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

J. K. Porter

J Anim Sci 1995. 73:871-880.

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 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 mass) 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 to livestock. Other compounds isolated
from the endophyte-infected fescue include: lysergic
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 their entire life cycle within the aerial portion of
their 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 and 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.
871
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acid amide (ergine), 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.
J. Anim. Sci. 1995. 73971-880
son and Garner, 19941, 3 ) on the chemistry and
biochemical origins of the compounds associated with
the 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 or
indirectly 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 et
al., 19841, the endophyte of perennial ryegrass
( Lolium perenne L. ).
Compounds Related to Acremonium-Infected
Fescue and Perennial Ryegrass
Ergopeptine alkaloids, primarily ergovaline (Figure
1, Tables 1 and 21, and the indole isoprenoid lolitrem
8 72
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], R1
and R2 substituents, and text for explanation).
alkaloids,primarilylolitremB (Figure 2, Table 2),
havebeenassociatedwith toxic A. coenophialum-
infected tall fescue and A. lolii-infected perennial
ryegrass, respectively. Both ergovaline and lolitrem B
have been isolated from laboratoryculturesand
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 tall fescue (Bush et al.,
H
PAXILLJNE
Figure 2. Structures of paxilline and lolitrem B
alkaloids isolated from A . Zolii-infected ryegrass.
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PORTER
R
R1 2
LOLINE H
cH3
N-ACETYLLOLINE CH
3 -3
N-FORMYLLOLINE CH0
CH3
N-ACETYLNORLOLINE H com3
N-METHYLLOLBE cH3 ?3
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 et al., 1989; Siege1 et al., 1990; Rowan,
1993). Although these compounds have been related
t o the insect-deterrent properties of the endophyte-
infected grassesratherthananimal 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 and
identification of the lolines and peramine, along with
the ergot and lolitremalkaloids.
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 majorclasses: the
clavine, lysergic acid, simple lysergic acid amides,
ergopeptine, and ergopeptamalkaloids (Berdeand
Schild, 1978; Perellino et al., 1992, 1993). The
ergopeptines,simple 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; Petroski and Powell, 1991).
Although ergovaline is the major ergopeptine alkaloid
in A. coenophialum-infected tall fescue (Yates 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 tothat of ergovaline (Richard A. Shelby,
 OF ENDOPHYTE
ANALYSIS TOXINS 873
Table 1. Major ions and atomic mass units (amu) for the ergopeptine (or ergot cyclol) alkaloids; AH, BH,
and CH arethe major ions (amu) resulting from isobutane chemical ionization mass spectrometry (CIMS)
(see Figure 1 for corresponding ions)

Group R2 m CH
a AH BH
Ergotamine PUP (R1 = -CH3)
Ergotamine -CH2Ph 581 245 268 315
Ergosineb -i-Bu 547 211 268 281
beta-Ergosine -sec-Bu 547 211 268 281
Ergovalineb -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 533 183 268 267
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-
carbinolamidehave biological activity (Berdeand
Schild, 1978; Oliver et al., 1993)and should be
considered (along with the minor ergot alkaloids)
where fescue toxicity is concerned. Ergonovine (Figure
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 indeeda
constituent of A. coenophialum-infected tall fescue.
Because ergonovine is found in conjunction with the
ergopeptine alkaloids in Clauiceps-infected wheat and
rye (Scott et al.,19921, it ispossible this alkaloid may
also be presentin endophyte-infected grasses.The
clavine
alkaloids chanoclavine(s), penniclavine,
elymoclavine, and agroclavine (Figure 6 ) have been
isolated from endophyte-infected tall fescue (Lyons et
al., 1986) and areprecursors in thebiosynthesis of the
simple lysergic acid amides andthe ergopeptines
(Floss, 1976; Garneret al., 1993; Porter, 1994.
Isolation and Identification. There are a variety of
procedures for the extraction, isolation, and identifica-
tion of the ergotalkaloids from A. coenophialurn-
infected tall fescue. Current methods of choice involve
extractionwith either an aqueous tartaric or lactic
acid solution (lactic acid seems to work best for the
extraction of ergovaline from the infected fescue

Table 2. Major alkaloidsa associated with

Acremoniumb-infected tall fescue (TF) and
perennial ryegrass (PRG) seed and forageC

Alkaloids
Endophyte-
grass Lolines Ergovaline Peramine Lolitrems
AC-TF~ 4
1,800-5,000 2-6 -
M-PRG~ - 5 5-40 5-10
~~

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; Figure 4.Peramine and its major ions (amu)resulting
Yateset al., 1990; Bush etal., 1982, 1993. from electron impact mass spectrometry.

