西贡蕉枯萎病生防木霉菌株gz_2的鉴定及生物学特性研究

:1001-4829(2010)05-1533-07:2010-10-12:(09RZBC3):(1983-),,,,,*gz2柯仿钢1,黄思良2*,付岗3,汪茜1,张欣4,黎起秦1(1.,530005;2.,473061;3.,530007;4.,571737):拮抗西贡蕉枯萎病菌的木霉菌株gz2是一株具有生防潜力的拮抗菌,依据其形态特征和ITS序列将其鉴定为哈茨木霉(TrichodermaharzianumRifai)gz2菌株的生物学特性测定结果表明,该菌株最适宜在马铃薯蔗糖琼脂(PSA)培养基上生长;蔗糖酵母膏分别为最佳碳氮源;菌丝最适生长温度范围为28~30;最适pH5.0~6.0;不同光照条件对该菌产孢影响明显对西贡蕉枯萎病的盆栽防治试验结果表明,gz2菌株对西贡蕉枯萎病具有一定的防效,其中每隔7d施1次gz2固体菌剂处理的防治效果最好,在伤根接种60d后,平均防效达到74.4%:西贡蕉;枯萎病;木霉菌株gz2;生物学特性;防治效果:S688.1:AIdentificationandBiologicalCharacteristicsofTrichodermaStraingz2,ABiocontrolAgentagainstSaigonBananaFusariumWiltKEFanggang1,HUANGSiliang2*,FUGang3,WANGQian1,ZHANGXin4,LIQiqin1(1.AgriculturalCollege,GuangxiUniversity,NanningGuangxi530005,China;2.NanyangNormalUniversity,NanyangHenan473061,China;3.MicrobiologyResearchInstitute,GuangxiAcademyofAgriculturalSciences,NanningGuangxi530007,China;4.EnvironmentandPlantProtectionInstitute,ChineseAcademyofTropicalAgriculturalSciences,DanzhouHainan571737,China)Abstract:Trichodermastraingz2isapotentialbiocontrolagentagainstFusariumwiltofSaigonbanana.ThestrainwasidentifiedasTrichodermaharzianumRifaibasedonitsmorphologicalcharacteristicsandsequenceanalysisoftheinternaltranscribedspacerregionoftheribosomalDNA(ITS).Thebiologicalcharacteristicsofstraingz2werestudied.TheresultsshowedthatthegrowthrateandconidianumberofthebiocontrolstrainwerefasterandmoreinPSAmediumthanthoseinothermedia.Sucroseandyeastextractweretheoptimumcarbonsourceandnitrogensourceforvegetativegrowthandsporulation,respectively.Theoptimumtemperatureformyceliumgrowthandconidialgerminationwas28-30,andtheoptimumpHvaluewas5.0-6.0.Lightconditionshadobviousinfluencesonsporulationsofthebiocontrolstrain.Theresultsofpotexperimentofstraingz2againstSaigonbananaFusariumwiltindicatedthatthecontroleffectofstraingz2againstSaigonbananaFusariumwiltwasbestwhensolidstraingz2wasusedonceinaweek.Theaveragecontroleffectreached74.4%60daysafterbeinginoculatedwithinjuredroot.Keywords:Saigonbanana;Fusairumwilt;TrichodermaStraingz2;Biologicalcharacteristics;Controleffects[Fusariumoxysporum.fsp.cubense(E.F.Smith)SnyderetHansen.]1[1~2],[3~6],,,[7],,,,[8]1gz2,gz215332010235Vol23No5西南农业学报SouthwestChinaJournalofAgriculturalSciences,111gz2,[9],4(PSA)12PSA(PDA)13gz21.3.1形态鉴定gz2PSA28,,,[10],1.3.2基因组DNA的提取PDA,[11]gz2DNA:0.1g,,1.5mL,750l,6530min;12000r/min10min,,1/103mol/LKAc,20min;12000r/min10min,,,20min;12000r/min10~20min,;400lddH2O5lRNAase,371h;/(11)40;12000r/min10min,,1/10KAc,20min,1;220min,12000r/min10min;-2075%3,5~10minTE1.3.3PCR扩增及纯化测序gz2DNA,ITS,[