Production of swainsonine by fungal endophytes of locoweed

Production of swainsonine by fungal endophytes of locoweed

Karen BRAUN#, Jennifer ROMERO, Craig LIDDELL$ and Rebecca CREAMER*

Department of Entomology, Plant Pathology, and Weed Science, New Mexico State University, Las Cruces, NM 88003, USA.E-mail : [email protected]

Received 17 March 2003; accepted 3 June 2003.

Consumption of locoweeds, legumes endemic in arid western USA, has long been associated with locoism, a diseaseof ruminant animals. To explore the relationship between fungi associated with locoweed and locoweed toxicity, 11

locoweed populations from various sites in New Mexico were assessed for endophytic fungi. Endophytes were isolatedfrom the leaves, stems, seeds, and flowers of eight populations of the toxic locoweeds Astragalus mollissimus, Oxytropislambertii, and O. sericea. Fungal cultures grew very slowly and sporadically produced subcylindrical conidia with very

dark transverse septa. All cultured endophytes produced the alkaloid swainsonine, which causes locoism. Endophyte-infected locoweed populations produced swainsonine, and the swainsonine level of endophyte strains in vitro was highlycorrelated with the swainsonine level of their host plant populations. The rDNA ITS from mycelia from four endophyte

isolates and b-tubulin encoding regions from mycelia of 18 fungal endophyte isolates were amplified using PCR and thenucleic acid sequences were analyzed. The nucleic acid sequences of the b-tubulin encoding regions were essentiallyidentical among all the endophytes regardless of plant genus and locations. Morphological evidence and sequenceanalysis of the ITS region suggest that the endophytes are most closely related to Embellisia. However, with the paucity

of Embellisia species represented in sequence databases, precise taxonomic placement will await further study.

INTRODUCTION

Locoweeds are perennial plants of the legume familyFabaceae commonly found in rangeland in the westernUSA. Consumption of the aerial plant parts of manylocoweed species in the genera Astragalus and Oxy-tropis causes significant economic losses to cattle,sheep, and horses due to locoism, a neurological disease(James & Panter 1989). Animals affected by locoismexhibit behavioral depression, a staggering walk, lackof muscular coordination, and have difficulty eating ordrinking. Locoweed toxicity is highly variable betweenlocoweed species, populations, individuals, and seasons(Gardner et al. 2001), and the mechanisms underlyingthe variability are unknown. Locoism has been tra-ditionally attributed to the production by Astragalusand Oxytropis of swainsonine (1,2,8-trihyroxyocta-hydroindolizidine), an alkaloid a-mannosidase in-hibitor (James & Panter 1989). Swainsonine has been

conclusively shown to cause locoism (Stegelmeier et al.1995). Swainsonine is very stable and can cause toxicreactions even when animals are fed plant tissues thathave been dried for many years (Ralphs et al. 1988).Swainsonine is produced by cultures of ‘Rhizoctonia ’leguminicola (Schneider et al. 1983), which is not atrue Rhizoctonia but a member of the Pleosporales(McGinn, Liddell & Pavia 1999, Liddell, unpub.), andthe entomopathogenic fungus Metarhizium anisopliae(Tamerler et al. 1998). From these fungi the swainso-nine pathway has been partially characterized (Wick-wire, Wagner & Broquist 1990).

Alkaloid-producing fungal endophytes are wide-spread among grasses (Powell & Petroski 1992) andtrees (Tan & Zou 2001), but have not been reportedfor legumes, which supposedly have the intrinsic abilityto produce the alkaloids themselves (Janzen 1971).Other bovine toxicoses such as festucosis or ryegrassstagger have been found to be caused by endophytesinfecting the forage plants. Seed-transmitted fungalendophytes are often mutualists that exchange chemi-cal protection against herbivores for nutrition, shelter,and dispersion provided by the host plant (Clay 1988).

In the course of research to identify pathogenicfungi that could infect locoweed, we found persistent,

* Corresponding author.# Current address: CEFyBO Centro de Estudios Farmacologicos

y Botanicos (CONICET), Serrano 669, (1414) Buenos Aires, Argen-tina.$ Current address: Artesian Therapeutics Inc., 22 Firstfield Road,

Gaithersburg, MD 20878, USA.

