Swainsonine-Containing Plants and Their Relationship to EndophyticFungiDaniel Cook,* Dale R. Gardner, and James A. Pfister

Poisonous Plant Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 1150 East 1400 North, Logan,Utah 84341, United States

ABSTRACT: Swainsonine, an indolizidine alkaloid with significant physiological activity, is an α-mannosidase and mannosidaseII inhibitor that alters glycoprotein processing and causes lysosomal storage disease. Swainsonine is present in a number of plantspecies worldwide and causes severe toxicosis in livestock grazing these plants. Consumption of these plants by grazing animalsleads to a chronic wasting disease characterized by weight loss, depression, altered behavior, decreased libido, infertility, anddeath. This review focuses on the three plant families and the associated taxa that contain swainsonine; the fungi that produceswainsonine, specifically the fungal endophytes associated with swainsonine-containing taxa; studies investigating the plant,endophyte, and swainsonine relationship; the influence of environmental factors on swainsonine concentrations in planta; andareas of future research.

KEYWORDS: swainsonine, endophyte, locoweed, Ipomoea

■ INTRODUCTION

Several species among three genera in the Fabaceae (Fabales)family, Astragalus, Oxytropis, and Swainsona, are toxic to grazinglivestock throughout the world.1−4 Many of the species withinthese genera are nontoxic and are important forages; however,several species are toxic to both livestock and wildlife. “Loco” isa Spanish word for crazy and has been used by the people ofNorth America for over a century to describe the neurologicdisease and behavior of poisoned animals, whereas “locoweed”has been used to describe the plants responsible for thepoisoning. De Soto and other Spanish explorers in thesouthwestern United States first noted locoism in their horses.As settlers and stockmen moved west on the American frontier,locoweeds along with other poisonous plants emerged as aserious livestock-poisoning problem. Due to the significantnumber of livestock losses attributed to locoism throughout thewest, the USDA Bureau of Plant Investigations established aresearch station at Hugo, CO, USA, in 1905.5 A subsequentseries of feeding and grazing experiments conducted by C. D.Marsh demonstrated that Astragalus and Oxytropis species werethe cause of locoism and documented many of the clinicalsymptoms associated with this neurologic disease.1,2,5

Animals intoxicated by Astragalus, Oxytropis, and Swainsonaspecies show similar clinical signs and pathology.6,7 Swainso-nine (Figure 1), an indolizidiine alkaloid, was first identified as

the bioactive principle of this neurologic disease in Swainsonacanescens,8 a legume native to Australia. Swainsonine was lateridentified as the active principle in Astragalus lentiginosus in theUnited States.9 Locoweeds are defined as Astragalus andOxytropis species that cause locoism and contain swainsonine,whereas Swainsona species that contain swainsonine are oftenreferred to as “poison peas”. Swainsonine, an alkaloid withsignificant physiological activity, is an α-mannosidase andmannosidase II inhibitor that alters glycoprotein processing andcauses lysosomal storage disease.8,10,11 Consumption of theseplants by grazing animals leads to a chronic neurologic diseasecharacterized by weight loss, depression, altered behavior,decreased libido, infertility, abortion, birth defects, anddeath.7,12 Swainsonine-containing plants are estimated tocause millions of dollars in livestock losses annually withinthe United States alone.13 Management solutions, intermittentdrought, and a greater awareness by livestock producers havelikely decreased the monetary losses; however, a recent costanalysis has not been performed.Swainsonine-containing Astragalus and Oxytropis species are

primarily found throughout the temperate regions of thenorthern hemisphere and South America, whereas Swainsonaspecies are found in Australia.5 Several species within eachgenus are reported to contain swainsonine (Table 1; Figure2).14−25 Detection methods used include chromatographycoupled with mass spectrometry,24 thin-layer chromatogra-phy,17 and a jack bean (Canavalia ensiformis) α-mannosidaseinhibiton assay.14,15 The verification of swainsonine insuspected plant species should be done using chromatographycoupled with mass spectrometry. Caution should be used about

Special Issue: Poisonous Plant Symposium, Inner Mongolia

Received: January 29, 2014Revised: April 23, 2014Accepted: April 24, 2014Published: April 24, 2014

Figure 1. Structure of the indolizidine alkaloid swainsonine.

