungus.
2. Grassesfrequentlyformsymbioseswithendophytic fungi belongingtothefamily Clavicipit-
aceae. 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 (Undifilim 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 Convolvula-
ceae 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 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 ranges
and that produce bioactive compounds are likely to be found as important mutualists in
additionalplants.
Key-words: defensive mutualism, ergot alkaloids, indole-diterpenes, lolines, peramine,
plant-herbivoreinteractions,swainsonine, symbiosis
including energetic (e.g. photosynthetic Chlorella in Anth-
Introduction
ozoans), nutritional (e.g. mycorrhizae), transport (e.g. pol-
Several classes of mutualisms have been recognized based lination) and defensive (Boucher, James & Keeler 1982;
upon the benefits exchanged between the partners Janzen 1985; Douglas 1994). In defensive mutualisms, one
partner provides protection from or resistance to one or
*Correspondenceauthor.E-mail:[email protected] more of its partner’s natural enemies. A classic example is
Published2013.ThisarticleisaU.S.GovernmentworkandisinthepublicdomainintheUSA.
JournalofAppliedEcology2013©BritishEcologicalSociety
2 D.G.Panaccioneet al.
thatoftheAnt-Acaciasysteminwhichvoraciousantspro- pletely dependent upon the host’s reproductive success
tect Acacia trees from megaherbivores (e.g. elephants) in (Ferdy&Godelle2005).
exchange for shelter in plant structures called domatia Thenaturalhostrangeofasymbiont(asopposedtothe
(Janzen 1966; Palmer et al. 2008). Defensive mutualisms range of species it could infect in a laboratory setting) is
involving microbial symbionts that produce protective another defining characteristic of endophytic fungi associ-
chemistry have repeatedly evolved in diverse taxa and can ated with defensive symbioses. Clavicipitaceous endo-
have resounding effects on host success as well as commu- phytes capable of vertical transmission are associated with
nity structure and dynamics (White & Torres 2009). For select taxa of the Poaceae and Convolvulaceae and have
example, marine bryozoans harbour bacterial symbionts narrow host ranges (Clay & Schardl 2002; Steiner et al.
that produce polyketide bryostatins, without which hosts 2011).Inmostinstances,agivenfungaltaxonisassociated
are vulnerable to fish predation (Lopanik, Lindquist & with a specific plant host species (Schardl 2010). In con-
Targett 2004; Lopanik, this issue), leaf cutter ants utilize trast, many horizontally transmitted endophytes represent
an actinomycete that produces antibiotics to ward off fungal genera that are ubiquitous in the environment and
yeasts that would otherwise degrade the ants’ fungal gar- are often associated with multiple host taxa (Rodriguez
den (Currie, Mueller & Malloch 1999), and aphids host et al.2009).
facultative bacterial symbionts that can provide resistance The defensive mutualism hypothesis does not exclude
to both abiotic (e.g. heat) and biotic (e.g. parasitoids) the possibility of negative effects to the host under certain
stressors (Oliver et al. 2010; Oliver, this issue). Defensive environmental conditions, especially since the host must
mutualism was proposed by Clay (1988) to describe the incur some due to endophyte infection. For example,
relationship between certain fungi and their grass hosts, in Cheplick, Clay & Marks (1989) observed reduced growth
which the fungi are afforded a habitat and carbohydrates in endophyte-infected tall fescue under low nutrient condi-
by their host plant and provide their host with protection tions.Turfvarietiesoftallfescueinfectedwithaparticular
frombioticstress(e.g.herbivory). Neotyphodium endophyte were more susceptible to root
Fungi that participate in defensive mutualisms typically disease caused by Pythium graminicola (Rodriguez et al.
are referred to as endophytes, even though some produce 2009). W€ali et al. (2006) showed that red fescue (Festuca
epiphytic structures. The term ‘endophyte’, however, also rubra) infected with Epichloe€festucae in subarctic regions
is used more broadly for any fungus, bacterium or other suffered more damage from the snow mould, Typhula
microorganism that colonizes living plants without caus- ishikariensis than did non-infected red fescue growing in
ing overt detrimentalsymptomsand typicallyhasnoobvi- these same areas. Despite the occasional example that
ous external signs of infection. Rodriguez et al. (2009) indicates a detriment of endophyte infection to the host,
proposed four classes of endophytic fungi that were completedependenceonthehostplantfortransmissionof
grouped according to criteria such as host range, tissues the endophyte provides a means for selecting fungi that
colonized, in planta colonization, in planta biodiversity, are beneficial to their host plants. Fungal endophytes that
mode of transmission and the nature of the benefits affor- are strictly vertically transmitted must be beneficial in
ded to the host. Two criteria we feel are diagnostic in order for the host plants to retain them (Ewald 1987). In
defining fungal endophytes that participate in defensive cases where negative effects have been seen, it does not
mutualisms are the capacity for vertical transmission of precludetheexistenceofsomepositivebenefitthathasnot
the endophyte via seed and a narrow host range, both of been measured that results in maintenance of the mutual-
which are characteristic of class 1 endophytes as described ism, such as reduced survival but increased regeneration
by Rodriguez et al. (2009), although those authors limited resulting in net positive population growth (Rudgers et al.
class 1 endophytes to species in the fungal family Clavici- 2012).