Mycol. Res. 107 (8): 980–988 (August 2003). f The British Mycological Society 980

DOI: 10.1017/S095375620300813X Printed in the United Kingdom.

slow-growing fungi in all toxic locoweed species. Thispaper reports the observations on those fungi, theirproduction of swainsonine, and their association withlocoweed toxicity.

MATERIALS AND METHODS

Locoweed sampling and fungal isolations

Locoweed species reported as toxic and non-toxic(Fox, Allred & Roalson 1998) from 11 populationswere collected from New Mexico and screened forendophytes. Locoweeds were identified by Kelly Allred(New Mexico State University). The locoweed plantspecies and their collection sites in New Mexico are:Astragalus agrestis (Colfax Co.), A. mollissimus (Jor-nada Experimental Range, Colfax Co.), A. humistratus(Lincoln Co., Catron Co.), A. lonchocarpus (ColfaxCo.), Oxytropis lambertii (Grant Co., Colfax Co.), andO. sericea (Colfax Co.). Endophytes were screenedfrom 240 plants and swainsonine-producing endo-phytes identified in eight of the 11 populations (Table 1).

Fungal isolations were performed from four sub-samples of leaves, stems, seeds, and/or flowers fromeach plant, resulting in 12–16 tissue samples per plantspecimen. Fresh or dried tissues were surface sterilizedfor 30 s in 70% ethanol, followed by 3 min in 1.05%v/v sodium hypochlorite and 1 min rinse in sterilewater. Tissues were then plated onto water agar. Theeffectiveness of surface sterilization was verified usinga modification of the imprint test (Schulz et al. 1999).After surface sterilization, an imprint of the tissue wasmade on acidified potato dextrose agar (PDA) plates.

A plant was considered endophyte-infected (E+)when any of the plated tissues yielded endophyticfungi within 30 d and endophyte-free (Ex) when they

did not. The hyphal tips of the recovered endophyteswere transferred onto PDA plates and maintained at18 xC. The 18 isolates listed in Table 1 have been pre-served as desiccated mycelia and stored at both 4 x andx80 x at the New Mexico State University Center forNatural History Collections (NMSU-CNHC).

Growth rate measurements of fungi were made fromPDA cultures growing at 25 x without light. Sporemeasurements were made from 2 month old cultures.Selected cultures that did not sporulate when grown onwater agar, PDA, or Czapek-Dox agar were artificiallyinduced by applying 10 ml per PDA plate of a 1 ml mlx1

solution of the fungicide propiconazole (SyngentaCrop Protection, Greensboro, NC).

Swainsonine identification and quantification

Dried locoweed plants (0.5 g D.W.) from previouslydescribed sources (Table 1) and endophytes (0.05–0.1 gD.W.) isolated from those plants (including all culturemedia) were extracted in 50 ml methanol using aSoxhlet apparatus for 4 h. Swainsonine concentrationand biological activity in the methanol extracts of 173plant and 77 endophyte samples were assessed by ana-mannosidase inhibition assay (Sim & Perry 1995).Results were assessed immediately afterward at 405 nmusing an EL-312e Microplate BioKinetics Reader(Biotek Instruments, Winooski, VT). Swainsonineconcentration was calculated from a standard curve.Swainsonine content of plant populations was com-pared to that of endophyte isolates using the generallinear hypothesis of SAS (SAS Institute, Cary, NC).The a-mannosidase inhibition assay provides relativeswainsonine values that can be used comparatively, butdoes not give absolute swainsonine values. The limit ofdetection of this method was 25 ng mlx1 swainsonine.

Table 1. Toxic fungal endophyte isolates identified from three locoweed species in New Mexico.