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 8 74 PORTER

LYSERGIC
AMIDE
LYSERGYLMETHYL-
ACID ERGONOVINE
CARBINOLAMIDE (ERGINE)

Figure 5. Examples of some of the lysergic

acid amide group of ergot alkaloids

[Testereci etal., 1990]), partitionchromatography been developed (Richard A. Shelby, Dept. of Plant
with chloroform or methylene chloride a t a nappropri- Pathology, Auburn Univ., Auburn, AL, unpublished
ate pH, column clean-up procedures using either data). Extraction of infected seed or grass with
silica, alumina, or an ion exchange resin, and identifi- alkalinemethanol, followedby filtration,anddirect
cation andanalysisusing co-chromatography (TLC HPLC analysis (fluorescence detection) with a mobile
and[or] HPLC) with ultraviolet or fluorescence detec- phase of either 60 or 70% alkaline methanol results in
tion. Mass spectrometry ( MS) also has been employed separation of ergovaline,ergovalinine, lysergic acid
for the identification, analysis, and quantification of
amide, and its isomer isolysergic acid amide (ergi-
ergotalkaloids.
nine), along with other minor ergopeptine alkaloids.
Theergotalkaloids are extremelysusceptible to
photolytic and air oxidation, hydration, andepimeriza- Moubarak etal. (1993) havereported a unique
tion at the C-8 position of the ergolene ring (Berde preparative method for the isolation and purification
and Schild, 1978; Garner et al., 1993; Porter, 1994). of largequantities of ergovaline. This procedure
Epimerization of the C-8 position occurs ineither involves a modification of the methods of Scott and
acidic or basic conditions and, therefore, the isolation Lawrence (19801, Testereci et al. (19901, and Rottin-
of the C-8 epimers (designated with the sufflxal -inine ghaus et al. ( 199 1):infected seeds are extracted with
[i.e., ergovaline vs ergovalininel) occurs in most
extraction 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- m3%
ingsmall volumes of extractsunder a stream of
nitrogen.Storingdriedconcentrates inambervials
undernitrogen at or below0°C will helpprevent \
\
further decomposition. Moubarak et al. (1993) have N N
successfully stored ergovaline at -4°C for up to 12 mo. H TI
Furthermore, ergovaline decomposes rapidly when CHANOCLAVINE I AGROCLAVINE
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 for the meaningful isolation
andquantitativeanalysis of the ergot alkaloids.
High-Performance Liquid Chromatography. Use of
HPLC with either ultraviolet or 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; ELYMOCLAVINE PrnCLAVINE
Testereci et al., 1990; Hill et al., 1991, 1993;
Rottinghaus et al.,1991; Moubarak et al., 1993; Zhang
et al., 1994). For example, a rapid, simplified HPLC Figure 6. Examples of some of the clavine group of
method for theanalysis of the ergot alkaloids as- ergot alkaloids isolated from A . coenophialum-infected
sociated with A. coenophialum-infected tall fescue has tall fescue.