pitaceae. In addition to vertical transmission and a narrow host
Hereditary symbionts that are strictly vertically trans- range, a third and striking feature of many plant-endo-
mitted are completely dependent on host reproduction for phytic fungus associations – particularly those that fit well
their own propagation and reproductive fitness. When a withthe definition of defensivemutualism – is the produc-
vertically transmitted symbiont benefits the host, it has an tion ofbioactive secondary metabolites bythe fungalsym-
indirect positive effect on its own fitness. Any symbiont bionts. Whereas fungi in general produce a wide array of
that is strictly vertically transmitted and puts its host at a secondary metabolites, we propose that bioactive metabo-
disadvantage would go extinct because its host would be lites are particularly important to vertically transmitted
outcompeted by non-infected conspecifics (Ewald 1987). fungi, which in the establishment of their fungus–plant
Vertically transmitted symbionts are essentially a trait of symbioses must have provided some immediate benefit to
the host and if one is ‘in the least degree injurious’, first the host plant. We describe here selected examples of such
principles would suggest it would be ‘rigidly destroyed’ by vertically transmitted endophytic fungi, the chemicals they
natural selection (Darwin 1859). Horizontally transmitted produce and the ways their chemicals contribute to the
symbionts, on the other hand, exploit the host’s ability to symbioses.Wealsoincludeexamplesofverticallytransmit-
survive and contact non-infected individuals and are often ted symbiotic fungi that produce noteworthy bioactive
more virulent as the symbiont’s reproduction is not com- chemicals in their symbiotic state but for which roles of
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JournalofAppliedEcology2013©BritishEcologicalSociety,FunctionalEcology
Bioactivealkaloidsoffungalendophytes 3
the chemicals in the symbioses have not been experimen- amine. Although each of these four classes of alkaloids is
tally established. In each case, the metabolites observed derived in some way from amino acid precursors, the four
originally appeared to be components of the plant or pathways are completely independent of one another. No
would have appeared as such if the symbiosis had not individual fungal isolate is known to produce representa-
alreadybeendiscovered. tives of all four classes; most epichloae produce metabo-
lites belonging to one to three of the chemical classes
(Schardlet al.2011).
Clavicipitaceousendophytes of grasses
FungiofthegeneraEpichloe€andNeotyphodiumgrowsym-
ERGOT ALKALOIDS
biotically with many cool season grasses. Neotyphodium
species are exclusively asexual and grow within the inter- Ergot alkaloids are a diverse family of secondary metabo-
cellular spaces of their grass hosts (Fig. 1a). Epichloe€spe- lites produced by certain epichloae, ergot fungi (Claviceps
cies represent the teleomorphic states of several spp.)and relatedspeciesofBalansia andPeriglandula (dis-
Neotyphodium species. In addition to the symbiotic phase cussed later), and in the opportunistic human pathogen
typicalofNeotyphodiumspecies,Epichloe€speciesarecapa- Aspergillus fumigatus. The biosynthetic pathway has been
ble of exiting their plant hosts via the formation of sexual studied extensively and recently reviewed (Lorenz et al.
reproductive stroma on plant inflorescences. Those sexu- 2009; Panaccione 2010; Wallwey & Li 2011). The diverse
ally reproducingEpichloe€species are thuscapable of verti- metabolites in the ergot alkaloid family can be grouped as
cal transmission through their asexual stage and clavines, simple amides of lysergic acid, or ergopeptines
occasional horizontal transmission when they reproduce based on their complexity and relative position in the
sexually. Schardl (2010) referred to representatives of the pathway (Fig. 2). Various ergot alkaloids interact as
twogeneracollectivelyasepichloae. agonists or antagonists at receptors for the monoamine
In addition to direct effects on herbivores, epichloid en- neurotransmitters serotonin, dopamine, adrenaline and
dophytes of grasses can significantly affect ecological noradrenaline. Resulting activities include vasoconstric-
communities. Neotyphodium coenophialum-infected tall tion, uncontrolled muscle contraction and disturbance in
fescue (Lolium arundinaceum) suppresses both plant (Clay the central nervous system and reproductive systems
& Holah 1999) and arthropod diversity (Finkes et al. (reviewedinLorenzet al.2009;Panaccione2010;Wallwey
2006), affects the outcome of competitive interactions & Li 2011). Numerous feeding studies with a variety of
(Clay, Marks& Cheplick 1993), alters plantsoil feedbacks mammals indicate that ergot alkaloids, at concentrations
(Matthews & Clay 2001), slows succession to forest at which they are found in endophyte-infected grasses,
communities (Rudgers et al. 2007), and disrupts relation- have significant detrimental effects on mammalian health
ships between diversity and ecosystem properties such as and reproduction (e.g. Hill et al. 1994; Filipov et al. 1998;
productivity(Rudgers,Koslow&Clay2004). Gadberry et al. 2003; Parish et al. 2003a,b). Ergot alka-
The epichloae collectively produce four classes of bioac- loids also affect insects and nematodes, which contain
tivemetabolitesintheirsymbioticassociationswithplants: homologousneurotransmitters.Activitiesininsectsinclude
ergot alkaloids, indole-diterpenes,loline alkaloids andper- feeding deterrence, delayed development and increased
(a) (b)
Fig.1.Class 1 endophytic fungi. (a) Ani-
line blue-stained hypha of Neotyphodium
(c) (d) (e)
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
ofI.asarifolia.(Photos:theauthors)
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JournalofAppliedEcology2013©BritishEcologicalSociety,FunctionalEcology
4 D.G.Panaccioneet al.
Fig.2.Diversificationofergotalkaloidsassociatedwithendophyte–plantsymbioses.Doublearrowsindicateoneormoreomittedinter-
mediates.Dashedarrowsindicateuncharacterizedsteps.Relevantenzymesassociatedwithcatalysisatbranchpointsareindicated.Atthe
first branch point, alternative formsof EasA formfestuclavine (not pictured)and agroclavine(Coyleetal. 2010); additional alternative
formsarehypothesizedtoproducecycloclavineandlysergolinPeriglandula-infectedIpomoeaspp.Atthesecondbranchpoint,combina-
tions of peptide synthetases Lps1, Lps2 and Lps3 are required to produce ergopeptines or simple amides of lysergic acid (Lorenz etal.
