[Objective] This study was conducted to investigate the pathogenicity of Plasmodiophora brassicae on cabbage grown under different temperature and soil pH conditions. [Method] The pathogenicity of P. brassicae were te...[Objective] This study was conducted to investigate the pathogenicity of Plasmodiophora brassicae on cabbage grown under different temperature and soil pH conditions. [Method] The pathogenicity of P. brassicae were tested at seven different temperatures and at six different soil pH values with the resting spore concentration of lx108 （spores/g） in the soil. The plant survival rate and incidence rate of clubroot were investigated after 90 d. [Result] The incidence rate of clubroot on cabbage among the different temperature sets varied in a descending order as follows： 30 ℃〉25 ℃〉20 ℃〉35 ℃〉15 ℃〉10 ℃〉5 ℃ at soil pH value of 6, indicating that the pathogenicity of P. brassicae was weak at 5 and 10 ~（3. The incidence rate increased with soil temperature increasing from 15 to 30 ℃, but decreased at 35 ℃. The incidence rates of clubroot were 80.36%, 100%, 65%, 10.77%, 3.23% and 0% at soil pH 4, 5, 6, 7, 8 and 9 at 25 ℃, respectively. The growth of cabbage was inhibited and the survival rate was reduced at pH 4.The incidence rates of clubroot were low at pH value of 7 and 8, and was 0% at pH 9. The Chinese cabbage grew better at pH value of 5 and 6, but had high incidence rates of clubroot. [Conclusion] The results revealed that the incidence rate of clubroot on cabbage was closely related to the temperature and soil pH.展开更多
Research progress was reviewed on the differential systems for physiologic races of Plasmodiophora brassicae Woron,including Williams,differential system and European clubroot differential（ECD） set.The existing prob...Research progress was reviewed on the differential systems for physiologic races of Plasmodiophora brassicae Woron,including Williams,differential system and European clubroot differential（ECD） set.The existing problems and countermeasures of the different differential systems were discussed,and a research status quo on the molecular identification and detection of clubroot pathogen in crucifers were introduced.展开更多
Five commonly-used reference genes： ACT （actin）, UBE （ubiquitin-conjugating enzyme）, RPL2 （ribosomal protein L2）, BRP II （RNA polymerase II subunit）, and NADH （nicotinamide adenine dinucleotide） were examin...Five commonly-used reference genes： ACT （actin）, UBE （ubiquitin-conjugating enzyme）, RPL2 （ribosomal protein L2）, BRP II （RNA polymerase II subunit）, and NADH （nicotinamide adenine dinucleotide） were examined using geNorm software as reference genes for RT-qPCR. Among the tested reference genes, ACT and UBE were the most stable in all samples. In parallel, expression analysis of nitrilases in Brassica juncea var. tumida, was performed to preliminarily investigate the molecular interactions between nitrilase and clubroot development at 10, 15, 20, 25, 30, and 40 d postinoculation （dpi） with a suspension of resting spores of Plasmodiophora brassicae. The results showed that different gene expressions of nitrilases were regulated during the initial periods of clubroot development. The expression level of BjNIT1 increased sharply from 20 to 40 dpi in infected roots while there were no remarkable changes in healthy roots. From 15 to 30 dpi, the expression levels of BjNIT2 and BjNIT4 in infected roots were lower than those in non-infected roots. Finally, BjNIT2 in treatment was down approximately to control at 40 dpi. Our results suggest that BjNIT1, which promoted overproductions of auxin, might be involved in P. brassicae infection of B. juncea.展开更多
A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA（rDNA） and internal transcribed spacer（ITS）.A pair of primers PBF1/PBR1 was designed based on the conse...A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA（rDNA） and internal transcribed spacer（ITS）.A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.展开更多
Increasing evidence has revealed that micro RNAs play a pivotal role in the post transcriptional regulation of gene expression in response to pathogens in plants. However, there is little information available about t...Increasing evidence has revealed that micro RNAs play a pivotal role in the post transcriptional regulation of gene expression in response to pathogens in plants. However, there is little information available about the expression patterns of mi RNAs and their targets in Chinese cabbage(Brassica rapa ssp. pekinensis) under Plasmodiophora brassicae stress. In the present study, using deep sequencing and degradome analysis, a genome-wide identification of mi RNAs and their targets during P. brassicae stress was performed. A total of 221 known and 93 potentially novel mi RNAs were successfully identified from two root libraries of one control(635-10CK) and P. brassicae-treated Chinese cabbage samples(635-10T). Of these, 14 known and 10 potentially novel mi RNAs were found to be differentially expressed after P. brassicae treatment. Degradome analysis revealed that the 223 target genes of the 75 mi RNAs could be potentially cleaved. KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis suggested that the putative target genes of the mi RNAs were predominately involved in selenocompound metabolism and plant hormone signal transduction. Then the expression of 12 mi RNAs was validated by quantitative real-time PCR(q RT-PCR). These results provide insights into the mi RNA-mediated regulatory networks underlying the stress response to the plant pathogen P. brassicae.展开更多
