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Lipid Composition of Botrytis cinerea and Inhibition of Its Radiolabelling by the FungicideIprodioneAuthor(s): Robert G. Griffiths, Jane Dancer, Elizabeth O'Neill, John L. HarwoodSource: New Phytologist, Vol. 160, No. 1 (Oct., 2003), pp. 199-207Published by: Blackwell Publishing on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/1514218 .Accessed: 13/05/2011 18:06

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Newf Phytologist Research

L i p i d composition o f B o t r y t i s c i n e r e a a n d i n h i b i t i o n o f it sradiolabelling b y t h e f u n g i c i d e i p r o d i o n eRobertG. Griffiths', aneDancer2,ElizabethO'Neill2andJohnL. Harwood''Schoolof Biosciences,CardiffUniversityPO Box 911, CardiffCF10 3US, UK, 2Bayer ropScience,14/20 ruePierreBaizet,BP 9163, F-69263Lyon,France

Author orcorrespondence:John L. HarwoodTel:+44(0)29 2087 4108Fax:+44(0)20 2087 4116Email: [email protected]: 20 March 2003Accepted: 30 May 2003doi:10.1046/j.1469-8137.2003.00848.x

Summary* Botrytis inerea s an important lantpathogen hat causesgreymould n over200 hosts. It is often controlledby dicarboximides,hich have variousproposedmechanismsf action, ncludingffectson lipids.Herewe haveexaminedheeffectof one dicarboximide,prodione, n lipidmetabolism.* B.cinerea,culturednmaltextractmedia,was challengedwithiprodione nditslipids xtracted, eparated y TLC, ndanalysedbyGLC.Lipidmetabolismwasfol-lowedusing[1-14C]acetate.* Triacylglycerolas the majornonpolar nd phosphatidylcholinehe main polarlipid n B. cinerea.Linoleate, ollowed by a-linolenate,werethe major attyacidsand most ipid lasseshadcompositions roadlyimilaro the total attyacidpattern.Iprodione, tconcentrationsausingacessation fgrowth 5 pM)causedadecreasein polar ipidbutnot totalnonpolaripid abelling.Within he nonpolar ipids,DAGwas better abelled.* The data show that iprodionehad a selective effect on lipidmetabolism.Thealteredpatternof labelling uggested thatcholine(ethanolamine) hosphotrans-ferasewouldbe worth nvestigatings a primaryite of action.Keywords:Botrytiscinerea,iprodione,dicarboximideungicide, ipids,choline-phosphotransferase.C New Phytologist (2002) 160: 199-207

IntroductionFungi are importantplant pathogens and Botrytis inereacausesgreymould in over200 hosts, ncludingeconomicallyimportantrops uchaspeasandbeans Isaac, 992).Fungicidesused to controlBotrytispeciesareoftenused for thecontrolof otherfungalpathogens f thecrop,butcontrol s difficultbecause he fungus nfectsvirtually veryplantgrowthstageas well as all plant parts (Maude,1980). Botrytiswas firstcontrolledbyprotectiveeedtreatment ndfoliar prays uchasdithiocarbarnatesnddicloran,but theseweresupersededbythebroad-spectrumenzimidazole-basedystemicungicidesin the 1960s, followed aterby thedicarboximides.Iprodione (3-(3, 5-dichlorophenyl)-N-isopropyl-imidazolidine-2,4-dione-dicarboximide) belongs to thedicarboximide class of fungicidesand was introduced n1974.The dicarboximidesreactiveagainst ungiof severalgeneranaddition o BotrytisEdlich& Lyr,1995;Pommer&Lorenz,1995).The mode of action of iprodioneand other

