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APPLIEDMicRoBIoLoGY,Apr. 1975, p 553-556Copyright i 1975 American SocietyforMicrobiology

Vol.29,No. 4Printed in U.SA.

Toxicity and Occurrence ofBalansia on Grasses from ToxicFescue Pastures

CHARLESW. BACON,* JAMES K. PORTER, Arm JOE D. ROBBINS

Field Crops Utilizationand Marketing Research Laboratory, Richard B. Russell AgriculturalResearch Center,Athens, Georgia 30604

Received for publication16 December 1974

Balansia epichloW(Weese) and B. henningsiana (Moell.)wer'e isolatedfromgrassesin toxicfescuepastures.B. epichloe,culturedin a syntheticmedium, wastoxicto chicken embryos. Thin-layer chromatography and ultravioletabsorptiondata indicated that in submerged culturethe fungus produced compounds withthe indoleor ergolinenucleus.

Tall fescue,Festuca arundinaceae Schreb. (F.elatior var. arundinacea [Schreb.] Celak.), a

pasture grass in many parts of the UnitedStates, occasionallybecomes toxic, and cattlegrazing this grassdevelop a condition known as"fescuefoot" (6 , 10). Clinical signs of thissyndrome have been described (19).These signsare similarin many repects to the gangrenous-type alkaloidpoisoning caused by the alkaloidsfrom Clavicepspurpurea as suggested by Woodset al . (18). Alkaloids from this ergothave beenruled out by other studies (6, 13), and theetiologicalagent remains unknown. The toxiccompound is generallyconsidered to be a vaso-constrictor(5 , 13), and the sporadic and sea-

sonalnature of the syndrome suggests that amycotoxin might be involved.Several fungi,isolated from toxic fescue pastures, producemycotoxins (9, 11, 20). These studies, however,did not prove that these fungi and their toxinscaused the syndrome.In recent investigationsof toxicfescue pas-

tures in Georgia two species ofBalansia wereobserved.This paper reports the isolation ofthese systemic phytopathogens from severalspeciesofgrassesin these toxicpastures and onthe in vitro toxicityof one species.Balansia epichloe(Wesse),obtained from

ascospores,had not previouslybeen culturedinthe laboratoryso we had to develop a mediumwhich favored germination and growth. Thismedium consistedof: malt extract (Difco),5 g;yeast extract (Difco),20 g; glucose,20 g; agar,20 g; and distilled water, 1,000ml. On thismedium germination (60to 85%) was completeafter 3 days. The fungus was storedon agarslants of this medium at 4 C in capped testtubes.Subcultures and inoculawere prepared from

these tubes by macerating the contents of onetube in 10 ml of sterile, distilled water. For

submerged cultureof the fungus a syntheticmedium was used:mannitol, 30 g; sucrose,20 g;KH2PO4,5 g; NH4NO3,2 g; MgSO4-7H2O,0.2g; CaCl2- 2H2O, 0.1 g; a trace element solution,5 ml (H . J. Vogel,Microb. Genet. Bull. 13:42-43, 1956);thiamin,0.1 ug/ml and distilled wa-ter, 1,000ml.The inoculum (1ml)was placed ina 2.8-liter Fernbach flask containing250 ml ofthe syntheticmedium, and incubated on a gyra-tory shaker (200 rpm, 1-cm circularorbit) for 28days at 24 to 28 C.For routineanalysis,the medium was sepa-

rated from the mycelium by centrifugation

(20,000x g, 15 min) and filtered through glasswool,and proteins were removed by ultrafiltra-tion with a hollow fiber concentrator (AmiconCorp., Lexington, Mass.). The dialysatewaspassed through a glasschromatographic column(18by 3 cm ID) packed with Porapak Q (80to100 mesh; Waters Associates Inc., Milford,Mass.).Under a pressure of 10 lb/in2the columnwas washed with 300 ml of distilled water andelutedwith 300 ml of methanol. One portionofthe methanol solutionwas used for chemicalanalysis;theotherwas used for toxicitystudies.The methanol solutionused for toxicitystud-

ies was evaporated to dryness on a rotaryevaporator (40C), and the residuewas dis-solved in 3 ml of CHCl,. The residueremainingafter CHCl8 removal was dissolvedin 3 ml ofdistilled water. The column bypass was freezedried, and 1 g was dissolvedin 3 ml of distilledwater.These fractionswere bioassayed for tox-icity with fertile White Leghorn eggs (17) byinjecting0.01,0.025,and 0.05ml on the air cellbeforeincubation.For controlsthe same vol-umes of uninoculated medium and CHC1, ex-

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FIG.1. B. epichloisporulatingon smut grass(S. poiretit);insert shows th e blackergot-likepseudomorphicascostromataon the adaxial leafsurfacesof thegrass.

