Development And Application Of A Loop-mediated Isothermal Amplification Method For Rapid Detection Of Pseudomonas Syringae Pv. Tabaci And Pseudomonas Syringae Pv. Angulata

Tobacco wildfire disease and tobacco leaf spot disease are two major bacterial diseases in tobacco production.The infected tobacco leaves are perforated,brown and fragile,therefore the usability of these leaves in cigarettefactory are highly reduced.It has a serious impact on tobacco growers,especially their income.Development of a method for rapid detection of Pseudomonas syringae pv.tabaci and P.syringae pv.angulata will provide reliable data support for disease prevention and treatment before the disease occurs.Loop-mediated isothermal amplification（LAMP）have the advantages of fast detection speed,low requirement on detection environment,strong specificity,and strong sensitivity.Because of these advantages,it has been widely used in medical,animal and plant disease detection,genetically modified food testing and other fields.Therefore,we use LAMP for the detection of tobacco wildfire disease and tobacco leaf spot disease.A LAMP method for rapid detection of P.syringae pv.tabaci andP.syringae pv.angulata was developed applicated.The main results are as follows:（1）P.syringae pv.tabaci and P.syringae pv.angulata was isolated and identified from different infected tobacco leaves.Finally,we obtained pure P.syringae pv.tabaci and P.syringae pv.angulata for the following research.（2）Mavue 2.3.1 softwarewas used for identify specific segment of P.syringae pv.tabaci.According to the selected specific segment,we designed and screened primers for P.syringae pv.tabaci.（3）LAMP primers for P.syringae pv.tabaci and P.syringae pv.angulata were respectively obtained.According to the specific segment of P.syringae pv.tabaci,LAMP primers were designed with software Primer Explorer V5.Due to the lack of genome data for P.syringae pv.angulata,we use PCR primers for P.syringae pv.tabacito amplify P.syringae pv.angulata.The PCR products were sequences and blasted with P.syringae pv.tabaci.Finally,we obtained the difference between P.syringae pv.tabaci and P.syringae pv.angulata.Base on this difference,we obtained a set of LAMP specific detection primers for P.syringae pv.angulata.（4）LMAP rapid detection system for tobacco wildfire and tobacco leaf spot were established and optimized.The reaction conditions such as the amount of BST DNA Polymerase used,the amount of dNTPs used,the concentration of Mg²⁺used,the concentration of betaine used,the ratio of internal and external primers,reaction time,reaction temperature,etc.were optimized.Finally,the optimum field LAMP detection working concentration was determined.（5）The reliability of the detection system is verified by combining LAMP detection system with field application.The reliability and sensitivity of the LAMP detection system finally determined by comparing with ordinary PCR and fluorescence quantitative PCR using field-sampling suspected diseased leaves and healthy leaves as controls.