Single Resting Spore Isolation Of Plasmodiophora Brassicae And Function Identification Of Clubroot Susceptibility Gene

Clubroot diseases caused by Plasmodiophora brassicae Woronin is spreading worldwide.The variation of physiological races of P.brassicae in field is rich.Single resting spore isolates are needed for researches.However,method and technology of single resting spore isolation is tedious and time-consuming.Clubroot resistance breeding is also further improved under the context of big data,but the current work has not yet made breakthrough progress,the performance of many disease-resistant accessions in the field is not durable.Aim to the two markable aspects.In this study,we first identifed the physiological races from Jiangsu and Zhejiang provinces.Subsequently,analysis of the ITS sequence differences of P.brassicae from different geographical origins has been carried out.After also observed its germination stimulating factors,our study established one novel single resting spore isolation technique based on the existing dilution method,droplet method,and the imprint method.The study did not continue a preference for dominant single disease resistance genes in clubroot resistance breeding.Because the germplasm obtained is always not durable.Taking into account the specificity of the P.brassicae,the study focued on possible susceptible genes involved in the early stage of P.brassicae infection.Mutant materials obtained from CRISPR/Cas9 gene editing technology and purchased from NASC are characterised for candidate S-genes for functional identification.The main findings are as follows:（1）Three physiological races of P.brassicae have been identified from Zhejiang and Jiangsu provinces.Results showed that the pathogen from Wenling city,Zhejiang was identified as ECD24/0/0 physiological race,P8 race of Williams system.The pathogen from Jinyun city,Zhejiang belonged to ECD24/16/30 physiological race,P1 of Williams system.The pathogen from Yixing city,Jiangsu belongs to the ECD28/31/31 physiological race,that is,the P4 physiological race of the Williams system.In addition,the pathogenic ITS regions and 18 S rDNA sequences in different hosts and different regions are not significantly different.ITS sequences or 18 S rDNA sequences analysis are not enough for physiological races identification so far.The ribosomal DNA of pathogen of Wenzhou city,Zhejiang and Qingpu district,Shanghai,is very different from that of other regions.（2）Microexamination of resting spores and the bait crops root exudate have a promoting effect on germination of resting spores.Concentration degree of spore contents is consistant with the extinction of the outermost wall structure,which maybe related to the maturation and germination of resting spores.The sampling period of gall is important for obtaining resting spore solution with high quality,and the resting spore could be stored at 4 °C.The 5 bait crops root exudate have a promoting effect on germination of resting spores,and the ryegrass root exudate has the best effect among them.（3）Established a novel set of stable and efficient P.brassicae single spore isolation technology,which is suitable for the separation of very small pathogenic microorganisms that cannot be cultivated in artificial culture medium.The operation is simple,the separation efficiency is high,and the requirements for instruments and equipment are low.The stability is strong,but the galls of the single resting spore-affected plants are small.The PCR detection with primers Plasmo-3 / Plasmo-4can be used as an effective method for screening single resting spore inoculation materials.（4）Screening of the candidate susceptible genes（S-genes）on clubroot.In this study,we obtained 10 candidate S-genes whose expression was up-regulated in the early stage of infection.At the same time,pBI121_Cas9_U6-26_MCS plasmid has successfully been constructed.Three lines(salk₀85849,salk₀63470 and wrky11（At2g31550）from 31 homozygous T-DNA insertion mutants and 4 large fragment knockout mutants exhibit significant clubroot resistance.（5）Functional analysis of the candidate S-genes of AT2G35930 and AT3G22970.Gene expression results indicated AT2G35930 was predominantly expressed in the hypocotyl,and GFP fusion proteins of both genes were distributed on the nucleus and cell membrane.Compared with Col-0,partial clubroot-resistance of at3g22970（salk₀85849）is repeatable.Compared with the no-load transformant of pBI121,both gene overexpressing plants were more susceptible to P.brassicae.The content of pathogens in root tissue of mutants was less than that of Col-0,and the stage of pathogen development was slightly delayed.In summary,AT2G35930 and AT3G22970 candidate S-genes are involved in supporting pathogenic development within the host.