桃黄化病病原植原体的鉴定及16SrDNA序列分析

2009,36(3):341-346ActaHorticulturaeSinica:2008-08-26;:2008-12-15:3E2mail:5787huangyun@sina1com1cn;Tel:[phone]SrDNA13,1,2,1(1,625014;2,028043):DAPI,;,40650nm,350nm,1016nm16SrDNADNAnested2PCR,115kb,16SrDNANecY2In1(nectarineyellows):;;;;;16SrDNA:S66211:A:05132353X(2009)0320341206TheIdentificationandSequenceAnalysisof16SRibosomalDNAofthePhy2toplasmasp.AssociatedwithPeachYellowDiseaseHUANGYun13,WANGJing1,ZHAOYan2qin2,andHEMiao1(1DepartmentofPlantPathology,SichuanAgriculturalUniversity,Yapan,Sichuan625014,China;2DepartmentofPlantProtec2tion,InnerMongoliaUniversityfortheNationalities,Tongliao,InnerMongolia028043,China)Abstract:ThephytoplasmaassociatedwithpeachyellowdiseaseoccurredinLongquanyiDistrictinSi2chuanProvincewasstudied.Thespecificfluorescencedotsforthepathogenwereobservedinphloemcellsofleafveinsunderafluorescencemicroscopebystainingwith4,62diamidino222phenylindolediacetate.Phyto2plasma2likeparticleswerealsofoundinthesametissuesunderatransmissionelectronicmicroscope.Itsdiam2eterwas40-650nm,350nmonaverageandthethicknessoftheunitmembraneofthepathogenwas10-16nm.The16SrDNAabout115kbwasamplifiedbynested2PCRwithuniversalprimersforPhytoplasmausingthetotalDNAextractedfromdiseasedpeachasthetemplates.Aphylogenetictreewasconstructedbasedonthesequencesof16SrDNA.TheresultshowedthatPhytoplasmaassociatedwithpeachyellowdiseasewascloselyrelatedtonectarineyellows(NecY2In1).Keywords:peach;peachyellowdisease;Phytoplasma;morphology;phylogenetics;16SrDNA,,,,1967Doi(,MLO;),,700,70(,2005),801000nm,,,10,DAPI,16SrDNA(Leeetal.,1998;Subandiyahetal.,2000;,2002,2005;©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.)DAPI,;16SrDNA,,111120073,4,50,525,,(Jujubewitches2broom)PCR()112112115%,4011molL-1(pH710)3;110gmL-14,6--2-(4,62diamidino222phenylindole,DAPI)1015min;Nikon280i,420nm(,1999)11212115mm215mm,(2006),,,,,H260011311311DNA3g,Lee(1993)NeimarkKirkpatrick(1993)DNA-201131216SrDNAPCR16SrDNA,Lee(1993)PCRR16mF2(52CATGCAAGTCGAACGGA23)R16mR2(52CTTAACCCCAATCATCGAC23),()DNA,DNA,PCR50L,10buffer(MgCl2)5L,0125molL-1MgCl25L,10mmol4dNTP8L,DNA20ng,10pmol2L,TaqDNA015L,2515LPCR:944min,501min,722min;94115min,50115min,72215min,35;940175min,501125min,72215min;7210min,45LPCR1%(0105LmL-1Goldview),254nm1131316SrDNAnested2PCRGundersonLee(1996)nested2PCR,R16F2n(52GAAACGACTGCTAAGACTGG23)R16R2n(52TGACGGGCGGTGTGTA2CAAACCCCG23)PCRPCR30DNAnested2PCR:955min;9530s,571min,721min,40;7210min11416SrDNA11411DNAPCR()GenBanklcl121108311412DNACLUSTALX118(alignment),Mega210,NeighborJoining,(bootstrap),1000,Diaporthevaccinii(outgroup)243©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(1),,(1)21112,,,100nm40650nm(2)(3,4)(3),1222nm,1016nm343©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(1993)R16mF2/R16mR2DNAPCRDNAPCR30,R16F2n/R16R2,nested2PCR:4115kbDNA(5),DNALee(1993),,5nested2PCR1:;2:;3:;4:;5:;6:;7:;M:DNAVFig.5Theagarousgelelectrophoresisofnested2PCRproduct1:Blankcheck;2:Healthpeach;3:Jujubewitchesp2broom;4-7:PeachyellowdiseaseoccurredinSwanWoods,UniteVillage,AppleVillageandHuaguoVillage;M:MarkerV.213PCR,nested2PCR,1211bp16SrDNABLASTGenBank,:JWB(jujubewitchesp2broom;GenBankAY197661),PY2In(peachyellows;GenBankAY197660)NecY2In1(nectarineyellows;GenBankAY332659)416SrDNA,JWB1,PY2In2,NecY2In13JWBPY2InNecY2In199%214ClustalX118,Mega210,NeighborJoining:NecY2In1(nectarineyellows;GenBankAY332659),6,616SrDNAFig.6Phylogenetictreebasedon16SrDNAsequencesBootstrapvaluesareshownonbranches.443©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(,1999)(,1994),,DAPI(1989),,100nm,(1998),,(1998);,,40650nm,300nm,(1993)80800nm,(1998)600nm,1016nm,(2001)913nm31216SrDNA16SrDNADNAnested2PCR,1211bpDNA,,PCR,,,nested2PCRnested2PCR2PCR,21,16SrDNA(RFLP)16SrRNA,(Lim&Sears,1989;Seemulleretal.,1998)NecY2In1(nectarineyellows;GenBankAY332659),,,ReferencesCaiHong,KongBao2hua,LiFan,ChenHai2ru,DuanYong2jia,LiuJin2yuan.2002.Detectionofphytoplasmaincactuswitchespbroomandse2quenceanalysisofITS16SrDNAfragment.ActaPhytopathologicaSinica,32(2):175-181.(inChinese),,,,,.2002.16SrDNA.,32(2):175-181.CaiHong,LiXiao2lin,KongBao2hua,ChenHai2ru.2005.CloningANDsequencesanalysisof16SrDNAofphytoplasmassociatedwithdifferentsymptomofgardenPetunia.JournalofHuazhongAgriculturalUniversity,24(1):1-4.(inChinese),,,.2005.16SrDNA.,24(1):1-4.GeQuan2qing,MaixnerM,WenFu2jiang.2005.AnalysisofanextrafragmentamplifiedbyP1/P7nestedU3/U5primersingrapevineyellows(stolbur)phytoplasmadetection.ActaPhytopathologicaSinica,35(2):97-103.(inChinese)543©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(stolbur).,35(2):97-103.GundersonDE,LeeIM.1996.Ultrasensitivedetectionofphytoplasmasbynested2PCRassaysusingtwouniversalprimerpairs.PhytopathMedit,35(3):144-151.JinKai2xuan,TianGuo2zhong,WangYue.1989.Studiesonquickde