【作者】 李金萍；

【导师】 李宝聚；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2013， 博士

【摘要】 十字花科蔬菜根肿病是由芸薹根肿菌（Plasmodiophora brassicae Woron）侵染引起的一种重要土传病害。近年来，十字花科蔬菜根肿病在我国云南、四川、湖北等主栽区大面积暴发流行，造成巨大的经济损失。本论文以十字花科蔬菜根肿病菌为研究对象，研究根肿病菌检测技术及畜禽粪便在根肿病菌传播过程中的作用。主要研究结果如下：1.创建了Hoechest33342-PI荧光双染检测新技术，检测不同介质中根肿菌休眠孢子活力。休眠孢子染色特征为，有活力的孢子发蓝色荧光，无活力孢子发红色荧光。最佳染色条件为，Hoechest33342的工作浓度为5μg/mL，染色的最佳时间为在37℃下孵育10min；PI的工作浓度为5μg/mL，染色时间为在4℃条件下染色15min。该技术适用于检测物理、射线及化学药剂作用引起的根肿菌休眠孢子失活，也可检测多种植物病原真菌活力，包括镰刀菌属(Fusarium)、葡萄孢属(Botrytis)、壳球孢属(Macrophomina)、疫霉属(Phytophthora)等17个属植物病原真菌。2.建立了十字花科蔬菜根肿病菌快速、高灵敏性检测技术。建立了基于根肿rDNA-ITS区序列的实时荧光定量PCR检测技术，最低检测限点为70个拷贝数/反应，相当于1000个休眠孢子·g-1土壤，其灵敏性比常规PCR至少高出2～3个数量级。采用蔗糖密度梯度离心法对样本中根肿菌休眠孢子进行富集，进一步提高了荧光定量PCR检测的灵敏性，检测限点为10个休眠孢子·g-1土壤，灵敏性较单独荧光定量PCR使用时提高了100倍。3.证明了畜禽粪便是十字花科蔬菜根肿病菌传播途径之一。采用荧光定量PCR技术检测带菌鸡粪及猪粪，平均带菌量分别为6.42×106个休眠孢子.g-1鸡粪和5.62×106个休眠孢子.g-1猪粪；Hoechst33342-PI复染法检测带菌鸡粪及猪粪中休眠孢子活力，有活力孢子比例分别为62.20%和68.89%；接种感病寄主植物后病情指数分别为47.22和52.78，三者结合证明了禽粪便可传播十字花科蔬菜根肿病菌。4.初步明确了畜禽消化道环境对根肿菌休眠孢子活力的影响。体外模拟畜禽消化道环境，评价高温（41℃和37℃）、高胆盐浓度（0.3%禽畜胆盐）、强酸环境（pH值2.0，3.0）处理后根肿菌休眠孢子存活率。根肿菌休眠孢子在鸡的消化道高温、高胆盐浓度及强酸作用后有活力孢子比例为53.24%，猪消化道环境作用后有活力休眠孢子比例为57.86%；接种感病寄主植物后病情指数分别为48.34和52.78，根肿菌休眠孢子对消化道的高温、高胆盐浓度及强酸环境具有很强的耐受性。5.采用高温堆肥技术可以杀灭带菌猪粪中的根肿菌，实现根肿菌无害化处理。整个堆肥过程中共进行2次发酵，温度均维持在55～68℃之间，持续时间均为10d左右，水分含量在55%左右，整个堆肥过程中堆体温度和水分含量有利于杀死根肿菌休眠孢子，在堆肥进行40d时可以完全杀死猪粪中的根肿菌休眠孢子，无活力孢子比例为100%，接种感病寄主植物不发病。发酵结束时堆肥pH值为8.32，呈微碱性适于农作物生长；毒素敏感植物种子的发芽率超过90%，完全符合植物生长的要求，对植物生长无抑制作用。本研究创建了Hoechest33342-PI荧光双染检测技术，用于检测不同介质中根肿菌休眠孢子活力，首次将荧光双染技术应用到植物病原菌活力检测领域，可为植物病原菌活力、致病力检测及杀菌剂活性筛选提供了新的技术手段；建立了十字花科蔬菜根肿病菌快速高灵敏性检测技术。该技术为进一步研究根肿病的流行规律及早期诊断和监测提供了理论依据和有效的技术手段。同时，通过体外模拟技术初步明确了畜禽粪便传播根肿病菌的机制，证明了畜禽粪便是十字花科蔬菜根肿病的传播途径之一。通过高温堆肥技术实现了猪粪中根肿菌无害化处理，该研究结果为根肿病防治策略的制定提供理论依据及技术支持。更多还原