2009;Ortel&Keller2009).Lysergicacidisbracketedtoindicatethatitisnottypicallyconsideredaclavine.
mortality (Clay & Cheplick 1989; Ball, Miles & Prestidge Notably, the knockout strain accumulated the same molar
1997;Potteret al.2008). quantity of ergot alkaloids as the wild type, but the alka-
The ergot alkaloid pathway is notable for its accumula- loids were restricted to earlier pathway intermediates and
tion of intermediates and spur products to concentrations spur products. Thus, the accumulation of both intermedi-
that approach or exceed the amounts of the pathway end ates and end products is beneficial to the fungus and
product (Panaccione et al. 2003; Panaccione & Coyle its grass host in resisting vertebrate and invertebrate
2005). Panaccione (2005) hypothesized that this ineffi- herbivorepressures.
ciency in turning over intermediates has been selected for The general significance of ergot alkaloids to
becausethoseaccumulatingintermediatesorspurproducts Lp1-infected perennial ryegrass was apparent from the
provide some benefit to the producing fungi (or its grass observation that perennial ryegrass containing a different
host,inthecaseofendophytes)thatdiffersfromthebene- knockout mutant, which was completely devoid of ergot
fit(s) provided by the pathway end product. Differences in alkaloids but still colonized by the fungus (Wang et al.
activities of clavine intermediates and spur products com- 2004), was strongly preferred by rabbits, even over the
pared to ergopeptines or the simple amides are apparent endophyte-free perennial ryegrass (Panaccione et al.
from direct exposure of bacteria and nematodes to these 2006b).Thus,withoutanyergotalkaloidsthisgrasswould
alkaloids in vitro (Panaccione 2005; Panaccione et al. be subject to increased herbivory and likely at competitive
2006a; Bacetty et al. 2009a,b). In a more natural setting, disadvantage compared to grasses containing ergot
studies with perennial ryegrass (Lolium perenne) and gene alkaloid-producingendophytes.
knockout mutants of the endophyte Epichloe€ typhin-
a 9 Neotyphodium lolii isolate Lp1 (hereafter simply Lp1)
INDOLE-DITERPENES
provide more support for this hypothesis. A knockout
mutant thataccumulated certain clavines butnotergopep- The indole-diterpenes represent another important class of
tinesorsimpleamidesoflysergicaciddeterredrabbitfeed- diversealkaloidsproducedbysomeepichloaeaswellasby
ing on infected grasses as well as or better than the wild certain Claviceps spp. and some members of the Tricho-
typeofthefungus(Panaccioneet al.2003,2006b).Incon- comaceae (e.g. Aspergillus and Penicillium spp.) (Saikia
trast, perennial ryegrass containing the same knockout et al. 2008). Indole-diterpenes have been studied inten-
endophyte had reduced insecticidal and insect feeding sively because certain members of this class of metabolites
deterrent properties compared to wild-type endophyte, have strong tremorgenic activity in mammals. For exam-
indicating a role for ergopeptines and simple amides of ple,thelolitremsproducedbyNeotyphodiumloliiinperen-
lysergic acid in these anti-insect traits (Potter et al. 2008). nial ryegrass cause ryegrass staggers (Gallagher, White &
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JournalofAppliedEcology2013©BritishEcologicalSociety,FunctionalEcology
Bioactivealkaloidsoffungalendophytes 5
Mortimer 1981; Gallagher et al. 1982, 1984), which can indole-diterpenescouldbebeneficialtotheirhostbyacting
resultinsignificanteconomiclosses. against insects, as has been demonstrated for the biogeni-
Similar to the ergot alkaloids, the indole-diterpenes of cally related yet structurally distinct compound nodulisp-
endophytesareverydiverse.Asimplisticviewofthediver- oric acid. Nodulosporic acid is produced in culture by
sification of indole-diterpenes can be based on the oxida- Nodulisporium sp. (an anamorphic fungus in the Xylaria-
tion and prenylation of intermediate terpendole I and its ceae that was isolated from an unidentified woody plant)
subsequent metabolites independently, resulting in differ- andhasgoodinsecticidalactivityagainstarangeofinsects
ent end products including terpendoles, lolitrems and (Byrne, Smith & Ondeyka 2002). The less commonly
janthitrems(Fig. 3). encountered but biogenically related janthitrems also may
Much of the early analysis of grass endophytes for be associated with insecticidal activity. Janthitrems accu-
indole-diterpenes focused intensively on lolitrem B. Evi- mulate in plants with N. lolii isolate AR37, an endophyte
dence for lolitrem B as the key tremorgenic toxin in strain that is included in some commercial varieties of
N. lolii-infected perennial ryegrass has come from animal perennial ryegrass because of its low tremorgenic activity.
feeding studies (e.g. Gallagher, White & Mortimer 1981; AR37-infected perennial ryegrass varieties are notably
Gallagheret al.1982)aswellasfromcomparisonsofnatu- resistant to the insect pest Wiseana cervinata (porina)
rally occurring isolates that vary in indole-diterpene pro- (Jensen & Popay 2004); however, a direct linkage of the
files (e.g. Bluett et al. 2005a,b). More recent genetic anti-insect activities of AR37 with the janthitrems has not
screening,facilitatedbyamorethoroughunderstandingof beenestablished.Severalotherindole-diterpeneshavebeen
indole-diterpenebiosynthesis,indicatedthatsomeepichloid isolated from the sclerotia of various Aspergillus spp.
endophytesthatdonotproducelolitremBstillproduceless (Trichocomaceae, Eurotiales), and these indole-diterpenes
complicated indole-diterpenes such as terpendoles (Gate- have been demonstrated to have anti-insect activities
nby et al. 1999; Young et al. 2009; Schardl et al. 2011). throughfeedingandtopicalassays(Gloer1995).
Interestingly,fungalendophytesproducingterpendolesbut The production of indole-diterpenes and ergot alkaloids
lacking lolitrem B have been successfully marketed in for- by certain representatives of two phylogenetically disjunct
age varieties of perennial ryegrass in New Zealand as less families, the Clavicipitaceae and the Trichocomaceae (and
toxic alternatives to traditional perennial ryegrass varieties very rarely by fungi outside these families), is remarkable.
