Identification Of Canola Clubroot Resistance In Hunan And Establishment Of Molecular Detection Method Of Plasmodiophora Brassicae

Canola clubroot is a soilborne disease caused by Plasmodiophora brassicae Woron.Canola clubroot occurs in all the main planting areas of canolaseed in China,and the occurrence area is increasing year by year,thus becoming one of the main diseases limiting the production of canolaseed.In this study,we conducted experiments to estimate the disease resistance of canolaseed cultivars planted in Hunan,and the Plasmodiophora brassicae in soil were quantitative and rapid detected using Real-time fluorescence quantitative PCR(q PCR)and LAMP detection methods.The main research results are as follows:1.The resistance of 50 canolaseed cultivars suitable for planting in Hunan was identified by the methods of greenhouse artificial inoculation and field natural induction.Results showed that twelve cultivars showed resistance to clubroot(disease index < 20),accounting for 24% of the tested cultivars.Among these diseaseresistance cultivars,Nanyou 868 and Zheyou 50 were immune to P.brassicae,and xiaoheza737 was highly resistant to P.brassicae.These varieties can be popularized and applied as disease resistant varieties.2.The qPCR was used to detect the content of P.brassicae in 12 field soil samples.Results showed that the number of resting spores in the tested soil samples was generally ranged from 105 to 106 resting spores/g soil.In the tenfold gradient dilution soil samples,103 resting spores /g soil was the threshold for the occurrence of canola clubroot.q PCR could quantitatively detect 2.44×102 copy numbers and 4.88×103 resting spores/g soil sample concentrations.The standard curve was drawn with a certain concentration of standard plasmid DNA,and the curve showed a negative correlation.The standard curve could be used to evaluate the initial number of P.brassicae in soil samples.The experiment confirmed that an efficient,fast and specific q PCR detection method for P.brassicae could be established.3.The LAMP reaction time and temperature(65℃ 50min)were optimized.The LAMP amplification reaction results were evaluated according to the color change of SYBR Green Ⅰ dye.Result showed that LAMP could detect at least 183 fg of the P.brassicae DNA,the sensitivity was 1000 times higher than that of conventional PCR.The LAMP based detection technology has the advantages of good specificity and high sensitivity,and can specifically detect P.brassicae.The whole test takes only 50 min,and a constant temperature water bath can complete the whole reaction system and achieve amplification without the need of complex instruments.