油菜根肿休眠孢子间接酶联免疫检测法的建立

Establishment of Indirect Enzyme-linked Immunosorbent Assay for Detecting Plasmodiophora brassicae Resting Spores

根肿病是由专性寄生菌——芸苔根肿菌感染所引起的一种严重影响十字花科植物生长的植物病害．本文针对根肿病缺乏简便、快速检测方法的问题，制备油菜根肿休眠破碎孢子抗血清，确定抗血清及酶标抗血清最佳工作浓度，优化间接酶联免疫反应条件；优化的抗血清和酶标血清的工作浓度分别为1：2000和1：1000，孢子的饱和包被浓度为10^7个／mL.确定休眠孢子稀释液的最低检测浓度为10个／mL，建立快速检测油菜根肿菌的间接酶联免疫吸附测定法，该方法稳定、可靠，板间误差和板内误差均小于10．0％．与9种土壤分离得到的真菌和7种土壤常见菌黑曲霉、粉红粘帚霉、立枯丝核菌、产黄青霉、绿色木霉、枯草杆菌、脓杆菌进行特异性实验，其中一种未知菌MI交叉反应率〉30％，其余在8．51％～24．88％之间，真菌的交叉反应率普遍高于细菌．对已知孢子浓度的土壤进行了检测，土壤样品休眠孢子浓度的最低检测限度为10^3个/g．

Club root is a plant disease caused by the infection of an obligate parasite, Plasmodiophora brassicae Woronin, and it seriously affects the growth of the plants of Cruciferae. Because there is lack of methods for detecting club root, the antiserum of ultrasonic disrupted resting spores is prepared. The optimum concentrations of the antibody and enzyme-labeled antiserum were determined, and the reacting conditions were optimized. The optimized concentrations of antibody against P brassicae and enzyme-labeled antiserum were 1/2000 and 1/1000, respectively. The saturated antigen concentration was 107 spores/mL. The limit of detection was 10 spores/mL. The indirect enzyme-linked immunosorbent assay （iELISA） for rapid diagnosis of P brassicae was established, and it was stable and reliable. The variation coefficients of intra-assay and inter-assay were lower than 10.0%, respectively. The specificity tests were done with 9 fungi isolated from soil and 7 soil-borne microbes： Aspergillus niger, Gliocladium roseum, Rhizoctonia solani, Penicillium chrysogenum, Trichoderma viride, Bacillus subtillus and Agrobacterium tumefaciens. The cross-reaction rate of one unknown fungus M1 was greater than 30%, and the rates of others ranged between 8.51%-24.88%. The cross-reactivies of fungi were higher than those of bacteria. The soil with a known spore concentration was tested, and the lowest concentration for detecting soil samples was above 10^3 spores/g.