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 OF ENDOPHYTE
ANALYSIS TOXINS
a 5% aqueous lactic acid solution andthe 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:methanol (4:1, vol/vol) solution.
Scott etal.
(1992) and Rottinghaus et al. (1993) have reported
additional extraction, clean-up, and HPLC procedures
for the analysis of the ergopeptine alkaloids in cereal
grains,flour,and feeds.
Thin-LayerChromatography. After the ergot
alkaloids have been extracted into a suitable organic
solvent, TLC on silica gel remains one of the most
powerful tools for the analysis and identification of
these compounds. The major advantages of TLC are
that severalsamplesmay be analyzed at the same
time, 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 (see 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 the TLC platesinmethylene chloride:
isopropyl alcohol (92:8, vol/vol) three times (drying
theplates between runs), 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 theplates
consistent with theknown standards, extraction of the
alkaloids from the silica using methano1:chloroform
(1:4 or 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) (Porter et al., 1979).Other solventsystems
used for the TLC analysis of these compounds are
listedin 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 withthe indole nucleus and gives the
ergopeptine alkaloids their characteristic bright, pale
blue fluorescence (UVlambda maxinmethanol at
approximately 315 and 242 nm). Those ergot
alkaloids devoid of the 9,lO-double bond give
characteristic dark blue absorption under 254 nm (UV
lambda max in methanol at approximately 292, 280,
275, and 222 nm) indicative of the indole nucleus.
Spraying the TLC plateswith a solution of p-N,N-
dimethylaminobenzaldehyde (van Urk's reagent;
Stahl, 1969) followed by spraying with a 1%sodium
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875
Table 3. Solvent systemsa effectively usedfor
thin-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)
CHC13:EtzNH (9O:lO)
Benzene:Dimethylformamide (86.5:13.5)
aAll systems (vol/vol) are in saturated atoms, in tanks lined
with Whatman No. 1 filter paper.
bPerellino e t al., 1993; Porter et al., 1974,1979,1981.
nitrite solution (water:ethanol, 1:l vol/vol) (Sprince,
1960) produces intense blue spots thatare 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 tartrate) provides a method for quantifying total
ergot alkaloids in crude extracts
(Michelonand
Kelleher, 1963). Individual ergot alkaloids may then
be 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 MS includeelectron
impact ( E I ) (Porter et al., 1979), chemical ionization
( CI) (Porter and Betowski, 1981; Porter et al., 1981),
andtandemmass (MS/MS) spectrometry (Plattner
et al., 1983; Yates et al., 1985; Lyons et al.,1986;
Porteret al., 1987).Under low resolutionelectron
impact (70 eV), the ergopeptinealkaloids pyrolyti-
cally decompose into the lysergic acid amide, the cyclic
peptide, and diketopiperazine fragments A, B, and C,
respectively (Figure1). Thesefragmentsthenun-
dergo E1 and produce spectracharacteristic of the
ergolene ring and the peptide portion of the parent
molecule. Fragments useful intheinterpretation of
these spectra occur at 70, 125, and 154 atomic mass
units (arnu), which are characteristic of the proline
moiety (Porteretal., 1979; Bianchi etal.,1982).
Theseions, in combination with the lysergic acid
amide ion at 267 amu (ionA, Figure l ) ,are indicative
of an ergopeptine alkaloid.
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 resolutionelectronimpact
mass spectrometry ( EIMS) (70 eV) in theanalysis of
a the ergopeptine alkaloids is the low abundance (i.e., 5
1%) of the molecular ion and the diagnostic fragment
ion B when R2 is an alkyl substituent (i.e., ethyl-, n-
butyl-,
isobutyl-, sec-propyl-, isopropyl-; Figure1,
Table 1). Although fragmentsassociatedwith the
ergolene nucleus are isobaric withthosefragments
related to the cyclic peptide ( B ) and diketopiperzine
876 PORTER
( C ) portions of the parent molecule (Porter et al., needed, and isomeric species (i.e., ergosine vs beta-
1981), ergovaline andits C-8 epimer ergovalinine ergosine; Figure 1, Table 1) can be distinguished.
produce a highabundance of ion 196 amu(ion C; However, MSNS analysis requires expensive in-
Porter et al., 1979). Spectral interpretationof complex strumentation not readily available to most laborato-
mixtures of the ergopeptines, however, become some- ries, and like CIMS, this systemcannotdistinguish
what more difficult. between epimeric ergopeptine
alkaloids.
Subse-
Isobutane chemical ionization massspectrometry quently, M S N S is not practical for routine screening
( CIMS) has been employed in theidentification of the of toxic or infected grasses. Field desorption MS also
ergopeptinealkaloids (Porterand Betowski, 1981; has been used in the identification of the ergopeptine