Plants which

yielded toxic

endophytes/Isolates sequenced

Host Locality plants tested Collection date b-tubulin ITS

Astragalus mollisimus Jornada, NM 15/20 June 1997 A24

A3

Jornada, NM 1/15 Sept. 1997 AA15

AA12

Jornada, NM 11/12 May 1998 AB3 AB9

AB 9

Jornada, NM 3/6 May 1998 C4 C4

C7

Colfax, NM 15/21 June 1997 N1-1

N1-23

N1-14

Oxytropis lambertii Grant, NM 12/15 Sept. 1997 B1 B1

B10

B9

Colfax, NM 19/23 June 1997 M8-6

O. sericea Colfax, NM 1/16 June 1997 L9 L12

L12

L14

K. Braun and others 981

Swainsonine chemical identity in the extracts of 11fungal samples was confirmed by GC-Mass Spec-trometry (Molyneux et al. 1995). Methanol extracts offungi were dried with N2, resuspended in pyridine, andderivatized with TMSI (N-methyl-N-trimethylsilyltri-fluoracetamide). TMSI derivatives were analyzed usinga Varian-Saturn 3400 ion trap instrument operatingat 70 eV equipped with a 30 mr0.25 mm diam, 1 mmfilm thickness, DB5MS fused silica column. The col-umn temperature was programmed from 120–275 x at5 x minx1. Chromatograms and mass spectra of thesamples were compared with an external swainsoninestandard (Sigma Chemical, St Louis, MO).

Fungal genomic DNA extraction

Eighteen fungal isolates cultured from eight locoweedpopulations were subjected to genetic analysis (Table 1).Two fungal isolates per locoweed population wereselected, with the exception of the Oxytropis lambertiilocoweed population from Colfax, NM, for whichonly a single isolate was available. An additional iso-late from each of the N, B, and L populations was in-cluded since these fungal isolates tended to grow slowerthan other isolates. Total DNA was extracted from2–6 months-old fungal cultures (grown on PDA) usingPlant DNAzol (Gibco BRL, Grand Island, NY) asdescribed in the product instructions. The 18 isolateswere used for b-tubulin amplification and four of thosewere used for ITS amplification (Table 1).

PCR amplification of the ITS region

PCR reactions were carried out in a total volume of50 ml containing 0.4 mM each dNTP, 2.5 U Taq DNApolymerase, 0.5 mM of each primer, 10 mM Tris-HCl,pH 9.0, 50 mM KCl, 0.1% Triton X-100, 3 mM MgCl2,and 5 ml DNA. ITS4 and ITS5 primers (White et al.1990) which amplify the rDNA ITS regions containingITS1 and ITS2 and the intervening 5.8S rDNA wereused for amplification.

Amplification was carried out in a Perkin-ElmerModel 480 thermal cycler (Norwalk, CT) with thefollowing parameters : 94 x for 30 min, then 30 cyclesconsisting of 94 x for 30 s, 50 x for 45 s, and 72 x for45 s. A final extension of 72 x for 10 min followed.

PCR amplification of the b-tubulin region

PCR reactions were carried out in a total volume of50 ml containing 24.8 ml H2O, 5 ml 10r buffer (100 mM

Tris-HCl, pH 8.3; 500 mM KCl, 0.01% gelatin), 1 ml10 mM dNTPs, 3 ml 50 mM MgCl2, 3 ml each of 5 mM

primers, 0.5 U Taq DNA polymerase, and 10 ml of 1:10dilution of purified DNA. The oligonucleotide primersused for amplification of the b-tubulin region were5k-TCCACCAGCTKYGAGAACTC-3k and 5k-ACCT-CCTTCATGGAGACCTT-3k. These primer sequenceswere developed using a multiple alignment of four

members of the Dothideales (Leptosphaeria nodorum,Venturia inaequalis, Pleospora herbarum, and Myco-sphaerella pini ).

Amplification was carried out with the followingparameters : 35 cycles consisting of 95 x for 60 s, 50 x for45 s, and 72 x for 60 s. A final extension of 72 x for5 min followed.

Sequence analysis of endophyte amplicons

Amplification products were separated by electro-phoresis on a 1.8–2% low melting agarose gel, thebands visualized with ethidium bromide and thespecifically amplified band of 700 kb (ITS primers) or400 kb (b-tubulin primers) excised. Fragments werethen purified from the agarose using a Wizard PCR-Preps DNA purification kit (Promega, Madison WI).Fragments were ligated into a pGEM-T Easy Vector

Table 2. Source of sequences used in the study.

Fungus

GenBank sequence accession nos.