Review

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the presence of swainsonine without this verification as theother methods are preliminary and not definitive.Swainsonine concentrations in locoweeds vary among

species, varieties, and populations, often ranging from notdetected to >0.2%.16,24 For example, in Astragalus amphioxysmean swainsonine concentrations were <0.001%, whereas inspecies such as Astragalus mollissimus var. mollissimus meanconcentrations were >0.1%.16 Additionally, in some varieties ofOxytropis lambertii and A. mollissimus, such as O. lambertii var.articulata, O. lambertii var. lambertii, and A. mollissimus var.thompsonii, mean swainsonine concentrations were <0.001% inall populations, yet in other varieties within these species meanconcentrations were >0.01%.16,24

The purpose of this review is to highlight past and currentresearch investigating (1) other swainsonine-containing plantsbesides those in the Fabaceae family; (2) fungi that produceswainsonine; (3) the plant, endophyte, and swainsoninerelationship; (4) environmental factors that influence swainso-nine concentrations; and (5) areas for future research.

■ OTHER SWAINSONINE-CONTAINING PLANTS

In addition to the Fabaceae family, swainsonine has beendocumented in two other plant families: the Convolvulaceae(Solanales) and the Malvaceae (Malvales) (Figure 2; Table 3).Swainsonine was identified in these plant families afternumerous episodes of livestock poisoning by these plants andsubsequent economic impact. Many of the species within thesefamilies are nontoxic and are important forages. Clinical signsand pathology of animals poisoned by these plants are similarto those of animals poisoned by swainsonine-containinglegumes.6,26−31 Two genera in the morning glory family,Ipomoea and Turbina, contain swainsonine. Seven taxa amongthese two genera, Ipomoea carnea, Ipomoea polpha, Ipomoeariedelii, Ipomoea sericophylla, Ipomoea sp. Q6 (aff. calobra),Ipomoea verbascoidea, and Turbina cordata, contain swainsonine(Table 2).26−30 Swainsonine concentrations also vary consid-erably in some of these species.30 These species are found inSouth America or Australia with the exception of I. carnea and I.verbascoidea. I. carnea can be found throughout the tropics

Table 1. Summary of the Astragalus, Oxytropis, and Swainsona Species Suspected of Causing Locoism and/or Reported To HaveSwainsonine, Swainsonine Detection Methods, the Associated Undif ilum Endophyte (If Known), and Endophyte DetectionMethods

detection

taxa country swainsonine detection endophyte culture PCR refs

A. allochrous USA, Mexico enzyme 14, 15A. amphioxys USA LC-MS Undif ilum sp. x 16A. asymmetricus USA TLC 17A. bisulcatus USA TLC, enzyme 14, 15, 17A. didymocarpus USA TLC 17A. drummondii USA enzyme 14, 15A. emoryanus USA TLC, enzyme, MS 17, 18A. f lavus USA TLC 17A. humistratus USA 19A. lentiginosus USA, Mexico TLC, LC-MS Undif ilum fulvum x x 16, 17A. lonchocarpus USA enzyme 14, 15A. missouriensis USA enzyme 14, 15A. mollissimus USA, Mexico TLC, enzyme, LC-MS Undif ilum cinerum x x 14−17A. nothoxys USA, Mexico 20A. oxyphysus USA TLC 17A. pehuenches Argentina, Chile TLC 21A. praelongus USA TLC/enzyme 14, 15, 17A. pubentissimus USA LC-MS Undif ilum sp. x x 16A. purshii USA 19A. pycnostachyus USA TLC 17A. strictus China GC-MS Undif ilum oxytropis x 22A. tephrodes USA enzyme 14, 15A. thurberi USA enzyme 14, 15A. variabilis China GC-MS, LC-MS Undif ilum oxytropis x x 22, 23A. wootoni USA, Mexico TLC, LC-MS Undif ilum sp. x x 16, 17O. besseyi USA 20O. campestris USA, Canada 20O. glabra China GC-MS, LC-MS Undif ilum oxytropis x x 22, 23O. glacialis China GC-MS Undif ilum oxytropis x 22O. kansuensis China GC-MS Undif ilum oxytropis x 22O. lambertii USA, Canada TLC, enzyme, GC-MS, LC-MS Undif ilum oxytropis x x 14, 15, 17, 24O. ochrocephala China GC-MS Undif ilum oxytropis x 22O. sericea USA, Canada TLC, enzyme, LC-MS Undif ilum oxytropis x x 14−17, 24O. sericopetala China GC-MS Undif ilum oxytropis x 22S. canescens Australia GC-FID, LC-MS Undif ilum sp. x x 25, 37S. galegifolia Australia GC-FID 25S. greyana Australia GC-FID 25