dicarboximidess not clearand therehave beenconflictingproposedmechanisms f action. Forexample, nhibitionofactivetransport Jespersetal., 1993) includingeffects onglucose e.g.Edlich& Lyr,1995)havebeenreported.Othershave ound glycerol ccumulation, ossibledue to alterationsin synthesiscausedby a cAMP-dependentprotein kinase(Pillonel& Meyer,1999). Serineproteinkinaseshavealsobeen nvoked n theactionof thedicarboximide,inclozolin,on UstilagomaydisOrthetal.,1995). narecentpaper, singiprodione-resistantutants,Cui etal. (2002) suggestedhataputativehistidinekinasemight beaspecific arget ordicar-boximideingicides.Theseauthors lsopointoutothermechan-ismsofaction,whichhavebeensuggested ydifferent uthors.Several f theeffectsofdicarboximidesn target rganismsseem oinvolve hecytosolicmembrane. s aresult thasbeensuggested hat the compoundscould affectthe membranedirectlyor through interferencewith the biosynthesisofmembraneipids Leroux& Fritz,1984;Edlich&Lyr,1995).Moreover,nanumber fstudies, lterationsnlipidmetabolism? NewPhytologist(2003)160:199-207 www.newphytologist.com 199


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New200 Research Phytologisthavebeennoted(Buchenauer, 976;Fritz tal., 1977;Hisadaetal., 1978; Pappas& Fisher,1979). Furthermore,everalreportshave found that lipid peroxidesareproducedas aresultof dicarboximidereatmentEdlich& Lyr,1992, 1995;Steel,1996;Choi eta/, 1997).However,thershavenotfoundthiseffect e.g.Ortheta/, 1992,1993;Cabral&Cabral,000).Nevertheless,here s a goodcorrelation etween he contentof intracellular ipid peroxides and the fungicide concen-trationwhen B. cinerea nd Mucormucedo retreatedwithdicarboximidesEdlich& Lyr,1992) and, thus, it has beenproposedhat ipidperoxidesespeciallyhoseoriginatingromthemitochondrialnnermembrane) re nvolvedn themodeof actionof dicarboximidesEdlich& Lyr,1995;Choi etal.,1997). This is supportedby the reduction n peroxides ndotherprotective ffectsby antioxidants uch asa-tocopherol(Edlich& Lyr,1992, 1995;Steel& Nair, 1993;Choi etaL,1996)althougha reversalf toxicity s not always pparent.

Therangeof fungiexamined or their ipidbiochemistrysquite imited Weete,1980;Lbsel,1988)withgreatermphasison fattyacidcomposition hanof lipidclasses r of lipidmeta-bolism. Severalgroups have determined the fatty acidsofB.cinereandotherAscomycetes.uch ungiproduce-linolenicacidrather han y-linoleniccid,which sfound n lower ungi.Palmitates themainsaturated cidwitholeateand inoleate sthemajorunsaturatedomponentsShaw, 965;Weete,1974;Wassef,1977;Lbsel,1988).However,hecomplexacyl ipidsof Botrytispp.and theirmetabolism asnot beenexamined.Clearly,n order o define urther nypossibleeffectof ipro-dione on thelipidsof Botrytis,uchknowledgewasneeded.Inthispaperwereport irstly omedetailsof thelipid com-positionof B. cinerea nd, secondly, ffectsof iprodioneontheradiolabellingf lipids rom[1-'4C] cetate.Theseexperi-ments revealed hat the fungicideproduced ignificantandqualitative lterations f lipidlabellingat iprodioneconcen-trations hatblockedradial rowth.

MaterialsandMethodsGrowthof B. cinerea culturesCultures of B. cinereawere maintainedon malt extract(Oxoid, Basingstoke,UK) medium,prepared y dissolving2% (w/v)maltextract n 11 distilledwaterwiththeadditionof 2%(w/v) agarandautoclaving.Cultureswere ncubated t20?C in continuous ight andsubcultured very4-5 weeks.For hegeneration f liquidcultures,porulatingB. cinerea(c. 1 weekold) grownon maltextract-agar edium(above)wasfloodedwith 5 ml of maltextractmedium.Thesurface ftheplatewasscrapedightly o create spore uspensionwith-out disturbinghemycelia.The suspensionwaspipetted ntoa flaskcontaining500 ml of maltextractmedium andincu-bated at 20?C in continuouslight with rotaryshakingat120 r.p.m.After 16-24 h, theliquid culturewascentrifugedat4225 gfor 10 min, thesupernatantecanted nd thepellet

resuspendedn maltextractmediumat 1g (wetweight)cells/4 ml medium orexperimentalnalysis.Radialgrowth inhibitionplate testsIprodionewasdissolvedn dimethylsulphoxideDMSO)andthelatterwasadded o afinalconcentrationf 0.25%(v/v) nwarmmalt extract-agarmedia.Aliquotswerepipetted ntoPetridishesandmycelial lugs,cutfromasporulating ulture,wereplaced n the centreof theagarplates.Radial rowthwasmeasured vera numberof daysand comparedo controls,whichhadDMSO(0.25% /v)butnofimgicideddedalthoughthe concentrations f DMSO used werefoundnot to affectgrowthorlipidmetabolism).