Porteretal.,1981). This method involves an ion- alkaloids (Bianchietal.,1982)and provides ex-
molecule reaction of the alkaloids in the presence of a tremely simplified spectra along with intense molecu-
reagentgas(isobutane).The ion-molecule reaction lar ions.
results in reduced fragmentations, as seen with low
resolutionEIMS, produces simplified spectra,and Quantification and Toxicity
thus circumvents interpretation difficulties of the
more complex spectra and those
resulting from Cornel1 etal.(1990) havereportedergovaline
mixtures of these alkaloids. Under isobutane CIMS, concentrations at 50 ng/g of infected grass is suffxient
the lysergic acid amide ( A ) , cyclic peptide ( B ) , and to produce some signs of fescue toxicosis incattle
diketopiperazine ( C ) molecules (Figure 1) abstract a stressed by heat. Spiers ( 1993 demonstrated similar
proton from a t-butyl cation (which is generated in the thermoregulatoryimbalances in rats dosed wither-
massspectrometer)andtheresulting ion-molecule govaline. Dyer (1993) and Oliver et al. (1993) have
reaction produces spectra containing three major ions reported analogous in vitro vaso-activities of ergova-
represented by AH, BH, and CH in Figure 1and Table line
and lysergic acid amide, respectively. Thus,
1. Theseions are 1 + amu greater than the parent although ergovaline isan exceptional marker as-
fragments A, B, and C (Porter and Betowski, 1981; sociated with A. coenophialum-infected fescue, ergova-
Porter et al., 1981; Plattner et al., 1983; Yates et al., line toxicosis in livestock most probably is augmented
1985). The major fragments used to identify the by the total concentration of the ergot, and possibly
known ergopeptine alkaloids by this method are other alkaloidsin the infected grass.
outlined in Table 1. Although ergovaline and its C-8 Ergovalineconcentrations in infected tall fescue
epimerergovalinine produce thesamethree major seeds and forages have ranged from 2 to 6 pg/g (Table
fragments at 268 (AH), 267 (BH), and 197 ( C H ) 2). Totalconcentrations of ergot alkaloids in A.
amu and cannot be distinguished by this method, both coenophialum-infected tall fescue vary with the season
compounds ( a s with the other ergopeptines and their andamount of nitrogenfertilization(Lyons et al.,
C-8 epimers)separate nicely using TLC and(or) 1986; Belesky et al., 1988; Lyons et al., 1990;
HPLC analysis (Porter et al., 1974, 1979; Yates and Rottinghaus et al., 1991; Arachevaleta et al., 19921,
Powell, 1988; Rottinghaus et al., 1991, 1993; Perellino and these are factors that should be considered prior
et al., 1993). Tandem mass spectrometry (Plattner et to sample collection and analysis for alkaloid content.
al., 1983) also has been employed in the separation Whethertherelative concentrations of individual
andanalysis of ergopeptine alkaloids from both ergot alkaloids vary with season or nitrogen fertiliza-
endophyte-infected tall fescue and perennial ryegrass tioniscurrentlyunknown.
(Yatesetal., 1985; Lyons etal., 1986; Rowan and LoIine Alkaloids. The loline alkaloids in A.
Shaw, 1987). In an overly simplified description, MS/ coenophialum-infected tall fescue are produced either
MS (which also is conducted inthe presence of a by the plant in response to the fungus and(or) as a
reagent and[orl a target gas) uses one stage of mass defense mechanism in response to insectherbivory
separation to isolate the individual alkaloids of that may involve interactions between both
the
interest in a crude extract; depending on the ioniza- endophyte and its host grass. Moreover, Petroski et al.
tion mode, this usually involves the molecular ion, a (1990) have suggested the lolines may be alleopathic,
protonated molecular ion, or a molecular anion;a thus improving the infected-grass’s ability to compete
second stage of mass separation is then employed to withothergrasses.Thechemistry, occurrence, and
analyzethe product and(or)daughter ions (also biological effects of the loline alkaloids and associated
dependent on thereagent and[orl targetgas).The endophyte-grass interactions
have been reviewed
fragmentationmechanisms for the ergopeptine (Powell and Petroski, 1992; Bush et al., 1993). The
alkaloidsutilizing MSMS withisobutaneasthe lolines are directly related toA. coenophialum-infected
reagent gas and argon as the target gas areanalogous grassesandhave not been identified in A. lolii-
to that described for isobutane CIMS and are infected grasses(Siege1 et al., 1990).
described indetail(Plattneret al., 1983). Major Capillarygaschromatography (GC) using either
advantages of this system are that complex mixtures flame ionization ( FID) and(or) MS detection ( MSD)
of these compounds can be analyzedwithoutprior (Yates et
al., 1990; Powell and Petroski, 1992;
cleanup, only small samples of extracted material are Tepaske et al., 1993) is the current method of choice

