【作者】 张林青；

【导师】 程智慧；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2008， 博士

【摘要】 大蒜白腐病是大蒜毁灭性病害，主要危害大蒜的根、鳞茎和叶，苗期直接造成田间缺株死苗，严重地块绝收，造成很大损失，直接影响大蒜高产稳产。目前，化学药物防治白腐病效果不明显，大量用药还会引起产品农药残留增加，降低食用安全性。要从根本上解决白腐病，必须选育抗病品种。但是，大蒜是花器退化的有性不育植物，品种老化和退化严重，又缺乏有效的育种途径，通过离体培养筛选抗病突变体可能是其快捷而有效的主要育种途径之一。本研究以主栽品种改良蒜和汉中红皮蒜为试材，在分离培养大蒜白腐病病菌的基础上，优化毒素培养体系、提取毒素并明确其主要组分，以白腐病病菌粗毒素作为抗性筛选的选择压，对大蒜愈伤组织抗病突变体进行筛选和分析，建立大蒜抗白腐病体细胞无性系突变体筛选体系，获得大蒜抗白腐病再生植株，并对再生植株的生理生化特性进行分析，对开辟以大蒜为代表的无性繁殖作物的育种途径，具有重要意义。由生产田间发病的大蒜植株上分离鉴定获得白腐小核菌（Sclerotium cepivorumBerk．），研究明确了病原菌的生物学特性。结果表明，菌丝的生长适宜条件为18．0℃、pH 4．0～5．0、持续光照培养。12 h黑暗12 h光照、15．0～18．0℃、pH 4．0～5．0条件下产菌核量最多。12．0～24．0℃、pH 4．0～9．0、空气湿度100％以上条件下适合菌核萌发。菌丝和菌核的致死温度分别为45．0℃和50．0℃。白腐病菌侵染可激活大蒜体内相关防御酶系，使PAL、SOD、POD和PPO 4种酶活性明显提高，抗病品种汉中红皮蒜的酶活性升高幅度大于感病品种改良蒜；而CAT酶活性却下降，抗病品种汉中红皮蒜的酶活性下降幅度低于感病品种改良蒜。防御酶活性高低与品种的抗性关系密切。以大蒜幼苗根系生长抑制率为指标，采用生物检测法筛选了大蒜白腐病菌产毒培养条件。结果表明，大蒜白腐病病菌的最佳产毒培养基为Fries液体培养基，培养温度为18．0℃，培养基pH值为5．0，在黑暗条件下连续振荡培养6 d时产生的粗毒素的毒性最强。用白腐病菌毒素处理汉中红皮和改良蒜幼苗后，2个品种叶片中SOD、POD活性均升高，而CAT活性降低；抗病品种的SOD、POD和CAT活性均高于感病品种，且SOD、POD活性峰值出现早，并以POD对毒素胁迫最敏感。2个品种在白腐病菌毒素处理后的O₂^-含量始终高于同期对照，而感病品种O₂^-含量在毒素处理24 h后均高于同期抗病品种。MDA含量变化趋势与O₂^-的变化基本趋势相似。因此，大蒜叶片的活性氧含量和保护酶活性与其抗病性密切相关。采用GC-MS分析表明，白腐菌粗毒素成分主要含有有机酸类（organic acids）、醇类（alcohols）、酯类（ester）、甾体类（Steroids）、杂环类化合物（heterocyclic compounds）。其中，月桂酸和邻苯二甲酸是致病毒素的有效成分，月桂酸毒性强于邻苯二甲酸。以2个大蒜品种茎盘外植体的愈伤组织为材料，大蒜白腐病病原菌粗毒素为筛选压力进行抗白腐病细胞无性系变异筛选。结果表明，大蒜白腐病病原菌粗毒素对大蒜茎盘愈伤组织诱导、生长和不定芽的分化具有明显的抑制作用，随着粗毒素浓度的升高，抑制作用增强；在粗毒素浓度50％条件下，筛选、鉴定获得了抗白腐病细胞无性系变异，并成功再生植株，获得小鳞茎。对离体筛选的大蒜抗白腐病突变体（突变型）和未经筛选的原品种（原始型）的愈伤组织经白腐病菌粗毒素处理后72 h内的苯丙氨酸解氨酶（PAL）、多酚氧化酶（PPO）活性以及木质素和富含羟脯氨酸糖蛋白（HRGP）含量的变化分析表明，毒素处理后2种愈伤组织的PAL和PPO活性均升高；突变型的PAL和PPO活性均明显高于原始型，且PAL、PPO活性峰值出现早，并以PAL对毒素胁迫最敏感。2种愈伤组织毒素处理后木质素始终高于同期对照，而突变型的木质素含量均高于同期原始型。HRGP含量的变化趋势与木质素的变化趋势基本相似。因此认为，HRGP的积累和细胞壁的木质化与大蒜的抗病性有关，离体筛选的大蒜抗白腐病突变体的抗病机制与一般抗性品种类似。更多还原

