草莓褐色叶斑病病原菌Pilidium lythri鉴定及室内防治药剂筛选.pdf

RESEARCHREPORTSdoi:10.16801/j.issn.1008-7303.2023.0063Identification of Pilidium lythri and screening of fungicides in vitroYANYitong1,SUNJunyuan1,LIFuxin1,ZHANGGuijun1,YANZhe1,HUANGZhongqiao2,GAOHuige2,BIYang*,1(1.Key Laboratory for Northern Urban Agriculture of Ministry of Agriculture and Rural Affairs,Beijing University of Agriculture,Beijing102206,China;2.Department of Plant Pathology,China Agricultural University,Beijing 100193,China)Abstract:Inrecentyears,strawberrytan-brownleafspotdiseasecausedbyPilidium lythriisaseriousnewdiseasethathasoccurredinChina.Between2015and2017,atotalof90symptomaticsampleswerecollectedfromChangpingDistrict,BeijingCity;ZhuchengCounty,ShandongProvince;andLoudiCity,HunanProvinceinChina,with30samplesfromeachdistrict.Twenty-sixfungalisolateswereidentifiedasP.lythribasedonmorphology,molecularbiology,andphylogeneticanalyses.ThisisthefirstreportofP.lythricausedstrawberrytan-brownspotinShandongandHunanProvincesinChina.Atpresent,therearenoregisteredfungicidesinChinaforcontrollingtan-brownspotdiseaseonstrawberrycausedbyP.lythri.Inthisstudy,thesusceptibilityof26isolatestoeightcommonlyusedfungicidesandnovelfungicideSYP-14288weredeterminedin vitro.TheresultshowsthatSYP-14288hasthestrongestinhibitoryactivityonthemycelialgrowthof26isolatesofP.lythri,withanaverageEC50valueof(0.330.06)g/mL,canbeusedasaneffectivefungicideforthecontrolofstrawberrytan-brownleafspotdisease.TheaverageEC50valueswerefrom3.92g/mLto72.58g/mLforepoxiconazole,difenoconazole,prochloraz,tebuconazole,chlorothalonil,andmyclobutanil.Amongthem,azoxystrobinhadlowertoxicity,andmancozebhadthelowestinhibitoryactivity.Theresultsofthisstudycanprovideanimportantreferencefortherationaluseoffungicidestocontrolstrawberrytan-brownspotdisease.Keywords:Pilidium lythri;strawberrytan-brownleafspotdisease;mycelialgrowth;SYP-14288草莓褐色叶斑病病原菌Pilidium lythri鉴定及室内防治药剂筛选闫奕彤1,孙浚源1,李福鑫1,张桂军1,闫哲1,黄中乔2,高慧鸽2,毕扬*,1(1.北京农学院农业农村部北方城市重点实验室，北京102206；2.中国农业大学植物病理学系，北京100193)摘 要：近年来，由病原菌 Pilidium lythri 引起的草莓褐色叶斑病，是一种在中国发生严重的新病害。作者在 2015 年至 2017 年间，从北京市昌平区、山东省诸城市和湖南省娄底市采集有症状的草莓样品共 90 株，每地 30 株，根据形态学特征、分子生物学及系统发育分析，其中 26 株分离物被鉴定为 P.lythri，并且山东和湖南系首次发现由 P.lythri 引起的草莓褐色叶斑病。鉴于目前中国还没有登记用于防治草莓褐色叶斑病的杀菌剂。本研究通过室内毒力测定法测定了26 株 P.lythri 菌株对 8 种常用杀菌剂和 1 种新型杀菌剂双苯菌胺(SYP-14288)的敏感性。结果表明：SYP-14288 对 P.lythri 菌丝生长的抑制活性最强，平均 EC50值为(0.330.06)g/mL，可将其作为防治草莓褐色叶斑病的首选药剂；氟环唑、苯醚甲环唑、咪鲜胺、戊唑醇、百菌清Received:December6,2022;Accepted:June28,2023;Published online:July11,2023.URL:https:/doi.org/10.16801/j.issn.1008-7303.2023.0063http:/ Journal of Pesticide ScienceE-mail:和腈菌唑对 P.lythri 的平均 EC50值在 3.9272.58g/mL 之间，其中菌株对嘧菌酯和代森锰锌的敏感性较低。该研究结果可为合理使用杀菌剂防治由 P.lythri 引起的草莓褐色叶斑病提供参考。