(Bluett et al. 2005a,b). Lolitrem B-deficient varieties may Whereas the known alkaloid-producing Clavicipitaceae
still induce minor tremoring in mammals, presumably due (order Hypocreales) all are associated with living plants,
to the presence of janthitrems or other indole-diterpenes, the alkaloid-producing Trichocomaceae (order Eurotiales)
buttheeffectsareminimal(Bluettet al.2005a,b). are primarilysaprotrophs onplant matter.Althoughergot
The observation that some non-tremorgenic epichloae alkaloids and indole-diterpenes are assembled from some
retain the ability to produce intermediates in the indole- common precursors, the biosynthetic pathways for these
diterpene pathway is interesting, considering their negligi- alkaloids are completely independent. The polyphyletic
ble anti-mammalian activity compared to the lolitrems. distribution of the two independent pathways among such
Young et al. (2009) speculated that the less tremorgenic diversefungicannotbeexplainedatpresent.
Fig.3.Diversificationofindole-diterpenesassociatedwithendophyte-plantsymbioses.Doublearrowsindicateoneormoreomittedinter-
mediates.Dashedarrowsindicateuncharacterizedsteps.LtmE/LtmJandLtmF/LtmKrepresentseparateprenyltransferase/monooxygen-
ase (respectively) combinations that work on opposite ends of members of the indole-diterpene family (Young etal. 2006, 2009). Each
combinationcanactonmultiplesubstrates.
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JournalofAppliedEcology2013©BritishEcologicalSociety,FunctionalEcology
6 D.G.Panaccioneet al.
Schardl et al. (2007) carefully reviewed studies on verte-
LOLINES
brate toxicity of lolines and concluded that anti-vertebrate
The loline alkaloids are a family of aminopyrrolozidine effectsoflolineswerelikelytobenegligible,becausestudies
alkaloids,derivedfromhomoserineandprolinejoinedina indicating such effects were either confounded by the
non-peptidic manner. Lolines have been most intensively presenceofergotalkaloidsinthesameplanttissuesorcon-
studied in endophytic Neotyphodium spp. but also have ductedwithexceptionallyhighconcentrationsoflolines.
beenreportedintheplantsAdenocarpusdecorticans(Faba-
ceae)(reviewedbySchardlet al.2007)andArgyreiamollis
(Convolvulaceae) (Tofern et al. 1999). Whether the lolines PERAMINE
intheAdenocarpusandArgyreiaspeciesareofplantorigin Peramine is the most widely distributed of the four classes
or derived from endophytic fungi has not yet been deter- of epichloae-derived secondary metabolites (Schardl et al.
mined. The biosynthesis and activities of lolines has been 2011), but its production is not known outside of the epi-
reviewedindetailbySchardlet al.(2007).Lolinesoccurin chloae (Clay & Schardl 2002; Tanaka et al. 2005). Its ori-
exceptionally high concentration in Neotyphodium coeno- ginas afungalmetabolitewasshownbyitsproductionby
phialum-infected tall fescue (Lolium arundinaceum) and isolated fungi in vitro (Rowan 1993), and more convinc-
alsoarefoundabundantlyandinvariableformsinseveral ingly by its disappearance from grass–endophyte symbiota
otherepichloae-infectedgrasses(Schardlet al.2007,2011). upon mutation of the relevant gene in the fungus (Tanaka
Variability among lolines is mainly generated by the pres- et al. 2005). Peramine is unique among the four major
ence or absence of methyl, formyl or acetyl groups on the classes of epichloae-produced alkaloids, in that it is a sin-
homoserine-derivedaminegroup(Fig. 4a). gle chemical as opposed to a family of chemicals, and it
The insecticidal and insect feeding deterrent activities of appears to be the product of a single multifunctional
lolines have been shown in a series of feeding experiments enzyme as opposed to a complex pathway (Tanaka et al.
with either endophyte-infected plants (e.g. Yates, Fenster 2005). Peramine is derived from a dipeptide possibly made
&Bartelt1989;Siegelet al.1990;Jensen,Popay&Tapper upofarginineandaprecursortoproline(Fig. 4b).
2009)orwithpurifiedlolines(e.g.Riedellet al.1991).Rie- Peramine is a strong feeding deterrent for Argentine
dell et al. (1991) also applied lolines topically to aphids stem weevil, an important pest of perennial ryegrass in
and noted that toxicity of lolines was comparable to that New Zealand, and several other insects (Clay, Hardy &
ofnicotine.Aconvincingdemonstrationofthesignificance Hammond 1985; Johnson et al. 1985; Rowan, Hunt &
of lolines to insect resistance in an endophyte-infected Gaynor 1986; Rowan, Dymock & Brimble 1990; Rowan
grass came from an elegant genetic study conducted by 1993). The anti-feeding effects of peramine are not univer-
Wilkinson et al. (2000) who observed co-segregation of sal,however,astheaphidRhopalosiphumpadiappearsnot
activity against two different aphid species and loline pro- tobedeterredbyitspresence(Johnsonet al.1985;Gaynor
duction in a genetic cross among Epichloe€festucae isolates &Rowan1986).Thesignificanceofperaminetodefending
in meadow fescue (Lolium pratense). Moreover, aphid plant material against herbivory by Argentine stem weevil
mortality increased with increasing concentrations of wasconvincinglydemonstratedinastudybyTanakaet al.
lolinesinplantscontainingloline-positiveprogeny. (2005) in which the gene encoding the multifunctional
In addition to the well-documented effect on insects, enzyme responsible for peramine biosynthesis was inacti-
lolines also are nematicidal (Bacetty et al. 2009a). The vated by knockout. Resulting peramine-deficient mutants
effects of lolines appear to be restricted to invertebrates. wereassusceptibletofeedingbytheArgentinestemweevil
asendophyte-freeplantsofthesamevariety.
Unlike the ergot alkaloids and indole-diterpenes, which
(a) arefoundmainlyintissuesthatarecolonizedbytheendo-
phyte (pseudostem or seeds), peramine is water soluble
and dispersed throughout the plant (Ball et al. 1997a,b;
Spieringet al.2002, 2005;Koulman et al.2007).Peramine
isfoundinfluidsexudedfromcutleavesofalltestedendo-
phyte-infected varieties of perennial ryegrass, tall fescue,
and Elymus sp. and in the guttation fluid of endophyte-
infectedperennialryegrassandElymussp.(Koulmanet al.
