读书报告12_磷酸对天竺葵青枯病的控制.ppt

controlofbacterialwiltofgeraniumwithphosphorousacidd j norman磷酸对天竺葵青枯病的控制 主要内容 一 前言二 材料与方法三 结果与讨论 一 前言 onceitbecomesestablishedinasusceptiblecrop southernbacterialwilt causedbyralstoniasolanacearumisveryhardtoeradicate therearetwoprimaryavenuesbywhichr solanacearummovesandgainsaccesstocrops oneisviawater andtheotheristhroughinfectedpropagationmaterials 由青枯菌引起的南方青枯病一旦侵染敏感作物 就很难根除 青枯菌侵染作物的两种途径是 通过水流侵染 通过被侵染物质传播 tocombatbacterialwilt resistantcultivarshavebeenrecommendedinareasoftheworldwherethepathogenisendemic insusceptiblegreenhousegrowncrops onlystrictsanitationhasbeensuccessfulinprohibitingplantinfestation 为了抵抗地方性青枯病 世界上很多地方都推荐种植抗性作物 种植在温室的敏感作物只有控制环境卫生才能抑制其侵染 geraniumsaresusceptibletoraces1and3ofr solanacearum mostgeraniumsproducedintheworldarevegetativelypropagatedinguatemala costarica columbia china andkenya inalltheselocations endemicpopulationsofr solanacearumexist therearenoknowntreatmentsthatareeffectiveinprotectinggeraniumplants 天竺葵对于青枯菌属1和3是敏感的 天竺葵广泛种植在世界各地如危地马拉 哥斯达黎加 哥伦比亚 中国和肯尼亚 但这些地方都存在地方性青枯病 目前还没有有效的方法保护天竺葵作物 thus theobjectiveofthisresearchwastodetermineifgeraniumplantscouldbeprotectedfrominfectionwithapplicationsofselectedbactericidesandchemicals 因此本实验的目的就是对天竺葵作物施以筛选过的细菌和化学药品来验证是否能保护作物免受侵染 二 材料与方法1screeningofproducts arace1 biovar1 strain r1b1 isolatedinflorida p673 frompothos epipremnumaureum linden andre blunt wasusedintheinitialscreeningofprospectiveproducts theisolatesfrompothosareofcentralamericanoriginandpossessabroaderhostrangethanstrainsendemicinflorida 在弗罗里达的黄金葛里分离出来的r1b1 p673 被用于开始预期产品的筛选 它是在在美国中部分离出来的菌属比弗罗里达的地方性菌属更原始 且具有更广泛的寄主 promisingproductswerelatertestedinenvironmentalchamberswitharace3 biovar2 strain r3b2 isolatedfromgeranium uw551 providedbyc allen universityofwis consin 更多的产品将会用r3b2 uw551 在环境检测箱中进行检测 r3b2有威斯康辛大学的艾伦提供 材料包括 三异丙基乙黄酰 苯并噻二唑 硫酸铜 氢氧化铜 铜锡 铜盐 双氧水 糖醛 苯烷基二甲基氯化铵 奥索利酸 starner 枯草芽孢杆菌 磷酸钾盐 theproductsweremixedinwateratthehighestlabeledratesrecommendedbythemanufacturersforplantproduction productsarenotlabeledforcontrolofralstonia theseproductswereappliedasadrenchthroughpottingmediumat50mlper9 cmpot intheinitialscreening productswereappliedevery7daysforatotaloffourapplications plantswereinoculatedwithr solanacearum3daysafterthefirstapplication productswerenotretestedunlessplantswereprotectedfrominfection 这些物质以浸液浇到盆栽介质中 每盆50ml 浇到9cm深处 在开始的筛选中每7天施一次 总共4次 第一次施用之后3天开始接种青枯菌 这些物质不再测定除非作物被侵染 productswerescreenedwithzonalgeraniumplants rootedgeraniumcuttingsweretransplantedinto9 cm diameterplasticpotscontaining85gofvergocontainermixa 60 canadianpeat 20 vermiculite and20 perlitebyvolume ph6 5 andfertilizedwith1 5gofosmocote15 9 12withmicronutrientsperpot 产品的筛选用带状天竺葵 其根要移栽到直径为9cm的塑料盆中 盆中有85g介质 60 碳泥 20 蛭石 and20 珍珠岩 ph6 5 和1 5g奥绿肥 15 9 12 plantsweregrownforaminimumof4weeksbeforetreatments experimentswereconductedingreenhouseswithtemperaturesmaintainedbetween18and32 candlightlevelsbetween285and380 mol m 2 s 1 plantswerearrangedinacompletelyrandomizeddesignwith10plantspertreatment 在处理之前 作物最少生长4周 该实验在温室中进行 温度 18到32 c 光照 285到380 mol m 2 s 1 实验完全随机区组 每个处理10株 