Analysis of Endophyte Toxins Fescue and Other Grasses Toxic to Livestock

8/8/2019 Analysis of Endophyte Toxins Fescue and Other Grasses Toxic to Livestock

http://slidepdf.com/reader/full/analysis-of-endophyte-toxins-fescue-and-other-grasses-toxic-to-livestock 1/12

J. K. Porter

Analysis of endophyte toxins: fescue and other grasses toxic to livestock

1995. 73:871-880. J Anim Sci

http://jas.fass.orgthe World Wide Web at:

The online version of this article, along with updated information and services, is located on

www.asas.org

by on January 8, 2011. jas.fass.orgDownloaded from

http://jas.fass.org/



http://www.asas.org/










Analysis of Endophyte Toxins: Fescue and Other Grasses

Toxic to Livestock

J. K. Porter

Toxicology and Mycotoxin Research Unit, Richard B. Russell Agricultural Research Center,

ARS, USDA, Athens, GA 30613

ABSTRACT: Research on livestock toxicoses

caused by Acremonium (endophyte)-infected grasses

strongly implicate the ergopeptine alkaloids with A.

coenophialum-infected fescue and paxilline and the

lolitrem alkaloids with A. lolii-infected perennial

ryegrass as the causative agents. Isolation, identifica-

tion, and detection of these toxins involves extraction

with appropriate solvents, clean-up procedures, and

chromatographic methods with known standards.

Thin- layer, high-performance liquid and gas chro-

matography along with ultraviolet and mass spectro-metric (i.e., electron impact, chemical ionization,

tandem ma ss ) characterizations have been reported.

These methods have varying degrees of success

depending on the matrix from which the alkaloids

have been extracted. Ergovaline is the primary

ergopeptine alkaloid isolated from cultures of A.

coenophialum and also from infected fescue grass and

seeds toxic t o livestock. Other compounds isolated

from the endophyte-infected fescue include: lysergic

acid amide (e rg in e) , the clavine class of ergot

alkaloids (chanoclavine I , agroclavine, elymoclavine,

penniclavine), the pyrrolizidine alkaloids (N-formyllo-

line, N-acetylloline, N-methlyloline, N-acetylnorlo-

line), and the unique pyrrolopyrazine alkaloid pera-

mine. The loline alkaloids and peramine have been

more associated with the insect-deterrent properties of

the endophyte-infected fescue than with livestock

toxicoses. Also, both peramine and the ergopeptine

alkaloids (ergovaline, ergotamine) have been isolated

from A. lolii-infected perennial ryegrass. More re-cently, paxilline and lolitrem B have been detected in

laboratory cultures of A. coenophialum isolated from

tall fescue. The ergot alkaloids in endophyte-infected

perennial ryegrass may be more related to decreased

animal productivity (weight gains, reproduction prob-

lems), whereas the lolitrems cause the staggers

syndrome. The detection, isolation, identification, and

analyses of these compounds from Acremonium-in-

fected grasses is presented.

Key Words: Acremonium, Endophytes, Alkaloids, Analytical Methods, Toxins, Grasses

Introduction

Toxicoses in livestock grazed on Acremonium spp.-

infected grasses have a pronounced negative economic

effect on animal production (Hoveland, 1993). The

Acremonium grass endophytes are taxonomically

aligned with the family Clavicipitaceae and live or

spend the ir en tire life cycle within the aerial portion oftheir grass host (Bacon and DeBatista, 1991). Several

reviews have been published 1 ) on the economic

perspectives with regard to livestock toxicoses(Stuedemann and Hoveland, 1988; Hoveland, 19931,

2 ) on mechanisms and mode of toxicity (Thompson

and Porter, 1991;Porter an d Thompson, 1992;Thomp-

‘Presented at a symposium titled “Fescue and Other Toxic

Grasses: Effects on Livestock Production” at the ASAS 85th Annu.

Mtg., Spokane, WA.

Received April 11, 1994.

Accepted October 5, 1994.

J. Anim. Sci. 1995. 73971-880

son and Garner, 19941, 3 ) on the chemistry and

biochemical origins of the compounds associated with

th e grass endophytes (Bush et al., 1993;Garner et al.,

1993; Rowan, 1993; Porter, 19941, and 4 ) on the

biological and agronomic aspects of endophyte-host

grass associations (Bacon, 1988; Bacon and Siegel,

1988; Siegel et al., 1990; Bacon and DeBattista, 1991;

Bacon and White, 1994). This review will focus on the

analyses of compounds that have been directly o rindirectly related to A. coenophialum Morgan-Jones &

Gams (Morgan-Jones and Gams, 19821, the endo-phyte of tall fescue (Festuca arundinacea Schreb.),

and A. lolii Latch, Christensen, & Samuels (Latch e t

al., 19841, the endophyte of perennial ryegrass

(Lolium perenne L. ).

Compounds Related to Acremon ium- In f ec t edFescue and Perennial Ryegrass

Ergopeptine alkaloids, primarily ergovaline ( Figu re

1,Tables 1 an d 21, and the indole isoprenoid lolitrem

871







872 PORTER

Figure 1. General structure of ergot peptide alkaloids

and their major ions resulting from electron impact (EI)

[fragments A , B, and C) and(or)chemical ionization (CI)

(fragments AH, BH, and CH) mass spectrometry (see

Table 1 for corresponding atomic mass units [amu], R 1

and R2 substituents, and text for explanation).

alkaloids,primarily olitremB (Figure 2, Table 2 ) ,

havebeen ssociatedwith toxic A. coenophialum-

infected tall fescue and A. lolii-infected perennial

ryegrass, respectively. Both ergovaline and lolitrem B

have been isolated from laboratoryulturesnd

grasses infected by these endophytes.

Other alkaloids occurring with the endophyte-host

grass associations are the loline alkaloids, primarily

N-acetylloline and N-formylloline (Figure 3, Table 2 )

in A. coenophialum-infected tal l fescue (Bus h et al.,

H

PAXILLJNE

Figure 2. Structures of paxilline and lolitrem B

alkaloids isolated from A . Zolii-infected ryegrass.

R 1cH3

CH3

cH3 ?3

R

H

2

LOLINE

N-ACETYLLOLINE CH3 -3

N-FORMYLLOLINE

N-ACETYLNORLOLINE H com3

N-METHYLLOLBE

CH0

Figure 3. Some of the major loline alkaloids isolated

from A . coenophialurn-infected tall fescue.

1982, 1993; Yates et al., 1990) and the pyrrolopyra-

zine alkaloidperamine (Figure 4,Table 2 ) in both

infected fescue and perennial ryegrass (Rowan et al.,

1986; Tapper e t al., 1989; Siege1 et al., 1990; Rowan,

1993). Although these compounds have been related

t o the nsect-deterrent properties of the endophyte-

infected grasses ather hananimal toxicoses, they

may augment the toxicity of the ergot and lolitrem

alkaloids, respectively. Therefore, a secondary empha-

sis of this review will be placed on the isolation andidentification of the lolines and peramine, along with

the ergot and lolitrem lkaloids.