关键词:Pilidium lythri；草莓褐色叶斑病；菌丝生长；双苯菌胺中图分类号：S482.2文献标志码：A 0 IntroductionStrawberry(Fragaria ananassaDuch.)isanimportantcommercialfruitcropthatiswidelygrownintemperateregionsworldwide.Overthepasttwentyyears,theplantingareaandyieldofstrawberryhavegrownrapidly.AccordingtothelateststatisticsfromFAOSTAT(https:/www.fao.org/faostat/zh/#home)in2020,strawberryplantingareashavereachedmorethan100000hectaresinChina.Meanwhile,strawberryproductionwasupto3000000tonsperyearinChina,rankingtopoftheworld1.Duringthegrowthperiod,strawberriesareaffectedbyavarietyoffungalpathogens,forexample,Botrytis cinereaPers.,Colletotrichumspp.,andVerticillium dahliaeKleb.2.Pilidium lythri,previouslynamedPilidium concavumandHainesialythri,hasbeenreportedtocausestrawberrytan-brownrotdiseaseworldwide.Strawberrytan-brownrotdiseasewasfirstreportedinJapan,andsincethenreportedinseveralothercountriessuchasPoland,Brazil,andBelgium3-7.InChina,strawberrytan-brownrotdiseasecausedbyP.lythrihasbeenreportedonlyinBeijingsofar8.Inadditiontostrawberry,P.lythricanalsoinfectmorethantenhosts,includingOlea europaea,Fallopia japonica,Hieracium caespitosum,Bergeniacrassifolia,Paeonia suffruticosa,Eucalyptussp.,Rosa rugosa,Prunus domestica,Paeonia lactiflora,Greyia radlkoferi,Aesculus hippocastanum,Vaccinium corymbosumandCornus mas5-6,8-9.P.lythriinfectsstrawberryleaves,stems,fruits,androots.Aftertheinitialacuteinfection,acircular,water-stainedbrownspotemergesinthecenterormarginofstrawberryleaves6-7.Theapplicationofchemicalfungicidesisaneffectivemethodformanagingstrawberrydiseases.However,thedevelopmentoffungicideresistancehascompromisedthiscontrolstrategy.Besides,consi-deringthesafetyoffreshstrawberry,therearealsosomelimitationstotheuseofchemicalfungicidesinthefield.Currently,themanagementofP.lythriheavilyreliesontheapplicationofeffectivefungicidesandhostresistance.However,thereislittleresearchonthechemicalcontrolofstrawberrytan-brownrotdiseasecausedbyP.lythri.ThepurposeofthisstudyistoidentifyandcharacterizeP.lythriassociatedwithtan-brownleafspotdiseaseofstrawberryinChinabasedonmorphological,molecularandpathogeniccharac-teristicsandtoevaluatetheinhibitoryactivityofazoxystrobin,chlorothalonil,epoxiconazole,difeno-conazole,mancozeb,myclobutanil,prochloraz,tebu-conazoleandSYP-14288againstthisfungusin vitro.1 Materials and methods 1.1 MediumPDA：200gpotato,20gdextrose,and15gagarin1000mLdistilledwater.1.2 FungicidesTechnical-gradeazoxystrobin(95%a.i.;SyngentaBiotechnologyCo.,Ltd.,Shanghai,China),chlorothalonil(98%a.i.;HenanChunguangAgrochemicalsCo.,Ltd.,Henan,China),difenoconazole(98%a.i.;HangzhouYulongAgrochemicalsCo.,Ltd.,Zhejiang,China),epoxiconazole(97.8%a.i.;HenanZhongzhouSeedTechnologyDevelopmentCo.,Ltd.,Henan,China),mancozeb(98.5%a.i.;HebeiHesenChemicalCo.,Ltd.,Hebei,China),myclobutanil(98%a.i.;HenanZhongyuanGermplasmFactory,Henan,China),prochloraz(97%a.i.;HangzhouQingfengAgrochemicalsCo.,Ltd.,Zhejiang,China),SYP-14288(97%a.i.;ShenyangResearchInstituteofChemicalIndustry,Shenyang,China),andtebuconazole(98%a.i.;JiangsuFengdengPesticideCo.,Ltd.,Jiangsu,China)weredissolvedrespectivelyindimethylsulfoxide(DMSO)tomakestocksolutionsandwerestoredat4inthedark.1.3 Diseased sample collection and pathogenisolationAtotalof90diseasedsampleswerecollectedrandomlyfromstrawberryfieldsexhibitingtypicalsymptomsoftan-brownrotdiseaseinChangpingNo.5YANYitongetal:IdentificationofPilidium