Production of the Alkaloid Swainsonine by a Fungal Endophyte inthe Host Swainsona canescensDaniel S. Grum,† Daniel Cook,*,† Deana Baucom,‡ Ivan W. Mott,§ Dale R. Gardner,† Rebecca Creamer,‡

and Jeremy G. Allen⊥

†USDA/ARS Poisonous Plant Research Laboratory, 1150 East 1400 North, Logan, Utah 84341, United States‡Department of Entomology, Plant Pathology, and Weed Science, New Mexico State University, Las Cruces, New Mexico 88001,United States§USDA/ARS Forage and Range Research Laboratory, Logan, Utah 84322, United States⊥Animal Health Laboratories, Department of Agriculture and Food, 3 Baron-Hay Court, South Perth WA 6151, Australia

*S Supporting Information

ABSTRACT: Legumes belonging to the Astragalus, Oxytropis,and Swainsona genera have been noted by ranchers in theAmericas, Asia, and Australia to cause a neurologic diseaseoften referred to as locoism or peastruck. The toxin in theselegumes is swainsonine, an α-mannosidase and mannosidase IIinhibitor. Recent research has shown that in Astragalus andOxytropis species swainsonine is produced by a fungalendophyte belonging to the Undif ilum genus. Here Swainsonacanescens is shown to harbor an endophyte that is closelyrelated to Undif ilum species previously cultured fromlocoweeds of North America and Asia. The endophyteproduces swainsonine in vitro and was detected by PCR and culturing in S. canescens. The endophyte isolated from S. canescenswas characterized as an Undif ilum species using morphological and phylogenetic analyses.

Toxic groups of legumes belonging to the Astragalus,Oxytropis, and Swainsona genera have been noted by

ranchers in the Americas, Asia, and Australia.1−5 Consumptionof some Astragalus and Oxytropis species by livestock in theAmericas results in a condition known as “locoism”, whileconsumption of some Swainsona species in Australia causeslivestock, most commonly sheep, to become “peastruck”.1−3

Clinical signs and pathology are similar in animals intoxicatedby locoweed species and Swainsona species.6 Consumption ofthese plants by grazing animals leads to a chronic diseasecharacterized by weight loss, depression, altered behavior,decreased libido, infertility, and death, resulting in significanteconomic losses on an annual basis.7 Swainsonine (Figure 1), atoxic indolizidine alkaloid, was first identified in the Australianlegume Swainsona canescens.8 Subsequently swainsonine was

identified as the toxin in Astragalus and Oxytropis species thatcause locoism.9

As well as occurring sporadically in the Fabaceae,swainsonine has been reported in two other plant families.Ipomoea carnea and Turbina cordata,10,11 both members of theConvolvulaceae family, and Sida carpinofolia,12 a member of theMalvaceae family, are all reported to contain swainsonine. Thesporadic occurrence of some natural products such asswainsonine in plants brings into question the origin andsignificance of these products as chemotaxonomic markers.Three possible mechanisms may explain their sporadicoccurrence in unrelated taxa:13 first, the biosynthetic pathwaysof a given natural product may have evolved multiple timesover evolutionary history; second, the genes responsible for thebiosynthesis of a particular natural product may have beenhorizontally transferred between unrelated taxa;14 and third, thenatural product may be produced by a microbe associated withthe plant.Fungi including Rhizoctonia leguminicola (Ceratobasidiaceae)

and Metarhizium anisopliae (Clavicipitaceae) are reported toproduce swainsonine.15,16 Additionally, fungal endophytes ofthe genus Undif ilum (Pleosporaceae) found in the generaAstragalus and Oxytropis (Fabaceae),17−19 previously described

Received: April 1, 2013Figure 1. Structure of the indolizidine alkaloid swainsonine.