Many Ri T-DNA-transformed roots were incited on seedling hypocotyl segments of cabbage, cauliflower and oilseed rape following genetic transformation with Agribacterium rhi-zogencs. The transformed roots were in vitro...Many Ri T-DNA-transformed roots were incited on seedling hypocotyl segments of cabbage, cauliflower and oilseed rape following genetic transformation with Agribacterium rhi-zogencs. The transformed roots were in vitro inoculated with the resting spores of Plasmodi-ophora brassicae on solid medium, in liquid medium and semi-solid medium respectively.On solid medium, the resting spores penetrated root hair cells, but the root cortex was not invaded so that no symptom of clubroot galls was developed. In liquid medium, the root cortex was invaded, giving rise to an abnormal division of root cortical tissues and gall formation. In semi-solid medium, the resting spores penetrated the cells of the cut surfaces of root segments and caused the cut surfaces to swell.展开更多
Clubroot is a prevailing soil-borne disease affecting rapeseed production worldwide.However,few clubroot resistant rapeseed accessions were available for breeding.Identification and introgression of new clubroot resis...Clubroot is a prevailing soil-borne disease affecting rapeseed production worldwide.However,few clubroot resistant rapeseed accessions were available for breeding.Identification and introgression of new clubroot resistant genes from closely related species by distant hybridization is an effective strategy.In the present study,9 radish(Raphanus sativus L.,2n=18,RR)lines resistant to Plasmodiophora brassicae pathotype 4 were used as donors to transfer clubroot resistance into a susceptible rapeseed(Brassica napus L.,2n=38,AACC)line by distant hybridization combined with embryo rescue.Nine intergeneric crosses were made but only 1(411×93039)produced F1 plants both from embryo rescue and natural seed-setting.Authenticity of triploid F1 hybrids(2n=28,ACR)were verified by flower color,cytological observation and molecular marker analysis,and 2 genuine F1 hybrids were identified.After chromosome doubling,these synthetic allohexaploid plants(2n=56,AACCRR)became partially fertile(pollen viability rate=35%)and were backcrossed with rapeseed parent to generate a BC1 population(2n=47,AACCR).Totally 178 BC1 plants were obtained,of which the majority(96.1%)were resistant to clubroot.These backcrossing progenies could be used for the breeding of new rapeseed varieties resistant to clubroot.展开更多
基金Supported by Science and Technology Project of Yunnan Province(2014RA061)Special Fund for Modern Agriculture Research System for Rape of Yunnan Province~~
文摘[Objective] This study was conducted to investigate the pathogenicity of Plasmodiophora brassicae on cabbage grown under different temperature and soil pH conditions. [Method] The pathogenicity of P. brassicae were tested at seven different temperatures and at six different soil pH values with the resting spore concentration of lx108 （spores/g） in the soil. The plant survival rate and incidence rate of clubroot were investigated after 90 d. [Result] The incidence rate of clubroot on cabbage among the different temperature sets varied in a descending order as follows： 30 ℃〉25 ℃〉20 ℃〉35 ℃〉15 ℃〉10 ℃〉5 ℃ at soil pH value of 6, indicating that the pathogenicity of P. brassicae was weak at 5 and 10 ~（3. The incidence rate increased with soil temperature increasing from 15 to 30 ℃, but decreased at 35 ℃. The incidence rates of clubroot were 80.36%, 100%, 65%, 10.77%, 3.23% and 0% at soil pH 4, 5, 6, 7, 8 and 9 at 25 ℃, respectively. The growth of cabbage was inhibited and the survival rate was reduced at pH 4.The incidence rates of clubroot were low at pH value of 7 and 8, and was 0% at pH 9. The Chinese cabbage grew better at pH value of 5 and 6, but had high incidence rates of clubroot. [Conclusion] The results revealed that the incidence rate of clubroot on cabbage was closely related to the temperature and soil pH.
基金Supported by the National Science and Technology Program of the Ministry of Science and Technology of China(2010BAD01B04)Research Fund of Department of Science and Technology of Sichuan Province(2008NG0003)the Genetic Engineering Fund of Department of Finance of Sichuan Province(2011JYGC06)~~
文摘Research progress was reviewed on the differential systems for physiologic races of Plasmodiophora brassicae Woron,including Williams,differential system and European clubroot differential（ECD） set.The existing problems and countermeasures of the different differential systems were discussed,and a research status quo on the molecular identification and detection of clubroot pathogen in crucifers were introduced.
