中英文---温度、湿度和剪口龄期对腐烂病菌和轮纹病菌自剪锯口侵染和发病的影响1-副本

温度、湿度和剪口龄期对腐烂病菌和轮纹病菌自剪锯口侵染和发病的影响EFFECTOFTEMPERATURE,WETNESSANDAGEOFPRUNINGWOUNDSONVALSAMALIANDBOTRYOSPHAERIADOTHIDEAINFECTIONFROMPRUNINGWOUNDSABSTRACTTHEEFFECTOFVALSAMALIVARMALIVMMANDBOTRYOSPHAERIADOTHIDEAINFECTIONFROMPRUNINGWOUNDSWASINVESTIGATEDBYTEMPERATURE,WETNESSANDAGEOFPRUNINGWOUNDSTEMPERATUREANDWETNESSHAVENOSIGNIFICANTEFFECTONVALSAMALIANDBOTRYOSPHAERIADOTHIDEAINFECTIONFROMPRUNINGWOUNDS,BUTTHEEXTENDEDDISTANCEOFVALSAPATHOGENHASVISIBLEDIFFERENCEININOCULATEDXYLEMATDIFFERENTTEMPERATUREMOSTLONGDISTANCEOFINOCULATEDPATHOGENISOLATEDATTWIGSEGMENTSWAS5CMAWAYFROMTHEINOCULATEDENDTHEINOCULATEDPRUNINGWOUNDSBYVALSAMALISHOWNOSYMPTOMDURINGTHATTIME,BUTSHOWGRADUALLYFROMMARCHINTHESECONDYEARFLESHPRUNINGWOUNDSHAVENORESISTANCETOBOTRYOSPHAERIADOTHIDEAINFECTIONWITHOUTWETNESSANDTHEOCCURRENCEOFINOCULATEDPRUNINGWOUNDSWAS100SOFLESHPRUNINGWOUNDSCANBEINFECTEDWHENSPORESOFVALSAMALIANDBOTRYOSPHAERIADOTHIDEASPREADINTOWITHRAINWATERINTHESPRINGKEYWORDSAGEOFPRUNINGWOUNDS,TEMPERATURE,WETNESS,EXTENDEDDISTANCEINTRODUCTIONVALSACANKERVALSAMALIMIYABEWHEREAS,INSOMEREGIONS,THERATEOFDISEASEDPLANTSWASALMOST100,ESPECIALLYINSHANDONG,HENANANDBEIJING国立耘ETAL,2009BOTRYOSPHAERIADOTHIDEAISNOTJUSTINFECTSTEMSANDALSOLEADALARGENUMBEROFPYCNIDIATOAPPEARONTHESURFACEOFINFECTEDBRANCHESALARGENUMBEROFSPORESPREEDINGWITHTHERAINCANINFECTAPPLEFRUITSANDFORMTHESYSTEMOFVERTICILVEINED,RESULTINGINFRUITSROTTHEINCIDENCEOFFRUITRINGROTWAS530ONUNBAGGEDFRUITS,ANDEVEN80INSERIOUSCASE中国苹果树上的两种重要枝干病害。腐烂病主要危害主干、主枝，导致皮层腐烂，造成死枝、死树，重者毁园，在中国是苹果树上的第一大病害，严重威胁苹果产业的发展。2008年调查，中国10个苹果主产省市的147个果园内，苹果树的总体发病率为527曹克强ETAL,2009。腐烂病在中国已出现四次大的流行，几乎毁灭了自50年代以来新建的苹果园。轮纹病主要危害幼树、侧枝，在枝干上形成疣状突起，称为病瘤，大量病瘤聚集在一起形成粗皮，病瘤和粗皮严重削弱树势。当枝条受水份胁迫或极度衰弱时，轮纹病菌在皮层内迅速扩展，导致皮层坏死，形成干腐症状，导致幼树和枝条枯死。2008年调查，中国7个苹果主产省市的88个果园内，枝干轮纹病的总体发病率为776，其中山东、河南和北京的危害相对较重，病株率几乎为100国立耘ETAL,2009。除危害枝干外，轮纹病菌在枝干上还能产生大量孢子，随雨水传播侵染果实，形成轮纹症状，造成果实腐烂。在非套袋的果实上，果实轮纹病的发病率达530，病重年份达80。APPLEVALSACANKERWASTHEFIRSTOCCURRENCEINHOKKAIDOOFJAPAN,AFTERSPREADINGINTOMAINLANDOFJAPANANDKOREAINSUCCESSIONIN1916,VALSACANKERWASINTRODUCEDFOLLOWINGWITHTHEINTRODUCTIONOFAPPLETHEOCCURRENCEOFTHEDISEASEWASMORESEVEREINXIONGYUEINLIAONINGPROVINCE（SAKUMA1990UHMANDSOHN1995SOFAR,VALSACANKERMAINLYOCCURREDINEASTERNASIAANDALSOINEVERYAPPLEPRODUCINGREGIONSINCHINA其中北部省区受害严重THEREWERETHREESPECIESOFVALSACANCAUSEDTHISDISEASETHEYAREREPECTIVELYVMALI，VMALICOLAZURBANDVPERSOONII（LEUCOSTOMPERSOONII）THECAUSALAGENT,VMALIMIYABEETYAMADA,CANBEALSODIVIDEDINTOTWOVARIETIESBELOWTHERANKOFSPECIESLEVEL,VMALIVARMALIVMMANDVMALIVARPYRIVMPTHEVARIETYOFVMALIVARMALIWASTHEPREDOMINANTPATHOGENOFAPPLETREEVALSACANKERINCHINA臧睿,2012,WANGETAL,2011苹果树腐烂病于1887年首先在日本的北海道发生，后陆续传入日本本岛和朝鲜。1916随苹果引种引入中国，19世纪20年代辽宁熊岳一带受害较重（SAKUMA1990UHMANDSOHN1995。目前主要分布于东亚地区，中国各苹果产区均有发生，其中北部省区受害严重。中国苹果树腐烂病菌由三个种组成，即VMALI，VMALICOLAZURB和VPERSOONII（LEUCOSTOMPERSOONII）。VMALI有VMALIVARMALIVMM和VMALIVARPYRIVMP两个亚种，其中VMM是导致中国苹果腐烂病的主要致病菌臧睿,2012,WANGETAL,2011。VMMISUSUALLYCONSIDEREDASWEAKLYPATHOGENICANDMAINLYINFECTSTHROUGHVARIOUSWOUNDSINCLUDINGPRUNINGWOUNDS,FROSTBITES,SUNSCALDSANDSOONWILLISON1936BIGGS1989KEPLEYANDJACOBI2000ADAMSETAL2006；CHENC1981WHEREASTHEPATHOGENALSOPARASITIZESTREESTHROUGHSOMEMICROWOUNDSSUCHASEYES,LEAFSCARSANDFRUITSCARSTHEPRUNINGWOUNDSARETHEMOSTSUSCEPTIBLESITESANDALSOTHEMOSTIMPORTANTBYTHECAUSALFUNGUSPARASITIZED高桑亮等，1972CAIXIAWANGOBSERVED967NEWCANKERSCARSINTHESPRINGOF2011WHEREAS,8004CANKERSCARSWEREDEVELOPEDFROMPRUNINGWOUNDS王彩霞ETAL,2014THEMYCELIUMOFTHECAUSALFUNGUSINFECTEDTHROUGHPRUNINGWOUNDSMAINLYPARASITIZESINTHEDUCKSOFXYLEMANDATTACHESTOTHESURFACEOFTHEPRUNINGWOUNDS藤田等，1981；藤田等，1985THECORTICALTISSUESAREDAMAGEDWHENTHEMYCELIUMOFTHECAUSALFUNGUSEXPANDSFORMTHEDUCKSOFXYLEMTOTHECORTICALTISSUESTHEPATHOGENCANSURVIVEATLEAST35YEARSINTHEXYLEMWHENTHECONDITIONSARESUITABLE,THEFUNGUSEXPANDSRAPIDLYANDLEADTHECORTICALTISSUESTOOUTBREAKTHEOUTBREAKFROMPRUNINGWOUNDSANDRECURRENCEOFOLDSCARSARECAUSEDBYTHEMYCELIUMOFTHEPATHOGENEXPANSIONINTHEXYLEM陈延熙,1951,王金友ETAL,1985苹果树腐烂病菌属于弱寄生真菌，主要从剪锯口、冻伤、日灼伤等伤口侵染WILLISON1936BIGGS1989KEPLEYANDJACOBI2000ADAMSETAL2006；CHENC1981，也可通过芽眼、叶痕、果痕等处的微伤口侵染，剪锯口是最易受腐烂病菌侵染的部位高桑亮等，1972，也是腐烂病菌的主要侵染孔口。2011年王彩霞等王彩霞ETAL,2012检查了967块新病疤，其中源自剪锯口侵染的病疤占8004。从剪锯口侵染的腐烂病菌，主要潜伏于木质部的导管内和附着于伤口表面藤田等，1981；藤田等，1985，并不能立即致病，当病菌在木质部导管内生长扩展到达皮层时，才能导致皮层发病。木质部内腐烂病菌至少能存活35年牟惠芳，刘开启,1994；王金友ETAL,1985，条件适宜时，木质部内菌丝迅速扩展，导致皮层发病。剪锯口发病、旧病斑复发都是木质部内的菌丝生长扩展所致陈延熙,1951,王金友ETAL,1985。