Note

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as Embellesia species,20 have been reported to be responsible forthe production of swainsonine.21 Endophytic Undif ilum speciesdescribed to date are vertically transmitted, and removal of theseed coat or fungicide treatment of seeds results in plants withlittle or no detectable swainsonine.22−24 Two chemotypes ofplants coexist within some Astragalus and Oxytropis popula-tions: chemotype 1 plants are defined as individuals containingswainsonine concentrations greater than 0.01% and quantita-tively greater amounts of Undif ilum, while chemotype 2 plantsare defined as individuals containing less than 0.01% (generallynot detected) and quantitatively smaller amounts of Un-dif ilum.25,26 Endophyte amounts are correlated with concen-trations of swainsonine in Astragalus and Oxytropis loco-weeds.27,28

Swainsona is a large genus of the Fabaceae family with allspecies being endemic to Australia and New Zealand. Thegenus Swainsona is closely related to the Astragalus andOxytropis genera of Asia and the Americas.29 Swainsonine-containing Astragalus and Oxytropis species have consistentlybeen found to be associated with fungal endophytes in thegenus Undif ilum.18,30 It seems probable that a fungal endophytemay be associated with Swainsona taxa that containswainsonine. Here we report the examination of the endemicAustralian species, S. canescens, for a fungal endophyte thatproduces swainsonine.Two species of Swainsona, S. canescens, which has a history of

poisoning sheep, and S. maccullochiana, which is anecdotallybenign,31 were examined for the presence of swainsonine andan endophyte. Swainsonine was detected at a concentration of0.6 mg/g in seeds of S. canescens, while it was not detected in S.maccullochiana seeds. A single fungal endophyte was success-fully cultured from six of eight S. canescens seeds, while nofungal endophytes were cultured from the 10 S. maccullochianaseeds examined. A fungal endophyte was cultured from seven ofeight S. canescens seed coats that were mechanically separatedfrom the embryo, while no fungal endophyte was cultured fromthe eight embryos. These results, where an endophyte can becultured from the seed coat but not the embryo, suggest thatthe endophyte resides primarily in the seed coat, which isconsistent with reports in Astragalus and Oxytropis that containswainsonine.22−24

The endophyte isolates from S. canescens all producedswainsonine in vitro as verified by GC-MS (Figure 2, Figure S1,Supporting Information)32 and LC-MS/MS (Figure S2,Supporting Information).33 Both the fungal biomass and theculture media contained swainsonine. The mean concentrationof swainsonine for the endophytes from S. canescens was 7.4mg/g, with a range of 3.7−11.9 mg/g (Table 1). Undif ilumspecies from Astragalus and Oxytropis species of North Americaalso produced swainsonine in vitro (Table 1). The fungalbiomass and the culture media contained swainsonine in theseisolates as well. The mean concentrations of swainsoninediffered among Undif ilum species from different hosts (p <0.001). For example, U. cinereum from A. mollissimus had amean concentration of 7.5 mg/g, U. fulvum from A. lentiginosushad a mean concentration of 16 mg/g, and U. oxytropis from O.sericea had a mean concentration of 1.9 mg/g (Table 1).Interestingly, the rank order of the concentrations ofswainsonine from the Astragalus and Oxytropis endophytesare similar to the concentrations found in each of the respectiveplant species.23 These results are also consistent with previousreports where in planta swainsonine concentrations correlated

with swainsonine concentrations of the correspondingendophyte isolated from the respective host.21,34

The endophyte isolates from S. canescens were identified asan Undif ilum species (Pleosporaceae) based on theirmorphology. The isolates were slow growing like otherUndif ilum species (Figure S3, Supporting Information).Germinating conidia from the isolates produced the distinctiveand diagnostic germ tubes that are characteristic of the genusUndif ilum, being wavy or undulating in their growth untilbranching (Figure 3).17 Furthermore, it was authenticated to bean Undif ilum species by the molecular analysis of the internaltranscribed spacer rDNA (ITS) (Figure S4, SupportingInformation). All isolates of the endophyte yielded identicalsequences of the ITS. The ITS sequences obtained from thethree isolates of the endophyte have been deposited withGenBank (JX674068, JX674069, JX674070). The endophyteisolates from S. canescens and seed purchased from B&T WorldSeeds were deposited at the Fungal Collection of theIntermountain Herbarium at Utah State University (UTC259540).Swainsonine concentrations in plants derived from seeds of

S. canescens purchased from B&T World Seeds and from thetwo voucher specimens of S. canescens collected in WesternAustralia ranged from not detected to 3.7 mg/g, with a mean

Figure 2. GC-MS chromatogram and mass spectrum of swainsonine,as the tri-TMS derivative (tR = 14.54 min) and key fragment ions. Thesample was from that produced from the endophyte of Swainsonacanescens.

