Construction And Utilization Of Plasmodiophora Brassicae Single Spore Isolation System

In recent years,clubroot has been occurring frequently in Chinese cabbage and other cruciferous crops,which seriously affected the yield and quality of cruciferous crops.Plasmodiophora brassicae usually exists as a mixture of several physiological races.The same source pathogen has different pathogenic types,therefore,it may be more valuable to purify and isolate the physiological race by using single spore separation.In this study,a system of single spore isolation was established and used to isolate the single spore in Shenyang and Shandong area,detected difference between different physiological races by PCR,predicted the approximate location of the ITS region of the Plasmodiophora brassicae rDNA.The specific results are as follows:In this study,a system of single spore isolation was established by optimizating inoculation method of Plasmodiophora brassicae single-spore,determinating inoculation seeds-age and changing plant culture procedures.The system is that single-spore being inoculated on 2-days-old roots using agarose plugs-frozen box inoculated method and the inoculated plants being cultivated with hydroponics-medium transit culture-soil culture.which can increase the infection rate to 47.86%.In this process,medium transit culture is necessary before water culture,which can increase the survival rate of the plant to 67%.Plasmodiophora brassicae single spore in the Shenyang and Shandong regions was isolated by the above optimized single spore isolation system,and the physiological race of was identified by Williams system with injected brassicae solution.Four physiological races(No.4,No 8,No 9,and No 11)were obtained from the Plasmodiophora brassicae collected in Shenyang area of China.Nine physiological races(No.1,No.2,No.4,No 8,No 9,No.11,No.13,No.15,and No 16)were obtained from the Plasmodiophora brassicae in Shandong area of China.It was found that the dominant physiological race was No.4 race in Shenyang and Shandong areas.Compared and analyzed three DNA extracted methods and directly bacteria solution for PCR detection,it was found that using the kit method or directly bacteria solution could eliminate the influence of plant host.Compared rDNA of Plasmodiophora brassicae and other fungus to predict approximate ITS location of Plasmodiophora brassicae.PCR results in 18 S,ITS-1,ITS-2 region showed different physiological races may have differences.As is shown above,Plasmodiophora brassicae usually exists as a mixture of several physiological races,each physiological race may have difference in 18 S and ITS region.