12],ITS1(5!TCCGTAGGTGAACCTGCGG3!)ITS4(5!TCCTCCGCTTATTGATATGC3!)PCR20,l10∀PCRbuffer2,ldNTP0.5,lITS10.6,lITS40.6,l15.1,lDNA1,lTaq0.2l:943min;9455s,5650s,72lmin,32;727min,DNAMarkerDL2000,120V30min,UNIQ10,1.3.4数据分析BioEdit,NCBIBLASTGenBankITS141.4.1不同培养基对菌株gz2菌丝生长和产孢的影响gz2(8mm)PSAPDA,28,2448h,72h10mL,,,31.4.2不同碳源对菌株gz2菌丝生长和产孢的影响,9,,,1.4.3不同氮源对菌株gz2菌丝生长和产孢的影响,10,,1.4.4不同碳氮比对菌株gz2菌丝生长和产孢的影响,()41424448410412,1.4.5不同温度对菌株gz2菌丝生长和产孢的影响5101520252830323540,PSA,1.4.6不同pH对菌株gz2菌丝生长和产孢的影响1mol/LHCl1mol/LNaOHPSApH4.05.06.07.08.09.010.011.0,8,1.4.7不同光照对菌株gz2菌丝生长和产孢的影响8mm3dgz2PSA,12h(11W1,201534西南农业学报23cm)12h31.4.8不同培养时间对木霉菌株gz2产孢的影响8mm3dgz2PSA,24h24h,815gz21.5.1木霉菌株gz2及西贡蕉枯萎病菌孢子悬浮液的制备gz2PSA,,(8mm),200mL500mL,287d,,106/mL,41.5.2木霉菌固体菌剂的制作(12),,1211h,106/g,7d,107/g1.5.3盆栽试验设计#gz2200g,1;∃gz2200g,7dgz2200g,3%106/mLgz210min,,106/mL30mL&gz210min,7d30mL,3∋,30mL,7d50050%200mL,3(1gz2rDNAITSPCRFig.1ElectrophoretogramofPCRproductsofrDNAITSofTrichodermaharzianumstraingz2CK,30mL)CK1,200g,30mL22cm,20cm,5~6,2~3d13,530d1(2009814),15d1:0:;1:25%;3:26%~50%;5:51%~75%;7:75%;9:,:=(∀)∀(%)=-∀10016Excel,DPS6.55221gz2gz2PSA,,,,,,,,,,22ITSgz2Fig.2PhylogenetictreebasedonITSsequencehomologyofstraingz2withtherelatedfungusderivedfromGenBankdatabase15355:gz2~41,,,,,,,,2.6~3.5(3.3)m∀2.2~3.2(2.6)m22ITSgz2DNAPCR,500bp(1),ITS592bp(GenBank:GU196282),GenBankITS,gz2TrichodermaharzianumRifaiITS99%MEGA4.0gz2GenBank13ITS,gz2T.harzianum(GenBank:AF194011)(2),gz2T.harzianum,gz2(T.harzianum)23gz21,gz2PSAPDA,PSA;PSAPDA,;;,6PSAgz2,gz224gz2gz29(2),,gz2;;25gz2gz210(3),,gz2,,1gz2Table1Effectsofdifferentculturalmediaonmyceliumgrowthandsporulationofstraingz2Medium(cm)Colonydiameter24h48h(∀106/mL)No.ofconidiaproducedMaltosemedium4.5cBC6.2cC12.3eEPDAmedium4.9bcBC9.0aA38.8cCPSAmedium5.0bcBC9.0aA98.6aACornmedium5.2bB7.5abA17.0deDECzapek∗smedium6.1aA9.0aA67.5bBOatmedium4.6cBC6.4bB27.3dCD:1%5%,Note:Thecapitalandsmalllettersinthesamecolumnindicatedsignificantdifferenceat0.01and0.05leve.lThesameasfollows.2gz2Table2Effectsofdifferentcarbonsourcesonmyceliumgrowthandsporulationofstraingz2Carbonsource(cm)Colonydiameter24h48h(∀106/mL)No.ofconidiaproducedMaltose1.5abA5.8abcdABC26.5bcdBCSucrose1.8abA6.4abAB39.7aALactose2.0aA6.3abcAB24.3cdBCTrehalose1.6bA5.3cdBC22.0dCMannite1.8abA6.2abcAB10.0fDEArabinose1.7abA5.6bcdABC29.3bBStarch1.6abA5.6abcdABC27.5bcBCXylitol1.8abA5.0dC15.7eDGlucose1.8abA6.6aA37.3aACarbonfree1.7abA6.0abcABC7.4fE1536西南农业学报233gz2Table3Effectsofnitrogensourceonmyceliumgrowthandsporulationofstraingz2Nitrogensource(cm)Colonydiameter24h48h(∀10