Table 1. Mean Swainsonine Production of DifferentUndif ilum Species Isolated from Astragalus, Oxytropis, andSwainsona Species

plant speciesfungal

endophytemean swainsonine

(mg/g) range SE

S. canescens Undif ilum sp. 7.4a 3.7−11.9 0.5A. mollissimus U. cinereum 7.5a 1.4−22.5 2.9A. lentiginosus U. fulvum 16a 4.9−32.6 4.3O. sericea U. oxytropis 1.9b 0.3−8.4 1.1a,bDifferent letters within column are statistically significant (p <0.05).

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concentration of 2 mg/g (Table 2). These highly variableconcentrations of swainsonine are similar to observations in

Astragalus and Oxytropis locoweeds in North America andChina.25,26,30,35 To determine if the swainsonine-producingendophyte isolated from seeds of S. canescens was present inplanta, first, an endophyte was cultured from S. canescenssamples containing swainsonine that was morphologicallysimilar to the seed-isolated endophyte, and the respective ITSsequences were amplified (Table 2). Second, the endophytewas detected by PCR through amplification of the ITS fromleaf samples of S. canescens. In each case the amplified PCRproducts were sequence verified to be identical to the isolatescultured from S. canescens seeds. The endophyte was detectedby PCR in all the S. canescens plants, including the one whereswainsonine was not detected, but it was cultured only fromplants with concentrations of swainsonine greater than 1 mg/g(Table 2). The endophyte was quantitated, and samples withswainsonine concentrations greater than 1 mg/g hadsignificantly more endophyte than the sample with nodetectable swainsonine (Table 2). These results are consistentwith reports in Astragalus and Oxytropis locoweeds in NorthAmerica and China concerning the two chemotypes of theplants.25,26,30,35 Swainsonine was not detected in plants derivedfrom seeds of S. maccullochiana (Table 2), nor was any

endophyte detected by culturing or Undif ilum detected byPCR.In conclusion, an endophyte was isolated from S. canescens

that produces swainsonine in vitro. This endophyte belongs tothe genus Undif ilum, but is morphologically and phylogeneti-cally distinct from other Undif ilum species so far described.Furthermore the data support that this Undif ilum species isvertically transmitted. It seems highly probable that Undif ilummay be associated with other Swainsona species that arereported to contain swainsonine, such as S. galegifolia,31 andthis merits further investigation. As swainsonine occurssporadically in two other plant families, the Convolvulaceaeand Malvaceae, it seems highly probable that a swainsonine-producing endophyte may be associated with these plants aswell. In fact, a fungal endosymbiont belonging to the fungalorder Chaetothyriales was recently isolated from Ipomoeacarnea, which produces swainsonine in vitro.36

■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. Acetic acid and ammonium

acetate were ACS grade and diluted with deionized water. All solventsused for LC-MS or GC-MS were HPLC grade. BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide; catalog no. 33155-U) waspurchased from Supelco Analytical. LC-MS analysis was performedusing a ThermoFinigan LCQ Advantage mass spectrometer equippedwith a Surveyor Plus autosampler, MS quaternary pump, and BetasilC18 reverse-phase HPLC column (100 × 2.1 mm) with a guardcolumn of equivalent phase. GC-MS analysis was performed with aThermoFinigan Polaris GCQ mass spectrometer and Trace GCequipped with an Agilent DB-5MS column (30 m × 0.25 mm).

Plant Material. Swainsona canescens F. Muell. and S. maccullochianaF. Muell seed lots were purchased from B & T World Seeds(Paguignan, 34210 Aigues-Vives, France). Locoweed seeds werecollected from populations of Astragalus mollissimus Torr. var. earleii(Fort Davis, TX, USA, N 30°33′30.2″, W 103°51′40.5″), A.lentiginosus Douglas var. araneosus (Wahwah Valley, UT, USA, N38°23′45.6″, W 113°17′28.66″), and Oxytropis sericea Nutt. (ParkValley, UT, USA, N 41° 54′ 15.4″, W 113° 20′ 54.9″).30 Voucherspecimens of S. canescens (NSW 886571 and NSW 886572) werecollected from Madoonia Downs Station, Coolgardie, WesternAustralia (S 31°31′06″, E 121°46′26″ and S 31°34′59″ E122°00′11″). Plants of Swainsona canescens (n = 6) derived from theabove-mentioned seeds were grown in the greenhouse with a 16 hphotoperiod and day/night temperatures of 25 °C/20 °C. Leaves fromthe plant were harvested and frozen at −80 °C. Subsequently, theharvested leaves were freeze-dried and ground. Swainsonine (1) andDNA were extracted from this plant material for further analyses.

