Single Spore Isolation And Molecular Identification Of Plasmodiophora Brassicae In Main Chinese Cabbage Producing Areas

Plasmodiophora brassicae is an obligate parasite on the roots of plants.Most cruciferous crops can be infected at any growth period.It can cause crop to be growed slowly,withered,and even death.Due to the diversity of P.brassicae physiological races,and the unstable and variable existence state,it is difficult to carry out resistant breeding.In this research,the single-spore isolation technology system and the Williams identification system were used to study on P.brassicae of several regions including Hubei province,Neimenggu province,Shanxi province,Beijing,Henan province,Liaoning province and Yunnan province et al.The pathogenicity,infection and defense enzyme of different physiological races were studied.It the differences of the physiological races were examined at the molecular level by camparing ITS sequences of P.brassicae.The specific results are as follows:1.In this study,we successfully isolated 1-16 races of Williams identification system.The race 5 was non-pathogenic one and race 13,race 16 have been successfully isolated in our previous research.Therefore,all the races(except the non-pathogenic race 5)of Williams system have been collected.Statistics results of single spores showed,race 4 was the dominant species in these tested areas2.In this research,we compares and analyzes the incidence and infection of ‘SN143' and ‘SN205' infected by those 15 physiological races,and found that the more physiological races,the stronger the pathogenicity;Same hosts infected by different races showed different infection degree;to the same race,the susceptible is more early infected than that of the resistant varieties.Among all these 15 races,race 4 has the strongest infection ability and pathogenicity.Due to the analysis of CAT activity,the highest peak was found on 12 d after inoculation,and the CAT activity is significantly higher in resistant/sensitive Chinese cabbage affected by the single spore races of P.brassicae than uninfected control.The CAT activity in the susceptible varieties returned to normal at 16 d after inoculation,but the resistant varieties continued to be significantly higher than control,indicating that the plants were resistant to P.brassicae infection by regulating CAT activity mainly occurred on about 12 d after inoculation.The role of CAT in disease resistant materials is more durable.3.Due to the difference analysis of POD activity,we found,from 12 d to 16 d after inoculation,the POD activity was significantly higher in infected susceptible variety by P.brassicae single-spore races than that in uninfected control.But to resistant varieties,the significantly increasing of POD activity was happened on the 8 d after inoculation,and then decreased rapidly on 12 d.It indicates that POD is mainly related to the repair of damaged plants.The stronger the repairing ability,the earlier the POD content reducing.In susceptible variety,POD content in race 4 was significantly higher than that in the other races at 12 d after inoculation,indicating that the POD content is related to races,and can be used to distinguish race 4 and other races.4.Due to the SOD activity analysis,it was found that,in susceptible cultivars,the SOD activity was significantly increased on 4d-8d after inoculation in infected plants by P.brassicae single-spore race than that in uninfected control.In resistant varieties,the activity of SOD was significantly increased in infected plants on the 8 d after inoculation and quickly returned to normal.It indicated that SOD can be used to detect the early infection,and the susceptible material could be infected earlier,so its SOD activity changed earlier.5.We found the CTAB method is better to be used to extract the DNA of P.brassicae than the diatomaceous earth method,because it can exclude the influence of plant hosts.The ITS sequences of 15 different physiological races were detected and analyzed,and the sequence difference can be found in ITS-1,ITS-2 and 18 S regions,but no difference was found on 28 S region.The band of ITSs are different for different races,so ITSs can be used for molecular identification of different races of P.brassicae.In short,15 races of P.brassicae were obtained through this study.CAT and SOD could be used as an auxiliary index for early screening of resistant varieties.POD had the ability to identify different races.ITSs can be used for molecular identification of different races of P.brassicae.