A. coenophialum-Infected Fescue

Ergot Alkaloids. The chemistry, biochemistry, and

pharmacology of the ergotalkaloids from Claviceps

spp.-infected grass seeds, cereal grains, and laboratory

cultures have been the focus of research from time

immemorial Bove, 1970; Berde and Schild, 1978).

The known “ergotalkaloids” produced by Clauiceps

spp. may be divided into five major lasses: th e

clavine, lysergic acid, simple lysergic acid amides,ergopeptine, and ergopeptam lkaloids (Berdeand

Schild, 1978; Perellino et al., 1992, 1993). The

ergopeptines, imple lysergic acid amides,and cla-

vines (Figures 1, 5, and 6 ) have been isolated from A.coenophialum-infected tall fescue (Yates et al., 1985;

Lyons et al., 1986; Petroskind Powell, 1991).

Although ergovaline i s the major ergopeptine alkaloid

in A. coenophialum-infected tall fescue (Ya tes et al.,

1985; Lyons et al., 1986; Yates and Powell, 1988;

Rottinghauset al., 19931, lysergic acid amide (or

ergine; Figure 5 ) can exist in concentrations approxi-

mately equal to hat of ergovaline (Richard A. Shelby,







ANALYSIS OF ENDOPHYTEOXINS 873

Table 1 . Major ions and atomic mass units (amu) for the ergopeptine (or ergot cyclol) alkaloids; AH, BH,and CH are he major ions (amu) resulting from isobutane chemical ionization mass spectrometry (CIMS)

(see Figure 1 for corresponding ions)

Group R2 m a AH BHH

Ergotamine PUP (R 1 = -CH3)

Ergotamine -CH2Ph 581 268 31545

Ergosineb -i-Bu 547 268 28111

beta-Ergosine -sec-Bu 547 268 28111Ergovalineb -i-Pr 533 268 267 197

Ergobine -Et 519 268 252 183

Ergoxinegroup (R1 = -C2H5)

Ergostine -CH2Ph 595 268 329 245

Ergoptineb -i-Bu 561 268 295 211

beta-Ergoptine -sec-Bu 561 268 295 211

Ergonine -i-Pr 547 268 281 197

Ergobutine -Et 533 268 26783

Ergotoxinegroup (R1 = -i-Pr)

Ergocristine -CH2Ph 609 268 343 245

alpha-Ergocryptine -i-Bu 575 268 309 211

beta-Ergocryptine -sec-Bu 575 268 309 211

Ergocornineb -i-Pr 561 268 295 197

Ergobutyrine -Et 547 268 281 183

aMW = molecular weight.

bFound in endophyte-infected grass and cultures.

Dept. of Plant Pathology, Auburn Univ., Auburn, AL,

personal communication). Lysergic acid amide proba-

bly results from the solvolytic cleavage of lyser-

gylmethylcarbinolamide (Figure 5 ) and is not consid-

ered a true naturalproduct (Groger et al.,1968; Floss,

1976). Both lysergic acid amide and lysergylmethyl-

carbinolamide ave biological activity (Berde nd

Schild, 1978; Oliver et al., 1993) nd should be

considered (along with the minor ergot alkaloids)

where fescue toxicity is concerned. Ergonovine (F igure

5 ), another simple lysergic acid amide, also has been

isolated from endophyte-infected fescue seeds

(Petroski and Powell, 1991). However, this compound

may be from Claviceps contamination of the seeds

(Yates and Powell, 1988) and additional studies are

needed to verify whether ergonovine is indeed

constituent of A. coenophialum-infected tall fescue.

Because ergonovine is found in conjunction with the

ergopeptine alkaloids in Clauiceps-infected wheat and

Table 2. Major alkaloidsa associated withAcremoniumb-infected tall fescue (TF) andperennial ryegrass (PRG) seed and forageC

Endophyte-

grass Lolines Ergovaline Peramine Lolitrems

Alkaloids

A C - T F ~ 1,800-5,000 2-6 -

M-PRG~ - 5 5-40 5-10

rye (Scot t et al., 9921, it ispossible this alkaloid may

also be present n endophyte-infected grasses.The

clavinelkaloids chanoclav ine(s), penniclavine,

elymoclavine, and agroclavine (Figure 6 ) have been

isolated from endophyte-infected tall fescue (Lyons et

al., 1986) and are recursors in thebiosynthesis of the

simple lysergic acid amides andhe ergopeptines

(Floss , 1976; Garner t al., 1993; Porter, 1994.

Isolation and Identification. There are a variety of

procedures for the extraction, solation, and identifica-

tion of the ergotalkaloids from A. coenophialurn-

infected tall fescue. Current methods of choice involve

extractionwith eitheran aqueous tartaric or lactic

acid solution (lactic acid seems to work best for the

extraction of ergovaline from the infected fescue

~~

aConcentrations are micrograms of alkaloid/gram of material.

bAc = A. coemphialurn; Al = A, lolii.

‘Gallagher et al. , 1985; Fannin et al., 1990; Siege1 et al. , 1990;Yateset al., 1990; Bush etal., 1982, 1993.

Figure 4.Peramine and its major ions (amu)resulting

from electron impact mass spectrometry.







874 PORTER

LYSERGICCIDMIDEYSERGYLMETHYL-

(ERGINE)

Figure 5. Examples of some of the lysergiccid

[Testereci et l., 1990]), partition hromatography

with chloroform or methylene chloride a t anappropri-

ate pH, column clean-up procedures using either

silica, alumina, or a n ion exchange resin, and identifi-cation andanalysisusing co-chromatography (TLC

and[or] HPLC) with ultraviolet o r fluorescence detec-

tion. Mass spectrometry ( M S) also has been employed

for the identification, analysis, and quantification of

ergot lkaloids.

Theergotalkaloids are extremely usceptible to

photolytic and airoxidation, hydration, andepimeriza-

tion at the C-8 position of the ergolene ring (Berde

and Schild, 1978; Garner et al., 1993; Porter, 1994).

Epimerization of the C-8 position occurs ineither

acidic or basic conditions and, therefore, the isolation

of the C-8 epimers (designated with theufflxal - in ine

[i.e., ergovaline vs ergovalininel) occurs in mostextraction procedures. Decomposition and epimeriza-

tion may be minimized by working under subdued or

yellow light, concentrating extracts in uucuo at room

temperature or less (i.e., S 25"C), and by concentrat-

ing mall volumes of extractsunder a stream of

nitrogen.Storingdriedconcentrates inambervials

undernitrogen at or below0°C will helpprevent

further decomposition. Moubarak et al. (1993) have

successfully stored ergovaline a t -4°C for up to 12 mo.