Fungal Cultures. Scarified seeds from all taxa, as well as leaf andstem material from S. canescens, were surface sterilized in a mixture of70% ethanol, 30% bleach, and 0.01% Triton X-100 (Promega,Madison, WI, USA) solution for 5 min, followed by a second stepof 30% bleach and 0.01% Triton X-100 for 5 min, and a third stepwherein the plant material was rinsed two times in sterile water. Seeds,leaves, and stems were placed on potato dextrose agar (PDA) (Becton,Dickinson and Company, Sparks, MD, USA) in polystyrene plates(Falcon) and placed in the dark at 24 °C.

Fungal cultures were subcultured, with a single pinpoint mass ofmycelium being placed onto the center of a preweighed sterile filterpaper that was placed on the surface of a PDA plate. Cultures weregrown for 41 days under conditions described above, and measure-ments recorded. After 41 days the culture plus filter paper and the agarwere removed separately and freeze-dried. The mass of the fungalculture was calculated by subtracting the mass of the filter paper alonefrom the mass of the culture plus filter paper. Swainsonine amountswere determined for the fungal culture plus filter paper and agar todetermine the amount produced by the endophyte.

Other subcultures of the endophyte from S. canescens were grownon water agar and PDA plates at room temperature with ambient light

Figure 3. Wavy germ tube from germinating conidia from anendophytic isolate from Swainsona canescens on V8 agar,; scale bar = 10μm.

Table 2. Swainsonine Concentrations and EndophyteDetection and Quantitation by PCR and Culturing inSwainsona canescens and Swainsona macculochiana

endophyte detection

species sampleswainsonine(mg/g) PCR culture pg/ng

Swainsonacanescens

plant 1 n.d.a + − <0.2plant 2 1.7 + + 62.4plant 3 2.0 + + 9.9plant 4 2.5 + + 23.4plant 5 3.2 + + 4.7plant 6 3.7 + + 115.7voucher NSW886571

2.5 + + /

voucher NSW886572

1.2 + + /

Swainsonamaccullochiana

plant 1 n.d. − − /plant 2 n.d. − − /plant 3 n.d. − − /plant 4 n.d. − − /

aNot detected (n.d).

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and then in sunlight for 2−4 weeks to examine culture growth andsporulation. Fungal specimens on water agar plates exhibitedsporulation when exposed to natural light, and the sporulatedspecimens were examined using a Nikon Optiphot microscope(Nikon, Tokyo, Japan). Spores were collected from the outer marginof growth. Spores were placed on V8 agar on microscope slides andkept in a humidity chamber. They were viewed daily, and germinationwas noted within a week.Swainsonine Analysis. Swainsonine was extracted using a

modification of the procedure described by Gardner and Cook.37

Dried plant material (50 mg), freeze-dried mycelia plus filter paper(whole), and freeze-dried agar (150 mg) were extracted in 2% aceticacid for 18 h with agitation. After extraction, samples were centrifugedand an aliquot from the extraction was diluted into 20 mM ammoniumacetate in a 1 mL autosampler vial. Samples were analyzed by LC-MS/MS to quantitate swainsonine as previously described.33 Forendophyte samples, total swainsonine (freeze-dried agar plus myceliaand filter paper) was calculated and expressed as a percent of themycelial mass. The detection limit of swainsonine was 0.01 mg pergram material dry weight using this extraction procedure. To verify theidentification of swainsonine in the fungal endophyte a 1.0 mL aliquotof the acetic acid extract was added to a Strata-XC (30 mg) SPEcolumn that was prerinsed with methanol and water (2 mL each). TheSPE columns were rinsed again with water and methanol (2 mL), andswainsonine was eluted with 3 mL of ammoniated methanol (1 to 5dilutions of methanol saturated with NH3). The extract wasevaporated to dryness. Pyridine (200 μL) and BSTFA silylationreagent (50 μL) were added, and the sample was heated (60 °C) for30 min. Samples were diluted to 1.0 mL with chloroform and thenanalyzed by GC-MS for swainsonine (tri-TMS derivative) using theGC-MS conditions previously described.32