ITS b-Tubulin

Fusarium inflexum 1808708

Exserohilum fusiforme 9501883

E. turcicum 9501887

E. rostratum 9501886

Bipolaris papendorfii 9501895

B. portulaceae 12620995

Curvularia inaequalis 6841013

C. brachyspora 6715398

Stemphylium astragali 18139902

S. lycopersicae 18139914

S. trifolii 18139921

Drechslera catenaria 12621018

D. andersenii 12621019

D. poae 12621016

Alternaria alternata 6715474 3688133, 3688131

A. radicina 7546957

A. brassicae 7546948 3688135

A. conjuncta 18034000

A. tenuissima 14279753

A. abutilonis 15209130

A. cheiranthi 7546942

A. smyrnii 7546941

A. japonica 7547959

A. photosticta 5669032

A. brassicicola 7546947 3688145

A. solani 3688167, 3688153, 3688163

A. linicola 3688139, 3688147

A. infectoria 3688137, 3688143

A. lini 3688141

A. protenta 14279366

A. porri 7546955

Lewia infectoria 756952

Ulocladium alternariae 7546970

Pleospora papaveraceae 3983413

Embellisia sp.

DAR 58941

6715397

Embellisia sp.

DAR 74619

6715399

E. protea 18034002

E. eureka 18034001

Swainsonine from locoweed endophytes 982

(Promega) and used to transform JM109 High Ef-ficiency Competent Cells (Promega).

Recombinant plasmids were purified from bacterialcells using WizardMinipreps DNA Purification System(Promega). Plasmids containing inserts of the correctsize were sequenced in both directions using an ABIautomated sequencer or a Lycor automated sequencerat the New Mexico State University BioinformationFacility. Three independent clones were sequencedfor each fungal isolate. Sequences were aligned usingClustalw. The GenBank accession no. for the ITS se-quence of the L12 endophyte isolate is AY228650, andthe b-tubulin encoding region of the C4 endophyteisolate is AY228651.

Phylogenetic analyses were performed using pro-grams contained in PAUP Phylogenetic Software(version 4.08; Sinauer Associates, Sunderland, MA).To aid in placing the endophytes within the Pleos-poraceae, the ITS sequences from the endophyteswere compared with those of 33 species (Table 2)within the family. The sequences of the b-tubulinencoding regions of the endophytes were comparedwith those of 12 Alternaria species and Fusarium in-flexum as an outgroup. For both sets of sequencecomparisons, phylogenetic trees were produced byparsimony analysis, using closest step-wise additionand branch swapping by tree bisection-reconnection,and distance analysis, using neighbor-joining withKimura 2-parameter distances. For each analysis,1000 bootstrap replicates were performed to assessstatistical support for resulting phylogenetic treetopology.

RESULTS

Swainsonine-producing endophytic fungi were isolatedfrom eight populations of three locoweed species,Astra-galus mollissimus, Oxytropis lambertii, and O. sericeaplants (Table 1). Although not every plant within thepopulations was infected, 95 isolates of endophyteswere recovered from the three locoweed species. Noendophytes were isolated from Astragalus agrestis,A. lonchocarpus, and A. humistratus. External sporesor mycelium of the endophytic fungi were not foundon living host plants, and endophyte infection did notinduce obvious damage or disease to the plants.

Endophytes were isolated from leaves, stems, andflowers and seeds when present, although not fromevery tissue type tested from each plant. Of the plantsfrom which endophytes were isolated, 72.9% of leaf,97.2% of stem, 93.1% of seed, and 100% of flowersamples yielded endophyte cultures. No endophyteswere isolated from the roots of plants that otherwiseyielded the fungi from other plant tissues ; however,rapidly growing fungi and bacteria could be isolatedfrom roots. Although routine culturing of endophyteswas accomplished from dried locoweed tissues, endo-phytes could also be isolated from fresh tissues.

Fungal cultures isolated from Astragalus plantswere initially white, turning greyish after 10–15 wk,with a dark undersurface, while isolates derived fromOxytropis plants were dark brown to black, with adark undersurface. Endophytes grew slowly (0.03–0.34 mm dx1) and had a thin (2–8 mm diam) septatemycelium. Sporulation on plates was sporadic in some

1 2

Figs 1–2. Endophyte isolate L12. Fig. 1. Mycelia and conidia. Fig. 2. Condia. Bars=10 mm.


isolates and absent in others. Spore size and shapewas somewhat variable. Conidia were dark, simple,subcylindrical, with 1–5 very dark transverse septa,14–60r6–13 mm (Fig. 1). The conidia were apicallyborne blastospores. The average (n=100) conidiallength of isolate L12 was 35 mm, and most conidia had2–4 septa (Fig. 2). Conidia produced upon fungicidetreatment were generally similar in size and shape tothose produced naturally.

