Preparation And Application Of Monoclonal Antibodies Against Candidatus Liberibacter Asiaticus And Dickeya Dadantii Of Stem And Root Rot Desease Of Sweet Potato

Plant bacterial disease is one of very important plant diseases.There are more than 500 plant bacterial diseases reported in the world and more than 150 bacterial diseases recorded in China.Plant pathogenic bacteria can harm crops,vegetables,flowers,fruit trees,forests and medicinal plants and so on.Among of them,the outbreaks of Citrus huanglongbing(HLB)caused by Candidatus Liberibacter asiaticus and Stem and root rot desease of sweet potato caused by Dickeya dadantii caused serious yield and economic losses.Development of rapid and sensitive pathogenic bacterium detection techniques is the key to the diagnoses,prevention and control of these two plant bacterial diseases.Serological detection techniques are simple,rapid,sensitive,specific and suitable for large-scale sample detection.Therefore,it has been widely used in the diagnoses and detections of plant bacterial diseases.But there is no report about specific and broad-spectrum monoclonal antibody(MAb)against Candidatus Liberibacter asiaticus and serological detection technology for Stem and root rot of sweet potato in the world.For this purpose,MAbs against Candidatus Liberibacter asiaticus and Dickeya dadantii were respectively produced,and effective serological technologies based on the developed MAbs were established for rapid and reliable detection of these two pathogenic bacteria in this thesis.These research achievements will provide technology and materiel for diagnosis and detection,forecast and early warning and scientific prevention and control of these two plant diseases.(1)Monoclonal antibodies against Candidatus Liberibacter asiaticus and their detection application:The Candidatus Liberibacter asiaticus-infcted citrus vein extract was used as an immunogen to immunize BALB/c mice.Via cell fusion,cell culture,antibody detection and cell cloning,six hybridoma cell lines(6A7,9A1,11H9,10G1,13F11 and 18F12)secreting sensitive and specific murine MAbs against Candidatus liberibacter asiaticus were obtained.The titers of ascitic fluids containing MAbs secreted by six hybridoma cell lines ranged from 10-6 to 10-7 by indirect-ELISA.All MAbs were isotyped as IgG1,kappa light chain.Western blot analyses indicated that three MAbs(6A7,13F11 and 18F12)could strongly react with a protein of approximately 115 kDa in Candidatus Liberibacter asiaticus-infected leaf tissues,MAbs 11H9 could strongly react with a protein of approximately 56 kDa in Candidatus Liberibacter asiaticus-infected leaf tissues.Western blot analyses of other two MAbs(9A1 and 10G1)showed no hybridization signal,so it was speculated that they were spatial configuration epitopes.According to the serological principle,two serological assays,antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA)and dot enzyme-linked immunosorbent assay(dot-ELISA)were developed for detecting Candidatus Liberibacter asiaticus using the developed MAbs.Among them,the sensitivity of ACP-ELISA based on MAb 6A7 or 10G1 was higher,and could detect Candidatus Liberibacter asiaticus in leaf crude extracts diluted at 1:10240(w/v,g/mL),while the ACP-ELISA based on MAb 9A1,18F12,11H9 or 13F11 could detect Candidatus Liberibacter asiaticus in leaf crude extracts diluted at 1:5120(w/v,g/mL).The dot-ELISA based on MAb 6A7 has the highest sensitivity,and could detect the bacteria in 1:10240 dilution(w/v,g/mL)of Candidatus Liberibacter asiaticus-infected leaf extracts.The dot-ELISA based on MAb 18F12 has a sensitivity of 1:5120 dilution(w/v,g/mL)of Candidatus Liberibacter asiaticus-infected leaf tissue extract,while MAb 10G1 has a sensitivity of 1:2560 dilution of Candidatus Liberibacter asiaticus-infected leaf tissue extract.Sensitivities of the dot-ELISAs based on MAb 13F11,9A1 or 11H9 were also up to 1:1280 dilution.A total of 125 citrus samples from Guangxi Zhuang Autonomous Region,Jiangxi Province,Zhejiang Province and Chongqing Municipality in China were screened for the presence of Candidatus Liberibacter asiaticus using the developed two assays,and the detection results showed that 83 of the 125 samples were infected by Candidatus Liberibacter asiaticus.Furthermore,the serological detection result was consistent with the result of PCR detection.Nucleotide sequencing and sequence blast of the PCR products showed that the positive samples tested by the serological assays were really infected by Candidatus Liberibacter asiaticus.(2)Monoclonal antibodies against Dickeya dadantii of Stem and root rot desease of sweet potato and their detection application:Using the pathogenic bacterium strain FY1710 of Stem and root rot of sweet potato as the immunogen,five hybridoma cell lines(25C4,16C10,9B1,6H4 and 9H10)secreting MAbs against Dickeya dadantii of Stem and root rot of sweet potato were prepared with the hybridoma technology.The titers of prepared ascitic fluids containing MAbs were up to 10-6 by indirect-ELISA.All these MAbs belong to IgG1 isotype and ? light chain.Based on the prepared MAbs,four serological detection assays,ACP-ELISA,TAS-ELISA,dot-ELISA and Tissue blot-ELISA were developed for sensitively and specifically detecting Dickeya dadantii of Stem and root rot of sweet potato.The sensitivity analyses revealed that the developed ACP-ELISA,TAS-ELISA,and dot-ELISA could detect the pathogenic bacteria of Stem and root rot of sweet potato diluted at 1:107,1:108 and 1:108(wet weight of bacteria/volume,g/mL),respectively.The specificity analyses indicated three serological assays had a positive reaction with different pathogenic strains of Stem and root rot of sweet potato,and a negative reaction with tested non-pathogenic strains.The developed tissue blot-ELISA can specifically detect the pathogenic bacteria of Stem and root rot of sweet potato in sweet potato plant tissues,and does not have any immune reaction with the healthy sweet potato plant Tissue.