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worldwide, whereas I. verbascoidea is an African species byherbarium record,32 although the report of its containingswainsonine comes from Brazil.29 Only a single species of theMalvaceae, Sida carpinifolia, contains swainsonine (Table 2).31

■ SWAINSONINE-PRODUCING FUNGIFor many years it was thought that swainsonine was a plant-derived secondary metabolite; however, recent researchsuggests otherwise. A swainsonine-producing ascomycete,

Undifilum oxytropis (Pleosporales), an endophyte associatedwith O. lambertii, Oxytropis kansuensis, and Oxytropis sericea, isreported to produce swainsonine (Table 1; Figure 2).33,34

Initial studies described this endophyte as an Embellisiaspecies.35 The Undif ilum genus (Pleosporales) is phylogeneti-cally related to the genera Alternaria, Embellisia, andUlocladium.34 On the basis of molecular and morphologicalcharacteristics, three unique Undif ilum species, Undifilumcinerum, Undifilum fulvum, and Undifilum oxytropis, have beenfound and identified among the three North Americanlocoweed taxa A. mollissimus, A. lentiginosus, and O. sericea,respectively (Table 1).34,36 This is in contrast to theswainsonine-containing Astragalus and Oxytropis speciesexamined to date in China, which all have the same endophytespecies, U. oxytropis (Table 1).22 A swainsonine-producingUndif ilum sp. has also been identified in the Australian speciesS. canescens (Table 1); molecular and morphological datasuggest it is a novel species as well.37 To date, the swainsonine-containing Astragalus, Oxytropis, and Swainsona spp. evaluatedfor the presence of an Undif ilum spp. by PCR are associatedwith an Undif ilum spp. (Table 1).16,22,37 Undif ilum spp. can becultured from seeds and above-ground plant tissues; however,they have not been cultured from below-ground tissues.33

Undif ilum spp. are primarily found in the seed coat as they canbe cultured from the seed coat but not the embryo.38 As aresult, plants derived from the embryo where the seed coat hasbeen removed contain little or no swainsonine.38 Usingmicroscopy, U. oxytropis was detected growing between cellsin vegetative tissues of O. sericea, providing further support thatit is an endophyte.39

Recently, a swainsonine-producing endosymbiont in theascomycete order Chaetothyriales was isolated from I. carnea(Table 1).40 This endosymbiont was cultured from seeds and

Figure 2. Photographs of swainsonine-containing plants: (A)Oxytropis lambertii; (B) Astragalus lentiginosus; (C) Astragalusmollissimus; (D) Swainsona galegifolia; (E) Ipomoea carnea; (F) Sidacarpinifolia. Photographs A, B, and C were provided courtesy andcopyright of Al Schneider, http://www.swcoloradowildflowers.com;photograph D was provided courtesy of Moonlight0551, http://commons.wikimedia.org; photograph E was provided courtesy of J. M.Garg, http://commons.wikimedia.org; and photograph F was providedcourtesy of Franklin Riet-Correa.

Table 2. Summary of the Ipomoea, Turbina, and Sida Species Reported To Have Swainsonine, Swainsonine Detection Methods,the Associated Endosymbiont (If Known), and Endophyte Detection Methods

detection

taxa country swainsonine detection endophyte culture PCR refs

I. carnea tropics worldwide GC-MS, LC-MS Chaetothyriales sp. x x 26, 40I. polpha Australia GC-MS 27I. riedelii Brazil LC-MS 28I. sericophylla Brazil LC-MS 28I. sp. Q6 (aff. calobra) Australia GC-MS 27I. verbascoidea Brazil and Africa LC-MS 29, 32T. cordata Brazil LC-MS 30S. carpinifolia Brazil LC-MS 31

Table 3. Important Characteristics of Plant−FungalSymbiota Producing Swainsonine Considered in ThisReview

Fabaceae Convolvulaceae Malvaceae

host clade eudicots eudicots eudicotshost growthform

herbaceous mostly woody vines(also shrubs, trees,herbaceous vines)

herbaceous

host genera Astragalus,Oxytropis, andSwainsona

Ipomoea and Turbina Sida

fungal order/family

Pleosporales/Pleosporaceae

Chaetothyriales/Chaetothyriaceae (?)