基金financially supported by the Natural Science Foundation of Chongqing Science and Technology Commission,China (2008BB1370)Fuling Agricultural Science Institute of Chongqing,China
文摘Five commonly-used reference genes： ACT （actin）, UBE （ubiquitin-conjugating enzyme）, RPL2 （ribosomal protein L2）, BRP II （RNA polymerase II subunit）, and NADH （nicotinamide adenine dinucleotide） were examined using geNorm software as reference genes for RT-qPCR. Among the tested reference genes, ACT and UBE were the most stable in all samples. In parallel, expression analysis of nitrilases in Brassica juncea var. tumida, was performed to preliminarily investigate the molecular interactions between nitrilase and clubroot development at 10, 15, 20, 25, 30, and 40 d postinoculation （dpi） with a suspension of resting spores of Plasmodiophora brassicae. The results showed that different gene expressions of nitrilases were regulated during the initial periods of clubroot development. The expression level of BjNIT1 increased sharply from 20 to 40 dpi in infected roots while there were no remarkable changes in healthy roots. From 15 to 30 dpi, the expression levels of BjNIT2 and BjNIT4 in infected roots were lower than those in non-infected roots. Finally, BjNIT2 in treatment was down approximately to control at 40 dpi. Our results suggest that BjNIT1, which promoted overproductions of auxin, might be involved in P. brassicae infection of B. juncea.
基金supported by the emarked fund for Moden Agro-Industry Technology Research System, China (CARS25)the National Natural Science Foundation of China (31201473)the Key Laboratory of Biology and Genetic Improvement of Horticulture Crops, Ministry of Agriculture, China
文摘A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA（rDNA） and internal transcribed spacer（ITS）.A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.
基金supported by the Excellent Young Scientist Foundation of Henan Academy of Agricultural Sciences(2016YQ11)the National Key Technology R&D Program(2012BAD02B01-3)+1 种基金the Specialized Scientific Research Fund of Henan Academy of Agricultural Sciences(20157805)the Excellent Technology Innovation Team of Henan Province
文摘Increasing evidence has revealed that micro RNAs play a pivotal role in the post transcriptional regulation of gene expression in response to pathogens in plants. However, there is little information available about the expression patterns of mi RNAs and their targets in Chinese cabbage(Brassica rapa ssp. pekinensis) under Plasmodiophora brassicae stress. In the present study, using deep sequencing and degradome analysis, a genome-wide identification of mi RNAs and their targets during P. brassicae stress was performed. A total of 221 known and 93 potentially novel mi RNAs were successfully identified from two root libraries of one control(635-10CK) and P. brassicae-treated Chinese cabbage samples(635-10T). Of these, 14 known and 10 potentially novel mi RNAs were found to be differentially expressed after P. brassicae treatment. Degradome analysis revealed that the 223 target genes of the 75 mi RNAs could be potentially cleaved. KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis suggested that the putative target genes of the mi RNAs were predominately involved in selenocompound metabolism and plant hormone signal transduction. Then the expression of 12 mi RNAs was validated by quantitative real-time PCR(q RT-PCR). These results provide insights into the mi RNA-mediated regulatory networks underlying the stress response to the plant pathogen P. brassicae.
文摘Many Ri T-DNA-transformed roots were incited on seedling hypocotyl segments of cabbage, cauliflower and oilseed rape following genetic transformation with Agribacterium rhi-zogencs. The transformed roots were in vitro inoculated with the resting spores of Plasmodi-ophora brassicae on solid medium, in liquid medium and semi-solid medium respectively.On solid medium, the resting spores penetrated root hair cells, but the root cortex was not invaded so that no symptom of clubroot galls was developed. In liquid medium, the root cortex was invaded, giving rise to an abnormal division of root cortical tissues and gall formation. In semi-solid medium, the resting spores penetrated the cells of the cut surfaces of root segments and caused the cut surfaces to swell.
基金Financial support from the National Key Research and the Development Program of China (2018YFD0200902, 2016YFD0100202)
文摘Clubroot is a prevailing soil-borne disease affecting rapeseed production worldwide.However,few clubroot resistant rapeseed accessions were available for breeding.Identification and introgression of new clubroot resistant genes from closely related species by distant hybridization is an effective strategy.In the present study,9 radish(Raphanus sativus L.,2n=18,RR)lines resistant to Plasmodiophora brassicae pathotype 4 were used as donors to transfer clubroot resistance into a susceptible rapeseed(Brassica napus L.,2n=38,AACC)line by distant hybridization combined with embryo rescue.Nine intergeneric crosses were made but only 1(411×93039)produced F1 plants both from embryo rescue and natural seed-setting.Authenticity of triploid F1 hybrids(2n=28,ACR)were verified by flower color,cytological observation and molecular marker analysis,and 2 genuine F1 hybrids were identified.After chromosome doubling,these synthetic allohexaploid plants(2n=56,AACCRR)became partially fertile(pollen viability rate=35%)and were backcrossed with rapeseed parent to generate a BC1 population(2n=47,AACCR).Totally 178 BC1 plants were obtained,of which the majority(96.1%)were resistant to clubroot.These backcrossing progenies could be used for the breeding of new rapeseed varieties resistant to clubroot.
