Quantitative Detection Of Verticillium Dahliae.Kleb.in Cotton Field Soil

Cotton Verticillium wilt?Verticillium dahliae.Kleb.?is a typical soil-borne disease and one of the most important diseases in the world.Its dormant structure can survive for 10 years or longer in soil and is also the main source of initial infection.The aim of this research were quantifing,locating and monitoring the dynamic of microsclerotia during cotton growing season in soil of cotton field apply on the selective culture medium method,verifying the relationship between the severity of disease and the quantity of microsclerotia in soil,and exploring the contribution accumlating microsclerotia on different resistant cotton.Verticillium dahliae microsclerotia were detected and quantified adopting the method of the specific primers of PCR,loop mediated isothermal amplification?LAMP?and loop mediated isothermal amplification of real-time fluorescence?RT-LAMP?.The aim was screening high sensitivity,good specificity,the system for qualitative and quantity Verticillium dahliae microsclerotia in soil.The system was beneficial to forecasting and risk assessment cotton Veticillium wilt.The main findings of this paper are as follows:1?The results showed that the germination rate of microsclerotia reached 88%for 5 days culturing on MSEA selective medium..The microsclerotia mainly existed in the tilling layer of 0²0 cm.The number of microsclerotia was related to the growing season of cotton in soil of cotton field,seedling stage was relatively low and the highest number in August.The incidence of cotton Verticillium wilt were correlated with the number of microsclerotia in soil.That is,the more serious Verticillium wilt of cotton,the more microsclerotia in soil.Cotton resistance is also influence the quantity of microsclerotia accumulated in soil susceptibility cotton varieties accumulating the more microsclerotia in soil.2?The system of LAMP wsa optimized for specific detection Verticillium dahliae.The optimization were as follows:d NTPs 0.6 mmol/L,Mg²⁺2mmol/L,0.8 mmol/L betaine,and primer is 12:1,reaction time50 min,reaction temperature is 65?,verified 10 times as high as the system sensitivity specificity primer PCR detection sensitivity,and the operation is convenient.3?A quantitative amplification method RT-LAMP was established for detecting microsclerotia of cotton Verticillium wilt.The standard curve of RT-LAMP was drawn.The regression equation was:Y=-3.7817X+43.156,the correlation coefficient R=0.955,and the determining coefficient R²=0.9968.The specificity of the five samples was evaluated.The amplified signal of target was obvious,and the melting temperature of the product was 86?±0.5?,without interference of primer dimer.Its sensitivity is5.8×10¹ soil/g,which is 10 times higher than that of specific primer PCR.It has the characteristics of little change of three times repeat and good repeatability of small coefficient of variation.4?The relationship between and of Verticillium wilt in soil was discussed by real-time fluorescence technique of loop mediated isothermal amplification.The results of quantitative microsclerotia density cotton Verticillium wilt in soil using RT-LAMP showed that there was a linear relationship between the microsclerotia density and disease index of cotton Verticillium wilt.The linear equation was:Y=0.3713X+8.3322,the correlation coefficient R=0.964,the determining coefficient was R²=0.9247,but there was a negative linear relationship between the disease index and Ct value.The linear equation is:Y=-0.1078X+15.627.The coefficient of correlation R=0.974 and the coefficient of determination R²=0.9482.The correlation between fluorescent quantitative Ct value and microsclerotia density was further demonstrated.The disease index was inversely proportional to the disease index,that is,the larger the fluorescent quantitative Ct value,the smaller the microsclerotia density and the disease index.Compared with the results of selective medium method,there were significant differences in the number of microsclerotia in the same soil.But the t value and average value detected by real time fluorescence quantification with ring-mediated isothermal method were higher than those by selective medium method,and the reliability and sensitivity were high.