BOTRYOSPHAERIADOTHIDEAMAINLYINFECTSFROMLENTICELSANDSTOMASOFBRANCHESANDPARASITIZESPREFERABLYTHROUGHPRUNINGWOUNDSONTHEPRUNINGWOUNDS,THEBDOTHIDEAMAINLYINFECTSCORTICALTISSUESANDLEADSTHECORTICALBLOWPRUNINGWOUNDSNECROSIS,FORMINGTHEDRYROTWITHTHEWIDTH2MM20MMTHEDRYROTSCABSONTHEPRUNINGWOUNDSHAVELITTLEINFLUENCEONTHETREEBODY,BUTCANPRODUCEALARGEOFSPORETHOSESPORESPREADFOLLOWINGWITHRAINWATERANDINFECTTHELOWERBRANCHESOFTHEAPPLETREEALARGEOFDRYROTSCABSCANMAKETHELOWERBRANCHESSERIOUSDISEASEAFTERTHEACCUMULATIONOF23YEARS轮纹病主要从枝条的皮孔和气孔侵染，便更容易从剪锯口侵染。在剪锯口上，轮纹病菌主要侵染皮层，导致剪锯口下方皮层坏死，形成宽度2MM20MM的干腐病斑。枝条剪锯口处的干腐病斑对树体的生长影响不大，但能产生大量孢子，在生长季节随雨水传播侵染下部枝条。经23的积累，导致下部枝干严重发病。VALSAMALIMIYABEANDBOTRYOSPHAERIADOTHIDEAINTHESCARSOFBRANCHESCANPRODUCECONSTANTLYCONIDIAANDASCOSPORETHECONIDIAANDASCOSPORERELEASEFOLLOWINGWITHRAINWATERANDREACHTHESUSCEPTIBLEPOSITIONWITHTHESPREADOFRAINANDAIRTHEFUNGUSISDIFFICULTTOERADICATEBYCONVENTIONALMETHODSWHENINVADESTHEHOSTTISSUESEXPERIMENTSHOWEDTHATTHEBESTCONTROLEFFECTOFSYSTEMICFUNGICIDESCOULDREACH55WHENBOTRYOSPHAERIADOTHIDEAINFECTEDBRANCHESFORTENDAYSANDCOULDNOTEFFECTIVELYCONTROLTHEINCIDENCEANDPREVALENCEOFDISEASE张高雷ETAL,2011FORTHEPREVENTIONANDCUREOFTHEAPPLEBRANCHESDISEASE,THEMEASUREOFOPTIMUMPERIODSPRAYINGPROTECTIONHASBEENATTEMPTEDTOCONTROLSINCE1950SHOWEVER,DUETOLIMITEDKNOWLEDGEEXISTSABOUTTHEOCCURRENCEANDEPIDEMICREGULARITYOFSTEMDISEASE,ASWELLASPOORPROTECTIONEFFECTOFFUNGICIDES,THECONTROLEFFECTEXPECTEDFAILEDTOBEOBTAININ1960S,ASTUDYFOUNDTHATVALSAMALIWASCHARACTERIZEDBYLATENTINFECTION,ANDTHEMETHODSPREVENTIONOFSTEMDISEASETURNSPRAYINGPROTECTIONTOSPRAYERADICATINGTHEFUNGUSINTHETREEPERMEABILITYFUNGICIDESWERESELECTEDATECHNOLOGYISUSEDTOERADICATETHEFUNGALPATHOGENICITYWHICHSURFACEANDSHALLOWOFTHETREESANDTOPREVENTDISEASEBYSPRAYINGORCOATINGSTRONGPERMEABLEERADICANTHOWEVER,THETECHNOLOGYCANNOTCONTROLEFFECTIVELYTHEVALSACANKERAFTERCARRIEDOUTTENYEARS枝干病斑内的腐烂病菌和轮纹病菌都能不断产生分生孢子和子囊孢子，随降雨释放，并随雨水或气流传播到达感病部位。病菌侵入寄主组织后，难以铲除。实验表明，轮纹病菌在枝干上侵染10天后，内吸治疗剂的防治效果最高能达到55，无法有效控制病害的发生与流行（张高雷2010）。对于苹果树枝干病害的防治，上世纪50年代曾尝试适期喷药保护的措施防治枝干病害，但由于对枝干病害发生和流行规律的认识不足，以及杀菌剂保护效果差等方面的原因，未能获得预期的防治效果。60年代研究发现，腐烂病具有潜伏侵染的特性，枝干病害的防治从喷药保护为主转向喷药铲除树体内潜伏的病菌为主，并筛选出福美砷等强渗透性的杀菌剂。通过在树体休眠期喷施或涂布强渗透性的铲除剂，树体表面和浅层潜伏病原菌，以达到防治病害的目的，然而，该项技术实施10年后，仍没有达到有效控制腐烂病的目的。