| Clubroot, also named meloidogynosis, is a worldwide soil-borne disease which is caused by the obligate biotrophic pathogen of Plasmodiophora brassicae Woronin. It’s one of the most serious diseases in crucifer all over the world. In practice, agricultural control, chemical control and biological control has been mainly used now. However, since the labor-intensive, long time and easily cause soil compaction of agricultural control. Chemical control can easily lead to pesticide residues. Biological control is much less mature and limited by biocontrol fungi, the overall efficacy of P. brassicae is unsatisfactory. Therefore, breeding of clubroot-resistance cruciferous cultivars is regarded as the most fundamental and safest approach to control clubroot. Unfortunately, the studies on clubroot-resistance inheritance are not so clear at present, and clubroot-resistance cultivars usually specify to the pathotypes as well. Moreover, the pathogen variation occur at rapidly, leading to the resistance of the newly bre 饿 d cultivars can’t last for a long time. Thus, in order to control clubroot effectively, it should be not only increase the intensity of selecting resistant varieties, which provides the novel resistance resources for breeding, but also illuminate the pathogensis of the pathogen and reinforce early detection of P. brassicae Woronin, and it will be possible to conduct integrated control to clubroot. In this study, we studied on appropriate method of indoor inoculation by comparing the incidence of different inoculation methods, and exploring the optimal disease vaccination system by orthogonal experiment. The clubroot-resistance cultivars from 130 cruciferous materials have been collected and screened by using inserting method, and the identification system of P. brassicae in root tissue by optical microscopy and PCR method have been established. Subsequently, seven different races of P. brassicae have been identified through the system of the European Clubroot Differential Sets, and the information of the rDNA fragment has been analyzed as well. The results are as follows:(1) Insertion method inoculated susceptible plant, the DI is highest, inoculation concentration is the key factor for severe disease. This experiment was conducted to study disease degree of the three effects of inoculation methods on susceptible plants.The results showed that insertion method>dipping method>injection method, disease index indicated a trend of decreasing. Meanwhile, to explore the optimal conditions of indoor inoculation using the orthogonal experiment, the main four factors are inoculation concentration, number of vaccination, inoculation quantity and inoculation time. The results showed that the inoculation concentration was the key elements of serious disease. Higher the inoculation concentration, the higher the disease. In addition, we studied the pathogenicity differentiation between fresh and frozen infected root. Result showed that the former DI refers to 72.04, the latter is only 27.78.Compared with frozen infected root, the fresh infected root is much more serious in DI.(2) Various differences in pathogenicity of pathogen. Resistance to clubroot disease of 130 accessions cruciferous crops from Jiangshan, Zhejiang Province; Shanghai Qingpu district; Wenzhou and Wenling,Zhejiang Province have been identified by the method of inserting inoculation. Results indicated that the pathogenicity of Wenling and Shanghai pathogens is much more severe than that of Jiangshan and Wenzhou. For Brassica campestris host, in addition to the weak pathogenic of Wenzhou region, three other areas of the pathogen pathogenicity are stronger. For the hosts of B. juncea,four areas where the pathogen pathogenicity are strong and as to B. oleracea host, four areas where the disease index of pathogen are rendering comparison big of differences. From ANOVA results, results showed that among trials and different species of pathogens, the p value are 0, equally, indicating that different pathogens and trials are very significant differences between different varieties.(3) Two highly and broadly resistant cultivars have been obtained from the result of resistance identification. In our experiment, resistance to clubroot disease of 36 varieties of B. campestris,78 varieties of B. oleracea,15 varieties of B. juncea and a variety of Eruca saliva Mill, in four different infected soil samples have been collected and screened by the method of insert inoculation. Disease resistance appraisal results showed that the different host species on the same soil source or different soil source resistance reaction were obviously different and the same host species react to different sources of bacteria resistant performance was inconsistent, or even diametrically opposed. We obtained 2 high-resistant varieties that are broad-spectrum, ’Xuejiansihao’ and ’271’,5 moderately resistant that are broad-spectrum, namely,’777-1’,’544-2’, ’Y38-4’and’573-12’, which are overall belong to B. oleracea.(4) The race of P. brassicae from Huoshaoping is ECD17/31/31, the race of P. brassicae from Lichuan is ECD16/4/0, the race of P. brassicae from Wenling is ECD17/15/15,the race of P. brassicae from Shanghai is ECD16/0/5,the race of P. brassicae from Linzhi, Shuanghe and Wenzhou is ECD16/0/0. P. brassicae in root tissue was identified by optical microscopy and PCR method, and the races have also been identified by the ECD system.Results showed that isolate from Huoshaoping was ECD17/31/31, isolate from Lichuan was ECD 16/4/0, isolate from Wenling was ECD17/15/15, isolate from Shanghai was ECD16/0/5, isolate from Linzhi,Shaunghe and Wenzhou were same as ECD16/0/0.(5) Three pair of primers have been developed to amplify P. brassicae rDNA ITS sequences, and select two paires conventional PCR primers which can be used to distinguish different P. Brassicae. With three developed primers designed in P. brassicae rDNA used to PCR amplification, then compared the amplification results with NCBI databases rDNA sequence, comparison result indicated that 1RF and 2RF(18SF-18SR)primers could be rapidly used to detect presence of P. brassicae in root tissues at the early stage. |
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