Furthermore, ergovaline decomposes rapidly when

extracted from non-freeze-dried plant tissues (Garner

et al., 1993). Thus, observing the correct precautions

during sample collection, handling,andpreparation

for analysis are crucial fo r the meaningful isolation

andquanti tative nalysis of the ergot alkaloids.

High-Performance Liquid Chromatography. Use of

HPLC with either ultraviolet o r fluorescence detection

is rapidly becoming the preferred method for routine

screening and analysis of the ergopeptine alkaloids in

endophyte-infected grasses (Yates and Powell, 1988;

Testereci et al., 1990; Hill et al., 1991, 1993;

Rottinghaus et al., 991; Moubarak et al., 1993; Zhang

et al., 1994). For example, a rapid, simplified HPLC

method for theanalysis of the ergot alkaloids as-

sociated with A. coenophialum-infected tall fescue has

ERGONOVINE

amide group of ergot alkaloids

been developed (Richard A. Shelby, Dept. of Plant

Pathology, Auburn Univ., Auburn, AL, unpublished

dat a) . Extraction of infected seed o r grass with

alkalinemethanol, followedby filtration,anddirectHPLC analysis (fluorescence detection) with a mobile

phase of either 60 o r 70% alkaline methanol results in

separation of ergovaline,ergovalinine, lysergic acid

amide, and its isomer isolysergic acid amide (ergi-

ni ne ), along with other minor ergopeptine alkaloids.

Moubarak et l. (1993) haveeported a unique

preparative method for the isolation and purification

of large uantities of ergovaline. This procedure

involves a modification of the methods of Scott and

Lawrence (19801, Testereci et a l. (19901, and Rottin-

ghaus et al. (199 1): nfected seeds are extracted with

m3%

\

N\

NH TI

CHANOCLAVINE I AGROCLAVINE

ELYMOCLAVINE PrnCLAVINE

Figure 6. Examples of some of the clavine group of

ergot alkaloids isolated from A . coenophialum-infected

tall fescue.







ANALYSIS OF ENDOPHYTEOXINS 875

a 5% aqueous lactic acid solution andhe ergot

alkaloids are adsorbed onto SM-2 Biobeads (BioRad,

Hercules, CA). Extraction of the Biobeads with

methanol, followedby an HPLC clean-up procedure

using a C-18 RP column (Vydac, Separations Group,

Hesperia,CA),and HPLC analysisusinggradient

elution with acetonitri1e:ammonium carbonate:metha-

no1 as the mobile phase results in pure ergovaline ( 2

95%). Zhang et al. (1994) have employed an amber-lite XAD-2 exchange resin for a rapid clean-up step

after extracting infected fescue seed with a 5% lactic

acid:methanol4:1, vol/vol) solution. cott et l.

(1992) and Rottinghaus et al. ( 19 93 ) have reported

additional extraction, clean-up, and HPLC procedures

for the analysis of the ergopeptine alkaloids in cereal

grains,lour, nd feeds.

Thin-Layerhromatography. After the ergot

alkaloids have been extracted into a suitable organic

solvent, TLC on silica gel remains one of the most

powerful tools for theanalysisand identification of

these compounds. The major advantages of TLC are

that severalsamplesmay be analyzed at the sametime, TLC does not involve expensive instrumenta-

tion, and it may be used as a complement to MS in the

confirmatory identification and analysis of mixtures of

epimeric alkaloids (s ee below). Perellino et al. (1993)

have reported a TLC procedure on silica gel for the

separation of most all of the known ergot alkaloids. By

developing t he TLC plates nmethylene chloride:

isopropyl alcohol (92:8, vol/vol) three imes (drying

theplates between run s) , thesealkaloids separate

into the isolysergic acid group (i.e. , -inine epimers),

the ergotoxine group, the ergoxine group, and a

mixture of the ergotamine and clavine groups.

Removal of the silica from thearea of theplatesconsistent with the nown standards, extraction of the

alkaloids from the silica using methano1:chloroform

(1:4 o r 1:1, vol/vol) (Porter et al. , 1974; Perellino et

al., 19931, and rechromatography in chloroform:

methanol (9:l or 4:1, vol/vol) separates ergovaline

from the clavine alkaloids (agroclavine and chanocla-

vines)(Porteret al., 1979).Other solventsystems

used for the TLC analysis of these compounds are

listedn Table 3.

Visualization of the ergot alkaloids on a TLC plate

may be accomplished with a hand-held W light at

254 and 366 nm. The9,lO-double bond in theD-ring of

the ergolene portion of the molecule (Figure 1) is

conjugated with he indole nucleus and gives the

ergopeptine alkaloids their characteristic bright, pale

blue fluorescence (UV ambda max nmethanol at

approximately15 and 242 nm). Those ergot

alkaloids devoid of the 9,lO-double bondive a

characteristic dark blue absorption under54 nm (UV

lambda max in methanol a t approximately 292, 280,

275, and 222 nm) indicative of the indole nucleus.

Spraying he TLC plateswith a solution of p-N,N-

dimethylaminobenzaldehyde (v an Urk's reagent;

Stahl, 1969) followed by spraying with a 1% odium

Table 3. Solvent systemsa effectively usedforthin-layer chromatography on silicagel for

separation and identification of ergot alkaloidsb

CH2Cl2:iso-PrOH (92:8; 90:lO; 75:25)

CHC13:MeOH (9O:lO; 80:20)

CHC13:MeOH ( 9 O : l O ) in a saturated NH3 atmosphere

CHC13:MeOH:NH3 (94:5:1)

Benzene:Dimethylformamide (86.5:13.5)

CHC13:EtzNH (9O:lO)

aAll systems (vol/vol) are nsaturatedatoms, in tanks lined

bPerellino et al., 1993; Porter et al., 1974,1979,1981.

with Whatman No. 1 filter paper.

nitrite solution (water:e thano l, 1:l vol/vol) (Sprince,

1960) produces intense blue spots that re also

characteristic of the ergot alkaloids. Colorimetric

analysis a t 590 nm of a crude alkaloid fraction relative

to a known standard (i.e.,ergonovine maleate; ergota-

mine ta rt ra te ) provides a method for quantifying total

ergot alkaloids in crudextractsMichelonnd

Kelleher, 1963). Individual ergot alkaloids may thenbe identified and quantified by a combination of TLC,

HPLC, and(or) MS.

Mass Spectrometry. Identification and quantitative

analysis of the ergot alkaloids isolated from infected

grassand from culturesusing M S includeelectron

impact (EI) (Porter et al., 1979) , chemical ionization

( CI) (Porter and Betowski, 1981; Porter e t al., 1981),

and andemmass (MS/MS) spectrometry (Plattner

et al., 1983; Yates e t al., 1985; Lyons e t al.,1986;

Porteret al., 1987).Under lo w resolutionelectron

impact (70eV) , he ergopeptinealkaloids pyrolyti-

cally decompose into the lysergic acid amide, the cyclic

peptide, and diketopiperazine fragments A, B, and C,respectively (Figure1) . These ragments henun-

dergo E1 and produce spectracharacteristic of the

ergolene ring and the peptide portion of the parent

molecule. Fragments useful in he nterpretation of

these spectra occur at 70, 125, and 154 atomic mass

unit s (arnu) , which are characteristic of the proline

moiety (Porteretal ., 1979; Bianchi etal.,1982).