lythriandscreeningoffungicidesin vitro1077District,Beijing;ZhuchengCounty,ShandongProvince;andLoudiCity,HunanProvince,Chinain2015-2017,with30samplesfromeachdistrict.Among26isolatesofP.lythri,oneisolate(BJ-4)wasfromBeijingCity,nineisolates(HNLD-1toHNLD-7,HNLD-9,HNLD-12)werefromHunanProvince,and16isolates(SD17-2toSD17-12,SD14toSD18)werefromShandongProvince(Table1).TheinfectedTable 1 Information of ITS and LSU gene sequence of P.lythri strains used for phylogenetic analysisSpeciesorstrainGenBankNo.ITSGenBankNo.LSUHostCountryReferenceCommonnameLatinnameP.lythriAY487094.1AY487095RoseRosasp.USA11P.lythriAY487097.1AY487098PeonyPaeonia suffruticosaJapan11P.pseudoconcavumKF777184.1KF777236.1AshtreeGreyia radlkoferiSouthAfrica12P.acerinumAY487088AY487089HorsechestnutAesculus hippocastanumNetherlands11P.acerinumAY487091.1AY487092gardensoilHortum terramNetherlands11P.eucalyptorumKT950854.1KT950868.1EucalyptusgrandisEucalyptus robustaFrance13Chaetomella raphigeraAY487076.1AY487077BlueberryVaccinium corymbosumUSA11BJ-4MH322003MH322002StrawberryFragaria ananassaChinaThisstudyHNLD-1MH322005MH322004StrawberryFragaria ananassaChinaThisstudySD17-3MH322016MH322014StrawberryFragaria ananassaChinaThisstudyHNLD-2MH322006_StrawberryFragaria ananassaChinaThisstudyHNLD-3MH322007_StrawberryFragaria ananassaChinaThisstudyHNLD-4MH322008_StrawberryFragaria ananassaChinaThisstudyHNLD-5MH322009_StrawberryFragaria ananassaChinaThisstudyHNLD-6MH322010_StrawberryFragaria ananassaChinaThisstudyHNLD-7MH322011_StrawberryFragaria ananassaChinaThisstudyHNLD-9MH322012_StrawberryFragaria ananassaChinaThisstudyHNLD-12MH322013_StrawberryFragaria ananassaChinaThisstudySD17-2MH322015_StrawberryFragaria ananassaChinaThisstudySD17-4MH322017_StrawberryFragaria ananassaChinaThisstudySD17-5MH322018_StrawberryFragaria ananassaChinaThisstudySD17-6MH322019_StrawberryFragaria ananassaChinaThisstudySD17-7MH322020_StrawberryFragaria ananassaChinaThisstudySD17-8MH322021_StrawberryFragaria ananassaChinaThisstudySD17-9MH322022_StrawberryFragaria ananassaChinaThisstudySD17-10MH322023_StrawberryFragaria ananassaChinaThisstudySD17-11MH322024_StrawberryFragaria ananassaChinaThisstudySD17-12MH322025_StrawberryFragaria ananassaChinaThisstudySD17-14MH322026_StrawberryFragaria ananassaChinaThisstudySD17-15MH322027_StrawberryFragaria ananassaChinaThisstudySD17-16MH322028_StrawberryFragaria ananassaChinaThisstudySD17-17MH322029_StrawberryFragaria ananassaChinaThisstudySD17-18MH322030_StrawberryFragaria ananassaChinaThisstudyP.lythriJQ790789.1_JapaneseknotweedFallopia japonicaUSA14P.lythriJX047867.1_MeadowhawkweedHieracium caespitosumFrance15P.lythriKF911079.1_StrawberryFragaria ananassaUSA6P.lythriJQ995228.1_StrawberryFragaria ananassaChina8P.lythriGU126750.1_PeonyPaeonia suffruticosaChina16P.lythriFM211810.1_ChinesecabbageBergenia crassifoliaFrance17P.lythriKJ908840.1_StrawberryFragaria ananassaPhilippines18P.lythriKF060281.1_EucalyptusEucalyptussp.Mozambique19P.lythriKF646103.1_RugosaroseRosa