河南省油菜菌核病菌对氯氟联苯吡菌胺及其复配剂的敏感性.pdf

由核盘菌 Sclerotinia sclerotiorum引起的菌核病(sclerotiniastemrot)是一种破坏性严重的病害，多发于油菜和许多其他阔叶作物，在农业生产中主要采用杀菌剂进行化学防治。氯氟联苯吡菌胺作为一种琥珀酸脱氢酶抑制剂(SDHIs)，对核盘菌的菌丝生长有较好的抑制作用。为建立河南省核盘菌对氯氟联苯吡菌胺的敏感性基线，采用菌丝生长速率法测定了 2015 年和2016 年从河南省不同地区采集分离的 119 株核盘菌对氯氟联苯吡菌胺的敏感性。结果表明：氯氟联苯吡菌胺对核盘菌菌丝生长的有效抑制中浓度(EC50)值范围为 0.04170.4732g/mL，平均 EC50值为(0.19680.1053)g/mL。EC50值频率分布范围窄且呈单峰曲线，平均 EC50值可以作为河南省核盘菌对氯氟联苯吡菌胺的敏感性基线。为明确氯氟联苯吡菌胺是否能与其他不同作用机制杀菌剂复配，采用菌丝生长速率法测定了核盘菌对氯氟联苯吡菌胺、多菌灵、咯菌腈、丙硫菌唑、菌核净、叶菌唑及其混合物的敏感性。结果表明，氯氟联苯吡菌胺、多菌灵、咯菌腈、丙硫菌唑、菌核净和叶菌唑对核盘菌 EC50值分别为 0.1256、0.1122、0.0229、0.0651、0.8057 和 0.0278g/mL。氯氟联苯吡菌胺与多菌灵、咯菌腈、丙硫菌唑、菌核净、叶菌唑(体积比1:1、1:3、1:5、3:1 和 5:1)复配剂的增效系数(SR)值范围为 0.543.57，表现为相加或增效作用。综上结果表明，氯氟联苯吡菌胺可以与多菌灵、咯菌腈、丙硫菌唑、菌核净、叶菌唑这 5 种不同类型的杀菌剂通过交替或复配使用，阻止和延缓核盘菌抗药性的进一步发展。研究结果可为油菜菌核病的防治和河南省油菜菌核病菌对氯氟联苯吡菌胺的敏感性监测提供科学依据。关键词:氯氟联苯吡菌胺；核盘菌；菌核病；敏感性基线；复配剂中图分类号：S482.2；481.4文献标志码：A 0 IntroductionSclerotinia sclerotiorumisoneofthemostdevastatingfungalpathogensintheworld,whichiscapableofattackingmorethan700plantspecies1.Sclerotiniastemrot,adestructivefungaldiseasecausedbythefungusS.sclerotiorum,presentsinoilseedrapeandmanyotherbroadleafcrops2.Itcanalsodamagecommercialcropssuchascrucifers,legumes,compositae,etc.3.TheinfectionofS.sclerotiorumbeginswhenascosporescolonize,bloomandinvadethestem4.S.sclerotioruminfectsallabovegroundpartsofoilseedrapeplants,includingleaves,stems,flowersandpods.Sclerotiniastemrotischaracterizedbynecrosisoftheplant,reducedpodnumbersandstuntedgrowth.Itmayalsoleadtoadecreasedyieldandqualityofoilseedrape5.Currently,sclerotiniastemrotismainlycontrolledbychemicals6-7.Themainmethodtocontrolsclerotiniastemrotistospraybenzimidazolefungicidesandthemixturesoffungicidesatthefloweringstageofoilseedrape8.However,withthelong-termuseofthesefungicides,S.sclerotiorumcausedveryobviousresistancetothesefungicidessuchascarbendazimandcyprodinil9-10.Asthedistributionoffungicide-resistantisolatesexpandseachyear,thecontrolefficiencyoffungicidesdecreaseseachyear,orevenbecomesineffectiveeventually,leadingtocontrolfailure11.Therefore,thereisanurgentneedtoscreenoutfungicideswithnewmodesofactionandbettereffectsonsclerotiniastemrot.Thesuccinatedehydrogenaseinhibitors(SDHIs)belongtothefungicidesofpyrazolecarbox-amidessuchaspenthiopyradandbixafen.TheSDHIsarecharacterizedbyabroadspectrumoffungicidalactivityandarealsoeffectiveagainstagronomicallyimportantAscomycetes12-13.Bixafen,discoveredbyBayerCropSciencein2001,hasshownhighinhibitoryactivityagainstmanycerealpathogensinfieldtrials14-15.Bixafenhasalongtimeofaction,varioustypes,highefficiencyandstrongselectivity.Ithasexcellentinternalabsorptionandconductivityabilities.Ithasboththerapeuticandpreventiveeffectsandinhibitstheproductionofsporesandthegrowthoffungalhyphae.AsthefungicideisnotcurrentlyregisteredonoilseedrapeinChina,bixafenhasnotbeenusedinS.sclerotiorum.Inapreviousreport,thesensitivityofS.sclerotiorumagainstanSDHIfungicideboscalidwasdetermined.TheEC50valuesofS.sclerotiorumtoboscalidrangedfrom0.0073to0.3880g/mL,andthemeanEC50valuewas(0.150.09)g/mL.Thefrequencydistributionwasunimodal16.However,therearefewNo.4LILuyietal:SensitivityofSclerotinia