DNA Extraction. DNA was extracted from lyophilized, groundplant material and mycelia (∼20 mg) using the DNEasy Plant Mini Kit(Qiagen Inc., Valencia, CA, USA) according to the manufacturer’sinstructions. DNA was quantified with the ND-1000 spectropho-tometer (Nanodrop Technologies, Wilmington, DE, USA).PCR Primers. The PCR primers ITS 538 and OR1a (5′ GTC AAA

AGT TGA AAA TGT GGC TTG G 3′)30,39 were used to amplify theinternal transcribed spacer region of the rDNA. Primers weresynthesized by Integrated DNA Technologies, Inc. (Coralville, IA,USA).Polymerase Chain Reaction (PCR). Thermal cycling conditions

for amplification of the ITS rDNA from fungal isolates as well as plantsamples were as follows: an initial denaturation step for 3 min at 94°C, followed by 40 cycles of 33 s at 94 °C, 50 s at 57 °C, and 30 s at 72°C, with a final extension of 5 min at 72 °C. Each reaction contained50 ng of fungal DNA or total DNA isolated from the plant. GoTaq(Promega Corporation, Madison, WI, USA) DNA polymerase wasused in reaction conditions recommended by the manufacturer. PCRproducts were resolved on a 1% agarose gel containing ethidiumbromide at 118 V for 20 min and visualized under UV illumination.Agarose gels were visualized and analyzed with a Kodak Image Station2000RT imager and its software (Eastman Kodak, Rochester, NY,USA).The fungal endophyte was quantitated by qPCR as described in

Cook et al.39 using a CHROMO 4 quantitative PCR detector (Bio-RadLaboratories Inc., Hercules, CA, USA). The limit of quantitation was0.2 pg of endophyte DNA per ng of total DNA. The primers usedwere ITS 5 (5′ GGA AGT AAA AGT CGT AAC AAG G 3′)37 andOR1a (5′ GTC AAA AGT TGA AAA TGT GGC TTG G 3′), whichamplify the ITS region of the rDNA region. Primers were synthesizedby Integrated DNA Technologies, Inc. (Coralville, IA, USA). A subsetof PCR products were verified to be Undif ilum via restriction enzymedigest, agarose gel electrophoresis, and sequencing.39

Sequencing. PCR products were prepared for sequencing with theQIAquick PCR purification kit (Qiagen Inc., Valencia, CA, USA)according to the manufacturer’s instructions. Amplified PCR productswere sequenced with the same forward and reverse primers used foramplification. DNA sequencing was performed at the Genomics Core

Facility, Center for Integrative Biosystems, Utah State University,Logan, UT, USA.

Phylogenetic Analysis. Sequences of the ribosomal ITS werecompiled using Sequencher v4.9 (GeneCodes Corporation, AnnArbor, MI, USA) for 15 Undif ilum, Alternaria, Embellisia, andUlocladium species (Table S1, Supporting Information). Alignmentof the sequences was made using CLUSTALW multiple sequencealignment,40 available online (www.genome.jp, April 26, 2012).Phylogenetic analyses were performed in PAUP 4.0 software (SinauerAssociates, Inc., Sunderland, MA, USA) with gaps treated as missingdata. Heuristic searches for the most parsimonious UPGMA41 treeswere conducted using random stepwise addition and tree bisection-reconnection branch swapping. Bootstrap statistical support wasgenerated by 1000 replications using 50% majority-rule consensustrees.

Data Analysis. All measurements and concentrations were logtransformed for statistical analysis. All comparisons were done usingANOVA in Sigma Stat 3.1 with a post hoc test of significance using theBonferroni correction. A p-value of <0.05 was considered to bestatistically significant. Data are presented as non-log transformed(mean ± the standard error) in tables.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information includes GC-MS and LC-MS/MSdata that support that the endophyte isolated from Swainsonacanescens produces swainsonine in vitro. Additionally, it shows aUPGMA bootstrap consensus tree that shows phylogeneticplacement of the endophyte among other Undif ilum species.Lastly, it contains a picture of the endophyte in contrast toother Undif ilum species isolated to date, comparing theirrelative growth rates. This material is available free of charge viathe Internet at http://pubs.acs.org.

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

■ ACKNOWLEDGMENTSThe authors would like to thank J. Roper and S. Larsen fortechnical assistance. The photo of Swainsona canescens in thetable of contents/abstract graphic was provided courtesy ofRoss McKenzie.

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