?

endophytetransmission

strictly verticaltransmission (?)

strictly verticaltransmission (?)

?

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plants and detected by PCR in plants containing swainsonine.Unlike the locoweed endophyte, this endosymbiont grows as anepiphyte on the leaf surface of plants containing swainsonine(Figure 3). The epiphyte/endosymbiont was not observed on

the surface of leaves from plants lacking swainsonine that werederived from fungicide-treated seeds. Plants derived fromfungicide-treated seeds did not contain swainsonine but rathercontained calystegines, nortropane alkaloids that are alsoglycosidase inhibitors. The calystegines are also found atsimilar concentrations in the control non-fungicide-treatedplants.40

Swainsonine has been detected in other phylogenticallydisjunct groups of Basidiomycetes and/or Ascomycetes.Rhizoctonia leguminicola (Cantharellales), a basidiomycete, is afungal pathogen of red clover (Trifolium pratense) that causesblack patch disease.41 R. leguminicola produces swainsonine aswell as the indolizidine alkaloid slaframine, which is responsiblefor the slobbers syndrome in horses.41,42 Recent data suggestthat this fungus is misclassified and is actually an ascomycete(R. Creamer, personal communication). Metarhizium anisopliae(Hypocreales), an ascomycete, produces swainsonine and is anentomopathogen that attaches to the outside of an insect,grows internally, and causes death.43 Lastly, a preliminary studysuggests two ascomycetes, Fusarium trinctum and a Schizo-phyllum sp., may also produce swainsonine.44 These fungi wereisolated from Chinese swainsonine-containing Astragalus andOxytropis species and their rhizosphere soils. More research isneeded to determine if F. trinctum and the Schizophyllum sp.produce swainsonine as there was no detailed analytical workperformed such as mass spectrometry coupled to liquid or gaschromatography.

■ PLANT/ENDOPHYTE RELATIONSHIPMultiple factors may influence the interaction of plant andendophyte and ultimately determine swainsonine concentra-tions in planta. Some considerations include, but are not limitedto, different plant parts and the phenological stages of the plant.

Other factors include, but are not limited to, different plantspecies, varieties, and populations as well as the respectiveendophyte associated with each.Secondary metabolites often differ among plant parts and as

a function of plant phenology. Swainsonine is found in all plantparts with greater concentrations in above-ground parts than inbelow-ground parts in Astragalus spp. and O. sericea (Figure4A).45,46 Furthermore, swainsonine concentrations vary season-

ally or as a function of phenology.47,48 In O. sericea, swainsonineconcentrations increase in above-ground tissues throughout thegrowing season until the plant reaches maturity (Figure 4A).47

Young newly developing tissues have the least amount ofswainsonine, whereas more mature tissues have moreswainsonine.47 As plants senesce, swainsonine concentrationsdecrease significantly.47 Likewise, the endophyte Undif ilum spp.is found in all plant parts, again with greater concentrations inabove-ground parts than below-ground parts with the exception

Figure 3. Photograph of Ipomoea carnea leaf showing theChaetothyriales fungal endosymbiont that produces swainsonine.

Figure 4. (A) Swainsonine concentrations (%) and (B) endophyteamounts (pg/ng total DNA) in different plant parts of Oxytropis sericeafrom Cuchara, CO, USA, as a function of phenological stage. Meanswainsonine concentrations and endophyte amounts ± the standarderror at the different phenological stages (stage 1, vegetative; stage 2,early flower; stage 3, late flower/early pod; stage 4, full pod/matureseed; stage 5, senesced) are shown. Adapted from data presented inCook et al.47

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of the root crown (Figure 4B).45,46 The root crown hasendophyte amounts more similar to those of above-groundparts, and because many locoweeds are perennial plants, thismay serve as a reservoir for the endophyte for the followingyear’s growth.45,46 In O. sericea, endophyte amounts increase inabove-ground parts throughout the growing season until theplant reaches maturity (Figure 4B).47 Swainsonine andendophyte amounts are highly correlated over the growingseason in O. sericea, indicating that endophyte amount plays acritical role in determining swainsonine concentration in theplant.47,48