(b)
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
Fig.4.Structures of (a) lolines and (b) peramine. Variation
amonglolinesderivesfromsubstituentsontheindicatednitrogen peramine-producing fungi) indicates the presence of
(Schardletal.2007). peramineinphloem.
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JournalofAppliedEcology2013©BritishEcologicalSociety,FunctionalEcology
Bioactivealkaloidsoffungalendophytes 7
and only four of those 15 produce both ergot alkaloids
DISTRIBUTION OF BIOACTIVE ALKALOID CLASSES
and indole-diterpenes (Table 1). The data show that
AMONG EPICHLOAE
anti-insect alkaloid classes are more likely to be present
The capacity to produce the four classes of bioactive alka- in plants containing epichloae endophytes than are anti-
loids varies among epichloae taxa. Two classes of epi- vertebratealkaloids.
chloae-produced alkaloids – the ergot alkaloids and the The distribution of lolines, ergopeptines (but not other
indole-diterpenes – have anti-vertebrate activities, whereas ergot alkaloids) and peramine among sexual stroma-pro-
three classes – the ergot alkaloids, lolines and peramine – ducingEpichloe€spp.(thosecapableofhorizontaltransmis-
have anti-invertebrate properties (with the anti-insect sion) compared to strictly asexual, vertically transmitted
activities of epichloae-derived indole-diterpenes still uncer- Neotyphodium spp. was investigated by Leuchtmann,
tain). In the list of grass–epichloae symbiota compiled by Schmidt & Bush (2000) who observed greater production
Schardl et al. (2011), there are 29 symbiota for which the oflolinesandergopeptinesintheverticallytransmitteden-
presenceofallfourclassesofendophytealkaloidshasbeen dophytes. The reduced level of anti-insect alkaloids in
tested. Among these 29 symbiota, 86% produce at least grasseshostingsexuallyreproducingepichloaeisconsistent
oneofthethree established anti-insectclassesofalkaloids, with the dependence of the sexual Epichloe€spp. on insects
and 48% have at least two classes of anti-insect alkaloids for spermatization, or transfer of gametes, among fungi of
(Table 1). The common toxic endophyte isolate of N. coe- oppositematingtypes.
nophialum is the only endophyte known to produce all
three classes of anti-insect alkaloids. The anti-vertebrate
Clavicipitaceousendophytes of Convolvulaceae
alkaloids are less common than the anti-insect compounds
among this same set of 29 symbiota for which data are ThegenusPeriglandulaconsistsofclavicipitaceousepibiot-
available. Approximately one-half (15 of 29) of the symbi- ic fungal symbionts of the Convolvulaceae (morning
otacontainatleastoneclassofanti-vertebratecompound, glories) that produce ergot alkaloids in the seeds and, in
Table1. Distribution of anti-vertebrate and anti-insect alkaloids among epichloae–grass symbiota in which all four classes of bioactive
alkaloidshavebeenassayed*
Fungus Hostplant Anti-vertebratealkaloids† Anti-insectalkaloids‡
Epichloe€elymi Elymuscanadensis ERG ERG,PER
Epichloe€festucae Festucalongifolia ERG,IDT ERG,PER
E.festucae Festucaovina ERG ERG,PER
E.festucae Festucarubrasubsp.commutata ERG ERG,PER
E.festucae F.rubrasubsp.commutata – PER
E.festucae F.rubrasubsp.rubra IDT –
E.festucae F.rubrasubs.rubra ERG ERG
E.festucae Loliumgiganteum ERG ERG,LOL
Epichloe€typhina Loliumperenne – PER
Neotyphodiumaotearoae Echinopogonovatus – LOL
Neotyphodiumcoenophialum Loliumarundinaceum ERG ERG,LOL,PER
N.coenophialum L.arundinaceum – LOL,PER
Neotyphodiumhuerfanum Festucaarizonica – PER
Neotyphodiumgansuense Achnatheruminebrians ERG ERG
Neotyphodiumlolii Loliumperenne ERG,IDT ERG,PER
N.lolii L.perenne IDT PER
N.lolii L.perenne IDT –
N.lolii9E.typhinaisolateLp1 L.perenne ERG,IDT ERG,PER
Neotyphodiumsiegelii Loliumpratense – LOL,PER
Neotyphodiumstarrii Bromusanomalus ERG ERG,PER
Neotyphodiumsp.E55 Poaautumnalis – LOL,PER
Neotyphodiumsp.E4074 Loliumsp. – LOL,PER
Neotyphodiumsp.E4078 Loliumsp. ERG,IDT ERG,PER
Neotyphodiumsp. Festucaparadoxa – PER
Neotyphodiumsp. Festucasubverticillata – –
Neotyphodiumsp. Hordelymuseuropaeus – –
Neotyphodiumtembladerae F.arizonica – PER
Neotyphodiumtyphinum Poaampla – PER
Neotyphodiumuncinatum L.pratense – LOL
*RefertoSchardletal.(2011)fordetailsonsymbiota.
†ERG,ergotalkaloids;IDT,indole-diterpenes.
‡ERG,ergotalkaloids;LOL,loines;PER,permine.
Published2013.ThisarticleisaU.S.GovernmentworkandisinthepublicdomainintheUSA.