sclerotiorumtobixafenanditsmixturesinHenanProvince947reportsontheapplicationofbixafentopreventandcontrolS.sclerotioruminHenanProvinceandotherareasinChina.TheaimofthisstudywastoinvestigatethebaselinesensitivityofbixafenagainstS.sclerotiorumobtainedfromoilseedrapefieldsinHenan,China.ToestablishthebaselinesensitivityofS.sclerotiorumtobixafeninHenanProvince,thesensitivitiesof119isolatesofS.sclerotiorumtobixafenweredeterminedbasedonthemycelialgrowthratemethod.Themixtureoffungicideswithdifferentmodesofactionandstructuraltypesisacommonandeffectivemethodinproduction.Itimprovesthecontrolefficiencyanddelaysthedevelopmentofresistance.Inordertodeterminewhetherbixafencanbemixedwithotherfungicides,thefungicideswithdifferentmodesofaction,includingthephenylpyrrolefungicidefludioxonil,DMIfungicidesprothioconazoleandmetconazole,benzomezolefungicidecarbendazimanddicarboxylicfungicidedimetachlonewerechosen.ThesensitivityofeachfungicidemixturetoS.sclerotiorumwascalculatedbasedonthesynergisticratio(SR)values.TheresultsofthisstudyprovideimportantinformationforthecontrolofS.sclerotioruminHenanProvinceandthemonitoringofthesensitivityofS.sclerotiorumtobixafen.1 Materials and methods 1.1 Isolate collectionsIn2015and2016,plotswiththemostserioussclero-tiumstemrotwereselectedfromJiaozuo,Luohe,Nanyang,Pingdingshan,Zhengzhou,ZhoukouandotherplacesinHenanProvinceandthesclerotiawerecollectedrandomlyatcertaindistancesfromtherootsandstemsofdiseasedplants.TheseplantshavenotbeenpreviouslyexposedtobixafenandotherSDHIfungicides.Threetofivesclerotiawereobtainedfromeachcollectedplot,thencutintosmallpieces.Aftersterilizationina0.1%sodiumhypochloritedisinfec-tionsolutionfor30sanddriedwithsterilefilterpaper,sclerotiaweretransferredtopotatodextroseagar(PDA:200gofpotato,15gofagar,and20gofdextroseperliterofdistilledwater)platesfor2daysat25inthedark.ThepurifiedandretainedsinglemyceliumwastransferredtoaPDAslant,cultivatedfor2days,andstoredat417.Inthisstudy,atotalof119non-duplicateisolatesofS.sclerotiorumwerecollectedfromtheseisolates,includingPingdingshan(n=24),Nanyang(n=11),Zhengzhou(n=33),Zhoukou(n=14),Jiaozuo(n=15),Luohe(n=12),andXuchang(n=10).1.2 FungicidesAllfungicidesusedinthisstudyweretechnicalgrade.Bixafen(98%a.i.)wassuppliedbyShandongWeifangRunfengChemicalCo.,Ltd.(Shandong,China).Carbendazim(98%a.i.)wassuppliedbyJiangsuRotamChemistryCo.,Ltd.(Suzhou,China).Fludioxonil(95%a.i.)wassuppliedbySyngentaCropProtectionCo.,Ltd.(Basel,Switzerland).Prothioconazole(97%a.i.)waskindlyprovidedbyShandongHailierChemicalCo.,Ltd.(Shandong,China).Dimetachlone(95%a.i.)wassuppliedbyJiangxiHeyiChemicalCo.,Ltd.(Jiangxi,China).Metconazole(95%a.i.)wasprovidedbytheJiangsuHuifengBiologicalAgricultureCo.,Ltd.