Environment and genotype of the plant and/or endophyte oran interaction of these factors may influence swainsonineconcentrations. Swainsonine concentrations differ spatiallyamong populations of O. sericea; for example, meanconcentrations were approximately 3 times greater in plantsfrom Cuchara, CO, USA (0.40%), compared to plants fromPark Valley, UT, USA (0.15%) (Figure 5A).47 The followingobservations suggest that concentration differences betweenthese populations are due to the genotype of the endophyte.First, differences in swainsonine concentrations betweenpopulations persisted when plants from these two locationswere grown in a common garden (Figure 5B).49 Second, therespective endophyte, U. oxytropis, from each populationproduced different amounts of swainsonine in vitro whengrown under common culture conditions (Figure 5C).49 Third,a cross inoculation experiment, wherein the endophyte fromone population was inoculated onto the embryo from the otherlocation, demonstrated that the endophyte genotype, not the

plant genotype nor differences in endophyte amount, wereresponsible for differences in swainsonine concentrationbetween the two populations (Figure 5D).49 Although nodifferences were observed in the internal transcribed spacersequence between endophyte isolates from the two popula-tions,49 other genotypic differences may be present that explainthe difference in swainsonine production.Data suggest that the interaction of the plant species and

endophyte genotype is a key factor in determining swainsonineconcentrations in planta. First, different Astragalus andOxytropis species had similar rank order swainsonineconcentrations in field collections and greenhouse-grownplants. For example, Astragalus wootoni had the highest meanswainsonine concentration followed by similar concentrationsin A. lentiginosus and A. mollissimus var. mollissimus, and thelowest concentrations were noted in O. sericea.16,50 Second,similar trends in rank order swainsonine concentrationsbetween field collections and greenhouse-grown plants wereobserved from the highest to lowest, respectively, in A.mollissimus var. mollissimus, O. sericea, A. mollissimus var.bigelovii, and A. mollissimus var. matthewsii or var. thomp-sonii.51,52 In the latter example the trends in rank orderswainsonine concentrations remained the same whether or notthe plants were grown under optimum conditions, under waterstress, or with a deficiency in nitrogen.51,52 It is not known ifthe respective endophyte from each of above species producesdifferent amounts of swainsonine; however, results observed forswainsonine production between the U. oxytropis isolates foundin the Cuchara, CO, USA, and Park Valley, UT, USA, O. sericea

Figure 5. Swainsonine concentrations (%): (A) field collections from two populations, Cuchara, CO, USA (C), and Park Valley, UT, USA (PV), ofOxytropis sericea; (B) common garden performed in greenhouse of two O. sericea populations; (C) common garden of fungal endophyte isolatedfrom two O. sericea populations; (D) common garden of cross inoculation experiment (location: plant/endophyte). Figure adapted from datapresented in Cook et al.49

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populations suggest this may be the case.49 Caution should beused not to extrapolate beyond the populations of thehighlighted taxa because other populations of these same taxamay have different mean swainsonine concentrations.Swainsonine concentrations differ among individuals within

toxic populations of Astragalus and Oxytropis spp.16,24

Swainsonine concentrations segregate into two groups, namely,chemotype 1 plants, which have swainsonine concentrations>0.01%, and chemotype 2 plants, which have concentrationsmuch <0.01% (generally near 0.001% or not detected).45,46

Initial observations showed a correlation between swainsonineconcentrations and the presence of the endophyte, Undif ilum,as it could be cultured from chemotype 1 plants and was notcultured from chemotype 2 plants.53 Further studies confirmedthese culturing results and demonstrated that the endophytecould be detected by PCR in both groups.16 These twochemotypes differed significantly in the amount of endophytethey had, further supporting the differences in swainsonineconcentrations of the respective host plants.45,46 Additionalstudies demonstrated that seeds collected from each corre-sponding chemotype also differed significantly in swainsonineand endophyte amounts as well.54

Endophytes can be horizontally and vertically transmitted.55

The following observations support the hypothesis thatUndif ilum spp. are vertically transmitted from seed to progeny.Progeny derived from seeds of Astragalus and Oxytropischemotype 1 plants had variable swainsonine and endophyteamounts consistent with both chemotype 1 and chemotype 2plants.50 Progeny derived from seeds of a chemotype 2 planthad swainsonine and endophyte amounts consistent withchemotype 2 plants.50 Chemotype 1 seeds and their resultingprogeny, with variable amounts of both swainsonine andendophyte, suggest that there is imperfect transmission of theendophyte, resulting in both chemotypes. Chemotype 2 seedsand their resulting progeny suggest that these seeds lack somecritical amount of endophyte necessary to produce sufficientswainsonine to be classified as a chemotype 1 plant.50