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 ANALYSIS OF ENDOPHYTE TOXINS
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 to analysis,a sulfonic
acid solid phase clean-up step of the forage extracts is
necessary to remove substances that interfere with the
assay.Thedetectionlimit for N-acetylloline and N-
formylloine is10 ng. Tepaske etal. ( 1993j 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 eV),
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
( 1992) reported a method for the separation of the
loline and the ergot alkaloids from endophyte-infected
tall fescue whereby an alkaloidal extract is subjected
to column chromatographyusingSephadex LH-20.
The loline alkaloids pass through the column and the
ergot alkaloids are recovered by exhaustive elution of
the LH-20 with methanol. Also, Petroski and Powell
( 199 1) 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
thesealkaloids ( a s with the ergot alkaloids)in
forages varywithseason, theamount of nitrogen
fertilization,grazingpressure,andtheamount of
insect herbivory (Bush et al., 1982, 1993; Belesky et
al., 1987; Eichenseer et al., 1991). Further studies are
needed to determinewhetherthe loline alkaloids
significantly contribute to fescue toxicosis in livestock.
A. lolii-Infected
Perennial
Ryegrass
Paxilline
andthe Lolitrern Alkaloids. Current
evidence suggests that the indole-isoprenoid lolitrems
are almost as diverse as the ergot alkaloids. Paxilline
and lolitrem B (Figure2 ) are the two major alkaloids
associatedwithperennialryegrassstaggers (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 (Miles et al., 1993). Theseadditionalminor
lolitremsmaycontribute to the overall toxicity of
paxilline and lolitrem B.
Gallagher et al. (1985) reported a n isolation and
screeningmethod for lolitrem B in A. lolii-infected
Downloaded from jas.fass.org by on January 8, 2011.
877
perennialryegrass. One gram of oven-dried, milled
grass wasextractedwith 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 mLj and 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.
Theamount of lolitremB inthe infected grass
necessary to elicit thestaggers syndrome is only 5
ppm (Gallagheretal.,1985).
Perumine. Although the correlation of peramine
(Figure 4 ) as the major insect deterrentin A.
coenophialum-infected tall fescue and A. lolii-infected
perennialryegrasshas been reported (Rowanand
Tapper, 1989; Tapper et al., 1989; Siege1 et al., 19901,
mammalian toxicity to permine has not been deter-
mined. The isolation and analysis of peramine from
infected grassespresents some unique problems be-
cause of the guanidino moiety attached to the
aliphatic side chain on the pyrrolopyrazine ring.
Tapper et al. ( 1989) have reported a method for the
analysis of both peramine and lolitrem B using atwo-
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-
tionswithhexaneandwater ( 3 mL each). The
aqueous and organic phases are separatedby centrifu-
gation. Lolitrem B was analyzed in the organic phase
as previously reported (Gallagher et
al., 19851,
whereasperamine,after minor cleanupusingion-
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 93to loo%, 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) isinthe carboxylic
acid form. After the aqueous phase is aspirated onto
the columns, theyare washedwith 80% aqueous
methanol ( 3 mL j , the columns separated, and pera-
mineeluted from the acid column withaqueous
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. ( 1986) have defined the characteris-
tic low-resolution massfragmentation ( 7 0 eV) of
peramine(Figure4).
878
As withthe loline alkaloids, furtherstudiesare
needed to establish whether peramine augments the
toxicities of the ergot and(or) lolitremalkaloids or
whether peramine causes a unique mammalian toxico-
sis of its own.
Summary
The major toxins associated with Acremonium-
infected grassesandwithanimal toxicoses arethe
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 arethe major
lolitrems in A. ZoZii-infected perennial ryegrass. Pera-
mine is the majorinsect deterrent in both infected
grasses,whereasthe loline alkaloidsseemto be
primarily associated with A. coenophialum-infected
tall fescue.
Identification and analysis of the ergot alkaloids in
endophyte-infected grasses are effectively accom-
plished by a combination of TLC and HPLC and the
use of EIMS and CIMS. High performance liquid
chromatography with either ultraviolet orfluorescence
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 are that retention
times are consistent with known standards and that
their characteristic mass spectraallow 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) perennialryegrass 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 consistentlyaddedtoour knowledge for
combatingthese problems. Improvements in extrac-
tion efficiency, chromatography, and instrumentation
have provided information on the number and classes
of alkaloidsassociatedwith fescue toxicosis and
ryegrass staggers in animals on these Acremonium-
infected grasses. Continued improvements with isola-
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PORTER
tion and chromatographic techniques should provide
sufficient quantities of toxins necessary for further
animaltesting.Thesestudiesshouldthen provide
answers to both the individual and combined activities
of the toxinsisolated from the infected grasses.
Moreover, chemical analyses and animal studies will
eventually define whether fescue toxicosis in livestock
results from the total concentration of the ergot
alkaloids andwhether toxicosis is augmented by
either or both the loline alkaloids and peramine in
infected tall fescue. Animal studies also will define
whetherthe lolitrem-induced ryegrassstaggersin
livestock isaugmented by the ergot alkaloids and
peraminein infected perennialryegrass.
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