tract of uninoculated medium were tested andfound to be nontoxic (background motalityranged from 0 to 5%).The remainder of the methanol solutionwas

concentrated and applied to thin-layerchroma-tography plates of silica gel GF 254 by themethod of Agurell (2). Chloroform-methanol(4:1, vol/vol)was the developing solvent.Afterdevelopment, the plates were examined underultraviolet l ightat 254 nm, and those bandsshowing a dark blue absorption were marked.The portion of the plate containing the spotswas sprayed with a 4-dimethylaminocinnamal-dehyde reagent (12). Ultraviolet absorptionspectra in ethanol were obtained on the Beck-mann spectrophotometer acta CIII.One toxicfescue pasture was observed for 1.5

years, and during July and August B. epichloewas noted sporulating on smut grass(Sporobolus poiretii [Roem. and Schult. ]

Hitchc.).This weed is a serious problem inpastures in the southeasternUnited States(16).The fungus also was seen on fescue (Festucasp.), love grass (Eragrostishirsuta [Michx.]Nees),and panicum (Panicum anceps Michx.).Cattlewere observed to graze all these grasses.This fungus was detectedin the grassesbyfindingthe black ergot-likepseudomorphic as-costromata on their adaxialleaf surfaces(Fig.1) . B. epichloewas noted in other toxic fescuepastures under investigation.Also in thesepas-tures, B. henningsiana (Moell.)was observedgrowing on abaxial leaf surfacesof Panicumanceps, two speciesof Andropogon, and on aspeciesof Eragrostis.The laboratorycultureofB. henningsiana is being investigated.SporulationindicatedthatB. epichloewas the

more abundant species. In agreement withDiehl (7), infectedplantsappeared healthy,butgrasses that were heavilyparasitizedby B.

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VOL.29, 1975

TABLE1. Toxicityof B. epichloeto chicken embryo

%ToxicFraction

100 25a 506

CHCls 25 70 100Water 0 0 40Column bypass 0 30 90

aDosage, microliterspe r egg. Each dosage is anaverage of two replications;each replicationconsistsof 20 eggs.

epichloe did not produce inflorescences.In theseplants all the leavesin a clone showed eithertheasexual ephelidialstage (Ephelis state) or theblack ascostromata. These fructificationswereephemeral.B. epichloewas toxic to the chichen embryo

(Table 1). Toxicity resided in the CHClI,water-soluble,and column bypass fractions.Thin-layer chromatography of the methyl alco-hol fraction revealed several bands that ab-sorbed dark blue under ultravioletlight at 254nm . When sprayed with 4-dimethylaminocin-namaldehyde reagent, severalspots gave thecharacteristiccolorreactionfor ergot-typealka-loids (2). Two of these major components (Rf0.45) were scraped into glass funnels (fritteddisk), and the silica gelwas elutedwith chloro-form-methanol (1:1, vol/vol).The eluate wasconcentrated to dryness under a stream ofnitrogen,and the residualmaterial was takenup in ethylalcohol.The ultravioletabsorptiondata of these two compounds (Table 2) weresuggestive of the indole (or ergoline)nucleuscharacteristicof the ergot-type alkaloids(3).We are investigatingthe structureand toxicityof these compounds. Balansiais a clavicipita-ceous fungus, and like Clavicepsmight beexpected to produce alkaloids.Some new ergotalkaloidshave been reportedfrom unrelatedgenera of fungi(1).These data suggest that in submerged culture

B. epichloecan produce indolecompounds invitro. The morphology and nutritionalrequire-ments of the fungus in the syntheticculturemediumwillbe reportedelsewhere.The in vivorelationshipof this fungus to the host'smetabo-lism during various seasons of the year isunknown. Perhaps the production of toxiccom-pounds is host or season dependent, since thevariety of toxic compounds produced by aspeciesof Claviceps is hostdependent (1). Thisreport is the first to indicatethat B. epichloeproduces toxicsubstances in vitro and suggeststhat the genus, because of its systemic nature,should be considered a potential hazard to

TAmZ 2. Ultravioletabsorptionofmajor compoundsisolatedfrom B. epichlog

Compounds R. Xmax (nm)"

A 0.67 221,273,281,289B 0.45 221,273,280,289

a Determined in ethylalcohol.