rugosaLithuania20P.lythriKC614564.1_EuropeanplumPrunus domesticaIran21Note:“_”indicatesnoLSUgenesequence.1078农药学学报Vol.25partswerecutintopieces(0.5cmindiameter)fromtheedgesoflesions,soakedintosodiumhypochlorite(1%,V:V)for30s,sterilizedwith70%(V:V)ethanolfor60sandwashedwithdistilledwaterfor3timeswith10seachtime.ThedetachedsamplesweredriedonsterilepaperandplacedonPDAplates,andincubatedat25inthedarkfor7days.Finally,theisolateswerepurifiedbysinglesporestoobtainpurecultures10andtransferredtoPDAslantscoveredwithsterilemineraloilforlong-termstorageunder20atBeijingUniversityofAgriculture.1.4 Morphological descriptionTheisolateswereinoculatedonPDAplatesat28inthedarkfor5days.Theshapeandcolorofthecolony,aerialmycelium,andconidiumwererecorded.Formicroscopicfeatures,Riddellslideculturetechniqueswereused22.Eachmicrostructurewasmeasured30timesandpicturesofrelatedpropertiesweretakenusinganOlympusdigitalcamera(DM-21)mountedonanopticalmicroscope(OlympusBX-41,OlympusOptics).1.5 Pathogenicity analysisThepathogenicityoftheisolateswasverifiedbyKochsPrinciple23.Detachedanduntreatedstrawberry(cv.Benihopp)fruitsandleaveswereusedasinoculationmaterials,whichwerecleanedwithsterilewater,surfacedisinfectedwith0.8%(V:V)sodiumhypochloritefor2minutes,rinsedwithsterilewaterthreetimes,anddried.Fiveleavesandfruitswereinoculatedwitheachisolatebyplacingthemycelialplugs(5mmindiameter)thatwerecutwithacorkborerfromthemarginofafive-day-oldcolonyofisolates.Anotherfiveleavesandfruitswereinoculatedwith5-mmPDAplugsasthecontrolgroup.Afterincubationinachamber(ShanghaiYihengTechnicalCo.,Ltd.,Shanghai,China)with80%relativehumidity,12hlight/12hdarknesscycleat28for5days,P.lythriwasreisolatedasdescribedabovetoconfirmthepathogenidentity.Theexperimentwasperformedtwice.1.6 Internal transcribed spacer(ITS)and largesubunit(LSU)sequence analysisSingle-sporeculturesdevelopedonPDAmediumwereusedforDNAextraction,usingtherapidgenomeDNAextractionkitpurchasedfromBeijingBiomedBiotechnologyCo.,Ltd.(Beijing,China).TheDNAconcentrationofeachisolatewasdeterminedbyNano-DropND-1000Spectrophotometer(NanoDropTechnologies)inthreereplicates,andasuitabledilutionwasprepared.Inthepolymerasechainreaction(PCR),thepartialregionsoftwoloci,ITSrDNAandLSUrDNA,wereamplifiedusingtheprimersITS5(GGAAGTAAAAGTCGTAACAAGG)+ITS4(TCCTCCGCTTATTGATATGC)24andLR0R(GTACCCGCTGAACTTAAGC)+LR7(TACTACCACCAAGATCT)25-26.AllPCRsareperformedinatotalvolumeof25L,consistingof50nggenomicDNA,1DreamTaqGreenPCRMastermix(Fermentas)and0.2mofeachprimer.TheLSUandITSPCRconditionsweredescribedbydeGruyteretal.27andWoudenbergetal.28,respectively.PCRproductswereconfirmedbyelectrophoresisona1.0%(V:V)agarosegelin1Tris-Borate-EDTAbufferandsequencedatBeijingLiuheBGICo.,Ltd.