Swainsonine identification and production

Endophytic fungi isolated from locoweeds producedswainsonine in vitro and in amounts ranging from471 to 18 000 mg gx1

D.M. Swainsonine was recoveredfrom cultures as early as 13 d after transfer and aslate as 178 d after transfer onto PDA. Mass spectraof the endophyte-produced swainsonine were identicalto those of published spectra (Molyneux et al. 1995)and the standard. Endophyte extracts (n=11) exhibitedthe same fragmentation pattern and retention time(14 min 38¡1 s) as the standard. Spectra of samplesand standard shared the signature fragments at m/z120, 185, 260 and 299 and the molecular ion at 374.

Plants from which endophytes were isolated (E+plants, n=79) contained significantly more swainso-nine (mean 869¡101 S.E.M. mg gx1

D.M.) than plantsfrom which endophytes could not be isolated (Explants, n=84) (mean 66¡6 S.E.M. mg gx1

D.M.)(P<0.0002). Locoweed populations having higherincidence of endophyte infection (E+ plants/totalplants) had higher mean swainsonine contents (n=10,P<0.003) (Fig. 3). Endophyte cultures that producedhigher levels of swainsonine in vitro (n=6, P<0.0001)were isolated from locoweed field populations withhigher swainsonine content (Fig. 4).

Sequence of ITS and b-tubulin regions

Nucleic acid sequence of the ITS rDNA regions fromendophyte isolates from Astragalus mollissimus differedfrom that of Oxytropis lambertii by three nucleotides(99% similarity) (data not shown). The sequences fromthe b-tubulin region were nearly identical (>99%similarity, 1 nucleotide change) among the differentendophyte isolates, irrespective of host and geographicorigins. Parsimony and neighbour-joining analyses ofITS sequences (Fig. 5) showed the endophytes to bemore similar to species of Alternaria or Embellisia(Ascomycota: Pleosporales, Pleosporaceae) than ana-morphs of other members of the Pleosporaceae includ-ing Drechslera, Bipolaris, Exserohilum, Stemphylium,and Curvularia. The endophyte sequences are notidentical to those available for other fungal species.Analyses of the b-tubulin encoding region also showedthat the endophytes are not identical to Alternariaspecies (Fig. 6) ; no Embellisia sequences were availablefor comparison.

DISCUSSION

This study showed that the fungal endophytes isolatedfrom locoweeds produce swainsonine. Although highlyvariable, the swainsonine content of locoweed popu-lations was correlated with endophyte infection. Thecorrelation between the swainsonine production ofan endophyte isolate in vitro and the swainsonine con-tent of the locoweed population in the field suggeststhat the endophyte at least partially controls locoweedtoxicity.

These results agree with those of Gardner et al.(2003) in which they compared swainsonine levels in16 populations of O. lambertii. They showed that

AmoR

Loc

owee

d lo

g S

W (

�g

g–1 D

.M.)

AmoJ

OlaROlaG

AmoJAmoJ

AmoJ

OserCAhuL

AloC2

4

0 50 100

y = 0.0126× + 1.7175R2

= 0.89

% of endophyte infection

Fig. 3. Endophyte infection and mean swainsonine content

in locoweed populations. Regression of mean swainsonine(SW) content against percentage of endophyte infectionin 10 locoweed populations. Ahu, Astragalus humistratus ;

Alo, A. lonchocarpus ; Amo, A. mollissimus ; Ola, Oxytropislambertii ; Ose, O. sericea ; C, Colfax; G, Gila; J, Jornada;L, Lincoln; and R, Raton.

3000

2000

1000

0

Mea

n lo

cow

eed

SW

(�

g g–1

D.M

.)

4000 8000 12000

y = 0.164x + 24.48R2

= 0.98

AmoJ

OlaG

AmoJ

OseC

AmoC

AmoJ

Endophyte SW in vitro production (�g g–1 D.M.)