twoadditionaltreatmentswereincludedineachtest anoninoculatedcontrol saline 8 5gnacl literappliedwithoutr solanacearum andadiseasecontrol noproductapplied inoculatedwithpathogen 每个实验增加两个处理 不接种控制 含有盐分 nacl 无青枯菌 病害控制 不施其他物质 但接种病菌 forinoculumproduction r solanacearumstrainsweregrownontriphenyltetrazoliumchloride tzc medium 11 for48h andcellswereharvestedandspectrophotometricallyadjusted a600 insalineto5 108cfu ml 接种青枯菌株的要在氯化三苯基四氮唑 tzc 介质中生长48小时 达到5 108cfu ml 的时候收获菌株 wiltsymptomswererecordedastheyoccurred bacteriawerereisolated2weeksafterlasttreatment 6weeksafterinoculation fromplantswithoutwiltsymptoms across sectionofstem1cminlengthwasremovedapproximately0 5cmabovethesoillineofeachgeraniumplant 萎蔫症状一出现就要记录下来 最后一次处理之后两周 细菌就要从没有萎蔫症状的植物中分离 取1cm茎 从离地面0 5cm开始取 通过处理把悬浮液加到tzc介质中培养然后进行检测 2furthertestingofpromisingproducts geraniumplantswerefoundnottobesystemicallyinfectedbyr solanacearumwhentreatedwitheitherk phite a i 53 mono anddi potassiumsaltsofphosphorousacid orstarner a i 20 oxolinicacid attheinitialtestrates 在初始的检测中 当施以53 的磷酸一钾或二钾或starner 天竺葵不能被青枯菌系统性侵染 todetermineeffectiveapplicationratesandintervalsfortheseproducts fourratesweretested vol vol 0 25 0 5 0 75 1 0 atthreeapplicationregimes oneapplication twoapplicationsat14 dayinterval andfourapplicationsat7 dayinterval bactericideswereapplied3daysbeforepathogeninoculationasintheinitialproductscreen 为了测定这些物质的有效性和周期 用4种比率 0 25 0 5 0 75 1 0 测定 用三种施用方式 一次性施用 两次施用间隔为14天 四次施用间隔为7天 杀菌剂在接种病原菌之前三天施用 eachtreatmentagaincontained10replicates usingther1b1strain fourtestswereconductedwithtwotestsateachinoculumconcentration 5 108cfu ml 3 108cfu gramsoil and1 107cfu ml 6 106cfu gramsoil 每个处理10个重复 用r1b1菌株 进行了两个接种测试 5 108cfu ml 3 108cfu gramsoil 1 107cfu ml 6 106cfu gramsoil twoadditionalcontrols fiveplantseach wereaddedatthelowerinoculumratetomonitorconcentrationsofr solanacearumwithinthepottingmedium thesecontrolsconsistedof i potscontainingpottingmediumwithoutageraniumplant yetinoculatedwithr solanacearum and ii potscontainingpottingmediumwithoutplantorr solanacearuminoculation additionalcontrolswerewateredlikeallothertreatments 在接种较低的盆栽介质中增加了两个处理 每个处理5株 只有盆栽介质 盆栽介质 天竺葵 盆栽介质 接种 与其它处理一样浇水 sixweeksafterfirstchemicalapplication acorkborerwasusedtoretrievea1 gsampleofpottingmediumfromthefirstfivereplicationsofalltreatments adilutionseriesofthesoilsamplewasdoneinsdwandreplicaplatedontomodifiedsemiselectivemedium typicalr solanacearumcolonieswerecountedafter48hgrowthat28 c bacteriawerereisolatedfromallgeraniumplants6weeksafterinoculation aspreviouslydescribed coloniesfromplateswithtypicalr solanacearumgrowthpatternweresuspendedinsaline 8 5g liternacl andinjectedintoparenchymatoustissueoftobaccoforhypersensitiveresponse hr followingprotocolsoflozanoandsequeira 12 在28 c下培养48小时后计算青枯菌群 接种6周后从天竺葵中分离细菌 如前面所述得到的悬浮液注入马铃薯的薄壁组织中来测其敏感性 分离茎和土壤 把马铃薯种到每个盆子中来验证青枯菌是否足够病害发展 马铃薯对于r1b1和r3b2菌属是十分敏感的 最少4周后就会出现症状 与前面相同 出现症状就进行分离 ther3b2strainwastestedinenvironmentalchamberssetona12 hday nightcycle 19 cnight 24 cday 310 mol m 2 s 1 twotestswereconductedataninoculumconcentrationof5 108cfu ml 3 