Quantitative Detection And Dynamic Analysis Of Ralstonia Solanacearum Of Tobacco In Soil By Real-Time PCR

Tobacco bacterial wilt is a soil-borne bacterial disease infected by Ralstonia solanacearum.This disease occurs mainly in tropical and subtropical areas which have a high temperature.But in recent years, tobacco bacterial wilt was found in all the tobacco-growing areas and the incidence of this disease is gradually increasing. Ralstonia solanacearum is easily to be influenced by environmental fators.It caused a devasting blow in tobacco production in some years. In our country this disease gradually extended to the noeth tobacco area due to various climate and other reasons in recent years.It has happened in Shandong,Henan,Shanxi and Liaoning provinces,which have had a significant damage in some areas.Ralstonia solanacearum survives from the winter in soil or compost. The living time varies in different conditions and is greatly influenced by temperature and humidity. In this research, the dynamic model of Ralstonia solanacearum in different environmental conditions was analyzed in order to forecast the disease.To do this, two gene-specific primers were designed and verified the specificity for Ralstonia solanacearum. Real-time fluorescent quantitative PCR technique was used to calculate the population of Ralstonia solanacearum of different environmental conditions.At the same time,disease incideace was recorded and disease index was calculated,and the air temperature and precipitation offield were monitored. The relationship between disease incideace of tobacco bacterial wilt and the population of Ralstonia solanacearum in soil,air temperature and precipctayion were studied.The main result are as follows:1.We design and verify the specificity of the primers.Based on conserved sequence segment of Flic gene, we designed two primer pairs: Flic-f1/ Flic-r1 and Flic-f2/ Flic-r2. Two fragments,188- and 192-bp,were amplified with template of Ralstonia solanacearum genomic DNA,while PCR using genomic DNA of other bacteria gave no fragment.The results showed that the primer pairs Flic- f1/ Flic- r1 and Flic- f2/ Flic- r2 could detected specificly Ralstonia solanacearum.We used the first primer pair to complete the follow-up experiment.2.We optimized the reaction conditions of conventional PCR and Real-time PCR,and detected the sensitivity of the paris of primers.Using the designed specific primers,we conducted experiments based on conventional PCR and dye SYBR Green Real-time PCR to optimize the optimum temperature.We optimized annealing temperature from 48? to 62? and chose 57.7 as the optimal annealing temperature.We seria diluted to obtain 10-fold serial dilutions of standaed DNA extracted from tobacco bacterial wilt.The sensitivity of primer pairs was detected with DNA of Ralstonia solanacearum after 10-fold serial dilutions,using conventional PCR and Real-time PCR.The result showed that the detection limit of conventional PCR was 1.0×10-4 ng/?L and that of Real-time PCR was 1.0×10-5 ng/?L.3. The Ct value standard curve of Ralstonia solanacearum genomic DNA was generated. The Ralstonia solanacearum genomic DNA was diluted into various concentrations.And real-time fluorescent quantitative PCR by Bio-Rad-i Q5 system was carried out. The mean Ct value and the standard deviation was obtained for each DNA sample. The standard curve was generated using Ct value versus log DNA amount(fg).We set the ct value of 15 to 30 and determination coefficient of 0.998.The results showed that amplification efficiency was 99.6%, the linear range can be up to 6 orders of magnitude. The derivative view had a single peak and no primer dimers were found.These results proved that the standard curve can be used for quantitative detection of the Ralstonia solanacearum in the soil.4. The disease index curve of tobacco bacterial wilt in field were draw and relationship between the temperature, precipitation and the quantity of Ralstonia solanacearum were analyzed. The incidence of tobacco bacterial wilt in six fields of three different county was recorded and disease index was calculated.The population of Ralstonia solanacearum in field were obtained by real time PCR technology. Analysis on relationship between the temperature, precipitation and the quantity of were carried out.The results showed that in the hot and humid conditions, the incidence of Tobacco bacterial wilt increase rapidly. Based on the experimental results, it has significant associations between the content of soil bacteria in six points of three places and average daily temperature of the three days. But have no obvious correlation to rainfall of the three days.