Theseons, in combination with the lysergic acid

amide ion at 267 amu (ionA, Figure l), re indicative

of an ergopeptinelkaloid.Using ergovaline for

example, in addition to 267 amu, the other two major

fragmentsassociatedwith ions B and C (i.e .,frag-

ments indicative of the methyl substituent a t R 1 and

the isopropyl substituent at R2, Figure1,Table 1 )

occur at 266 and 196 amu, respectively. The major

disadvantagewith low resolutionlectronmpact

mass spectrometry ( EIMS) (70 eV) in the nalysis of

the ergopeptine alkaloids is the low abundance (i.e., 5

1%) of the molecular ion and the diagnostic fragment

ion B when R 2 is an alkyl substituent (i.e., ethyl-, n-

butyl-,sobutyl-, sec-propyl-, isopropyl-; Figure,

Table 1). Although fragments ssociatedwith the

ergolene nucleus are isobaric with hose ragments

related t o the cyclic peptide ( B ) and diketopiperzine







876 PORTER

( C ) portions of theparent molecule (Porter et al.,

1981), ergovaline and ts C-8 epimer ergovalinine

produce a highabundance of ion 196 amu(ion C;

Porter et al., 1979). Spectral interpretationof complex

mixtures of the ergopeptines, however, become some-

what more difficult.

Isobutane chemical ionization mass pectrometry

( CIMS) has been employed in the dentification of the

ergopeptine lkaloids (Porte r nd Betowski, 1981;Porteretal.,1981). This method involves an ion-

molecule reaction of the alkaloids in the presence of a

reagentgas isobutane).The ion-molecule reaction

results in reduced fragmentations, as seen with low

resolutionEIMS, produces simplified spec tra, nd

thus circumvents interpretation difficulties of the

more complex spectra andhoseesulting from

mixtures of these alkaloids. Under sobutane CIMS,

the lysergic acid amide ( A ) , cyclic peptide ( B , and

diketopiperazine ( C ) molecules (Figur e 1) abstract a

proton from a t-butyl cation (which is generated in the

mass pectrometer)and he esulting ion-molecule

reaction produces spectra containing three major ionsrepresented by AH , BH, and CH in Figure1 and Table

1. These ons are 1 + amu greater han he parent

fragments A, B, and C (Porte r and Betowski, 1981;

Porter et al., 1981; Plattner et al., 1983; Yates et al.,

1985) . The major fragments used to identify the

known ergopeptinelkaloids by this method are

outlined in Table 1.Although ergovaline and its C-8

epimerergovalinine produce thesame hree major

fragments at 268 (A H) , 267 (B H) , and 197 (C H )

amu and cannot be distinguished by this method, both

compounds ( a s with the other ergopeptines and their

C-8 epimers) epara te nicely using TLC an d(o r)

HPLC analysis (Porter et al., 1974, 1979; Yates andPowell, 1988; Rottinghaus et al., 1991, 1993; Perellino

et al., 1993). Tandem mass spectrometry (Platt ner et

al., 198 3) also has been employed in the separation

andnalysis of ergopeptine alkaloids from both

endophyte-infected tall fescue and perennial ryegrass

(Yatesetal. , 1985; Lyons etal. , 1986; Rowan and

Shaw, 1987). In an overly simplified description, MS/

MS (which also is conducted in he presence of a

reagent and[orl a target g as) uses one stage of mass

separation to isolate the individuallkaloids of

interest in a crude extract; depending on the ioniza-

tion mode, this usually involves the molecular ion, a

protonated molecular ion, or a molecular anion;asecond stage of mass separation is then employed to

analyzehe product and(or )daugh ter ions (also

dependent on the eagent and[orl targetgas).The

fragmentationechanisms for the ergopeptine

alkaloids tilizing MSM S withsobutane she

reagent gas and argon as the target gas are nalogous

to that described for isobutane CIMS andre

described indetai l Plattner t al., 1983). Major

advantages of this system are that complex mixtures

of these compounds can be analyzedwithoutprior

cleanup, only small samples of extracted material are

needed, and isomeric species (i.e., ergosine vs beta-

ergosine; Figure 1, Table 1) can be distinguished.

However, MSNS analysisequires expensive in-

strumentation not readily available to most laborato-

ries, and like CIMS, this systemcannotdistinguish

between epimericrgopeptinelkaloids.ubse-

quently, M S N S is not practical for routine screening

of toxic or infected grasses. Field desorption M S also

has been used in the identification of the ergopeptinealkaloids (Bianchi t l.,1982) nd provides ex-

tremely simplified spectra along with intense molecu-

lar ions.

Quantification an d Toxicity

Cornel1 et l. 1990) haveeported rgovaline

concentrations at 50 ng/g of infected grass is suffxient

to produce some signs of fescue toxicosis incattle

stressed by heat. Spiers ( 19 93 demonstrated similar

thermoregulatory mbalances in rats dosed wither-

govaline. Dyer (1 99 3) an d Oliver e t al. (1993 ) have

reported analogous in vi t ro vaso-activities of ergova-linend lysergic acid amide, respectively. Thus,

although ergovaline is n exceptional marker as-

sociated with A. coenophialum-infected fescue, ergova-

line toxicosis in livestock most probably is augmented

by the tota l concentration of the ergot, and possibly

other alkaloidsn the infected grass.

Ergovalineconcentrations in infected tall fescue

seeds and forages have ranged from 2 t o 6 pg/g (Table

2) . Total oncentrations of ergot alkaloids in A .coenophialum-infected tall fescue vary with the season

andamount of nitrogen ertilization Lyons et al.,

1986; Belesky etl., 1988; Lyons etl., 1990;

Rottinghaus e t al., 1991; Arachevaleta e t al., 19921,and these are factors that should be considered prior

to sample collection and analysis for alkaloid content.

Whetherheelative concentrations of individual

ergot alkaloids vary with season or nitrogen fertiliza-

tions urrentlyunknown.

LoIine Alkaloids. The loline alkaloids in A.

coenophialum-infected tall fescue are produced either

by the plant in response t o the fungus an d(o r) as a

defense mechanism in response to insectherbivory

that may involve interactionsetweenothhe

endophyte and its host grass. Moreover, Petroski e t al.