(Yancheng,China).Fungicidesweredissolvedintherespectivesolvents,toobtain10000g/mLstocksolutions(Table1).Allstocksolutionswerestoredat4.1.3 Sensitivity to bixafenInordertoestablishthebaselinesensitivitytobixafen,themyceliumgrowthinhibitionof119isolatesofS.sclerotiorumwasdetermined18.All119isolatesofS.sclerotiorumweretransferredfromthePDAslantsto9-cmPDAplatesandculturedat25inthedarkfor2days.The5mmmycelialplugswerecutfromtheedgeofa2-day-oldcolonyofeachS.Table 1 Concentrations of tested fungicides used in the studyFungicideConcentrations/(g/mL)Solventbixafen00.006250.0250.100.401.60acetonecarbendazim00.050.100.200.400.800.1mol/LHClfludioxonil00.050.100.150.200.25methanolprothioconazole00.006250.01250.0250.050.10acetonedimetachlone00.006250.01250.0250.050.10methanolmetconazole00.200.400.801.603.20acetone948农药学学报Vol.25sclerotiorumisolateandtransferredtoPDAplatescontainingbixafenatconcentrationsof0.00625,0.025,0.10,0.40and1.60g/mLandablanksolventcontrolwithoutfungicidewasset.After2daysofincubationat25inthedark,thediameterofeachcolonywasmeasuredintwoperpendiculardirections.Theaveragevaluesobtainedfromtheabovemeasure-mentwereusedtocalculatethemycelialgrowthrateaccordingtothefollowingformula(1):I/%=DckDtDck0.5100(1)Iistheinhibitionrate;Dckistheaverageofthediameterofthecontrolstrain,cm;Dtistheaverageofthediameterofthetreatedstrain,cm.TheEC50valuewascalculatedbasedonthelinearrelationshipbetweenthelog10ofeachconcentrationandtheprobabilityofinhibition.BasedonEC50value,thebaselinesensitivityofS.sclerotiorumtobixafenwasestablished.FishersprotectedLSD(P=0.05)wasusedtocomparethemeanEC50values18.Foreachgroupofexperiments,threegroupsofrepetitionsweresetandtheexperimentwasrepeatedthreetimes.1.4 Synergistic interaction in mixtures.Thesensitivityofthebixafenmixtureswithcarben-dazim,fludioxonil,dimetachlone,prothioconazole,ormetconazoleagainstS.sclerotiorumwasdeterminedbythedegreeofinhibitiononS.sclerotiorumbydiff-erentconcentrationgradientsandratiosofconcentra-tionofthefungicidemixturesabove.IsolatesofPDS1604werealsocarriedoutaccordingtotheabovemethods.Theexperimentcombinedbixafenandcarbendazim,fludioxonil,dimetachlone,prothiocon-azole,metconazoleaccordingto(1:1,1:3,3:1,1:5,5:1,V/V).Inthisstudy,5-mm-diameterplugsweretakenfromtheedgeofeachcolonywithapuncherandinoculatedonPDAmediumcontainingeachconcentration(Table2).After2daysofincuba-tionat25inthedark,thediameterofeachcolonywasmeasuredintwoperpendiculardirections.TheEC50valuewasobtainedaccordingtothemeasure-mentandcalculationmethoddescribedabove.Thetheoreticalinhibitionconcentrationofeachmixture(EC50(Exp)wasobtainedbyformula(2)19.TheSRwascalculatedaccordingtotheformula(3).WhenSR1.5,theinteractionisdefinedassynergistic.When1.5SR0.5,theinteractionisdefinedasadditive.WhenSR0.5,theinteractionisdefinedasanta-gonistic20.TheSRvaluewasusedtojudgetheinhibitionofeachratiooffungicidestoS.sclero-tiorum.Threegroupsofreplicatesweresetforeachgroupofexperimentsandtheexperimentwasrepeatedtwice.EC50(Exp)=a+baEC50(A)+bEC50(B)(2)SR=EC50(Exp)EC50(Obs)(3)A,Barecombinedfungicides;a,bistheproportionofthefungicideinthemixture;EC50(Exp)isthetheoreticalsuppressedmediumconcentration;EC50(Obs)istheactualmeasuredmediumconcen-tration.1.5 Statistical analysisDPSsoftware(version7.05;ZhejiangUniversity,Hangzhou,China)wasusedtoobtaintheregressionequationoftoxicity,theEC50value,andthe95%confidencelimit.ThebioassayoptioninDPSwasusedtodeterminetheEC50valueofS.sclerotiorum.TheresistanceratioswerecalculatedastheEC50valueoftheS.sclerotiorumisolates21.Thestatisticaldifferencesbetweensensitivityofdifferentisolateswereanalyzedviatheleastsignificantdifference(LSD)testatP=0.05.Inordertominimizeerrors,thesetestswererepeatedtwice,andtheaverageofbothtestswastakenasthefinalresult.2 