| Viral and bacterial diseases are two important diseases that harm crops.In recent years,a viral disease caused by Tomato mottle mosaic virus?ToMMV?has been prevalent and harmed solanaceous crops in Yunnan,Hainan and Shandong provinces,resulting in huge yield and economic losses.Kiwifruit bacterial canker caused by Pseudomonas syringae pv.Actinidae?Psa?has caused a devastating harm in kiwifruit planting areas in China,which seriously affects our nation's poverty alleviation policy and rural revitalization.In order to grasp the epidemic regularity of these two diseases and establish a scientific prevention and control strategy,it is urgent to establish efficient and rapid detection methods.For this purpose,monoclonal antibodies?MAbs?against ToMMV and Psa were prepared in this thesis,and serological methods for specific and sensitive detection of these two pathogens were developed using the obtained MAbs.The prepared MAbs and the developed test methods in this study will provide detection techniques and reagents for detection,diagnosis,early warning and scientific prevention and control of these two diseases.The research results are as follows:?1?Using purified ToMMV virions as the immunogen,six hybridoma cell lines?i.e.2D6,9C12,26A10,3A4,23A4 and 17B11?secreting anti-ToMMV MAbs were obtained by the hybridoma technique.Indirect-ELISA titers of ascites containing MAbs secreted by prepared hybridoma cells ranged from 10-66 to 10-8,and the isotypes of the six MAbs were determined to be IgG2a and?light chain.Western blot results showed that the all six MAbs could react with the 18 KDa capsid protein of ToMMV in infected tomato leaf tissues,but did not react with the extract from healthy tomato plant tissues.The analysis results of specificity demonstrated that all prepared MAbs could only react with ToMMV-infected tomato leaf tissues,but not with TMV-,ToMV-,CGMMV-,or CMV-infected plant leaf tissues or healthy tomato leaf tissues.Three serological assays,i.e.ACP-ELISA,dot-ELISA and Tissue print-ELISA for the specific and sensitive detection of ToMMV were established using the MAbs.Specificity analysis results of three developed serological assays indicated that ToMMV was specifically detected in ToMMV-infected tomato plant tissues,and no positive result was obtained when TMV-,ToMV-,CGMMV-,or CMV-infected plant leaf tissues and healthy tomato leaf tissues were used as the tested samples.The detection endpoints of the ACP-ELISA and dot-ELISA methods were 1:81,920 and1:40,960?w/v,g/mL?with the crude extract fromToMMV-infected tomato leaf tissues,respectively.Tissue print-ELISA was the most rapid and practical detection technique among the three established serological assays.The test results of field samples showed that the three newly established serological assays could be applied to easily,quickly,accurately and effectively monitor ToMMV in large-scale field samples.?2?Using the pathogenic bacterium of Kiwifruit bacterial canker as the immunogen,three MAbs?i.e.6C5,14H5 and 18A4?were prepared with the hybridoma technique.Indirect-ELISA titers of ascites containing MAbs secreted by obtained hybridoma cells ranged from 10-66 to 10-7,and the isotypes of the three MAbs were determined to be IgG1 and?light chain.The results of specificity and sensitivity analyses of MAbs by ACP-ELISA and dot-ELISA indicated that the three MAbs could not react with six non-pathogenic bacterium strains?i.e.SCF1,SCF2,SCF3,SCF4,SCF5,and SCF6?,but could strongly react with Shaanxi,Sichuan,Yunnan,and Jiangxi strains of Pseudomonas syringae pv.actinidae?Psa?of Kiwifruit canker.Results of sensitivity analysis demonstrated that the detection endpoints of the developed ACP-ELISA and dot-ELISA based on the prepared MAbs were 9.0×102and 3.60×103 CFU/mL interacting with Psa of Kiwifruit canker.The test result of field-collected samples indicated that the developed MAbs and the established serological assays could accurately detect Pas of Kiwifruit canker in field samples.The prepared MAbs and the newly developed serological methods will provide techniques and reagents for the detection,diagnosis,resistance breeding and scientific prevention and control of this disease. |
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