(1 99 0) have suggested the lolines may be alleopathic,

thus improving the infected-grass’s ability to competewithothergrasses.Thechemistry, occurrence, and

biological effects of the loline alkaloids and associated

endophyte-grassnteractionsave been reviewed

(Powell and Petroski, 1992; Bush et al., 1993). The

lolines are directly related toA. coenophialum-infected

grasses ndhave not been identified in A. lolii-

infected grassesSiege1 et al., 1990).

Capillarygaschromatography ( G C ) using either

flame ionization ( FID) an d( or ) MS detection ( MSD)

(Yatestl., 1990; Powell andetroski, 1992;

Tepaske et al. , 1993) is the c urrent method of choice







ANALYSIS OF ENDOPHYTE TOXINS 877

for the analysis of the loline alkaloids. Extraction of

ground seed (approximately 5 g) withmethylene

ch1oride:methanol:ammonia (75:25:0.5, vol/vol/vol),

followed by filtration, provides extracts ready for GC/

FID or GC/MSD analysis. Forage samples may be

extractedsimilarly, but prior t o analysis,a sulfonic

acid solid phase clean-up step of the forage extracts is

necessary t o remove substances th at interfere with the

assay.Thedetection imit for N-acetylloline and N-formylloine is10 ng. Tepaske etal. (1993 have

described a similar GC/MSD procedure for the analy-

sis of the loline alkaloids in bovine urine and plasma.

Although the loline alkaloids do not provide a well-

defined molecular ion in the mass spectrum ( 70 e V) ,

their separation under GC conditions and characteris-

tic mass fragmentation patterns (Powell and Petroski,

1992) allows for their unequivocal identification and

quantification.

Petroskiet al. (1990)and Powell andPetroski

(19 92 ) reported a method fo r the separation of the

loline and the ergot alkaloids from endophyte-infected

tall fescue whereby an alkaloidal extract is subjectedto column chromatographyusingSephadex LH-20.

The loline alkaloids pass through the column and the

ergot alkaloids are recovered by exhaustive elution of

th e LH-20 with methanol. Also, Petroski and Powell

(1991 employed counter current chromatography for

the separation of milligram quantities of N-methyllo-

line, N-acetylloline, and N-formylloline from an endo-

phyte-infected tall fescue seed.

Total lolines (defined as N-formyl- and N-acetyllo-

line) occurring in endophyte-infected tall fescue seed

(3,263 pg/g) and forage (1,723 pg/g) were quantified

using GC/FID (Yates et al., 1990). Concentrations of

theselkaloids ( a s with the ergot alkaloids)nforages varywith eason, theamount of nitrogen

fertilization,grazingpressure, ndhe mount of

insect herbivory (Bush et al., 1982, 1993; Belesky et

al., 1987; Eichenseer et al., 1991). Further studies are

needed t o determinewhetherhe loline alkaloids

significantly contribute to fescue toxicosis in livestock.

A. lolii-Infected erennial yegrass

Paxillinendhe Lolitrern Alk aloi ds. Current

evidence suggests that the indole-isoprenoid lolitrems

are almost as diverse as the ergot alkaloids. Paxilline

and lolitrem B (Figure ) are the wo major alkaloidsassociatedwith erennialyegrasstaggers (Gal-

lagher et al.,1985; Weedon and Mantle, 1987; Miles et

al., 1992; Fletcheretal., 1993; Pennet al., 1993).

Studies involved with determining the biosynthesis of

paxilline and lolitrem B resulted in the identification

of alpha-paxitriol, lolitriol, and the lolitrems A, C, D,

and E (Milesetal.,1993). Theseadditionalminor

lolitremsmay ontribute to the overall toxicity of

paxilline and lolitrem B.

Gallagher et al. (19 85) reported a n isolation and

screeningmethod for lolitrem B in A. lolii-infected

perennial yegrass. One gram of oven-dried, milled

grass was xtractedwith ch1oroform:methanol ( 5 0

mL; 2:1, vol/vol) for 1 h. One milliliter of the extract

was dried under nitrogen, reconstituted in methylene

chloride (2 mL j an d subjected to a cleanup step on

silica. The eluent (100 pL) from the silica was then

analyzed by HPLC on a Zorbax Silica column

(DuPont , Wilmington, DE ), using methylene chloride:

acetonitrile (80:20, vol/vol) as the mobile phase.Recovery of lolitrem B was 93 to 97% with detection

limits at .5 ng(fluorescencedetection).ThisHPLC

method has been used t o screen up to 80 sampledd.

The mount of lolitremB inhe infected grass

necessary t o elicit thestaggers syndrome is only 5

ppm (Gallagher t l., 985).

Perumine. Although the correlation of peramine

(Figure 4 ) as the majornsect deterrentn A.

coenophialum-infected tall fescue and A. lolii-infected

perennial yegrasshas been reported (Rowanand

Tapper, 1989; Tapper et al., 1989; Siege1 et al., 19901,

mammalian toxicity to permine has not been deter-

mined. The solation and analysis of peramine frominfected grassespresents some unique problems be-

cause of the guanidino moiety attached t o th e

aliphatic side chain on the pyrrolopyrazine ring.

Tapper et al. (19 89 ) have reported a method for the

analysis of both peramine and lolitrem B using awo-

phase extraction system in which 100 mg of freeze-

dried, ground grass is first extracted with methanol:

chloroform (3 mL) and followed by concurrent extrac-

tionswith exane ndwater ( 3 mL each). The

aqueous and organic phases are separatedby centrifu-

gation. Lolitrem B was analyzed in the organic phase

as previously reported (Gallaghertl., 19851,

whereasperamine,after minor cleanupusing on-exchange chromatography, was analyzed in the aque-

ous phase by HPLC with a mobile phase of acetoni-

tri1e:guanidinium formate (pH = 3.7). Recovery of

peramine is 93 o l oo%,with detection limits at 1pg/g

of infected grass. Alternatively,peraminemay be

analyzed in the aqueous phase, after an ion-exchange

cleanup stepwith two minicolumns connected in

series. The first column (BioRad AG 2 x 8, 200-400

mesh) is in thehydroxide form and the second column

(Analytichem Bond Elut CBA) is n he carboxylic

acid form. After the aqueous phase is aspirated onto

the columns, they re washedwith 80% aqueous

methanol (3 mL , the columns separated, and pera-mine luted from the acid column with queous

methanol and formic acid. Peramine is then analyzed

byTLC using ch1oroform:methanol:acetic acid:water

(20:10:1:1, vol/vol/vol/vol) as the developing solvent

(Tapperetal.,1989).Fanninetal. 1990)have

reported a rapid, sensitive, reverse-phase TLC method

for theanalysisand quantification of peramine in

crude extracts of several endophyte-infected grasses,

and Rowan et al. ( 19 86) have defined the characteris-

tic low-resolution massragmentation (70 eV) of

peramineFigure ).







878 PORTER

As with he loline alkaloids, furtherstudiesare

needed to establish whether peramine augments the

toxicities of the ergot an d( or ) lolitremalkaloids or

whether peramine causes a unique mammalianoxico-

sis of its own.

