Establishment And Application Of Real-time PCR For Monitoring Cucumber Target Leaf Spot

As a new airborne disease that has recently become popular in cucumber growing areas in Heilongjiang Province,cucumber target leaf spot has the characteristics of spreading rapidly,easy confusion with other cucumber diseases and controlling difficultly,which seriously threatens cucumber production.Therefore,the prediction and monitoring of cucumber target leaf spot is of great significance for disease prevention and control.In this paper,the occurrence of cucumber target leaf spot in Heilongjiang Province was investigated,and a fluorescence quantitative PCR system for cucumber target leaf spot was established and optimized used for monitoring the infection dynamics and the disease epidemic in field,the results were as follows:1.The occurrence of cucumber target leaf spot in Heilongjiang Province was investigated.Among the 17 villages,Damin Village,Xiaohuali Village and Dahuali Village in Qiqihar were the only ones without the occurrence of cucumber target leaf spot,and the others were all harmful.Mudanjiang and Jiamusi areas had a higher incidence with an average disease index of 25.8,while Qiqihar and Harbin had a lower incidence with an average disease index of 8.34.The areas with more severe infection were Xiaowufuma Village,Linjiang Village and Hengli Village in Jiamusi,with disease index of 32.7,46.5 and 32.9,respectively.2.A pair of specific primers CAF2?5'-GCCTCGAGCTGTTTTCCGTAAGT-3'?/CAR2?5'-CCGATCATGATACTGGCAGTGGTC-3'?was designed based on the actin sequence of Corynespora cassiicola,then a standard curve was constructed by positive standard DNA and the fluorescence quantitative PCR reaction system were optimized.The results showed that when the annealing temperature was 60°C,the amount of DNA template was 3?L and the amount of primer was 0.2?L(10 mmol·L^-1),the system was the most stable.The DNA detection range of C.cassiicola was1.36×10^-7-13.6 ng·?L^-1,which was 10³ times higher than the sensitivity of common PCR.Verifying the repeatability and stability of the PCR system between groups and within groups,the coefficients of variation were both less than 2%.3.The dynamic changes of latent infestation of C.cassiicola in cucumber cotyledons at different time after inoculation were detected by fluorescence quantitative PCR.The results showed that at the time of 4 h-8 h?Cq value<30?,the infiltration of the highly susceptible and susceptible varieties'leaves began to increase,and at the time of 8 h-12 h?Cq value<30?,the infiltration of the medium-resistant,resistant and highly resistant varieties'leaves began to increase with the infection rate gradually increased.When the Cq value was less than 21,the disease level was 1;the Cq value<19,the disease level was 2;the Cq value<17,the disease level was 3;the Cq value<14,the disease level was 4.4.Fluorescence quantitative PCR was used to monitor the occurrence and prevalence of cucumber target leaf spot in field.After the spores were collected in the field,the quantitative results were consistent with the real-time fluorescent quantitative PCR method and the micro-counting method.The feasibility of using fluorescent quantitative PCR technology for monitoring cucumber target leaf spot was proved.The results provide a technical method for the prediction and prevention of cucumber target leaf spot.