INRECENTYEARS,WITHDEPTHUNDERSTANDINGOFVALSAMALIMIYABEANDBOTRYOSPHAERIADOTHIDEAINCIDENCEANDEPIDEMICLAWSASWELLASTHEEMERGENCEOFNEWEFFICIENTFUNGICIDE,THEAUTHORTHINKSTHEPREVENTIONOFBRANCHESDISEASESHOULDSTARTFROMSAPLINGSTAGETHEBASICMEASURESISPROTECTINGPRUNINGWOUNDSANDBRANCHESFROMTHEPATHOGENINFECTIONTHEFOUNDATIONMEASURESISSCARAPINGSCABSTIMELYERADICATINGTHESOURCEOFFUNGUSFORPREVENTINGTHENUMBEROFINFECTEDFUNGUSACCUMULATINGINORCHARDTHEPRUNINGWOUNDSARETHEIMPORTANTORIFICESBYVALSAMALIMIYABEANDBOTRYOSPHAERIADOTHIDEAINFECTEDTHEPROTECTIONOFPRUNINGWOUNDSSHOULDFROMSEEDLINGANDSAPLINGSTAGETOCONTROLEFFECTIVELYVALSAMALIMIYABEANDBOTRYOSPHAERIADOTHIDEAINFECTEDSOTHATTHEOCCURRENCEANDEPIDEMICOFSTEMDISEASEISCONTROLLEDEFFECTIVELYTHEOBJECTIVESOFTHISSTUDY,BASEDONMANUAL,WERETODEFINITEITHECONDITIONOFTEMPERATUREANDMOISTUREONINFECTIONOFVALSAMALIMIYABEANDBOTRYOSPHAERIADOTHIDEAFROMPRUNINGWOUNDSIITHEEFFECTOFTEMPERATUREONTHEVALSAPATHOGENSGROWTHANDEXTENSIONINXYLEMTHEEFFECTOFAGEOFPRUNINGWOUNDSONPATHOGENINFECTIONTHEINFORMATIONABOUTPRESENTSTUDYCANPROVIDETHEORETICALBASISANDDATAFORTHEEPIDEMICFORECASTINGANDPREVENTIONTECHNOLOGYMAKINGOFTHETWOBRANCHESDISEASES近年来，随着对腐烂病和轮纹病发病与流行规律认识的深入，以及新型高效杀菌剂的出现，作者认为防治枝干病害需要从幼树期开始，以保护剪锯口和枝干不受病菌侵染为基本措施，并及时刮治病斑，铲除菌源，以防止内的侵染菌原量积累为基础。剪锯口是腐烂病菌和轮纹病菌的重要侵染孔口，从苗期和幼树期开始，保护好剪锯口，有效控制腐烂病菌和轮纹病菌从剪锯口侵染，就能有效控制枝干病害的发生与流行。本研究拟在人工控制条件下的接种试验，研究明确1）腐烂病菌和轮纹病菌从剪锯口侵染所需要的温度和湿度条件；2）温度对腐烂病菌在木质部内生长扩展的影响；3）剪锯口的龄期对病菌侵染的影响；为两种枝干病害的流行预测和制订防治技术提供依据和数据。1材料和方法MATERIALSANDMETHODS211病原菌及孢子繁殖PATHOGENANDSPOREPROPAGATIONDISEASEDBARKWITHTYPICALSYMPTOMSOFVALSACANKERWERECOLLECTEDFROMLEIYATOWNBAISHUICONTRYSHAANXIPROVINCEPATHOGENISOLATION,BETWEENTHESYMPTOMLESSANDAPPARENTLYINFECTEDAREA,SAMPLESWERETAKENANDTRANSFERREDTOFRESHPLATESOFPDAPOTATODEXTROSEAGARTHEFUNGALVALSAMALIISISOLATEDANDOBTAINEDFROMTHECULTIVATEDDISEASEDTISSUESASINGLEMYCELIUMWASOBTAINEDFROMTHEDISTALENDOFMYCELIAONTHEEDGEOFCOLONYBYCAPILLARYWITHADIAMETEROF08MM,NUMBERINGOFLXS080901HYGROMYCINBPHOSPHOTRANSFERASEGENEANDGREENFLUORESCENCEPROTEINGENEWEREINTEGRATEDINTOTHEGENOMEOFVMALIBYTHEMETHODOFATUMEFACIENSMEDIATEDTRANSFORMATION,ANDTHENOBTAINEDTHETRANSFORMANTLXS08090116COMPAREDWITHWILDTYPESTRAINLXS080901,THETRANSFORMANTLXS08090116HAVENODIFFERENCEONTHEBIOLOGICALCHARACTERISTICS,THEPATHOGENICITYANDTHESCARSYMPTOMSOFAPPLEBRANCHESTHETRANSFORMANTPREVALENCESITUATIONCONSISTENTSWITHNATURALLYINFECTEDINFIELDWANGETAL,2013DURINGTHISSTUDY,THISWILDTYPESTRAINSANDTRANSFORMANTSWERESTOREDANDGROWNONPOTATODEXTROSEAGARPDAMEDIUMAT4C2009年8月自陕西省白水县雷牙乡采集苹果树腐烂病病枝，切取病健交界处的病组织转入马铃薯葡萄糖琼脂培养基（PDA）中，分离获得腐烂病菌VALSAMALI。