animals grazing on parasitizedforagegrasses.This endophytic systemic parasite is a bettercandidate for the cause of fescuefoot than thesaprophytic fungi studied to date. ThatBalansia may be a problem in forageshas beenreportedfrom India, where cattle and goatsshowed signsindistinguishablefrom ergotpoi-soning of Clavicepspurpurea after grazingonlovethron grass (Andropogon aciculatus) para-sitizedwith a speciesofBalansia (14).Clavicepssp. was not found in that study.The involvement of Balansia in the fescuefoot syndrome is complicated by the generalelusivenature of the fungus and fescue growthrequirements. Cool temperatures favorableforthe growth of fescuemay suppress sporulationof the fungus.In the spring,we found onlya fewclonesof fescuegrassshowing the ascostromataof the fungus.Earlierstudiesindicatedthat thefungus will not sporulatewhen grassesare keptat cool temperatures (lowerthan 20 C) butcontinue to grow and invade new tissue as itshost grows (7). This suggests that parasitizedgrassesgrowing in their northern extremes orduring cool seasons would not show signs offungalsporulation,and thus go unnoticed.Thefungion warm-season grasses(smut grass,pani-cum, etc.) were heavily sporulating.Perhaps amore valid indicatorfor parasitism would be atest for chitinas proposed by Ride and Drysdale(15) or that suggested by Diehl (7 , 8). Thesignificanceof Balansia has been superficiallyexamined (4, 8).It parasitizes10 tribes of theAmerican Gramineae, many members ofwhichare economically important foragecrops (8). Inmore detailedinvestigationsof this genus,weare studying its economic importance to thegrowth of foragegrassesand to the animalsgrazingtheseparasitizedgrasses.

LITERATURE CITED1. Abe, M., and H. I izuka. 1971.Alkaloid and steroid

productionby microorganisms, p. 175-200. In K. Saka-guchi,T. Uremura, and S. Kinoshita (ed.), Biochemi-cal and industrialaspectsof fermentation. KodanshaLtd.,Tokyo.

2. Agurell,S. 1965.Thin-layer chromatography and thin-layerelectrophoretic analysisof ergotalkaloids.Rela-tions between structure,Rm value and electrophoreticmobilityin th e clavineseries. Acta Pharm. Suec.

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2:367-374.3. Agurell,S., and E. Ramstad. 1962.Analysis of clavine

alkaloidsof Pennisetum ergot. Lloydia 25:67-77.4. Atkinson, G. F. 1905. The genera Balansia an d

Dothichloein theUnited Stateswith a consideration oftheir economic importance. J. Mycol. 11:248-267.

5. Bruckner, G., and D. R. Jacobson. 1973. Bioassay ofsub-fractionsof toxic Festuca arundinaceae, p.114-123. In G. B. Garner, H. Tookey, and D. R.Jacobson (ed.), Proceedings of the fescue toxicityconference.UniversityofMissouri,Columbia, Mo.

6. Cunningham, I. J. 1949. A note on the cause of tall fescuelameness in cattle.Aust.Vet.J. 25:27-28.

7. Diehl,W. W. 1939.Identityand parasitism of a speciesofDothichloi. J. Agric. Res. Washington, D.C.58:947-954.

8. Diehl,W. W. 1950.Balansia and Balansiae in America, p.1-82.Agr.monogr. no. 4, U.S. Department ofAgricul-ture,Washington, D.C.

9. Ellis, J. J., and S. G. Yates.1971.Mycotoxins of fungifrom fescue.Econ. Bot.25:1-5.

10. Goodman, A. A. 1952.Fescue footin cattle in Colorado. J.Am. Vet. Med. Assoc.121:289-290.

11. Keyl, A. C., J. C. Lewis, J. J. Ellis, S. G. Yates,and H. E.

Tookey. 1967.Toxic fungi isolated from tall fescue.Mycopathol. Mycol. Appl. 31:327-331.12. Krebs, K. G.,D. Heusser, and H. Wimmer. 1969.Spray

reagents, p. 869.In E. Stahl (ed.), Thin-layer chroma-tography. Springer-VerlagInc., New York.

13. Jacobson, D. R.,W. M. Miller,D. M. Seath, S. G. Yates,H. L. Tookey, and I. A. Wolff.1963.Nature of fescuetoxicityand progress toward identificationof the toxicentity. J.Dairy Sci. 46:416-422.

14. Nobindro, U. 1934. Grass poisoning among cattle andgoats in Assam. Indian Vet. J. 10:235-236.

15. Ride,J. P., and R. B. Drysdale.1972.A rapidmethod forthe chemical estimation of filamentous fungiin planttissue. Physiol.Plant Pathol.2:7-15.

16. Smith, J. E., A. W. Cole, and V. H. Watson. 1974.Selectivesmut-grass controland forage quali tyre-sponse in bermudagrass-dallisgrasspastures. Agron. J.66:424-426.

17. Verrett,M. J. , J. Marliac,and J. McLaughlin, Jr. 1964.Use of the chicken embryo in the assay of aflatoxintoxicity. J. Assoc.Off.Anal. Chem. 47:1003-1006.

18. Woods, A. J., J. B. Jones, and P. G. Mantle. 1966.Anoutbreak of gangrenous ergotismin cattle. Vet. Rec.78:742-748.

19. Yates,S. G. 1962.Toxicity of tall fescueforage:a review.Econ. Bot.16:295-303.

20. Yates, S. G. 1971.Toxin-producing fungi from fescue

pasture, p. 191-206.In S. Kaids, A. Ciegler,and S. J.Ajl (ed.), Microbial toxins, vol.7. Academic Press Inc.,New York.

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