108cfu gramsoil allotherexperimentalparametersandcontrolswerethesameaspreviouslydescribed r3b2在环境检测箱内放12h后测定 两个实验的接种浓度为5 108cfu ml其它的实验参数和控制条件都与前面相同 antibacterialefficacyofcompoundscontainingphosphorusmanyproductscontainingparecurrentlysoldintheornamentalplantindustryasfertilizersorfungicides commoningredientsincludep2o5 h3po3 andh3po4withmono and ordi potassium wetestedtheefficacyforcontrollingbacterialwiltbyp2o5 h3po3 h3po4 andkcl 很多含磷产品在工厂都以肥料或菌剂来买 一般有p2o5 h3po3 kh2po4 k2hpo4 我们用p2o5 h3po3 h3po4 和kcl来测它们对青枯菌的控制效率 reagentgradekclandthethreepchemicalsbufferedwithcaco3wereusedinthefirsttest andthepchemicalsonlybufferedwithkohwereusedinthesec ondtest weusedfourconcentrationsofp 0 032 0 063 0 095 and0 127 foreachofthethreechemicalsinthetwotests andthefirstfourconcentrationsofk 0 054 0 108 0 162 and0 216 weremadefromkcl 第一个实验中 用caco3来缓冲kcl和三种含磷化合物 第二个实验中用koh来缓冲含磷化合物 四种p的浓度为0 032 0 063 0 095 0 127 两个处理的三种含磷物质浓度一样 4种k的浓度为0 054 0 108 0 162 0 216 asintheinitialproduct screeningsolutionswereappliedona7 dayintervalwithfourapplications again plants 10perreplication wereinoculatedwithstrainr1b13daysafterthefirstcompoundapplication twoweeksafterlastapplication allplantsweresampledforr solanacearumaspreviouslydescribed 在起始筛选中 筛选溶液将会以7天的间隔施用在四个处理中 作物接种r1b1菌 三天后施用第一次 最后一次施用之后两周收集所有植株来详细描述青枯菌 wemeasuredtheinvitroeffectofphosphorousacidonthegrowthofbothr1b1andr3b2strains flaskscontaining40mlofthefollowingsolutionswereused i sdw ii sdw pat0 127 ash3po3 iii sdw k phiteat1 iv nutrientbroth difco containing5gsucrose nb v nb pat0 127 ash3po3 and vi nb k phiteat1 我们在试管中进行了磷酸对r1b1和r3b2的控制实验 分6组 分别为 灭菌水 灭菌水 0 127 h3po3 灭菌水 1 磷酸钾 营养液 营养液 0 127 h3po3 营养液 1 磷酸钾concentrationsofviablecellsineachflaskwereenumeratedon0 1 7 14 21 and28days countswerediscontinuedwhenconcentrationsofcellsreached109cfu ml 在适当条件下进行培养 在0 1 7 14 21 和28天计算其存活的细菌数量 当其浓度达到109cfu ml 不在计算 safetyofk phitetocrop todetermineifphytotoxicityoccurredwiththek phiteproduct anexperimentwassetupaspreviouslydescribedusingfourconcentrations vol vol 0 50 0 75 1 and2 with10plantspertreatment dryweightsofbothrootsandfoliageofallplantsweredeterminedattheendofthetest inaddition wemeasuredmediumphandsolublesaltsforfiveplantsofeachtreatmentandperformedacompletefoliaranalysis 为了确定k phite对作物是否安全 我们进行了如前面所述4个浓度 0 50 0 75 1 和2 每个处理10株 实验最后测所有植株的根系和叶片 每个处理测5株的介质浓度和盐溶液 对叶片进行完整的分析 results screeningofproducts furthertestingofpromisingproducts efficacyofphosphorouscompounds safetyofk phitetocrop screeningofproducts intheinitialtesting nearlyalltheproductssloweddiseaseprogress however theydidnotprotecttheplantsfrominfection exceptforbenzothiadiazole actigard oxolinicacid starner andpotassiumsaltsofphosphorousacid k phite datanotshown furthertestingofbenzothiadia zolewasdiscontinuedduetoleafabscission thisabscissionoccurredevenatratesaslowas5 l liter 在开始的测试中 所有的物质都能减慢病害发生的过程 但不能保护作物免受侵染 除了苯并噻二唑 奥利索酸和磷酸钾盐 而苯并噻二唑即便在很低的浓度下也会损害叶片 所以不再继续实验 furthertestingofpromisingproducts lowratesandintervalsofstarnerwereineffectiveinprotectingplantsfrominfection highratesabove0 5 ona7 