RadiolabellingLiquidcultures 4 ml) of B. cinereamadeup as describedunderGrowth of Cultures)wereincubatedwith fungicidesolution for 1 h before the addition of 37 KBqof [1-14C]acetate(2.18 GBq mmol-[) (AmershamPharmaciaBiotech,LittleChalfont,UK). Cultureswere henincubatedor24 h (unlessotherwisestated)at 20'C in continuous ight with rotaryshaking at 120 r.p.m. Cultureswere subsequently ilteredthrough Whatman no. 1 filterpaper(WhatmanInt. Ltd,Maidstone,UK) usinga Buchner unnelunder suctionandwashedquicklywith three3 ml aliquotsof freshmedium toremove nyunincorporatedadiolabel.amples fthewashingsweretakenfor scintillation ountingto enableuptake o bedetermined. hemyceliaand filterpaperwere aken or ipidextraction ndanalysis.

Lipidextraction and analysisLipidswereextracted y a modificationKates,1986)of themethodof BlighandDyer(1959).Themodification seshotpropan-2-ol n order o inactivateipiddegradativenzymeswhich, otherwise, can generateartificialproducts (Kates,1986).Cultureswere iltered nd the filterpaperandmyceliawerecut into smallpiecesand immersed nto hot (700C)propan-2-ol.Aftervortexing, he mixturewasleft to extractfor 15 min beforecentrifuging t 1000 g for 10min. Thesupernatantwas decantedand the pellet was washedtwicemore with hot propan-2-oland then with propan-2-ol-chloroform 1: 1, v/v). The supernatantswere combined,taken o dryness, esuspendedn chloroform-methanol1: 1,v/v) andprocessed urther Bligh& Dyer, 1959).Lipid classeswereseparated yTLC using silicagel G60plates (E. Merck,Darmstadt,Germany)usingchloroform-methanol-aceticcid-water170: 30: 20: 7, byvol., system(i))orchloroform-methanol-ammoniumydroxide60: 25: 4,byvol.,system ii))assolvent orpolar ipids.The usual olventused oseparate eutralipidclasseswaspetroleum ther 60-800Cb.p)-diethylther-aceticcid(80: 20: 1, byvol.,system

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NewPhytologist Research 201(iii)).In order o separate iacylglycerolsromsterolsadequa-tely,a double developmentwas used (system iv)).The firstsolvent, unto threequarterseight,wasdiethyl ther-toluene-ethanol-aceticcid (40: 50: 2: 0.5, by vol.).Afterdrying,asecondsolventof diethylether-hexane6: 94, v/v)wasused.

Lipidbandsseparated yTLCwerevisualized y sprayingtheplatewith8-anilino- napthalenesulphoniccid n anhy-drousmethanol(0.2%,w/v) and viewingunderUV light.Incorporationf radioactivity as measured y scrapinghebandsand countingin Opti-fluorscintillant PackardBio-scienceBV,Groningen,TheNetherlands) singa 1209Rack-Betacounter WallacOy,Turku,Finland).Quenchcorrectionwas made by the external tandard, hannelsratio method.Lipid bandswere routinely dentifiedby comparisonwithstandards Sigma,Poole, UK) but werealso morefullyana-lysedusingdifferentialtainingmethods Kates,1986).

FattyacidanalysisTotal ipidextracts rseparatedipidclassesweresubjected oacid-catalysedethanolysis ndtheresultantattyacidmethylesters FAMEs) nalysed y (radio)GLC.Separation as n aglasscolumn(1.5 m x 3 mm internaldiameter) ackedwith10% SP-2330 on 100/120 mesh Supelcoport SupelcoUK,Poole, UK) at 180'C. Standard atty acids (Nu-ChekPrep.,Elysian,Mn 56028, USA) wereused to compareretentiontimesroutinely.Further onfirmation f their dentitieswasmadebyAgNO3-TLCndGLCon anonpolarSE-30) olumn.Forradioactiveamples, RaytestRagagasflow proportionalcounter(LabLogic, heffield,UK) was used. Quantificationwasmadeby using Ramona LabLogic)oftware.

