| In recent years,bacterial spot of tomato disease occurred in a large area in many provinces of China,causing serious economic losses to local farmers.In this study,the pathogen samples of tomato bacterial spot disease were identified,and the quantitative detection method of live cells was established,and applied to the quantitative detection of live bacteria in the diseased body.The main findings are as follows:(1)Collection of bacterial spot of tomato and pathogen identification.From 2016 to 2018,71 samples of suspected bacterial spot diseases of tomato were collected from vegetable production areas such as Beijing,Xinjiang,and Shandong.Through pathogenicity tests and molecular biological identification,the pathogenic bacteria that caused the bacterial blight of tomato were identified as Pseudomonas syringae pv.tomato(Pst)and 55 Pst strains were preserved.(2)quantitative PCR detection system for tomato bacterial spot was established.Using the pathogenicity-related gene HrpZ of Pst as the target sequence,specific primers Pst3F/Pst3R were designed and a target fragment with a size of 161 bp was specifically amplified.quantitative PCR detection system for Pst bacteria was established.Using a quantitative PCR detection system,the detection limit of the simulated contaminated seeds was 4.21 cfu/g seed,and the level of the diseased leaf was detected as4.15×102 cfu/g leaves.The quantitative PCR assays and routine isolation and identification of 63 samples of tomato bacterial spot disease collected from fields were performed respectively.The results of the two methods were consistent.The established Pst quantitative PCR detection system has the characteristics of strong specificity and high sensitivity,and can quickly and accurately detect the content of Pst in seeds and diseased tissues.(3)Propida monoazide(PMA)PMA was combined with qPCR to establish a quantitative detection technique for Pst live cells.It was clarified that the optimal concentration of PMA pretreatment was 10μmol/L.The best treatment time was dark incubation for 20 min and exposure for 10 min.Using PMA-qPCR technology,the dynamic changes of pathogenic bacteria in 0-30 days were studied under the conditions of 25%,50%,75%,90%and other humidity treatments.The results showed that the greater the humidity of air and soil,the faster the death rate of pathogenic bacteria.After treatment for 30 days under90%air and 90%soil moisture conditions,the number of pathogens in the disease was reduced from initial6.43×10~5 cfu/g and 9.32×10~5 cfu/g to 2.30×10~1 cfu/g and 1.27×10~1 cfu/g,respectively.Differences in mortality rates of pathogens were not significant under different air humidity and soil moisture conditions.In this paper,the quantitative detection technique of Pst living cells combined with PMA-qPCR was established and applied to the detection of pathogens in seeds,tissues and diseased bodies,which provided a rapid detection method for the early diagnosis of the disease and provided a theoretical basis for the ecological prevention and control of the disease. |
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