dayintervalprovidedprotectionforthemajorityoftheplants however atinoculationrates of3 108cfu gramsoiland6 106cfu gramsoil systemicinfectionswereoccasionallyobservedinplantstreatedwith0 75and1 starner duetothehighratesandintervalsrequiredfordiseaseprotection testingofstarnerwasdiscontinued 低比率和间隔的starner没有保护作用 高于0 5 虽有效 但作物还是会出现偶然的系统性侵染 所以对starner不再进行实验 k phitewasfairlyeffectiveinprotectinggeraniumplantsfrominfectionat3 108cfu gramsoil fig 1 andveryeffectiveinprotectingplantsat6 106cfu gramsoil 在接种浓度为3 108cfu gram时 磷酸钾的保护作用是有效的 在6 106cfu gram时保护效果非常明显 具体情况如图一所示 populationsofr solanacearumwerenotdetectableinthesoilafterpottingmediumwasdrenchedwithk phiteforeithertwoorfourapplications fig 2 ther1b1strainwasmorepersistentthanther3b2strainwhilether3b2strainwasundetectableatthattime 在两次或四次施用磷酸钾的处理中检测部到青枯菌 r1b1比r3b2更持久 r1b1在接种后6周还可以检测到青枯菌但r3b2 fig 1 effectsofk phiteapplicationoncontrolofralstoniasolanacearuminfection efficacyofphosphorouscompounds k phiteeffectivelyprotectedgeraniumplantsfrominfectionwithther1b1strainatallfourappliedrates fig 3 sinceitisaproductofpotassiumsaltsofphosphorousacid threepchemicals p2o5 h3po3 andh3po4 weretested 如图3所示 磷酸钾能的4个处理都能保护作物免受侵染 而其它含磷物质却不可以 fig 3 relativeeffectsofk phiteandphosphoruscompoundsincontrolofralstoniasolanacearum strainp673 r1b1 thelong terminvitrogrowthcurvesweresimilarforbothbacterialstrainstested infigure4 resultsaredisplayedforuw551 phosphorousacideitherinreagentgradeorproductforminhibitedcellreplicationinnbandsdw fig 4 两种细菌的长期试管生长曲线都相似 r3b2菌属结果如图所示 safetyofk phitetocrop therewasasignificant p 0 5 increaseinthegeraniumshootdryweightof27 withthe1 k phitetreatmentrelativetothewatercontrol allothershootdryweightswerenotsignificantlydifferentfromthewatercontrol ironlevelsdecreasedfrom122 5ppmto45 7ppm r20 58 leaveswerelessgreenonplantstreatedwith1 k phitethanonplantstreatedwithwaterinordertomaintainhealthygeraniumplants thek phiteproductshouldnotbedrenchedatratesabove1 onaprolongedbasis 与空白对照相比 用1 磷酸钾处理的天竺葵地上部干重明显增加了27 别的处理与空白对照无显著性差异 但磷酸钾高于1 时 叶片中铁的含量从122 5ppm减低到45 7ppm 叶片发黄 所以磷酸钾的比率不能高于1 fig 4 growthofralstoniasolanacearumuw551inflaskscontaining40mlofthefollowingsolutions steriledistilledwater sdw sdw 0 127 pash3po3 sdw 1 k phite 53 mono anddi potassiumsaltsofphosphorousacid nutrientbrothdifcocontaining5gsucrose nb nb 0 127 pash3po3 and nb 1 k phite discussion themajorityofthegeraniumssoldintheglobalnorth butwherer solanacearumisendemic inordertoexcluder3b2fromthesegeraniumproductionacilities rigoroussanitationmeasuresarebeingimplemented 24 however completeeradicationofbacterialwiltisdifficultgivenanenvironmentconducivetobothhostandpathogen thus inadditionosanitationandresistantcultivars growersneedothercontroloptions 天竺葵的主要产地是北半球 但存在地方性青枯病 为了控制天竺葵作物r3b2菌 对生产设施进行了严格的控制 然而一般很难完全根除 因此 除了控制环境和栽培抗性品种 种植者也需要其他控制 thisstudyshowedthatmostofthetestedproductsslowedtheprogressionofbacterialwiltongeranium butdidnotprotecttheplantsfrominfectionandsubsequentdeath onlyphosphorousacidprotectedplantsfrominfection itappearsthattheprotectionoccursinthesoilandrootmatrix aspathogencellscouldnotbecultu