ResultsLipid ompositionFattyacid analysis f total lipid extracts f B.cinerea,whichhad been grown n malt extractmediumfor 24 h, revealedtotal content of about31 pg of total fatty acidsper mg dryweightof cells. The main fatty acidspresentwerelinoleate

Table 1 The major fatty acid composition of Botrytiscinerea grownin malt extract medium

Fatty acid Composition (wt. %)Myristic (14 0) tracePalmitic (16: 0) 11.1, 14.1Stearic (18: 0) 2.8, 7.2Oleic (n-9, 18 :1) 11.1,12.1Linoleic (n-6, 18: 2) 43.9, 46.1a-Linolenic (n-3, 18 3) 25.6, 26.2Fungiwere grown for 24 h after subculturingunder the conditionsdescribed under the Materialsand Methods section. Resultsshowaverage values (n = 2 replicates)from 2 separate preparations.Trace


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> ~ ~ ~ ~ ~ ~ ~ ~~iilas5(b)

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(b) ~~~~~~~~~~~~~~~~~~~~~~~Fig.Fattyacidcomposition f lipid_ 160 -classes of Botrytisinerea.otal ipid(1) E::l 4:0 classeswere separated y TLCsystem-& 140 -E 16:0 (i) forpolar ipids A]andsystem(iii) or?C 2 Efl1 8:0 nonpolar ipids B]) see the Materialsnd

co 4-

. 12 * l 1 8:1 Methods ection)and individualands

c- 60 18-3,a-linolenate.Lipidabbreviations:0 D t l DAG,diacylglycerol;iPtdGly,

>~~~~~r5~iidcase

ca 40 - Idiphosphatidylglycerol (cardiolipin); AG,* 20 2 | 1 3 | | ~~~~~~~~~~~~~~monoacylglycerol;EFA, onesterified(b) |- n 3 n fatty acids; PtdOH, phosphatidicacid;0 ~~~~~~~~~~~~~~~~~~~phosphatidylethanolatdlns,Polar! DAG/ NIEFA TAG Sterol phosphatidylinositol,tdSer,MAG (sterols) esters phosphatidylserine; TAG, riacylglycerol.Resultssasmeans SDs for two separateLipid classes experiments.

describedwas found to give the bestquantitative xtractionwith the lowest NEFA levels. For radiolabelled ipids, thelevelsof 14C-NEFAwere not reducedby immediateextrac-tionof the incubationmediumwithout filtration,ndicatingthat the method usedwas efficient.The major phosphoglyceridewas phosphatidylcholine,representingbout26% of the totalacyl ipids.Phosphatidyli-nositol (PtdIns) nd phosphatidylserinePtdSer) id not sep-arate rom eachother n the normal olventsystemusedbut,fromthe results f specific olour tains,phosphatidylinositolwas he maincomponent.PhosphatidylethanolaminePtdEtn)wasthe secondmost abundantphosphoglyceride. hosphati-dylglycerol PtdGly)was not detected.The major onpolaripidclasses, AGandDAG, hadafattyacidcomposition Fig. 1) whichresembled he compositionof the totallipids (Table1) exceptthat a-linolenate wasless

prevalent.By contrast,the major phosphoglyceride, hos-phatidylcholine PtdCho),was enriched n polyunsaturatedfatty acids.Saturated atty acids (especiallypalmitate)weremoreenriched n PtdInsandNEFAcompared o total ipids.When comparing he total polarwith the totalnonpolar ip-ids, theincreased ercentage f linoleateanda-linolenateandlowerpercentage f palmitateand oleatein the polar ipidswerealsoseen clearly Fig. 1).Effectsof iprodione n growthof B.cinereaDicarboximide ungicideshavebeen reported o affect ipidmetabolism e.g. Buchenauer,976) andweused prodione sanexampleof thisgroupof compounds. prodionenhibitedradialgrowthwith significant ffectsbeing noted at 0.5 FiMandabove Fig.2).A statisticallyignificantnhibition f radial