用直径08MM的毛细管，自菌落边缘打取菌丝的端部转入PDA中，获得单菌丝分离菌株，编号为LXS080901。用农杆菌介导转化法ATMT将携带潮霉素B磷酸转移酶基因HPH和绿色荧光蛋白表达基因GFP转入腐烂病菌，获得转化子LXS08090116。经离体和活体测定，转化子LXS08090116和野生菌株LXS080901的生物学性状、致病性、以及在苹果枝干上形成的症状都没有差异，与田间自然发病情况一致（WANGETAL,2013）。2个菌株都保存于4C的冰箱中。FORINOCULATION,STRAINSSTOREDWERETRANSFERREDTOFRESHPLATESOFPDAANDCULTUREDAT25CINDARKNESSFOR35DAYSTHEFRESHINOCULUMWASOBTAINEDFROMTHEEDGEOFVMALIPLATESUSING5MMPUNCHANDINOCULATEDINTOTHEBARLEYCOMMEDIUMBARLEYHONEYPEPTONEAT25CINDARKNESSFORABOUT15DAYSTHEFUNGIPRODUCEDALARGENUMBEROFPYCNIDIATHEBARLEYPRODUCEDPYCNIDIAWERETRANSFERREDTO9CMDIAMETERPETRIDISHESOF1WATERAGARANDCULTUREDAT25CINDARKNESSFOR3DAYSTHEPYCNIDIARELEASEDCONIDIATHEFRESHCONIDIAWEREPLACEDINTOPUREWATERANDMADECONIDIASUSPENSIONWHICHCONTAINEDABOUT106SPORESPERMILLILITERFORINOCULATIONONTHISSTUDY,WILDTYPESTRAINSLXS080901WEREUSEDTOINOCULATEDOUTDOORTRANSFORMANTSLXS08090116WEREUSEDTOINOCULATEDVITROTWIGSINDOORTHEMATERIALSINOCULATEDBYTRANSFORMANTSWEREDESTROYEDAFTERTHEENDOFEXPERIMENT接种前，将保存菌种从冰箱中取出，接种于PDA培养基中，25C下黑暗培养35天，取菌落边缘直径5MM新鲜菌饼，接种于大麦粒培养基（配方，消毒），25C恒温箱中培养15天，大麦粒上可产生大量分生孢子器。取产生分生孢子器的大麦粒，转入含有1水琼脂直径为9CM培养皿中，25C下保湿培养3天，分生孢子器中溢出大量橘黄色分生孢子角赵红文献。挑取新鲜分生孢子角置于纯净水中，配成106个/ML孢子悬浮液用于接种枝条。本研究中室外接种全部用野生菌株LXS080901，室内离体枝条接种用转化子LXS08090116。转化子接种的材料在试验结束后全部销毁。ONEYEAROLDFUJIAPPLETWIGSWITHTYPICALSYMPTOMSOFBOTRYOSPHAERIADOTHIDEAWERECOLLECTEDFROMTHEORCHARDSOFSHUIMUTOUVILLAGELAIYANGCITYSHANDONGPROVINCEIN2008DISEASEDSAMPLEUSEDFORISOLATIONWASCULTUREDONPDAFROMTHECANKERMARGINBETWEENNECROTICANDAPPARENTLYHEALTHYTISSUESTOOBTAINBOTRYOSPHAERIADOTHIDEATHEMYCELIUMISOLATIONWEREINOCULATEDINTOFUJIAPPLEYOUNGFRUITSTOPRODUCEPYCNIDIAUNDERUVLIGHT冷伟锋ETAL,2009SINGLECONIDIAWERETRANSFERREDTOFRESHPDAPLATESATTOOBTAINPUREISOLATESLXS030101BYPRESSACAPILLARYWITHTHEDIAMETEROF1MM董娟华ETAL,2009MYCELIAWERESTOREDANDGROWNONPOTATODEXTROSEAGARPDAMEDIUMBEFOREUSINGAT4COMPAREDWITHWILDTYPESTRAIN,THEPUREISOLATESHAVENODIFFERENCEONPATHOGENICITYANDSHOWEDTYPICALSYMPTOMSOFRINGROTWHENINOCULATEDINTOTWIGSANDFRUITSOFFUJIAPPLE2008年自山东莱阳市水沐头村苹果园内剪取带有典型轮纹病瘤的一年生富士枝条，切取轮纹病瘤转入PDA进行组织分离，获得组织分离的轮纹病菌（BOTRYOSPHAERIADOTHIDEA）。将组织分离菌丝转接到富士苹果幼果上，黑光灯下诱导产孢冷伟锋ETAL,2009，通过毛细管挑取单孢董娟华ETAL,2009转入PDA中培养，获得单孢分离菌株，编号为LXS030101，保存于4C的冰箱中。单孢菌株回接富士苹果果实和枝条，都能表现典型的轮纹病症状，与野生菌株的致病性无差异。