Results and analysis 2.1 Sensitivity to difenoconazoleThesensitivityof119S.sclerotiorumisolatestobixafenwasdeterminedbasedonmycelialinhibition.Byanalyzingtheresultsofthesemycelialgrowthinhibitionofisolates,theEC50valuesofbixafentothe119isolatesofS.sclerotiorumrangedfrom0.0417to0.4732g/mLandthemeanEC50value(meanSD)was(0.19680.1053)g/mL(Table3).Thefrequencydistributionrangecurvewasunimodalwithanarrowrange(Fig.1).TheaverageEC50valueobtainedcanbeusedasthebaselinesensitivityofS.sclerotiorumtobixafeninHenanProvince.2.2 Synergistic interaction in mixturesTheresultsshowedthattheEC50ofthebixafen,carbendazim,fludioxonil,prothioconazole,dimeta-No.4LILuyietal:SensitivityofSclerotinia sclerotiorumtobixafenanditsmixturesinHenanProvince949chloneandmetconazoleagainstS.sclerotiorumwas0.1256,0.1122,0.0229,0.0651,0.8057,and0.0278g/mL,respectively,indicatingthatthetestfungicideshavehighinhibitoryactivityonthegrowthofS.sclero-tiorum.Themixturecombiningbixafenwithcarben-dazim,fludioxonil,sclerotium,prothioconazole,ormetconazoleaccordingto(1:1,1:3,3:1,1:5,5:1,V/V)fivetreatmentsshowsthattheSRvaluesagainstTable 2 Concentrations of fungicides mixtures used to determine the sensitivity of S.sclerotiorumMixturesVolumeratiooffungicideFungicideconcentration/(g/mL)bixafen:carbendazim1:100.006250.01250.0250.050.101:300.006250.01250.0250.050.101:500.006250.01250.0250.050.103:100.01250.0250.050.100.205:100.006250.01250.0250.050.10bixafen:fludioxonil1:100.0080.0160.0320.0640.1281:300.0020.0040.0080.0160.0321:500.0040.0080.0160.0320.0643:100.0040.0080.0160.0320.0645:100.0040.0080.0160.0320.064bixafen:prothioconazole1:100.006250.0250.100.401.601:300.006250.01250.0250.050.101:500.006250.0250.100.401.603:100.006250.01250.0250.050.105:100.006250.0250.100.401.60bixafen:dimetachlone1:100.006250.01250.0250.050.101:300.006250.0250.100.401.601:500.006250.0250.100.401.603:100.0500.1000.200.400.805:100.006250.01250.0250.050.10bixafen:metconazole1:100.006250.0250.100.401.601:300.006250.0250.100.401.601:500.01250.0250.050.100.203:100.006250.0250.100.401.605:100.006250.0250.100.401.60Table 3 Sensitivity of S.sclerotiorum to bixafen in Henan Province in 2015 and 2016SourceNumberofisolatesEC50valuerange/(g/mL)bAverageEC50value/(g/mL)c20152016TotalPingdingshan1410240.0417-0.40930.18020.1213aZhengzhou1518330.0540-0.34070.22240.0636aJiaozuo15150.0878-0.44200.26160.1117aZhoukou14140.0525-0.42620.21730.1203aNanyang11110.0710-0.35800.20760.1086aXuchanga10100.1094-0.47320.20380.1070aLuohe12120.0626-0.24120.13760.0540bTotal69501190.0417-0.47320.19680.1053aaMeansthatnosampleswerecollected.bEC50:effectiveconcentrationfor50%inhibitionofmycelialgrowth.cMeansinacolumnfollowedbythesamelowercaseletterwerenotdifferentaccordingtoFishersleastsignificantdifference(LSD)(P=0.05).950农药学学报Vol.25themycelialgrowthofS.sclerotiorumrangedfrom0.54to3.57(Table4).Amongthem,thehighestSRvalueof3.57wasobtainedbytheconcentrationratioexperimentofbixafenanddime