Seed endophyte amounts and/or a plant endophyte bygenotype interaction may be important factors in determiningswainsonine concentrations in chemotype 1 and 2 plants. Thefollowing observations suggest that seed endophyte amounts atgermination are a major determinant in the production ofswainsonine and thus the classification of a plant as chemotype1 or 2. First, plants derived from chemotype 1 seeds, in whichthe amount of endophyte was reduced by seed coat removal orfungicide treatment, had swainsonine and endophyte amountsconsistent with those of chemotype 2 plants.54 Results from theseed coat removal experiment were consistent with previousresults reported by Oldrup et al.38 Second, plants derived fromUndif ilum-inoculated chemotype 1 and chemotype 2 embryoshad swainsonine and endophyte amounts consistent with thoseof chemotype 1 plants.54 Similar results were observed for bothAstragalus and Oxytropis species. If a plant endophyte bygenotype interaction was a key determinant, one would haveexpected that plants derived from an inoculated chemotype 2embryo would have had swainsonine and endophyte amountssimilar to those of a chemotype 2 plant.54

■ INFLUENCE OF ENVIRONMENTAL FACTORS ONSWAINSONINE CONCENTRATIONS

As swainsonine is an alkaloid, one may suspect that nitrogenavailability could influence its production. Astragalus andOxytropis species are legumes and are known for forming

symbioses with nitrogen-fixing bacteria, thus altering thenitrogen status of plants.56 Swainsonine concentrationsincreased in plants inoculated with one strain of Rhizobiumbut not others, suggesting an interaction between the twoclasses of symbionts.56 The Rhizobium-inoculated plants withthe positive swainsonine response had more leaves, moreclusters, and greater leaf biomass. These changes inswainsonine and plant physiological measures likely resultedfrom increased nitrogen availability to the plant andendophyte.56 However, others have shown that when nitrogenwas supplied through fertilizer to locoweed species withdifferent mean swainsonine concentrations, there were noconsistent differences in swainsonine concentrations acrosslocoweed taxa either at deficient or optimum nitrogenamounts.51 All species regardless of mean swainsonineconcentration responded positively to nitrogen in terms ofbiomass and leaf photosynthesis and pigments.51 Additionally,other experiments showed no differences in swainsonineconcentrations in O. sericea or U. oxytropis when grownunder optimum or deficient nitrogen conditions.38 Theseresults suggest that a given endophyte may produce a definedconentration of swainsonine per unit biomass notwithstandingnitrogen availability. It is critical to note that the total pool ofavailable swainsonine is higher in plants under optimumnitrogen conditions because plant biomass is increased.Water status of the plant may influence swainsonine

concentrations. Swainsonine concentrations were reported tobe significantly greater compared to controls in O. sericea plantsand U. oxytropis isolates cultured from these plants that werewater-stressed.38 Further studies by Vallotton et al.52

demonstrated that swainsonine concentrations increased insome species in response to water deficit but not in others suchas O. sericea. Swainsonine concentrations increased only in taxawith medium and high swainsonine concentrations. In thespecies where swainsonine concentrations did change, concen-trations changed only after the third water deficit period.Furthermore, in the various plant species there was littleevidence for a growth trade-off in physiological measures suchas shoot and root biomass, relative growth rate, or photo-synthetic rate from taxa differing in mean swainsonineconcentrations. The differential effect of water stress in O.sericea may be a result of plants being grown in tissue culture onartificial media in one experiment, whereas in the otherexperiment the plants were grown in soil in pots.38,52

It is unclear if herbivory from insects or large ungulatesinfluences swainsonine production. Swainsonine concentrationsdid not change in response to clipping used to simulateherbivory.57 There are no reports investigating the bioactivity ofswainsonine against insects, fungi, or bacteria, with theexception of one preliminary study that suggested swainsoninehad no effect on a native weevil herbivore of locoweeds.58

Lastly, swainsonine appears not to deter grazing by largeungulates, as animals take 2−3 weeks to show clinical signs andcontinue grazing locoweeds after becoming intoxicated.59

■ FUTURE DIRECTIONSIn the past 15 years, knowledge concerning swainsonine andfungal relationships has changed dramatically. In summary, thefollowing observations support the assertion that swainsonine isa fungal-endophyte-derived secondary metabolite and is notproduced by the host plant in the legume genera and I. carnea:(1) Legume species and I. carnea that contain swainsonine areinfected with Undif ilum species22,33−37 or a Chaetothyriales