BEFOREINOCULATION,MYCELIUMOFBOTRYOSPHAERIADOTHIDEASTOREDWASTRANSFERREDTOPDAFOR57DAYSTHEFRESHINOCULUMWEREINOCULATEDINTO60DAYSFRUITSOFFUJIAPPLETHOSEFRUITSINOCULATEDSHOWEDTYPICALSYMPTOMSAFTER35DAYSANDCULTIVATEDUNDERUVLIGHT365NMTHEFRESHCONIDIAWEREPLACEDINTOPUREWATERANDMADECONIDIASUSPENSIONWHICHCONTAINEDABOUT104SPORESPERMILLILITERFORINOCULATION接种前，将轮纹病菌从冰箱内取出，在PDA培养基上培养57天，然后打取菌饼转接到富士苹果60日龄的幼果上。接种幼果35天后发病，然后置于黑光灯（波长365NM）下诱导产孢。挑取新产生的分生孢子，用纯净水配置成104个/ML孢子悬浮液备用。12露温和露时对腐烂病菌分生孢子自剪锯口侵染的影响12EFFECTOFTEMPERATUREANDWETNESSONINFECTIONPRUNINGWOUNDSBYVALSACONIDIATHEASSAYSHOWEDEFFECTSOF7KINDSOFTEMPERATUREAND7KINDSOFWETNESSONINFECTIONPRUNINGWOUNDSBYVALSACONIDIATHESEVENKINDSOFTEMPERATUREWERE5C、10C、15C、20C、25C、30CAND35CBY7THERMOSTATSCONTROLLED（BLUEPARDMGC250BP2,SHANGHAIBLUEPARDINSTRUMENTSCOLTD）RESPECTIVELYTHOSETHERMOSTATSWEREOPENEDANDSETTHEAPPROPRIATETEMPERATUREBEFORE12HOURSINOCULATIONTHESEVENKINDSOFWETNESSWEREKEPTFOR0H、1H、3H、6H、12H、24HAND48HAFTERINOCULATION,RESPECTIVELYALLASSAYSCONTAINED49COMBINATIONSOFTEMPERATUREANDWETNESS,ANDEACHCOMBINATIONTREATMENTCONDUCTEDTHREETWIGS试验测试了7个露温和7个露时对腐烂病菌分生孢子自剪锯口侵染的影响。7个露温分别为5C、10C、15C、20C、25C、30C和35C，分别由7台恒温箱控制（BLUEPARDMGC250BP2,SHANGHAIBLUEPARDINSTRUMENTSCOLTD）。恒温箱接种前12小时开启，并设定相应温度。7个露时处理分别为接种后保湿0H、1H、3H、6H、12H、24H和48H。全部试验共49个露温与露时组合，每个组合处理3个枝条。ONEYEARHEALTHYTWIGSWERECUTFROMFIVEYEARFUJIAPPLETREESBEFOREAPPLEBUDINMARCHBYDISINFECTEDFRUITSHEARSTWIGSWERECUTINTO20CMLONGSEGMENTS,KEEPINGPRUNINGWOUNDSTIDYANDINSERTEDINTOTISSUECULTUREVESSELSWITHDISTILLEDWATERTHEBASEOFTWIGSWEREIMMERSEDINTHEWATERFOR2CMTHEDISTILLEDWATERWASREPLACEDWATEREVERY3DAYSTHEHYDROPONICTWIGSWERESHIFTEDINTHERMOSTATSWITHRELEVANTTEMPERATUREANDPRECONDUCTEDFOR12HOURSFORINOCULATION,CONIDIASUSPENSIONOFVALSAMALILXS08090116WASSPRAYEDEVENLYONTOTHEPRUNINGWOUNDSOFTWIGSBYHANDEDSPRAYERUNTILLFLOWEDDOWNAFTERINOCULATION,EACHCOMBINATIONTWIGSWERECOVEREDBYPLASTICBAGSBESIDES0HOFWETNESSANDPLACEDINTOTHERMOSTATSWITHRELEVANTTEMPERATURETHEPLASTICBAGSWEREREMOVEDWHENWETNESSFINISHEDCONDUCTED,BUTTHOSETWIGSWERESTILLCULTIVATEDINTHERMOSTATSDURINGINASSAYS,THERELATIVEHUMIDITYRHWASSETBETWEEN50AND80THEDEWONTHESURFACEOFPRUNINGWOUNDSOFTWIGSWASEVAPORATEDINABOUTTWENTYMINUTESINFECTEDTWIGSWERETAKENOUTAFTERINFECTION72HOURSTHEINOCULATIONENDOFTWIGSWEREIMMERSEDIN75ALCOHOLFOR10STOKILLPATHOGENWHICHFAILEDTOINFECT,ANDTHENTWIGSWERESHIFTEDIN25ANDRH75OFTHERMOSTATSTOCULTIVATEFOR7DAYS3月份苹果萌芽前，自5年生的富士苹果树上剪取一年生的健康枝条，用消毒的枝剪，将枝条截成20CM枝段，保持剪口整齐，3个一组，插入组培瓶内，加入蒸馏水，使枝条基部浸入水中2CM，每3天更换一次蒸馏水。