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NewPhytologist Research 20340 -35 - 0.5 PM-* 1 PM/30 - 2 3ME25 -0 0M

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Fig.2 Inhibitionf the radial rowthof U i as p n of t.iaBotrytis inereabydifferentoncentrationsf 0 t-testriprodioneungicide.Results how means 0 12 3 4 5 6? SD(n = 3) for radial rowth nthepresenceofiprodione. Time(d)

Table3 Effect f increasingoncentrationsf iprodione n the labelling f Botrytis inerea ipidsrom 1-14CacetateUptakeandincorporationf radiolabel%)

IprodionepM) 0 0.5 1 5 10 50Uptake 94.8 ? 2.4 95.9 ? 0.5 96.4 ? tr 97.3 ? 0.2 92.0 ? 1.2 87.4 ? 0.9Incorporation 21.5 ? 1.3 21.3 ? 2.0 20.6 ? 0.2 17.5 ? 0.1 14.9 ? 0.2* 14.4 ? 0.7*Results how means? SD(3 independent xperiments).Uptakesas percentage f total radiolabel dded.Incorporations as percentage flabel akenupbythefungi. r? 0.05. *, significantlyifferentromcontrols noiprodionexposure)byStudent's -test(P< 0.05).

Table Effect f 10 pM prodione n the uptakeof [1_14ClacetatentoBotrytis inereaandincorporationf its radiolabelnto ipidswithtimeUptakeof radiolabel%)

IprodionepM) 2 h 4 h 8 h 12 h 24 hNone(control) 63.5 ? 6.3 93.0 ? 3.9 97.0 ? 0.2 96.5 ? 0.3 97.6 ? 0.310PM 71.5 ?2.8 89.0 ?6.9 96.5 ?0.4 96.3 ?0.7 94.9 ?1.2Incorporation%of radiolabelakenup)tprodionepM) 2 h 4 h 8 h 12h 24 hNone(control) 14.1 ? 1.5 20.2 ? 1.7 19.7? 2.9 16.7 ? 1.2 19.4? 1.310PM 10.0?1.6* 9.4?3.5* 12.8?2.1* 11.7?0.9* 11.8?1.0*Results how means SD(3independent xperimentsxcept or4 h incorporationheren = 2). *, significantlyifferentrom ontrol P < 0.05)byStudent's -test.

growthwasobserved tallconcentrationsfteronly2 days.At5 pMorhigheroncentrations,herewas carcelynygrowthwithiprodioneand theeffectswerestatisticallyignificant t 24 h.

Iprodionenhibitsipid ynthesisAninhibitionof growthbyiprodionewas seenat0.5 1M and1 pMconcentrationsy 2-3 days Fig.2) althoughB. cinereathenappearedo recover ndby6 days herewereno statisti-cally ignificant ifferencesromuntreated ontrols.However,theinhibitionof growthat5 pMiprodione rabovepersisted

(Fig.2). Accordingly,t was of interestto see whethertheinhibition f lipid abelling ccurredn thesameconcentrationrange. In Table3 it can be seen that lipid labellingwassignificantly educedwith5, 10 and 50 pIMprodioneovera24-h period,but not at the lower prodione oncentrations.Significant nd persistent rowth nhibitionby iprodione(Fig.2) was found at 5 pM or 10 pM concentrations ndthereforewe routinelyused theseconcentrationsor furtherexperimentsn lipid metabolismsee heDiscussion ection).In a time-course xperiment p to 24 h, the effectof 10 pMiprodionewas statisticallyignificant hroughout Table ).