【Abstract】 Garlic white rot,a damaging disease of garlic,mainly harms the roots,bulbs,and leavesof garlic.It may cause death of young plants and influence garlic production in fields,withgreat subsequent losses or even no harvest during severe incidence.At present,chemicalcontrol is the major method to control white rot but it is not so effective,and overuse offungicides may also result in chemical residuals in garlic products which may be a health risk.To solve this problem,the usual way is to develop a resistant variety.However,culturedgarlic is sterile,and it is very difficult or even impossible to achieve sexual hybridization,which is necessary for traditional plant breeding.The selection of somatic mutants is one ofthe important alternative breeding strategy for garlic.The biological characteristics of white rot pathogen,culture conditions for theproduction of crude toxin,components of curde toxin,selection of cell mutant lines resistantto white rot,physiological and biochemical characterization of garlic somatic mutants weresystematically studieed in this thesis.It is important for opening up a new breeding waytaking garlic as the representative of the asexual reproduction crops.White rot,caused by Sclerotium cepivorum Berk.is a ruined disease of garlic in thecourse of the production.The research on biological characteristics of Sclerotium cepivorumBerk.may provide a theoretical basis for the prevention and control of the disease.The effectsof temperature,pH,light,atmospheric relative humidity（RH）on mycelium growth andsclerotia germination were studied in this experiment.The results showed that mycelium grewwell when cultured at 18.0℃and pH 4.0～5.0.Light was beneficial to mycelium growth.Conditions such as 15.0～18.0℃,pH 4.0～5.0,12 hour light/12 hour dark were beneficial tosclerotia production.The sclerotia germinated well when cultured at 12.0～24.0℃,pH 4.0～9.0,and under RH 100%.The lethal temperature ofmycelial growth and sclerotia germinationwere 45.0℃and 50.0℃,respectively.The changes of defensive enzymes after inoculation by Sclerotium cepivorum Berk.intwo different resistant cultivars were studied.After obvious symptom appearing,the changesof defensive enzymes were determined in health and sick plants.The inoculation of pathologycould activated the activities of defensive enzymes in garlic.The results suggested thatactivities of PAL,SOD,PPO and POD were increased after inoculating regardless of in resistant cultivars or susceptible cultivars,and the enzyme activities in resistant cultivarHanzhong Red Skin were higher than those in susceptible cultivar Gailiang.However,theactivities of CAT was lower after inoculating regardless of in resistant cultivars or susceptiblecultivars,and the activities of CAT in susceptible cultivar dropped more than that in resistantcultivar.Resistance of the different garlic cultivars was significantly correlated with thedefensive enzymes.The activity of defensive enzymes is thought closely related to the diseaseresistance of cultivars.The relation between crude toxin production and culture conditions was examined bytesting the toxicity of crude toxin with garlic root growth inhibition rate.The results showedthat the optimum conditions for toxin production were Fries liquid medium at pH 5 and at18℃,with successive dark periods and shaking during culturing.Production of crude toxinreached the highest level on the 6th day of culturing.The dynamic changes of defensive enzyme activities,content of superoxide radical（O₂^-）and malonaldehyde（MDA）in leaf of cultivars Hanzhong Red Skin and Gailiang treated bycrude toxin of Sclerotium cepivorum Berk.were investigated at seedling stage.The resultssuggested that the activities of superoxide dismutase（SOD）and peroxidase（POD）,werestimulated and increased treating by crude toxin,and the activities of catalase（CAT）werelower after treating.The enzyme activities in resistant cultivar Hanzhong Red Skin werehigher than that in susceptible cultivar Gailiang.The peaks of the activities of SOD and PODin the resistant cultivar appeared earlier than that of the susceptible cultivar and POD wasmost sensitive to the toxin coercion.The results also showed that the content of O₂^-inresistant cultivar was lower than that in susceptible cultivar,after treating for 24 hours.Thedynamic changes of the content of MDA were basically in conformity with that of O₂^-.Theseresults indicated that the content of reactive oxygen species（ROS）and activities of protectionenzymes in leaf had a close relationship with disease resistance in garlic.GC-MS was used to analyse the components of curde toxin produced by Sclerotiumcepivorum Berk.The results suggested that the major components of curde toxin were organicacids,alcohols,ester,steroids and heterocyclic compounds.Dodecanoic acid and dibutylphthalate were active components of pathogenic toxin;toxicity of dodecanoic acid wasstronger than dibutyl phthalate.The somatic variant clones of disease resistance were screened by in vitro culture ofgarlic stem base explants in the medium with pathogen crude toxin added.The resultsshowed that the crude toxin had clearly negative effect on callus induction and growth.It alsoextremely inhibited differentiation of adventitious buds.The inhibitory effects becameintensified with the toxin concentration increased.Some somaclonal variation lines and plantlets resistant to Sclerotium cepivorum Berk.were obtained at crude toxin concentrationof 50%.Changes of activities of phenylalanine ammonia-lyase（PAL）and polyphenol oxidase（PPO）and contents of lignin and hydroxyproline-rich glycoprotein（HRGP）in the selectedmutant and the original callus were investigated 72 h after crude toxin treatment.The resultsshowed that the activities of PAL and PPO were stimulated and increased by crude toxintreatment.The enzyme activities in mutant callus were higher than that of original callus.Thepeaks of the activities of PAL and PPO in mutant callus appeared earlier than that in theoriginal callus and PAL was most sensitive to the toxin coercion.The results also showed thatthe content of lignin in original callus was lower than that in mutant callus.The dynamicchanges of the content of HRGP were basically in conformity with that of lignin.Theseresults indicated that the accumulation of HRGP and lignin deposition in cell wall had a closerelationship with disease resistance in garlic.These results suggested that in vitro screenedgarlic somatic mutants resistant to white rot had the similar resistant mechanism as thegeneral resistant varieties.更多还原