Summary

The major toxins associated with Acremonium-

infected grassesandwithanimal toxicoses are he

ergot and lolitrem alkaloids. Ergovaline and lysergic

acid amide (and[or] lysergylmethylcarbinolamide) are

the major ergot alkaloids in A. coenophialum-infected

tall fescue. Paxilline and lolitremB are he major

lolitrems in A. ZoZii-infected perennial ryegrass. Pera-

mine is he major nsect deterrent n both infected

grasses,whereashe loline alkaloidseemo be

primarily associated with A. coenophialum-infected

tall fescue.

Identification and analysis of the ergot alkaloids in

endophyte-infected grassesre effectively accom-plished by a combination of TLC and HPLC and the

use of EIMS and CIMS. High performance liquid

chromatography with either ultraviolet or luorescence

detection is the preferred method for routine screening

of endophyte-infected plants for the ergopeptine

alkaloids, the lolitrems, and peramine.The loline

alkaloids in infected tall fescue are more effectively

analyzed by GC with FID or MSD. The advantages of

GC/MSD analyses for the lolines ar e th at retention

times are consistent with known st and ard s and tha t

their characteristic mass spectra llow for unequivocal

identification and quantification.

Animal toxicosis caused by consuming A. coenophia-Zum-infected tall fescue has been reported to occur at

levels of 50 ng of ergovaline/g of grass, whereas A.

ZoZii-infected perennial ryegrass produces the staggers

syndrome at 5 Fg of lolitrem B/g of grass. The diverse

number of biologically active alkaloids that appear in

the endophyte-infected grasses no doubt will reduce

the overall quantity necessary to produce either tall

fescue and( or ) perennial yegrass toxicosis in live-

stock. Therefore, the total concentrations of the ergot

and( or) lolitrem alkaloids should beconsidered where

livestock are grazed on infected grass pastures.

Implications

Chemical analysis of endophyte toxins in Acremo-

nium-infected fescue and other grasses toxic to live-

stock has consistentlyadded oour knowledge for

combating hese problems. Improvements in extrac-

tion efficiency, chromatography, and instrumentation

have provided information on the number and classes

of alkaloidsssociatedwith fescue toxicosis and

ryegrass staggers n animals on these Acremonium-

infected grasses. Continued improvements with isola-

tion and chromatographic echniques should provide

sufficient quantities of toxins necessary for further

animal esting.These tudies hould hen provide

answers t o both the individual and combined activities

of the toxinssolated from the infected grasses .

Moreover, chemical ana lyses and animal studies will

eventually define whether fescue toxicosis in livestock

results from th e t o t a l concentration of the ergot

alkaloids andwhether toxicosis is augmented byeither or both the loline alkaloids and peramine n

infected tall fescue. Animal studies also will define

whetherhe lolitrem-induced ryegrass taggersn

livestock isaugmented by the ergot alkaloids and

peraminen infected perennialyegrass.

LiteratureCited

Arachavaleta, M,, C. W . Bacon, R. D. Plattner, C. S. Hoveland, and

D. E. Radcliffe. 1992. Accumulation of ergopeptide alkaloids in

symbiotic tall fescuegrown under deficits of soi l water and

nitro gen fertilizer. Appl. Enviro n. Microbiol. 583357.

Bacon, C. W. 1988. Procedure for isolating the endophyte from tallfescue and screening isolates or ergot alkaloids. Appl. Environ.

Microbiol. 54:2615.

Bacon, C . W ., an d J. DeBattista. 1991. Endophytic fungi of grasses.

In: D. K. Arora, B. Rai, K. G. Mukeji, andG. R. Knudsen (Ed.)

Handbook of Applied Mycology. Soils and Pla nts . Vol. 1. p 231

Marcel Dekker, New York.

Bacon, C. W., an d M. . Siegel. 1988. Endophyte parasitism of tall

fescue. J. Prod. Agric. 1:45.

Bacon, C. W ., an d J. F. White, Jr. 1994. Biotechnology of Endophytic

fungi of grasses. CRC Press, Boca Raton, FL.

Belesky, D. P., J. D. Robbins, J. A. Stuedemann, S. R. Wilkinson,

an d 0. . Devine. 1987. Fungal endophyte infection-loline

derivative alkaloid concentra tionof grazed tall fescue. Agron. J.

79:217.

Belesky, D. P, , J. A. Stuedemann, R . D. Plattner, and S. R. Wilkin-

son. 1988. Ergopeptin e alkaloids in graze d tall escue. Agron. J80:209.

Berde, B., andH. 0 . Schild. 1978. Ergot Alkaloids andRelated

Compounds. Handbook of Experimental Pharmacology. Vol. 49

Springer-Verlag, New York.

Bianchi, M. L ., N. C . Perellino, B. Gioia, and A. Minghetti. 1982

Production by Claviceps purp urea of two new peptide ergo

alkaloids belonging to a new series containing alpha-aminobu-

tyric acid. J . Nat. Prod. (Lloy dia) 45:191.

Bove, F. J. 1970. The Story of Ergot. S. Karger, New York.

Bush, L. P, , P. L. Cornelius, R.C. Buckner, D. R. Varney, R. A.

Chapman, P. B. Burrus, C. W. Kennedy, T. A. Jones, andM. .

Saun ders . 1982 . Association of N-acetylloline de rivativ e and N-

formylloline derivative with Epzchloe typhina in all fescue.

Crop Sci. 22:941.

Bush, L. P., F. F. Fannin, M. R. Siegel, D. L. Dahlman, and H. R.

Burton. 1993. Chemistry, occurrence and biological effects ofsaturated pyrrolizidine alkaloids associated withendophyte-

gras s nteractions. Agric. Ecosyst. & Environ. 44:81.

Cornell, C. N., J. V. Lueker, G. B. Garner, and J. L. Ellis. 1990.

Estab lishin g ergovaline evels or escue toxicosis, withand

without endopara sites, under controlled climatic conditions. In

S. S. Quisenberry and R. E. Joost (Ed.) Proc. Int. Symp.on

AcrernoniurnlGrass Interactions, Nov. 3, 1990, New Orleans

L A . p 75. Louisiana AgriculturalExperimentStation,Baton

Rouge.

Dyer, D. C. 1993. Evidence that ergovaline a cts on sero tonin recep-

tors. Life Sci. 53:223.

Eichenseer, H., D. D. Dahlman, and L. P. Bush . 1991. Influence of

endophyte infection, plantgendarvestnterval on







ANALYSIS OF ENDOPHYTETOXINS 879

Rhopalosiphum padi survival and its relation to quantityf N-

formyl and N-acetyl loline in tall fescue. Entomol. Exp. Appl.

60:29.

Fanni n, F. F., L. P. Bush, M. R. Siegel, and D. D. Rowan. 1990.

