| 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. |
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