【Abstract】 Clubroot of Crucifers is a soil-borne disease caused by P. brassicae. The outbreaks have been widespread in the main growing areas in China, such as Yunnan, Sichuan and Hubei. The widespread outbreaks have caused huge economic losses. P. brassicae rwas taken as the researching object, which included the detection technology of P. brassicae and the role of livestock and poultry manure in the transmission of P. brassicae.. The main results are as follows:1. A fluorescence microscopic method was developed to directly assess the pathogenic activity of P. brassicae resting spores. Resting spores were stained with a mixture solution of two fluorescences (Hoechest33342and propidium iodide), and were observed by fluorescence microscopy (phase contrast, UV filter set, oil immersion). The fluorescent staining characteristic of the spores showed that the live spores exhibited intense blue fluorescence, while the dead spores displayed red fluorescence. The optimum staining conditions were that, the work concentration of Hoechest33342was5μg/ml, incubated for10min at37℃; PI was5μg/mL, incubate for15min at4℃. This technology was also used to detect the gross changes in the pathogenic activity of the spores which were induced by heat, UV light and chemical treatments. Besides for the detection of P. brassicae, this method can also be applied to detect a variety of other plant pathogens, including17genus, such as Fusarium, Botrytis, Macrophomina, Phytophthora, and etc.2. A rapid and highly sensitive technology used for the direct detection and quantification of P. brassicae was developed. In addition, the SYBR Green I Real-Time PCR assay with P. brassicae ribosomal DNA and internal transcribed spacer (ITS) was also an important result. The detection limit was0.0234fg.μL-1when tested with P.brassicae genomic DNA. The detection limit in soil samples corresponded to1000resting spores-g-1soil, was higher than that of the conventional PCR by at least2-3orders of magnitude. The variation coefficient of Ct values of diluted standard DNA was less than4%, which indicated a good reproducibility. Using the sucrose density gradient centrifugation to gather the resting spores of P. brassicae in soil samples improved the detecting sensitivity of fluorescence quantitative PCR. Thus, the detection limit can reach to10resting spores-g-1soil after gathering, and the sensitivity of the assay was100fold higher than that of the real-time polymerase chain reaction (PCR).3. Livestock and poultry dung are the main transmission routes of P. brassicae. Using the fluorescence microscopic method, quantitative PCR, DNA of P. brassicae, the causal agent of clubroot, was detected and quantified in livestock and poultry dung and bioassay. Quantifiable levels of infested livestock and poultry dung were6.42×106resting spores-g-1poultry dung and5.62×106resting spores-g-1livestock dung, respectively; the vast majority of resting spores on these samples were viable, as determined by Hoechst33342-PI fluorescence dye staining. The percentage of viable P. brassicae resting spores was62.20%and68.89%in chicken and pig manure, respectively. The bioassays of infested chicken and pig manure can cause consistent clubroot symptoms, disease index were47.22and52.78. These results suggested that infested chicken and pig manure might cause dissemination of this pathogen. 4. The mechanism of livestock and poultry dung for dissemination of P. brassicae was preliminarily explained. Survival rates of P. brassicae resting spores were evaluated in vitro through simulating the high temperature (41℃and37℃), high concentration of bile salts (0.3%livestock bile salts) and acidic conditions (pH2.0,3.0) of chicken and pig gastrointestinal tract environments. The survival rate was53.24%in the simulative digestive tract of chicken with high temperature, high concentrations of bile salt and acid. The survival rate was57.86%in the simulative digestive tract of pig with high temperature, high concentrations of bile salt and acid. The susceptible plants inoculated with the treated P. brassicae resting spores in the simulative digestive tracks of chicken and pig showed obvious clubroot symptoms, and disease indexes were48.34and52.78, respectively. The results indicated that resting spores were strongly tolerant to high temperature, high concentrations of bile salt and also in acidic condition.5. P. brassicae in pig manure can be eradicated through high temperature composting technology. The whole composting process included two fermentations. For each fermentation, the pig manure was incubated at60℃-70℃for10days with high moisture levels (55%w/w moisture content). Both temperature and moisture content were critical for eradication of P. brassicae spores. The spores were totally extracted from composted clubroot affected residues after incubating for40days, and the percentage of dead resting spores was100%. After inoculated with these spores, the host didn’t show any clubroot symptoms in greenhouse plant bioassay. After two fermentations, the pH value of composting materials was8.32, slight alkaline which was suitable for the plants. The germination rate of Chinese cabbage seed can over90%, meeting the requirements of the plant growth and had no inhibitory effect on plant growth.A new technology of Hoechest33342-PI double dye fluorescence detection was developed to detect the vitality of resting spores in different media. Moreover, a fluorescence microscopy method was developed to directly assess the pathogenic activity of P. brassicae resting spores. It is the first time that the technology has been adopted in detecting the activity of plant pathogens; establishing the detection of P. brassicae with fast and high sensitivity technology successfully is also an important result. The application of these procedures will contribute to a better understanding of the epidemiology of clubroot and help us to control this disease.At the same time, by means of the in vitro simulation technology, the transmission mechanism of P. brassicae. depends on livestock and poultry manure, was previously known. We have also proved that livestock and poultry dung is a significant way of the transmission routes of P. brassicae. Moreover, through high temperature compost technology, the P. brassicae in the livestock and poultry dung can completely be killed. In summary, the results can provide the basic of theory and technical support to the establishment of the control strategies to Clubroot of Crucifers.更多还原