Study on Pathogenicity Difference of Plasmodiophora brassicae Under Different Temperature and pH Conditions

[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.

Research Progress of Differential Systems for Physiological Races of Plasmodiophora brassicae Wor

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.

Expression of Nitrilases in Brassica juncea var. tumida Tsen in Root Galls Caused by Plasmodiophora brassicae

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.

Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

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.

Genome-wide Investigation of micro RNAs and Their Targets in Brassica rapa ssp. pekinensis Root with Plasmodiophora brassicae Infection

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.

Distribution of Rapeseed Clubroot Disease in Hubei Province and Evaluation of Yield Loss

[Objective] The aim of this study was to reveal the distribution of rapeseed clubfoot disease in Hubei Province and to assess the yield loss caused by the pathogen, Plasmodiophora brassicae. [Method] Field surveys were conducted in Shayang, Dangyang, Zhijiang, Yidu and Changyang of Hubei Province during 2009- 2011. Clubfoot disease of rapeseed samples from the surveyed locations were con- firmed by PCR detection and plants infection experiment. The factors of yield and plot yields influenced by clubroot disease were determined in plot experiment. [Re- salt] Rapeseed clubroots were confirmed in Zhijiang and Dangyang and not found in Shayang and Yidu. Clubroot was found in cruciferous vegetables but not in rapeseed d in Changyang. Infection of P. brassicae significantly decreased of the first effective branch numbers of rapeseed, effective pod number per plant, seed number per pod and 1 000-grain weight. Yield of infected rapeseed decreased by 56.4% over non-infected control. [Conclusion] Rapeseed clubroot disease mainly distributed in Zhijiang and Dangyang of Hubei Province. The disease had an economic impact on rapeseed production.

Two adjacent NLR genes conferring quantitative resistance to clubroot disease in Arabidopsis are regulated by a stably inherited epiallelic variation

Clubroot caused by the protist Plasmodiophora brassicae is a major disease affecting cultivated Brassica-ceae.Using a combination of quantitative trait locus(QTL)fine mapping,CRISPR-Cas9 validation,and extensive analyses of DNA sequence and methylation patterns,we revealed that the two adjacent neigh-boring NLR(nucleotide-binding and leucine-rich repeat)genes AT5G47260 and AT5G47280 cooperate in controlling broad-spectrum quantitative partial resistance to the root pathogen P.brassicae in Arabidopsis and that they are epigenetically regulated.The variation in DNA methylation is not associated with any nucleotide variation or any transposable element presence/absence variants and is stably inherited.Vari-ations in DNA methylation at the Pb-At5.2 QTL are widespread across Arabidopsis accessions and corre-late negatively with variations in expression of the two genes.Our study demonstrates that natural,stable,and transgenerationally inherited epigenetic variations can play an important role in shaping resistance to plant pathogens by modulating the expression of immune receptors.

WeiTsing: a new face of Ca^(2+)-permeable channels in plant immunity

Plants employ pattern-and effector-triggered immunity(PTI and ETI)to synergistically defend invading pathogens and insect herbivores.Both PTI and ETI can induce cytosolic Ca^(2+)spikes,despite in different spatiotemporal patterns,to activate downstream Ca^(2+)-dependent immune signaling cascades.While multiple families of Ca^(2+)-permeable channels at the plasma membrane have been uncovered,the counterparts responsible for Ca^(2+)release from intracellular stores remain poorly understood.In a groundbreaking paper published recently by Cell,the authors reported that WeiTsing,an Arabidopsis endoplasmic reticulum(ER)-resident protein that was specifically expressed in the pericycle upon Plasmodiophora brassicae(Pb)infection,could form resistosome-like Ca^(2+)-conducting channel and protect the stele of Brassica crops from Pb colonization.As the channel activity of WeiTsing was indispensable for its immune function,the findings highlight a previously underappreciated role of Ca^(2+)release from intracellular repertoire in promoting plant disease resistance.