Fig. 4. In vitro swainsonine production of endophyte strainscompared to the locoweed populations from which they were

isolated. Regression of mean swainsonine (SW) content (mg/gD.M.) (D.M.=dry matter) of 6 locoweed populations againstthe in vitro SW production of their associated isolated endo-

phyte strains. Error bars are the standard error of the rawdata. Abbreviations as Fig. 3.


high (>0.01%) and low (<0.01%) levels of swain-sonine in plants were highly correlated with endophytepresence or absence, respectively. Ralphs, Welsh &Gardner (2002) found swainsonine in O. lambertii var.bigelovii populations throughout the southwest areasof plant distribution, but not in the northeast areas.They also found that within a population collectedfrom a particular site, the swainsonine levels were quitevariable, which is consistent with the results found inthis study. The evidence presented in all these studiessuggest that endophyte infection contributes to thepopulation level variability in swainsonine productionof locoweed plants in addition to the variability sug-gested to be attributed to environmental factors orplant genetic traits (Fox, Allred & Roalson 1998).

Swainsonine and endophytes were isolated fromAstragalus mollissimus, Oxytropis lambertii, and O. ser-icea, which had all been previously reported to containswainsonine (Allison 1984). No endophyte or swain-sonine was detected from A. humistratus or A. agrestisneither of which had been reported to be toxic. How-ever, while A. lonchocarpus has been reported to con-tain swainsonine (Smith, Allred & Kiehl 1992), wedid not isolate endophytes or swainsonine from the14 plants tested.

The general conidial morphology (including thedark pigmented septa) of the endophytes was mostsimilar to that of Embellisia (Simmons 1983, 1990).While the conidial size and shape, and relative lack oflongitudinal septa were similar to some Embellisia

Drechslera poa

Drechslera andersenii

Drechslera catenaria

Bipolaris papendorfii

Bipolaris portulaceae

Curvularia inaequalis

Curvularia brachyspora

Exserohilum turcicum

Exserohilum fusiforme

Exserohilum rostratumStemphylium astragali

Stemphylium lycopersicae

Stemphylium trifolii

Alternaria brassicae

A. brassicicola

Ulocladium alternariae

Pleospora papaveraceae

A. japonica

A. tenuissima

A. radicina

A. cheiranthi

A. alternata

A. abutilonis

A. smyrnii

A. photosticta

A. protenta

A. porii

Embellisia sp. DAR 74619

Embellisia sp. DAR 58941

Embellisia eureka

Endophyte from Oxytropis sericea L12

Endophyte from Astalagalus mollisiumus AB9

Lewia infectoria

A. conjuncta

Embellisia protea

5 changes

5483

100100

10097

96100

7560

8789

98100

999299

99

Fig. 5. Phylogenetic tree generated from parsimony and neighbour-joining analyses of ITS and 5.8S ribosomal genes of thefungal endophytes from locoweeds and selected members of the Pleosporaceae. The numbers at the branch nodes indicatethe confidence values for the presence of the branch obtained from bootstrap analyses using 1000 replicates; the top number is

that obtained from parsimony, and the bottom that from neighbour-joining analysis.


species, the host (locoweeds) and production of atoxic secondary metabolite (swainsonine) differedfrom described species. The results of the sequenceanalyses suggest that the endophytes collected inthis work may be related to Alternaria (Pryor & Gil-bertson 2000, Chou & Wu 2002) or Embellisia species;however, insufficient sequences are represented insequence databases to make a precise taxonomicplacement.

The differences in colony color between endophytesisolated from Astragalus and Oxytropis locoweeds andthe consistent 3 base pair differences between ITSsequence of isolates from the two different locoweedssuggest that these fungi may be different species orsubspecies. However, the lack of natural sporulation of

many of the isolates has not permitted a definitivedetermination.