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0 Control^ 300- E Iprodione6x 250-xE)-c 200 -C Lco 1502- 100 __,-;,g0

100*

Unknown Ptdins/ PtdCho PtdEtn PtdOH/ Non(PtdSer) (DiPtdGly) polarsLipidClasses

(b) 35001Control

^ 300 0 Iprodionex 250-E_ 200

. Fig.3 5 pMIprodionenhibits.? 150 - .* * phosphoglycerideabellingrom100 [1-14C]acetateutincreaseshatofdiacylglycerol.a)Phosphoglycerideo 50 100;00;l * *+ fractionseparatedbyTLCusing olvent

- 50 :. 1 T Fi . (i) (see the Materialsand Methods section)0 2.;.;t:-- - - -, -- -(b) Nonpolaripids eparatedbyTLC singPolar MAG Sterols DAG/ TAG solvent iv).Results s means? SD(3Polar MAG Sterols DAG/ TAG independent xperiments). , significantlyNEFA differentromcontrolbyStudent's -testLipidClasses (P < 0.05).Ascanbeseen, -I4C]acetate wasrapidlyakenup by the tissueandlipid labelling olloweda similar ime-coursen controltissues.The rapidequilibration f total lipid labellingwithavailableadiolabelledrecursor Table ), togetherwith theconstantpatternof lipid class abelling Griffiths, 001), sug-gestthatthe specificradioactivity f the acetatepool rapidlybecomesdilutedwith timeand alsothattherewas slow turn-overof radioactiveipids.Iprodione had no significant effect on the uptake of[1-_4C]acetateby B. cinereaat concentrations p to 10 pM(Table3) atanytimeup to 24 h (Table ), despite hesecon-centrations ausingprolongedcessationof growth(Fig.2).Thus, the effectsof the fungicideon lipid labellingweredearlynot due to loweravailabilityf radiolabelledrecursor.IprodioneausesspecificalterationsnlipidmetabolismThe inhibitionof totallipidlabellingwas then examined nmoredetailto see if this wasjust a nonspecific eduction n

total metabolismbecausegrowth was being preventedorwhethermorespecific ffectswerebeing elicited.First, we examined fatty acid biosynthesis.The majorlabelled attyacidsproducedby B. cinereaby 4 h werepalmi-tate (22%), oleate (36%) and linoleate (32%) with smalleramountsof stearate5%) andlinolenate 3%).5 pM iprodi-one did not causeany significant hange n thelabellingpat-tern (datanot shown). By 24 h, the patternof labelled attyacids n untreatedB. cinereawas17%palmitate, %stearate,25% oleate,46% linoleateand 9% linolenate ndicating hatincreaseddesaturation ook place with time, as would beexpected Harwood, 1988). 5 pM iprodionecauseda smallincreasen the relative adiolabellingf linoleate (to 56%).This was balancedby a decreasen the relativeabellingofoleate(datanot shown).Thesedata ndicate hat the inhibi-tion of lipid abellingbyiprodione Table3) isnot due to anyspecific eductionn activity f one of thecomponentpartsofthefattyacidbiosynthesismachinerye.g.a specific ondensingenzymeordesaturase).

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C10 ,UM^ 350 - El 0.5 PM?) El 1 PM300 - 1MMU 10 PME 2550

Fig.4 Concentration-dependencef the 200effectsof iprodione n the labelling f .Ototalpolar ipids ndnonpolaripid lasses c 150from(1-14C]acetatesdiscussednthe ?text (see alsoFig.3) iprodione nly O 100increasedhe labelling f DAGwithin hecombinedDAG/sterol and.Separation - 50of lipidsbyTLCwith method iii) seethe Materialsnd Methods ection). 0Means? SD(3 independent xperiments)shown.*, significantlyifferent romcontrol Polar DAG/sterols NEFA TAGbyStudent's-test (P< 0.05). Lipid classes

We then examined ipid classlabelling.The total lipidextractswere separatedby TLC using solvents suitablefornonpolar r polar ipidanalysis.n order o separateufficientmaterialor further adio-GLC nalysis if required)we hadto use one-dimensional LC andno individual olventcouldseparate lltherelevantipidclassesn one experiment.How-ever, by using differentsolventsystemsand separateTLCanalyses,we wereable oseparate ll of themajorabelled ipidclasses uccessfully.Separation f polar ipids by TLC (Fig.3a) revealed hatlabelling f allclasseswasreducedwhereas hatof totalneutrallipidswas ncreased thoughnot significantly). orsimplifica-tion, some bands are shown with combined componentsbecausehe standard olar ipidsolvent see heMaterials ndMethods ection)did not separate ll phosphoglyceridesde-quately.However, ndependentTLCs usingalternativeol-vent systemsrevealed hat very little phosphatidylserinerdiphosphatidylglycerolas labelled datanot shown).Frac-tions containinghese ipids hereforeontained ssentially lltheir radioactivityn phosphatidylinositolnd phosphatidicacid, respectively.

