刺槐心材腐朽菌的鉴定及其生物学特性

388Vo.l38No.820108JOURNALOFNORTHEASTFORESTRYUNIVERSITYAug.2010:,,19612,,:2009914:高国平信雯郑雅楠高拓祁金玉张伟魏振宏(,,110161)通过形态学和分子生物学方法鉴定沈阳地区刺槐心材腐朽病病原菌(编号为P1),并在室内培养条件下,采用生长速率法研究了温度pH值光照碳源和氮源对病原菌菌丝生长和菌落形态的影响结果表明:病原菌P1为槐生多年卧孔菌(Perenniporiarobiniophila(Murr.)Ryv.);菌丝生长适宜温度为20~25,适宜pH值为5~6,光照对菌丝生长无显著影响此外,P1对碳源的利用以蔗糖为最佳,氮源以酵母膏最佳刺槐;病原菌鉴定;生物学特性;ITSrDNAS792.26IdentificationandBiologicalCharacteristicsofLocustHeartwoodRotFungi/GaoGuoping,XinWen,ZhengYanan,GaoTuo,QiJinyu,ZhangWe,iWenZhenhong(SchoolofForestry,ShenyangAgriculturalUniversity,Shenyang110161,P.R.China)//JournalofNortheastForestryUniversity.-2010,38(8).-60~62Heartwoodrotfungus(P1)ofRobiniapseudoacaciainShenyangwasidentifiedbymorphologicalandmolecularbiologicalmethods.Andtheeffectsoftemperature,pHvalue,light,andcarbonandnitrogenresourcesonmyceliumgrowthandcolonymorphologywerestudiedbygrowthratemethod.ResultsshowedthatP1wasidentifiedasPerenniporiarobiniophil(Murr.)Ryv.Theoptimumtemperatureforthemyceliumgrowthwas20~25degreesC,andtheoptimumpHvaluewaswithintherangeof5~6.Illuminationhadnoobviouseffectonmyceliumgrowth.Themyceliumgrewbestonmediawithcanesugarascarbonsourceoryeastextractasnitrogenresources.KeywordsRobiniapseudoacacia;Identification;Biologicalcharacteristics;ITSrDNA(Robiniapseudoacacia),[1],,,30a[2-3],13%~23%,,[4-6],,1:(30~40a),:,,(P1),,[2,7]DNASDSCTAB[8]ITS4/ITS5rDNAITSPCRITS4:5!TCCTCCGCTTATTGATATGC3!;ITS5:5!GGAAGTAAAAGTCGTAACAAGG3![9]10∀buffer5L,10mmol/LdNTP2L,25pmo,lDNA1L(200ng),Taq2U,50mL945min;9440s,5040s,721min,35;7210min1%TaKaRaAgaroseGelDNAPurificationKitVer.2.0Genbank,BLASTClustalX(version1.81)ITS,MEGA(version4.1),[10-12]:(d=0.5cm)PDA,,5,6d[13],()PDA,5101520253035pHNa2HPO4(0.2mol/L)(0.1mol/L)NaOH(0.2mol/L)(0.2mol/L)PDApH,pH,pH3.04.05.06.07.08.09.010.011.0pHPDA,25PDA,2512h/12ha.()2g,2g0.1g05g1g20g1000mL,pH6,[14],25b.(LDL)2g20g0.1g1.5g3g20g1000mL,pH622.1菌种鉴定2.1.1形态特征:,1,,,,,,,,100cm,6.0cm,2.0cm,,10mm,,12mm,,5/mm::,,,,,:,,,,,,,4,,,30m:,,,,(5.8~7.0)m∀(4.0~6.0)m(1)1A.;B.;C.P1[1,2,7](Perenniporiarobiniophila(Murr.)Ryv.),,P1(P.robiniophila(Murr.)Ryv.)2.1.2分子生物学鉴定结果P126srDNAITS,572bp(2)2P126SrDNA.MDDL2000NAMarker;1.PCR;+.;-.P126srDNAITSGenBankBLAST,(Perenniporiarobiniophila(Murr.)Ryv.),P185%9,9P13,103:P1(Perenniporia)(Trametestrogii)(Dichomitussqualens)P1,(87%),,P1(Trametestrogii)(Dichomitussqualens)(50%),,P1,GenBank(GenBank),(Perenniporia),,P1(P.robiniophila(Murr.)Ryv.),(Basidmiycota),(Basidmiycetes),(Aphyllophorales),(Polyporaceae),(Perenniporia)3ITS,1000,50%2.2病原菌的生物学特性2.2.1温度5~35,20~25,25,5,,,25,,3542.2.2pH值pH3.0~11.0,(5)pH5~6pH11,;pH3,2.2.3光照,3,,(1);618:45pH1/cm/cm6.75#0.121.50#0.13,7.05#0.491.53#0.18,6.83#0.271.53#0.162.2.4碳源,(2),,6d8.38cm;,6d3.77cm,,2/cm/cm/cm/cm8.38#0.08a1.89#0.15a7.60#0.76a1.75#0.18a8.07#0.18b1.82#0.19ab6.94#0.20b1.49#0.12c7.85#0.49b1.74#0.32bc6.81#0.31b1.42#0.16c7.44#0.34c1.66#0.15cd6.79#0.32b1.51#0.11bc7.29#0.14c1.58#0.18d6.73#0.29b1.54#0.22bc6.10#0.09d1.43#0.12e6.57#0.23bc1.60#0.10b5.20#0.29e1.34#0.25e6.28#0.18c1.52#0.14bc3.77#0.15f0.90#0.09fL5.89#0.14cd1.39#0.12cCK3.30#0.11g0.81#0.09gDL5.65#0.26d1.08#0.14dCK3.16#0.19e0.72#0.08e:0.052.2.5氮源(2),;,1.08cm,;,3(Perenniporiarobiniophila(Murr.)Ryv.),(Basidimycota),(Basidimycetes),(Aphyllophorales),(Polyporaceae),(Perenniporia)20~25,,pH5~6;;,,,,,,[15][16-18],(),SotomeK[19],,,GenBank,,GenBank[1]DaiYC,NiemelT,KinnunenJ.ThepolyporegeneraabundisporusandPerenniporia(Basidiomycota)inChina[J].AnnBotFennic,i2002,39:169-182.[2],.[J].,2005,33(1):88-90.[3]ZhaoJD,ZhangXQ.ThepolyporesofChina[J].BiblMyco,l1992,14:510-524.[4],,.[J].,2000,13(1):15-22.[5].[M].:,2005.[6]N~nezM,RyvardenL.EastAsianpolypores2.Polyporaceaeslato[J].SynopsisFungorum,2001,14:462-465.[7],.:3()[M].:,1998:204-205.[8],,,.DNASDSCTAB[J].,1997,19(3):100-103.[9]WhiteTJ,BrunsT,LeeS.AmplificationanddirectsequencingoffungalribosomalRNAgenesforphylogenetics[M].NewYork:AcademicPress,1990:315-322.[10],,,.rDNAITS[J].,2008,44(2):160-164.[11],,,.1[J].:,2008,36(8):165-169.[12],,,.8ITS[J].:,2009,31(1):90-96.[13].[M].:,1998:140-141.[14],,.[M].:,2005:230-232.[15]TabataM,HarringtonTC,ChenW,eta.lMolecularphylogenyofspeciesinthegeneraAmylostereumandEchiondontium[J].Mycoscience,2000,41:585-593.[16],,.[J].,2001,28(4):224-226.[17],,,.[J].,2008,27(6):873-883.[18],,.[J].,2008,27(3):325-334.[19]SotomeK,HattoriT,OtaY.SecondreportofPolyporuslongiporusanditsphylogeneticposition[J].Mycoscience,2009,50:415-420.6238