将水培枝条转入相应温度的恒温内预处理12小时。接种时，从恒温箱中取出枝条，用手持喷雾器，将腐烂病菌LXS08090116菌株的分生孢子悬浮液均匀喷撒到枝条的剪口上，直至剪口处有孢子悬浮液流下为止。接种后，除露时处理为0H的枝条不套塑料袋，其他露时处理的枝条，每个组合的枝条套一个塑料袋，然后放回到相应温度的恒温箱中。至处理露时结束，解除套在枝条上的塑料袋，再放回到恒温箱中继续培养。试验期间，恒温箱内的相对湿度为5080之间，解袋后，剪口表面的露水约在20分钟内蒸发。接种72小时后，将全部枝条取出，将接种端浸入75的酒精保持10秒钟，杀死剪口表面未能侵染的病菌，然后转入温度25C、相对湿度为75的人工气候箱中再培养7天。FINISHEDTREATMENT,THOSEINOCULATEDTWIGSWERETAKENOUTANDCUTINTOFIVESEGMENTSINTURNFROMTHEINOCULATEDENDIN1CMUNITSBYDISINFECTEDFRUITSHEARSEVERYSEGMENTWASCUTINTOFOURPIECESEQUALLYCENTERINGAROUNDTHEPITHBYCROSSINGMETHODTHOSEPIECESOFTISSUESWEREIMMERSEDIN75ALCOHOLFOR2MINANDWASHEDWITHSTERILEWATERFORTHREETIMES,TRANSFERREDTOPDAMEDIUMAT25EACHPLATECONTAINEDFOURPIECESTISSUESISOLATEDTISSUESWEREINSPECTEDWHETHERTHEMYCELIAGREWFROMTHENEXTDAYTOTHETENTHDAYIFTHEMYCELIAGREWFROMISOLATEDTISSUES,THEMYCELIAWASPICKEDANDOBSERVEDBYFLUORESCENCEMICROSCOPETHEMYCELIAWEREINFERREDTOBETHEINOCULATIONVALSAMALIANDSPREADTOTHEISOLATEDTISSUESWHENSHOWEDGREENFLUORESCENCETHENUMBERANDRATEOFINFECTEDTISSUESWERECOUNTEDINTHEEVERYDISPOSALATTHESAMEDEPTHOFTWELVESEGMENTSTHISASSAYWASREPEATEDTHREETIMESATDIFFERENTTIMESEGMENTTHERMOSTATSWEREDISTRIBUTEDRANDOMLYEVERYREPEATEDTIME处理结束将枝条取出，用小刀剥除皮层，然后自接种端开始以1CM为单位，用消毒枝剪（每剪切一次，用75的酒精擦拭消毒20秒）从每个接种枝条上依次截取5个枝段，然后以髓部为中心按十字交叉法，将每个茎段平均分成4块枝条组织，用75的酒精浸泡30秒钟，用消毒2分钟，用75的酒精浸泡2分钟，经蒸馏无菌水漂洗3次，转接到PDA培养基中，每皿4块，在25C下培养。从第2天开始，每天检查每块分离组织内是否有菌丝长出，直到第10天结束。若分离组织内有菌丝长出，挑取菌丝在荧光显微镜下（荧光波长为）观察，若菌丝内有绿色荧光，则推断该菌丝为接种腐烂病菌的菌丝，接种病菌已生长扩展至该部分组织内。检查结束，统计每个处理同一深度12个枝段中带菌组织的块数和计算带菌百分率。全部试验在不同时间重复3次。每次重复恒温箱随机分配。13露温和露时对轮纹菌分生孢子自剪锯口侵染的影响13EFFECTOFTEMPERATUREANDWETNESSONINFECTIONPRUNINGWOUNDSBYBOTRYOSPHAERIADOTHIDEACONIDIATHEASSAYSHOWEDEFFECTSOF6KINDSOFTEMPERATUREAND5KINDSOFWETNESSONINFECTIONPRUNINGWOUNDSBYBOTRYOSPHAERIADOTHIDEACONIDIATHESIXKINDSOFTEMPERATUREWERE5C、10C、15C、20C、25C、30CAND35CBYSIXTHERMOSTATSCONTROLLED（BLUEPARDMGC250BP2,SHANGHAIBLUEPARDINSTRUMENTSCOLTD）RESPECTIVELYTHEFIVEKINDSOFWETNESSWEREKEPTFOR0H、1H、3H、6H、12H、24HAND48HAFTERINOCULATION,RESPECTIVELYALLASSAYSCONTAINED30COMBINATIONSOFTEMPERATUREANDWETNESS,ANDEACHCOMBINATIONTREATMENTCONDUCTEDTHREETWIGS试验测试了6个露温和5个露时对苹果轮纹病菌分生孢子自剪锯口侵染的影响。