Separation f nonpolar ipids by TLC showed that theapparentncreasen labellingof this fractionwas confined oa bandcontainingDAGandNEFAs Fig.3b).No significantincreasen labellingwasseen n any othernonpolaripidfrac-tion. Becauseof the potential mportanceof this finding ntermsof interpretinghe effectsof iprodione, urther xperi-mentswereconductedat different oncentrationsf the fun-gicideand usinga nonpolar ipid solvent hatwas capableofseparatingDAGsfromNEFAs.The originalnonpolar ipid solventsystem (system(iv))hadbeen the double-development roceduren order o sep-arateadequately terols,whichare mportant ompoundsofmany fungi. However,the labellingof this class was notincreased y 5 pIMprodione nd, n fact,wasslightly educed

(Fig.3b). Therefore,we wereableto use solventsystem(iii)(see heMaterials ndMethods ection)whichseparates on-esterifiedattyacidswell (althoughdiacylglycerolsndsterolsmigrate imilarly).Concentrations f iprodioneat 5 FiMorabove ignificantlyeducedhe absolute abelling f polar ip-ids(phosphoglycerides).omparedo controls herewerealsostatisticallyignificantncreases n DAG and a reduction nTAG labelling(Fig.4). As discussedabove,the increase nDAG labellingmayactually avebeenslightlygreater ecausethat of the co-migrating terolswas reducedmoderatelyby5 FiM prodione Fig. 3b).DiscussionThe lipidcontentof B. cinerea rown n maltextractmediumfor 24 h was about3.1% of the dry weightand was of thesame orderas that reported or B. tulipae clerotiaand withsderotia ndmycelia f thedosely elatedAscomycetecierotiniasclerotiorumSumner& Colotelo,1970). However,t shouldbe borne n mindthatthe lipid compositionof fungicanvarydependingon theirgrowth tage,environmental onditionsandmedia ompositionWeete, 980;L6sel, 988).The otal attyacidprofileofB. cineraagreedwellwith data orAscomycetesin general Shaw,1965) and B. tulipae Sumner& Colotelo(1970).However, ur results evealedomewhathigher aluesfora-linolenateand ess inoleate hanfora previous tudyofB. cinerea Shaw,1965). This may be due to the differentgrowthmediaused,as discussed bove seeL6sel, 1988).Previous tudieswith Ascomyceteshaveshown that Ptd-Chois themajorpolar ipid,followedbyPtdEtn Shaw,1965;Hendrix& Rouser,1976;Kushwaha tat, 1976;Letoublonet al., 1982). Ourdata(Table2) are n agreement.We alsoonlyfoundlow amountsof phosphatidylserinePtdSer).Forthe nonpolar ipids,TAGwasthe majoracyllipidbut withsignificantmountsof DAG (Table ). However,he amounts

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and percentagedistributionof nonpolar ipidsin fungicanbe extremely ariable Ldsel,1988) due no doubt, to growthconditions ncludingthe culture's ge (Weete, 1980; Lbsel,1988).Iprodione ffectivelynhibitedradialgrowthof B. cinerea,in keepingwith its use as afungicide or thisspecies.The effi-cacyof iprodioneandits cleareffecton lipid biosynthesisedus to study ts activity n detail.We found an IC50 or iprodi-one on radialgrowthof between1 and 5 jiM (Fig.2). Thiscompares o valuesof 1 jiM reported or growthon maltextract garmedium Pappas& Fisher, 979)and1.8 jiM onpotato/dextrose gar Steel& Nair, 1993).