Analysis of peramine in fungal endophyte-infected grasses by

reversed-phasehin-layer chromatography. J . Chromatogr.

503:288.

Fletcher, L. R., I. Garthwaite,and N. R. Towers.1993. Ryegrass

stagge rs in the absence of lolitrem B. In: D. E. Hume , G.C.M.

Latch, and H.S. Easton ( E d . ) Proc. 2nd Int. Symp. on Acremo-niumiGrass Interactions. Feb. 4-6, 1993, Massey University. p

119.AgResearch, Palmerston North, New Zealand.

Floss, H. G. 1976. Biosynthesis of ergot alkaloids and related com-

pounds. Tetrahedron 32:873.

Garner, G. B., G. E. Rottinghaus, C. N. Cornell, and H. Testereci.

1993. Chemistry of compounds associated with endophyte/g rass

interaction: Ergovaline- andrgopeptine-related alkaloids.

Agric. Ecosyst. & Environ. 44:65.

Gallagher, R. T., A.D. Hawkes, and J. M. Stewart. 1985. Rapid

determination of the neurotoxinolitremB in erennial

ryegrass by high-performanceiquid chromatography with

fluorescencedetection. J . Chromatogr. 321:217.

Groger, D., D. Erge, and H. G. Floss. 1968. Uber die he rfi un ft der

seitkette im d-lysergsaure-methlcarbinolamide.. Naturforsch.

Sect. B. ChemSci. 23B:177.

Hill, N. S . , W. A. Parrott , andD. D. Pope. 1991. Ergopept ine alkaloid

production by endophytes n a common tall fescue genotype.

Crop Sci. 31:1545.

Hill, N. S., . E. Rottinghaus , C. S. Agee, and L. M. Schultz. 1993.

Simplified sample preparation for HPLC analysis of ergovaline

in all fescue. Crop Sci. 33:331.

Hoveland, C. S . 1993. Importa nce and economic significance of th e

Acremonium endophytes to performance of animals and grass

plant. Agric. Ecosyst. & Environ. 44:3.

Latch, G.C.M., M. J. Christensen,and G. J. Samuels. 1984.Five

endophytes of LoZium an d Festuca in New Zealand . Mycotaxon

20:535.

Lyons, P. C., J . J .Evans, and C. W. Bacon. 1990. Effects of fungal

endophyte Acremonium coenophialum on nitrogen accumula-

tion and metabolism in tall fescue. Plant Physiol. (Roc kv) 92:

726.

Lyons, P. C., R. D. Pla ttn er, and C. W. Bacon. 1986. Occurrence of

peptide and clavine ergot alkaloids in tall fescue grass. Science

(WashDC) 232:487.

Michelon, L. E., and W. J . Kelleher. 1963. The spectrophotometric

determina tion of ergot alkaloids. A modified procedure employ-

ing paradimethylaminobenzaldehyde.Lloydia (Cinci) 26:192.

Miles, C. O., S. C. Munday, A. L. Wilkins, R. M. Ede, A. D. Hawkes,

P. P. Embling, and N. R. Towers. 1993. Large scale isolation of

lolitrem B, structu re determ ination of some minor lolitrems,

and tremorgeni c activities of lolitrem B and paxilline in sheep.

In : D. E. Hume, G.C.M. Latch, a nd H. S. Easton ( E d. ) Proc.

2nd Int. Symp. on AcremoniumlGrass Interactions. Feb. 4-6,

1993, Massey University. p 85. AgResearch, Palmerston North,

New Zealand.

Miles, C. O., A. L. Wilkins, R. T. Gallagher, A. D. Hawkes, S. C.Munday, a nd N. R. Towers. 1992. Synth esis and tremorgenicity

of paxitrolsand lolitriol: Possible biosynthetic precu rsor s of

lolitrem B. J. Agric. Food Chem. 40:234.

Morgan-Jones, G., and W. Gams. 1982. Notes on Hyphomycetes,

XLI. An endophyte of Festuca arundin acea an anamorp h of

Epichole typhina, new taxa in one o r two sections of Acremo-

nium. Mycotaxon 15:311.

Moubarak, A. S ., E. L. Piper, C. P. West, and Z. B. Johnson. 1993.

Inte raction of purified ergovaline from endophyte-infected tall

fescue with synaptosomalATPaseenzyme system. J. Agric.

Food Chem. 41:407.

Oliver, J . W., L. K. Abney, J. R. Str ickland, and R.D. Linnabary.

1993. Vasoconstriction in bovine vasculature induced by the

tall fescuealkaloid ysergamide. J . Anim.Sci. 71:2708.

Penn, J., I. Garthwaite, M. J. Christensen, C. M. Johnson, and N . R.

Towers. 1993. The importance of paxilline in creening for

potentially tremorgenic Acremonium isolates. In: D. E. Hume,

G.C.M. Latch, and H . S. Easton (E d. ) Proc. 2nd Int. Symp. on

AcremoniumiGrass In terac tions. Feb. 4-6, 1993, Massey

University. p 88. AgResearch, Palmerston North, New Zealand.

Perellino, N. C., J. Malyszko, M. Ballabio, B. Gioia, and A. Min-

ghetti. 1992. Direct biosynthesis of unnatu ral ergot alkalo ids. J.

Nat. Prod. (Lloydia) 55:424.

Perellino, N. C., J. Malyszko, M. Ballabio, B. Gioia, and A. Min-

ghetti. 1993. Identification of ergobine, a new natural peptide

ergotalkaloid. J . Nat. Prod. (Ll oydi a) 56:489.

Petroski, R. J. , D. L. Dornbos, Jr., and R. G. Powell. 1990. Germina-

tion and grow th inhibition of annual ryegrass (Lolium mul-

tiflorum L. ) nd alfalfa (Medicago sat iva L.) by loline

alkaloids and ynthe tic N-acetylloline derivatives. J. Agric.

Food Chem. 38:1716.

Petroski, R. J. , an d R. J. Powell. 1991. Prepar ative separa tion of

complex alkaloid mixture by high-speed countercurrent chro-

matography. In: P. A. Hedin (Ed.) Naturally Occurri ng Pest

Bioregulators. Am. Chem. Soc. Symp. Series #449. p 426.

Plattner, R. D., S. G. Yaks, andJ. K. Porter. 1983. Quadrupole mass

spectrometry/mass spect rometry of ergot cyclol alkaloids. J.

Agnc. Food Chem. 31:785.

Porter, J. K. 1994. Chemical constituents of gras s endophytes. In : C.

W. Bacon and J . F. White, J r . (Ed .) Biotechnology of En-

dophytic Fungi of G rasses. Chap. 8. CRC Press, Boca Raton,

FL.

Porter, J. K., C. W. Bacon, and J. D. Robbins. 1974. Major alkaloids

of a Claviceps isolated from toxic bermuda grass. J. Agric. Food

Chem. 22:838.