6个露温处理分别为10C、15C、20C、25C、30C和35C，分别由6台恒温箱控制。5个露时处理分别为接种后保湿0H、1H、4H、12H和24H。全部试验共30个露温和露时组合，每个组合接种3个枝条。THEHEALTHYCURRENTTWIGSWITH40CM60CMLONGWERESELECTEDFROMFIVEYEARVIGOROUSFUJIAPPLETREESDURINGJULYANDAUGUSTTHETIPOFTWIGSWERECUTFROMLIGNIFIEDSITETOFORMPRUNINGWOUNDSTWIGSWERECUTINTO15CMLONGSEGMENTSANDINOCULATEDAFTERPRUNEDFOR35DAYSONTHETREESTHREETWIGSWERECONTAINEDINEVERYGROUPANDPRECONDUCTEDFOR12HOURSINTHERMOSTATSWITHCORRESPONDINGTEMPERATUREFORINOCULATION,CONIDIASUSPENSIONOFBOTRYOSPHAERIADOTHIDEALXS030101WASSPRAYEDEVENLYONTOTHE35DAYSOLDOFPRUNINGWOUNDSBYHANDEDSPRAYERUNTILFLOWEDDOWNAFTERINOCULATION,EACHCOMBINATIONTWIGSWERECOVEREDBYPLASTICBAGSBESIDES0HOFWETNESSANDPLACEDINTOTHERMOSTATSWITHRELEVANTTEMPERATURETHEPLASTICBAGSWEREREMOVEDWHENFINISHEDTREATMENTSOTHATTWIGSWEREDRIEDNATURALLY7、8月份，自5年生旺盛生长的富士苹果树上，选取40CM60CM长的当年生健康枝条，从已木质化的部位剪除梢部，形成剪锯口。让修剪枝条在树体上生长35天后，剪取长度为15CM的枝段，用于接种。接种枝条每3个一组水培，置于相应温度的恒温箱中，预处理12小时。接种时，用手持喷雾器，将轮纹病菌LXS030101菌株的分生孢子悬浮液均匀喷撒到35日龄的剪锯口，直至剪口处有孢子悬浮液流下。接种后，除露时为0小时的处理外，其他露时处理的枝条，每个枝条套一个塑料袋，放回恒温箱中。到保湿处理时间结束，解去塑料袋，让接种枝条在恒温箱中自然晾干。INFECTEDTWIGSWERETAKENOUTAFTERINFECTION72HOURSTHEINOCULATIONENDOFTWIGSWEREIMMERSEDIN75ALCOHOLFOR10STOKILLPATHOGENWHICHFAILEDTOINFECT,ANDTHENTWIGSWERESHIFTEDIN30ANDRH75OFTHERMOSTATSTOCULTIVATEFORTHREEWEEKSPATHOGENISOLATION,CORTICALTISSUESBETWEENTHESYMPTOMLESSANDAPPARENTLYINFECTEDWERECUTFROMTHETENTWIGSSELECTEDRANDOMLYANDTRANSFERREDTOPDAWHENSHOWEDBROWNNECROTICSPOTONTHEINOCULATEDENDOFTWIGSDISEASEDTWIGSISCHARACTERISTICEDBYTHEWIDTHOFBROWNNECROTICSPOTLONGERTHAN5MMTHISASSAYWASREPEATEDTHREETIMESATDIFFERENTTIMESEGMENTTHERMOSTATSWEREDISTRIBUTEDRANDOMLYEVERYREPEATEDTIME接种后72小时，将全部接种枝条取出，接种端在75的酒精中浸泡10秒，以杀死表面未能侵染的病菌，然后转入温度30C，相对湿度为75的恒温箱中，再培养3周。当枝条皮层自接种端出现褐色坏死斑后，随机选取10个枝条，切取病健交界处的皮层转入PDA中，分离轮纹病菌。当褐色坏死斑的宽度长于5MM时，记为枝条发病。全部试验在不同时间重复3次，每次重复恒温箱随机分配。14温度对腐烂病菌在木质部内扩展的影响14EFFECTOFTEMPERATUREONEXTENDINGOFVALSAMALIINXYLEMTHEASSAYWASSETSEVENKINDSOFTEMPERATUREWHICHWERE5C、10C、15C、20C、25C、30CAND35CSIXTWIGSWEREINOCULATEDINEVERYKINDOFTEMPERATURETHETEMPERATUREWASSET12HOURSBEFOREINOCULATION试验设置7个处理温度5C、10C、15C、20C、25C、30C和35C，每个温度接种6个枝条。温度由恒温箱控制，试验前12小时开机并设定相应温度。ONEYEARHEALTHYTWIGSWERECUTFROMFIVEYEARFUJIAPPLETREESBEFOREAPPLEBUDINMARCHBYDISINFECTEDFRUITSHEARSTWIGSWERECUTINTO20CMLONGSEGMENTSANDKEPTPRUNING