(Beijing,China).1.7 Phylogenetic analysisThesequencesoftheisolatesobtainedinthisstudyweresplicedandcomparedbyDNAMAN6.0(LynnonBiosoft,USA).TheassembledsequenceswereusedasaqueryforBLAST(MegaBLASTfromNCBI).Thesubjectswithhighsimilarity,includingex-typeandex-epitypesampleswerethendownloadedfromGenBank(Table1).MrBayesVer.3.2.1wereusedtobuildtheBayesianInference(BI)phylogenetictreesforITSandLSUgenesamplifiedintheaboveisolates.ThephylogenetictreewasrootedwithChaetomellaraphigeravoucherBPI843541(GenBankNo.AY487077).1.8 Fungicide sensitivity testsFungicidesensitivitywasdeterminedbythemycelialgrowthratemethodin vitro29.Mycelialplugs(5mmindiameter)weretransferredfromtheleadingedgeofanactivelygrowingcolonyontoaseriesofPDAplatesamendedwithserialconcentrationsofazoxy-strobin,chlorothalonil,difenoconazole,epoxiconazole,myclobutanil,mancozeb,prochloraz,SYP-14288,ortebuconazole(Table2).DMSO-amendedPDAwasincludedinthecontrolgroup.ThefinalDMSOconcentrationinallplatesinthisstudywasadjustedto0.1%(V:V),whichdidnotaffectthegrowthofP.lythri.Afterincubationat25inthedarkfor8days,thecolonydiameterwasmeasuredbycrossmethod.Eachtreatmentwasreplicatedfourtimes,andtheexperimentwasconductedtwice.Themedianeffectiveconcentration(EC50)wascalculatedbyNo.5YANYitongetal:IdentificationofPilidium lythriandscreeningoffungicidesin vitro1079regressingthepercentageofgrowthinhibitionagainstthelogfungicideconcentration.1.9 Data analysisStatisticalanalyseswereperformedusingtheSASpackage(SASInstitute,Cary,NC)Ver.9.2.TheEC50valuesforeachisolatewerecalculatedbyusingPROCREGforlinearregression.MeanvalueswerecomparedusingFishersleastsignificantdifference(LSD)testatasignificancelevelof=0.05.2 Results and analysis 2.1 Morphology and cultural characteristicsThecolonyofthetestedisolateswascircularwithregularedgesandexhibitedwhite-to-cinnamonsparseaerialmyceliumonPDAmedium.Inaddition,acreamy-to-browncircleappearedaroundthecenterofthecolony(Fig.1a,b).Conidiawereunicellular,hyaline,smooth,andallantoidwithalittlebentpointingatbothsides.Thesizeoftheconidiawas8-12minlengthand2.0-2.5minwidth(Fig.1c).Thesexualstagewasnotobserved.2.2 Pathogenicity analysisFieldsymptomsofstrawberrydiseasecausedbyP.lythriweresimilartoanthracnose.Intheearlystageofthedisease,circularandbrownspotsareformedatthecenteroredgeoftheleaves.However,unlikeanthracnosefruitrotwithorangeconidialmasses,thefruitrottedbyP.lythriproducespinkconidialmasses(Fig.1d,e).The26isolateswereinoculatedonhealthystrawberryleavesandfruits.Attheonsetofinfection,thespotsonthemarginorinthemiddleofleaveswereverysmall,round,andwater-soakedlesions.Thespotsenlargedgraduallyupto1-3cmindiameter,andthecolorrangedfrombrowntodarkbrown,withpinksporemassesformingatthelatestage(Fig.1f).Onthefruitsurface,whitehyphaeappearedattheearlystage,andawhitelayerofmoldformedatthelatestage(Fig.1g).Thepathogenwasreisolatedandpurifiedfromdiseasedleavesandfruits.Thecolonycharacteristicsobtainedwerethesameasthoseoftheoriginallyisolatedpathogen.2.3 Phylogenetic analysisThreeisolateswereusedforthephylogeneticanalysisbasedonITSandtheconcatenatedITSandLSUTable 2 Initial concentration and series dilutionconcentrations of fungicidesFungicideSeriesdilutionconcentrations/(103g/mL)azoxystrobin50,100,150,200,250,300chlorothalonil5,10,20,30,40,50difenoconazole1,2,3,4,5,10epoxiconazole1,2,4,6,8,10myclobutanil10,25,50,75,100,