While the locoweed endophytes appear to be mostclosely related to Embellisia species, Embellisia hasnot been reported to produce toxic secondary metab-olites. In contrast, Alternaria species produce toxins(Logrieco et al. 1990), affecting plants (Scheffer 1992)and animals (Woody & Chu 1992). Plant pathogenicAlternaria species are capable of producing both hostspecific (Otani & Kohmoto 1992) and non-host specific(Rotem 1994) toxins. Toxic secondary metabolite pro-duction in plant-infecting Alternaria species has alsobeen used in identification and classification (Ander-son, Krøger & Roberts 2002). The toxins produced byAlternaria (Visconti & Sibilia 1994) differ significantly

Alternaria solani CBS 161.77

A. linicola 3A

A. solani CBS 111.44

A. solani CBS 110.41

A. brassicicola

Endophyte fromAstragalus mollisimus AA15

Endophyte fromOxytropis lambertii L12

A. infectoria 4B

A. infectoria 4A

A. brassicae

A. alternata 1A

A. alternata 1E

A. lini

Fusarium inflexum

10 changes

6679

9897

63

64

5555

9899

90

69

86

74

97

97

6655

A. linicola 3B

Fig. 6. Phylogenetic tree generated from parsimony and neighbour-joining analyses of the b-tubulin gene of the fungalendophytes from locoweeds and selected Alternaria and related species. The numbers at the branch nodes indicatethe confidence values for the presence of the branch obtained from bootstrap analyses using 1000 replicates ; top number

obtained from parsimony, bottom number from neighbour-joining analysis.


from swainsonine, which for Rhizoctonia leguminicolawas shown to be produced from pipecolic acid (Harriset al. 1988). However, Alternaria contains many un-described species that show host-specificity and pro-duce toxins (Simmons 1999).

Although there are no reports of endophytic Embel-lisia species, Alternaria species are known to occuras endophytes in a variety of plants (Petrini, Muller& Luginbuhl 1979, Carroll 1988, Petrini 1991). WhileAlternaria has not been reported as an endophyte onlocoweed, A. alternata has been described as endo-phytic on legumes (Larran et al. 2002). Alternaria hasalso been reported as an epiphyte and saprophyte(Dickinson & Bottomley 1980), and opportunisticpathogen (O’Donnell & Dickinson 1980, Rotem 1994)on legumes.

To the best of our knowledge, this is the first reportof an alkaloid-producing fungal endophyte from alegume. Many endophytes produce some type of sec-ondary metabolite or enzyme necessary for the col-onization of plant tissues (Petrini et al. 1992, Schulzet al. 1999). Alkaloid-producing fungal endophytesare widespread among grasses (Powell & Petroski1992), but have not been reported for legumes, whichwere thought to produce the alkaloids themselves(Janzen 1971).

The alkaloid toxins produced by grass endophytes(Clay 1988) and rugulosin, a toxin produced by afungal endophyte of conifer needles (Miller et al. 2002)have insecticidal properties. Similarly, swainsonine isan aphid feeding deterrent (Dreyer, Jones & Molyneux1985) and is toxic to vertebrates (James & Panter 1989).The strong relationship found between swainsonineproduction by locoweeds and endophyte infectionsuggests that the endophyte could be providing thelocoweed plants alkaloid protection against insect andanimal feeding.

The isolation of the endophytes from locoweed seedand flowers and the lack of sporulation of the endo-phytes when inhabiting living locoweed is consistentwith the biology of seed or maternally transmittedendophytes (Clay 1988), which are known to producehigh levels of alkaloids in the host grasses (Siegel et al.1990). Moreover, the loss of reproductive abilities hasbeen related to the acquisition of a mutualistic habit(Law & Lewis 1983), although some mutualistic endo-phytes still sporulate (Carroll 1988). However, manyendophyte-host interactions are thought to be betterconsidered as mutual antagonism based on the sec-ondary metabolites each partner produces (Schulzet al. 1999). Maternally transmitted endophytes arethought to have long evolutionary histories of associ-ation with their host plants (Clay 1988, Schardl et al.1997), which may be reflected, in this case, by theassociation between locoweed populations and endo-phyte strains having different swainsonine-producingabilities. Additional research is needed to understandthe association between the locoweed endophytes andtheir hosts.

ACKNOWLEDGEMENTS

We thank David Graham for his assistance in collection of locoweed

samples, Dave Perry for advice on various stages of this work, and

David Smith for help in the statistical analyses. We also thank Tracy

Sterling and Barry Pryor for their critical reading and suggestions in

manuscript preparation. We thank the New Mexico State University

Agricultural Experiment Station for support of this work.

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Corresponding Editor: B. Schulz


Production of swainsonine by fungal endophytes of locoweed

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