| Clubroot, which is caused by Plasmodiophora brassicae Woron, is one of worldly diseases and have caused serious economic loss in the cruciferous crops. Given the Plasmodiophora brassicae is soil borne disease and its resting spore survival time is long and the traditional method of prevention and cure was little effect, breeding resistant cruciferous vegetables cultivars was one of the more effective way to control root diseases. Therefore, effective methods are needed to screening disease-resistant varieties, and need to know the host disease resistance mechanism. In this study,on the basis of previous studies and in four kinds of Chinese cabbage as the experimental material,the disease resistance identification method and biochemical mechanism of Chinese cabbage were studied. The research contents including pots experiment,the observation of root-hair infection rates and cortex colonization amounts and RT-qPCR detection of P. brassicae DNA content(PbDNA); Exploring and analyzing the invertase activities,defense enzymes(CAT/SOD/PPO/POD)activities,MDA activity and the contents of soluble protein and the soluble sugar by the infection of P. brassicae.1. The measurement of resistance or susceptibleThe results were as follows: Zaoshuchangjiang5(82.5%, 36.2), Hualiangzao 5(66.7%, 37.5) and CR-Chunmei(68.9%, 36.0) were resistant; CR-Yingxiong(52.5%, 16.5) was susceptible.2. The establishment of the early screening methods disease-resistant varieties(1)Study on root-hair infection and cortex colonization of four kinds of ChineseCabbagesWith the method of solution culture, root-hair infection rates and cortex colonization amounts were investigate. The peak of root-hair infection rates of susceptible Cultivars significantly higher than that of resistant variety; The cortex colonization amounts of susceptible varieties by 16dpi(day post inoculation) were also significantly higher than that of resistant variety; Correlation analysis indicated the maxmum value of R2 among root-hair infection rate at the 8th dpi with diseased rate in pots was 0.911 and the maxmum value of R2 among cortex colonization amounts at the 16 th with diseased rate in pots was 0.980. So we can estimate the resistance or susceptible by root-hair infection and cortex colonization.(2)The study of PbDNA in the rootWith the method of solution culture and qPCR, the peak of PbDNA of Zaoshuchangjiang5, Hualiangzao 5, CR-Chunmei and CR-Yingxiong were respectively at 10th(lgPbDNA=6.28fg)dpi, 12th(lgPbDNA=6.13fg)dpi, 14th(lgPbDNA=6.4fg)dpi and 14th(lgPbDNA=5.67fg) dpi; The Significant difference of PbDNA between three kinds of susceptible varieties and resistant variety at 6th, 10 th and 16 th. Correlation analysis indicated the value of R2 among root-hair infection rate at the 6th dpi with PbDNA at the 6th dpi and among PbDNA at the 6th dpi with diseased rate in pots were respectively 0.840 and 0.877. So we can detect the PbDNA by qPCR and track early development state of P. brassicae within the host root and screen resistant varieties.Diseased rate in pots need lots of space, take a long time, and cannot explore root development in the host root; Microscopic observation and qPCR can track P. brassicae breeding conditions within the host root and don’t need a large space, but Microscopic observation was very trouble, subjective factor was serious, stability was poor and easy to cause a larger error; qPCR was more rapid, convenient operation and small error, so future study can can track P. brassicae breeding conditions within the host root by qPCR and find out the major differences of P. brassicae proliferation from different varieties and provide theoretical basis for the research of resistant mechanism and was expected to pass the technology directly screen resistant varieties.3. Invertase activity analysis by the infection of P. brassicaeIn two kinds of Chinese cabbage as the experimental material, testing the invertase activities using the method of solution culture, pots experiment, and Folin-Phenol. The results showed that: On one hand, the invertase activities of treatment samples were higher than their control samples, except individual time, and there are significant difference during the two stage of 12 dpi and 17 dpi between the treatment samples and their control samples. It was speculated that the change of the invertase activities can significantly influence the process of Clubroot; On the other hand, for Zaoshuchangjiang5, the invertase activities of treatment sample and control sample both enhanced firstly, but for CR-Yingxiong, the invertase activities of treatment sample and control sample gradually reduced, so from the beginning to show resistance to P. brassicae. So the study showed that the changes of invertase activit y was related to host resistance to P. brassicae.4. Study of related biochemical index of Chinese cabbageIn two kinds of Chinese cabbage as the test material, testing the activities change of CAT/SOD/PPO/POD / MDA and the content changes of soluble protein and soluble sugar using the method of solution culture. The results were as follows:(1)The CAT and SOD activity of resistant variety were higher than susceptible variety in the early stage of the test and in the late test period respectively. The CAT and SOD activity were positively correlated with disease resistance, so the CAT and SOD activity can be used as auxiliary means for the resistance identification to Clubroot after inoculation. Because of the PPO and POD activity had no obvious relationship with disease resistance, so the PPO and POD activity can not be used as auxiliary means for the resistance identification to clubroot after inoculation.(2)In the study, the MDA activity peck value of susceptible variety was higher than that of resistant varieity after inoculation, therefore the MDA content can be used as auxiliary means for the resistance identification to clubroot after inoculation. The soluble protein content of susceptible variety went up than that of control sample, but the soluble sugar content of resistant variety went up and down than that of control sample. Therefore the soluble protein content can not be used as auxiliary means of the resistance identification for Chinese cabbage to clubroot after inoculation. Both resistant varieity and susceptible variety contained lower content of soluble sugar than that of control sample after inoculation, but CR-Yingxiong contained higher content of soluble sugar than that of Zaoshuchangjiang5, so the the soluble sugar content can be used as auxiliary means for the resistance identification to clubroot after inoculation.In conclusion: Detecting the PbDNA by qPCR was the best way to screen resistant varieity; Invertase activity was related to host resistance to P. brassicae; The CAT、SOD、MDA avtivity and the soluble sugar content may be used as auxiliary means of the resistance identification for Chinese cabbage to clubroot after inoculation, but the PPO activity, POD activity and the soluble protein content may not be used as auxiliary means of the resistance identification for Chinese cabbage to clubroot after inoculation. |
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