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endosymbiont,40 respectively. (2) Plants derived fromfungicide-treated swainsonine-containing legume and I. carneaseeds have no detectable swainsonine or have concentrations<0.001%.40,54 (3) Undif ilum species and the Chaetothyrialesisolate that were isolated from their respective hosts produceswainsonine in vitro.22,33,34,37,49 Additional studies on nativelegumes demonstrate further that swainsonine is a fungal-endophyte-derived secondary metabolite. First, plants derivedfrom Astragalus and Oxytropis embryos where the seed coat wasremoved have no detectable swainsonine or have concen-trations <0.001%.38,54 Second, plants derived from Undif ilum-inoculated embryos have swainsonine concentrations >0.01%.54

There are yet unanswered questions that would be importantto solving poisoning problems worldwide. The identification ofplant taxa containing swainsonine defined to date are primarilya result of episodes of livestock poisoning that result in theplant screening for swainsonine. A systematic examination forswainsonine in taxa from the six plant genera that containswainsonine, namely, Astragalus, Oxytropis, Swainsona, Ipomoea,Turbina, and Sida, would be valuable to assess future risk. Amodified swainsonine extraction method that was recentlypublished60 allows for the rapid extraction of hundreds ofsamples simultaneously and could be used to accomplish thisobjective. Additionally, herbarium specimens in which a smallamount of material is removed from a voucher specimen couldbe screened rather than using field collections. This wouldfacilitate the LC-MS screening of hundreds of taxa in a shortamount of time, as verified by a recent paper that showsswainsonine can be detected in herbarium samples.23 This samematerial could be screened for the presence of thecorresponding fungal endophyte. Furthermore, the presenceor absence of swainsonine could be validated in the legumeslisted in Table 1. This approach would strengthen thefoundation for future work on genetic and environmentalfactors related to swainsonine and endophyte concentrations.The biosynthesis of swainsonine should be investigated to

identify and characterize the genes that encode the proteins inthe swainsonine biosynthetic pathway. To facilitate this work,the biochemical pathway for swainsonine biosynthesis has beeninvestigated in the fungi R. leguminicola and M. anisopliae.61,62

Swainsonine is derived from lysine, which is converted intopipecolic acid. Two precursors of swainsonine in the R.leguminicola biosynthetic pathway were also detected in theshoots of Diablo locoweed (Astragalus oxyphysus).61,63 As aresult, the investigators proposed that the biosynthetic pathwayof swainsonine in R. leguminicola is similar to the pathway inlocoweeds.63 Subsequently, in Astragalus and Oxytropis species,swainsonine has been shown to be produced by Undif ilumspecies,33,34,37 which supports the presence of a commonpathway in fungi. Investigators have recently developed atransformation system to genetically manipulate U. oxytropisthat will facilitate this work.64 These same investigators alsoknocked out the saccharopine reductase gene from U. oxytropis,a protein involved in lysine metabolism, and showed that thefungus had increased swainsonine amounts.65

A seed-transmitted endophyte is responsible for thebiosynthesis of swainsonine in the legume and morning gloryfamilies; thus, methods may be developed to disrupt thetransmission of this endophyte to seeds. The swainsonine-containing locoweed and morning glory species are nativeplants, so following the grass model by developing germplasmthat lacks the endophyte or has a nontoxic version of theendophyte is not an option, as these native range plants are not

planted in a pasture system like tall fescue and perennialryegrass.The preponderance of evidence to date indicates that

swainsonine is found in plants due to an interaction betweenthe host and a fungal endosymbiont. Additional research couldbe performed demonstrating that Chaetothyriales spp. areassociated with other swainsonine-containing morning gloryspecies as this has been demonstrated only for I. carnea.Research could also be performed to determine if the presenceof swainsonine in Sida carpinifolia is due to a fungalendosymbiont. We anticipate that a fungal endosymbiont willbe found in association with other swainsonine-containingmorning glory species and S. carpinifolia. Lastly, we anticipatethat whenever swainsonine is found in a given plant taxa,swainsonine will be associated with a fungal endosymbiontresponsible for its production.

■ AUTHOR INFORMATIONCorresponding Author*(D.C.) Phone: (435) 752-2941. Fax: (435) 753-5681. E-mail:daniel.cook@ars.usda.gov.NotesThe authors declare no competing financial interest.

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