Porter, J. K., C. W. Bacon, andJ. D. Robbins. 1979.Ergosine,

ergosinine, and chanoclavine I from EpichZoe typhzna. J. Agric.

Food Chem. 27:595.

Porter, J. K. , C. W. Bacon, J . D. Robbins, and D. Betowski. 1981.

Ergot alkaloid identification in clavicipitaceae systemic fungi of

pasturegrasses. J . Agric. Food Chem. 29:653.

Porter, J . K., C. W. Bacon, R. D. Plattner, andR. F. Arrendale. 1987.

Ergot peptide alkaloid spectra of Clauiceps-infected tall fescue,

wheat,andbarley. J. Agric. Food Chem.35359.

Porter, .K. ,and D. Betowski. 1981. Chemical onization mass

spectrometry of the ergot cyclol alkaloids. J.Agric. Food Chem.

29:650.

Porter, J . K., andF. N. Thompson, Jr . 1992. Effects of fescue

toxicosis on eproduction in livestock. J . Anim. Sci. 70:1594.

Powell,R. G., and R. G. Petroski. 1992. The loline group of pyrrolizi-

dine alkaloids. In : S. W . Pelletier (Ed . ) The Alkaloids: Chemi-

cal and Biological Perspectives. Vol. 8. p 320. Springer-Verlag,

New York.

Rottinghaus, G. E., G. B. Garner, C. N. Cornell, and J. L. Ellis. 1991.

An HPLCmethod for quant itating ergovaline inendophyte-

infected tall fescue: seasonal variation of ergovaline levels in

stems with leaf sheaths, leaf blades and seed heads. J. Agric.

Food Chem. 39:112.

Rottinghaus, G. E., L. M. Schultz, P. F. Ross, and N. S. Hill. 1993.

An HPLCmethod for the detection of ergot ingroundandpelleted foods. J. Vet. Diagn. Invest. 5:242.

Rowan, D. D. 1993. Lolitrems, peramine and paxilline: Mycotoxins

of ryegrassiendophyte interaction. Agric. Ecosyst. & Environ.

44:103.

Rowan, D. D., M. B. Hunt, and D. L. Gaynor. 1986. Perami ne, a

novel insect feeding deterrent from ryegrass infected with the

endophyte Acremonium loliae. J. Chem. Soc. Chem. Commun.

935.

Rowan, D.D., and G. J. Shaw. 1987. Detection of ergopeptine

alkaloids in endophyte-infected perennial ryegrass by tandem

massspectrometry . N.Z. Vet. J . 35:197.

Rowan, D. D., an d B. A. Tapper . 1989. An efficient method fo r the

isolation of peramine, an insect feeding deterrent produced by

the fungus Acremonium Zolii. J . Nat. Prod. (Lloydia) 52:193.







880 PORTER

Scott, P. M ,, and G . A. Lawrence. 1980. Analysisof ergot alkaloids in

flour. J. Agric. Food Chem.28:1258.

Scott, P. M., .A. Lombaert, P. Pellaer s, S. Bacler, and J. Lappi.

1992. Ergot alkaloids in grain foods sold in Canada. J.AOAC

Int. 75:773.

Siegel, M. R., G.C.M. Latch, L. P. Bush, F. F. Fannin, D. D. Rowan,

B. A. Tapper, C. W. Bacon, and M. C. Johnson. 1990. Fungal

endophyte-infected grasses: alkaloidaccumulation andaphid

response. J. Chem. Ecol. 12:3301.

Spiers, D. E. 1993. Fescue toxicosis: the environment al stres s con-

nection. J. h i m . Sci. 7l(Suppl.1):116(Abstr.).

Sprince, H. 1960. A modified Ehrlich benzaldehyde reagent for

detection of indoles on paper chromatograms.J . Chromatogr. 3:

97.

Stahl, E. 1969. Thin Layer Chromatography, A Laboratory Hand-

book. No. 73. pp 127, 869.Springer-Verlag, New York.

Stuedemann, J. A, and C. S . Hoveland. 1988. Fescue toxicity: His-

tory and mpact on animalagriculture. J. Prod.Agnc.1:39.

Tapper, B. A., D. D. Rowan, and G.C.M. Latch. 1989. Detection and

measurement of the alkaloid peramine in endophyte-infected

grasses. J. Chromatogr. 463:133.

Tepaske, M. R. , R. G. Powell, and R. J. Petroski. 1993. Quantitative

analysis of bovine urine and blood plasma for loline alkaloids.

J. Agric. Food Chem.41:231.

Testereci, H., G. B. Garner , G. E. Rottinghaus, C. N. Cornel, and

M.P.A. Andersen. 1990.Method for large scale solation ofergovaline from endophyte-infected tall fescue seed ( Festuca

arundinacea). In : S. S.Quisenbeny andR. E. Joost (E d. ) Proc.

Int. Symp. on AcremoniurnlGrass Interactions. Nov. 3, 1990,

New Orleans, L A . p 100. Louisiana AgriculturalExperiment

Station,Baton Rouge.

Thompson, F. N., and G. B. Garner. 1994. Vaccines and pharmaco-

logic agen ts to alleviat e escue toxicosis. In: C. W. Bacon and J.F. White J r. Ed. ) Biotechnology of Endophytic Fungi of

Grasses.Chap.9. CRC Press, Boca Raton, FL.

Thompson, F. N., and J. K. Porter. 1991. Tall fescue toxicosis in

cattle: Could there be apublic health problem. Vet. Hum .

Toxicol. 32:51.

Weedon, C. M., and P. M. Mantle. 1987. Paxilline biosynthes is by

Acremoniun loliae; a step towarddefining the origin of lolitrem

neurotoxins. Phytochemistry (OxF) 26:969.

Yates, S. G., R. J. Petroski, and R. G. Powell. 1990. Analysis of loline

alkaloids in endophyte-infectedfescue by capillary gas chro-

matography. J. Agric. Food Chem. 38:182.

Yates, S. G., R. D. P lattner, and G. B. Garner. 1985. Detection of

ergopeptine alkaloids inendophyte infected, toxic K-31 tall

fescue by mass spectrometry hass spectrometry . . Agric. Food

Chem.33:719.

Yates, S . G., and R. G. Powell. 1988.Analysis of ergopeptine

alkaloidsn endophyte-infected tall fescue. J . Agric. Food

Chem. 36337.

Zhang, Q., D. E. Spiers, G. E. Ro ttinghaus, and G. B. Garner. 1994.

Thermoregulatory effects of ergovaline isolated from endophyteinfected tall fescue seed on ra ts . J. Agric. Food Chem. 42:954.







Citations http://jas.fass.org#otherarticles

This article has been cited by 15 HighWire-hosted articles:

http://jas.fass.org/#otherarticles




Documents

Analysis of Endophyte Toxins Fescue and Other Grasses Toxic to Livestock