JournalofAppliedEcology2013©BritishEcologicalSociety,FunctionalEcology
8 D.G.Panaccioneet al.
some cases, the foliage of infected plants (Markert et al. amides and ergopeptines) have each been reported from
2008; Steiner et al. 2011). The two species described of the Convolvulaceae (Eich 2008), including the ergopeptine
Periglandula are fungi that form systemic infections in the ergobalansine (Jenett-Siems, Kaloga & Eich 1994), origi-
above-ground parts of their host plants and are vertically nally discovered in the clavicipitaceous fungi Balansia ob-
transmitted(Steineret al.2006).Epiphyticmyceliaarevis- tectawhichformepiphyticinfectionsofCenchrusechinatus
ible to the naked eye on young leaves (Fig. 1c). Steiner (Sandbur Grass) (Cyperaceae) (Powell et al. 1990). In
et al. (2011) have stated that there is no evidence that the addition to ergot alkaloids known from other Clavicipita-
fungi ever penetrate the host plant; however, the seed ceae, unique ergot alkaloids have been discovered in the
transmissibility of the fungi indicates that there must be Convolvulaceae, notably cycloclavine from Ipomoea hilde-
some internal growth of the fungus. Workers demon- brandtii, an African shrub (Stauffacher et al. 1969). All
strated that this fungus was responsible for ergot alkaloid reports of ergot alkaloids from the Convolvulaceae have
production when they observed that treatment of Ipomoea come from the speciose tribe Ipomoeeae (ca. 900 species)
asarifolia with fungicides resulted in the loss of epiphytic and show no clear phylogenetic pattern (Eich 2008); how-
mycelia concomitantly with loss of detectable ergot alka- ever, the phylogeny of this large family is still not clearly
loidsinthefoliage(Kuchtet al.2004).Unlikeotheroccur- resolved. Both major clades within the Ipomoeeae (Stefa-
rences of plant-associated Clavicipitaceae, Periglandula novic,Krueger&Olmstead2002)haveergotalkaloid-posi-
speciesaretheonlyclavicipitaceousfungiknowntoassoci- tive representatives, and there is variation within sections
ate with a dicotyledonous host (Steiner et al. 2006). These withrespecttothepresenceofergotalkaloids(Eich2008).
‘endophytes’ also appear to have a narrow host range: the Expanded sampling of the Convolvulaceae for ergot alka-
two described Periglandula species are chemically dissimi- loidswillsurelyrevealmorespeciesinfectedbyPeriglandu-
lar and occur on different host plants (Steiner et al. 2011). la. Extrapolation from available data would suggest
A third, undescribed, Clavicipitaceae from Ipomoea tri- upwards of 250 species of ergot alkaloid-positive
color, which does not form epiphytic mycelia, was detect- Ipomoeeae.
able via PCR and groups with described Periglandula Interestingly, loline and ergot alkaloids have been
species, yet it is also phylogenetically distinguishable detected in the seeds and foliage of one species, Argyreia
(Ahimsa-Mulleret al.2007). mollis (Tofern et al. 1999), suggesting Periglandula may
PriortothediscoveryofPeriglandula species,theoccur- also produce other classes of secondary metabolites found
renceofergotalkaloidsintheConvolvulaceaewasthought in other Clavicipitaceae. There have been no published
to be a case of convergent evolution or horizontal gene reports from the Convolvulaceae of the other two major
transfer (Steiner,Hellwig &Leistner 2008).Although pub- classes of clavicipitaceous secondary metabolites – indole-
lished evidence for Periglandula species colonization has diterpenes and peramine – although we do not know of
been provided for only three species of Convolvulaceae, anystudies that explicitlytested for them. There are,how-
ergot alkaloids are known to occur in many more species ever, reports of tremorgenic symptoms in livestock caused
in this diverse plant family (Eich 2008), each of which by grazing of foliage from ergot alkaloid-positive Convol-
likely harbours a species of Periglandula. Whereas ergot vulaceae, specifically caused by sheep feeding on Ipomoea
alkaloids were discovered in grasses due to their influence muelleri in Australia (Gardiner, Royce & Oldroyd 1965)
on agriculture (Lyons, Plattner & Bacon 1986), their dis- and sheep and cattle feeding on I. asarifolia in Brazil
covery in the Convolvulaceae followed from the work of (Araújo et al. 2008). Livestock grazing on grasses contain-
ethnobotanist Richard Schultes in Central America in the ing indole-diterpenes also suffer from tremorgenic symp-
late1930s(Schultes 1941;Schultes1969).Hereportedthat toms (Belesky & Bacon 2009). Because the tremoring
theseedsofTurbinacorymbosa(hosttoP. turbinae),called symptoms were caused by grazing on these two ergot
‘ololiuqui’bytheAztecs,andtheseedsofI. tricolor,called alkaloid-positive species (one of which is host to
‘badoh negro’ by the Zapotecs, were consumed ritualisti- P. ipomoeae),thepossibilitythatsomePeriglandulaspecies
cally for divination. Two decades later, Albert Hofmann produceindole-diterpenesshouldbeinvestigated.
isolated the lysergic acid amides ergine and lysergic acid The ecological effects of Periglandula infection or ergot
a-hydroxyethylamide from T. corymbosa (Hofmann & alkaloidpresenceintheConvolvulaceaehasyettobestud-
Tscherter 1960; Hofmann 1961), which were likely respon- ied. As Periglandula species are closely related to Neoty-
sibleforthehallucinogeniceffectsoftheseplants. phodium species endophytes and share some important
The discovery of ergot alkaloids in a dicotyledonous characteristics(e.g.verticaltransmissionandproductionof
plant spurred several studies on the occurrence of ergot ergot alkaloids), their presence also may confer similar
alkaloids in the Convolvulaceae; Eich (2008) has critically benefits to their convolvulaceous hosts such as resistance
reviewed this work. In the genus Ipomoea (ca. 500 spp.), to herbivory (Clay & Schardl 2002). Unlike the cool sea-
79 species have been screened for ergot alkaloids and 23 son pooid grasses which host epichloae, there is striking
(29%)areunambiguouslypositive(Eich2008).Thegenera variationinlifehistoryandhabitatevenamongthelimited
Argyreia, Stictocardia and Turbina also have ergot alka- number of known ergot alkaloid-positive Convolvulaceae.
loid-positive representatives (Eich 2008). Moreover, the They include herbaceous twining vines (e.g. I. tricolor),
three major types of ergot alkaloids (clavines, lysergic acid woody lianas (e.g. Argyreia nervosa), sprawling vines (e.g.