When we used concentrations f iprodionewhich wereeffectiveagainstgrowthwithin 24 h (5 jiM and above)wefound that,althoughuptakeof thelipidprecursor1-14C]ace-tatewasunaffected,ncorporationf itsradioactivityntolipidswas reduced ignificantly Tables3 and4). These concentra-tions also produceda sustainedreduction n radialgrowth(Fig. 2). This suggested hatthe inhibitionof lipidbiosynthe-sis might be of relevance o the mechanismof iprodioneactionon growth.However, he coincidenceof two changesdoesnot, of course,meanthat there s a causeand effectrela-tionship.Moreover,tcouldbearguedhat t wasthecessationof growth hat causeda reduction n lipid labellingand notvice versa.Nevertheless,he rapid uptakeof [1 14C]acetateinto the fungi was unimpaired Table3), exceptatveryhigh(50 PiM)prodione Table ) whichsuggestshatsomegeneralmetabolic unctionswerestill active.If the inhibitionof lipid biosynthesiss importantor pro-dione'smechanismof action then one might expectto seesomespecific ffects n metabolism, uchashasbeenobservedfor heactions f otherpesticidese.g.Harwood, 989).Indeed,changeswereseen at the levelof the basicKennedypathwayof complex ipid assembly seeGurretal., 2002). Effectsonthe productionof the majormembranephosphoglycerides(Fig. 3a) could account for cell breakage Edlich & Lyr,1992, 1995) or transport ffects Jespers t al., 1993;Jespers& DeWaard,1994) and other specific alterations een inintracellularmembranes Choi etal., 1996, 1997) duringfungicidal ctivity. ndeed,Orth et al. (1993)havereportedspecificantagonist ffectof PtdChoagainst heactionsof thefungicide olclophos-methyln U. maydis.In quantitative erms, he biggesteffect of iprodione s toreduce helabelling f themainmembraneipidsPtdChoandPtdEtn,and increase hatof DAG. A simple nterpretationfthis observations that the choline-and ethanolamine hos-photransferasesrenhibited. hese nzymes re hemain ourcesof PtdChoand PtdEtn n fungi (Gurret aL,2002). However,there areotherimbalancesn the flux though the Kennedypathway,because he labellingof other phosphoglyceridessalsoreduced Fig.3a) and, moreover, he rise n DAG label-ling does not increaseTAG production Fig.3b).

Nevertheless,urresultsmust be considered n relation othe convincingrecentevidence that iprodione targetsthe

coiled-coilregionof a putativehistidinekinase(Cui et al.,2002). Mutational changes n the gene were shown to co-segregate ith dicarboximidendosmotic ensitivity.t is alsorelevanthathyperosmotictressn S.cerevisideauses naccu-mulationof glycerol sa compatibleolute(Nevoigt& Stahl,1997) and increasesn intracellularlycerolhavebeen notedas a result f dicarboximidehallengen severalensitiveungi(e.g. Fujimura t al., 2000). Thus, although oci other thanosmosensinggenescan confer dicaboximide esistancee.g.Orth et al., 1994;Ramesh tal., 2001) andabnormal lycerolaccumulationmay not be essential for fungicidetoxicity(Fujimuratal., 2000), we haveto considerwhether mpair-ment of an osmosensinghistidinekinase(Cui etal., 2002)could accountfor the specificeffects on lipid metabolismseen. There aretwo reasonswhy this may not be so. First,changes n glycerol evels,as a resultof the osmoregulation,would not be likely o selectively lter he labelling f specificlipids becauseglycerol nters he Kennedypathway or lipidassembly t the beginning ndwouldprobably ffectall ipidsproduceddownstream. econd, he effectsarerelatively apid(Table ), so that a sustained lteration f lipid labellingpat-terns,as aresult f changed eneexpressionmaynot havehadtime to occur.However,he factthathistidinekinases rewellknown o beconnectedwithsignalling athways, hich ncludethose suggested or at least one lipid metabolising nzyme(delta-12 attyaciddesaturase:uzuki tal., 2000) means hata direct ink between he lipideffectsseen in ourworkandhisidine inase xpression/activityouldbeusefuillynvestigated.We haveshown n this study hat prodioneproduces pe-cific effectson lipid metabolismn B. cinereaat concentra-tions comparablewith its sustained inhibition of radialgrowth.The notable nhibitionof phosphoglyceride iosyn-thesisand the importanceof such molecules n membranefunction mean that activityagainst the choline- and eth-anolamine hosphotransferaseshouldbe investigatedurtheras a site of action orthis fungicide.

AcknowledgementsWethankheBBSRCCASEtudentshipoRG.G) andAgrEvo(UK)Ltd.(now BayerCropScience) or financial upport.

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