Published2013.ThisarticleisaU.S.GovernmentworkandisinthepublicdomainintheUSA.
JournalofAppliedEcology2013©BritishEcologicalSociety,FunctionalEcology
Bioactivealkaloidsoffungalendophytes 9
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
beachesworldwide whereitgrowsasapioneer speciesjust
abovethehightideline(Devall1992).
Fig.5.Proposedpathwayforswainsonineandslaframinebiosyn-
While many studies in epichloae-grass systems demon-
thesis.PathwayisbasedonstudiesconductedinRhizoctonialegu-
strate increased resistance to foliar herbivory, resistance to
minicola(Harrisetal.1988b).
seedpredationmaybeanimportantdimensionofthePeri-
glandula-Convolvulaceae symbioses. Some studies have
indicatedthatalkaloidscanbepresentintheseedsbutnot lysosomal storage disease and altered glycoprotein synthe-
the foliage (Chao & Der Marderosian 1973; Jirawongse, sis(Hartley1971;Dorling,Huxtable&Vogel1978).
Pharadai & Tantivatana 1977). Convolvulaceae seeds can Recently, a fungal endophyte, Undifilum oxytropis
be large, often >5 mm in diameter (Verdcourt 1978), and (Pryor et al. 2009), previously described as an Embellesia
presumably represent a large investment of plant species (Wang et al. 2006), was reported to produce
resources. Additionally, many species are highly parasit- swainsonine in locoweeds (Braun et al. 2003). The Undifi-
ized by bruchine beetles (Coleoptera: Bruchinae) (Reyes, lum genus (Pleosporaceae) is closely related to the genera
Canto & Rodriguez 2009). The larvae of these beetles can Alternaria, Embellesia and Ulocladium (Pryor et al. 2009).
boreintotheseed,consumethecotyledonsorembryoand Undifilum species are only associated with swainsonine-
leave a characteristic circular exit hole. Reports of bru- containing Astragalus and Oxytropis species with one
chineparasitismratesrangefrom4to85%inI. pes-caprae exception,Undifilumbormuelleri,apathogenofthelegume
(Wilson 1977; Devall & Thien 1989), and 34 to 100% in Securigeravariathatdoesnotcontainswainsonine.Undifi-
I. leptophylla, a shrub found in the short grass prairies of lumspecieshavebeenfoundtobeassociatedwithswainso-
the Central United States (Keeler 1980, 1991). Whereas nine-containing Astragalus and Oxytropis species in North
several factors may contribute to this variation, especially America and China (Pryor et al. 2009; Yu et al. 2010;
visitation by ants to extra floral nectaries (Keeler 1980), Baucom et al. 2012). Like many epichloae and the known
populationdifferencesinergotalkaloidcontentmayplaya Periglandula species, Undifilum species associated with
role. Other possibilities are that the specialized bruchines locoweeds are vertically transmitted and have no apparent
associated with ergot alkaloid-positive Convolvulaceae sexual stage (Oldrup et al. 2010; Ralphs et al. 2011).
haveovercome theplant’sacquireddefencefromPeriglan- Undifilum species also appear to have a narrow host range
dula or that there is ongoing co-evolution between the asdifferentplantspeciesareassociatedwithuniqueUndifi-
plant–fungalsymbiotaandthebeetles. lumspecies(Pryoret al.2009;Baucomet al.2012).
In addition to the legumes, swainsonine occurs sporadi-
cally in two other plant families, the Convolvulaceae and
Endophytes of locoweedsand related taxa
the Malvaceae. In the Convolvulaceae, some Ipomoea and
Several species in the legume genera Astragalus, Oxytropis Turbina species are reported to contain swainsonine, for
andSwainsonahavebeenfoundtobetoxictograzinglive- example, I. carnea and T. cordata (de Balogh et al. 1999;
stock in the Americas, Asia and Australia (Marsh 1909; Dantas et al. 2007), while in the Malvaceae, Sida carpino-
Marsh & Clawson 1936; Gardiner, Linto & Aplin 1969; folia is reported to contain swainsonine (Colodel et al.
Huang, Zhang & Pan 2003). Locoism, a neurologic dis- 2002). Like the legumes, swainsonine was identified in the
ease, was first noted by the Spanish conquistadors, and plantspeciesassociatedwiththesefamiliesduetolivestock
again during the settlement of Western North America by poisoningandsubsequenteconomicimpact.
pioneers (Marsh 1909; Marsh & Clawson 1936; Jones, Swainsonine is also reported to be produced by two
Hunt & King 1997). Clinical signs and pathology of loco- phylogenetically disjunct fungi, Rhizoctonia leguminicola
ism are similar in animals intoxicated by locoweed species (Ceratobasidiaceae) and Metarhizium anisopliae (Clavici-
and Swainsona species (James, Van-Kampen & Hartley pitaceae) (Schneider et al. 1983; Patrick, Adlard & Kesha-
1970; Panter et al. 1999). Swainsonine (Fig. 5), a trihydr- varz 1993). Rhizoctonia leguminicola is a fungal pathogen
oxyindolizidine alkaloid, was first identified as the active of red clover (Trifolium pratense) that causes black patch
principle in Swainsona canescens, a legume native to Aus- disease in the plant. Metarhizium anispoliae is an entomo-
tralia (Colegate, Dorling & Huxtable 1979), and subse- pathogen that attaches to the outside of an insect, grows
quently identified as the active principle in locoweeds internallyandcausesdeath.Therolesswainsonineplaysin
(Molyneux & James 1982). Swainsonine inhibits the eitherofthesebiologicalsystemshavenotbeenelucidated.
enzymes a-mannosidase and mannosidase II resulting in Like the ergot alkaloids, swainsonine appears to be more
Published2013.ThisarticleisaU.S.GovernmentworkandisinthepublicdomainintheUSA.
JournalofAppliedEcology2013©BritishEcologicalSociety,FunctionalEcology
10 D.G.Panaccioneet al.
widely distributed in fungi other than seed-transmitted matures (Cook et al. 2012). Finally, swainsonine concen-
endophytes. trations are greatest in floral parts and seeds (Grum et al.
Thebiosynthesisofswainsoninehasbeeninvestigatedin 2012),consistentwiththeoptimaldefencetheory.
the fungus R. leguminicola (Harris et al. 1988b). Swainso- There are few studies regarding the ecological role of
nine is derived from lysine which is converted into pip- swainsonine and how it responds to environmental
ecolic acid. Two precursors of swainsonine in the fungal changes. Swainsonine concentrations do not change in
biosyntheticpathwayweredetectedintheshootsofDiablo respondtoclippingusedtosimulateherbivory,nordoesit
locoweed(Astragalusoxyphysus)(Harriset al.1988a,b);as detergrazingasanimalsbecomeprogressivelymoreintoxi-
aresult,Harriset al.(1988a)proposedthatthebiosynthet- cated (Ralphs et al. 2002; Pfister et al. 2003). In fact ani-
ic pathway of swainsonine in R. leguminicola is similar to mals take 2–3 weeks to show clinical signs and continue
the pathway in locoweeds, where swainsonine was later grazinglocoweedsafterbecomingintoxicated(Pfisteret al.
foundtobeproducedbyUndifilumspecies. 2003). Activity of swainsonine against insects, fungi or
Supportforthefactthatswainsonineisafungal-derived bacteria has not been definitively tested in published stud-
secondary metabolite in locoweeds is based on the follow- ies, although preliminary results show that swainsonine
ing observations: (i) locoweed plants infected with Undifi- has no effect on some insect species (Parker 2008).
lum species contain swainsonine; (ii) plants derived from Legumes are known for forming symbioses with N-fixing
Astragalus and Oxytropis embryos in which the seed coat, bacteria and investigators found that swainsonine concen-
the primary location of Undifilum, was removed have no trations were greater in plants inoculated with one strain
detectable swainsonine, or have concentrations less than of Rhizobium but not others (Valdez Barillas et al. 2007),
0001% (Oldrup et al. 2010; Grum et al. 2012); (iii) plants suggestinganinteractionbetweenthetwoclassesofsymbi-
!
derived from fungicide-treated Astragalus and Oxytropis onts. An alternative interpretation is that the improved
seeds have no detectable swainsonine or have concentra- nitrogen status of the host may have increased substrate
tions less than 0001% (Grum et al. 2012); (iv) Undifilum availability for swainsonine production; however, no con-
!
species isolated from locoweeds produce swainsonine in sistent differences in swainsonine concentrations were
pure culture (Braun et al. 2003); (v) plants derived from observed in locoweed plants, whether nitrogen deficient or
embryos that were inoculated with Undifilum have swains- adequate, when nitrogen was supplied through fertilizer
onine concentrations greater than 001% (Grum et al. (Delaney et al. 2011). Lastly, swainsonine concentrations
!
2012); and (vi) rats fed U. oxytropis developed lesions and were shown to increase slightly in response to water stress
clinical signs similar to those fed swainsonine-containing in some locoweed species but not others (Vallotton et al.
Oxytropislambertii(McLain-Romeroet al.2004). 2012).
Swainsonine concentrations vary greatly among species, It has not been determined whether swainsonine is
varieties and populations. For example, Astragalus species plant- or fungal-derived in the legume Swainsona canes-
generally have greater swainsonine concentrations than do cens,intheConvolvulaceousgeneraofIpomoeaandTurbi-
Oxytropis species in North America (Ralphs et al. 2008) na, or in Sida carpinfolia, a species of the Malvaceae
whiledifferentvarietiesofO. lambertiivarygreatlyintheir family. The presence of swainsonine in these species may
swainsonineconcentrations(Gardner,Molyneux&Ralphs be a case of convergent evolution or horizontal gene
2001). Additionally, in toxic populations of locoweeds, transfer; however, due to the sporadic occurrence of
two chemotypes of plants have been identified, namely swainsonine in these genera, it seems probable that a
chemotype one plants, which contain swainsonine concen- swainsonine-producingfungalendophyteisassociatedwith
trations >001%, and chemotype two plants, which have these taxa that contain swainsonine. In fact, recent
!
concentrations <001% (generally near 0001% or not research suggests that a fungal endophyte is present in the
! !
detected) (Cook et al. 2009, 2011). These two chemotypes swainsonine-positive taxa, S. canescens and Ipomoea car-
differ significantly in the amount of endophyte they nea (Cook and Beaulieu, unpublished data). The fungal
contain which may explain the difference in swainsonine endophyte associated with S. canescens appears to be a
concentrations(Cooket al.2009,2011). novel Undifilum species, while the endophyte associated
Swainsonine and endophyte amounts have been investi- with I. carnea appears to belong to an Ascomycete family
gated in different plant parts at different phenological not related to the Pleosporaceae family that contains
stages(Cooket al.2012).Swainsonineisfoundinallplant Undifilum. Preliminary data suggest that these endophytes
parts although concentrations are greater in above-ground produce swainsonine in vitro, are vertically transmitted,
partsthaninbelow-groundparts(Cooket al.2009, 2011). andhaveanarrowhostrange.
Endophytic Undifilum species also are found in all plant A comparison of the locoweed/swainsonine-producing
parts with only small quantities found in the root (Cook endophyte system(s) to those involving clavicipitaceous
et al. 2009, 2011). The root crown appears to be a major endophytes reveals some similarities but also significant
reservoir for the endophyte during the following year’s differences (Table 2). (Note that the Convolvulaceae are
growth as many locoweeds are perennial plants (Cook interesting in that individual species have symbioses with
et al. 2009, 2011), and swainsonine and endophyte Periglandula species, presently undescribed swainsonine
amounts increase in above-ground parts as the plant producers, or in some cases no endophytes.) First, in the
Published2013.ThisarticleisaU.S.GovernmentworkandisinthepublicdomainintheUSA.
JournalofAppliedEcology2013©BritishEcologicalSociety,FunctionalEcology