Brassicae Infection Research Articles

A Combined mRNA and microRNA Transcriptome Analysis of B. oleracea Response to Plasmodiophora brassicae Infection

Clubroot disease, caused by the pathogen Plasmodiophora brassicae, is a serious disease that poses a critical threat to cabbage production. However, the molecular mechanism of the microRNAs (miRNAs) involved in the cabbage’s response to P. brassicae infection remains to be elucidated. Here, the mRNA and miRNA expression profiles of cabbage in response to a P. brassicae infection were analyzed. In the transcriptome analysis, 2217 and 5552 differentially expressed genes (DEGs) were identified at 7d and 21d after inoculation, which were enriched in MAPK signaling, plant–pathogen interaction, plant hormone signal transduction, and phenylpropanoid biosynthesis pathways. BolC02g057640.2J, BolC09g006890.2J, BolC02g013230.2J, BolC06g006490.2J, BolC03g052660.2J, BolC07g052580.2J, and BolC04g044910.2J were predicted to be significantly involved in the defense response or plant–pathogen interaction through co-expression network analysis. Small RNA data analysis identified 164 miRNAs belonging to 51 families. miR1515, miR166, miR159, and miR9563 had the greatest number of members among the miRNA families. Integrated analysis revealed 23 miRNA–mRNA interactions related to a P. brassicae infection. The target genes of differentially expressed miRNAs (DEMs) revealed the NAC, ARF, TCP, and SPL transcription factor members that probably participate in the defense response. This study provided new insights into the miRNA-involved regulatory system during the process of disease infection with P. brassicae in cabbage.

Bioinformatics and functional analysis of EDS1 genes in Brassica napus in response to Plasmodiophora brassicae infection

Enhanced Disease Susceptibility 1 (EDS1) is a key regulator of plant-pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) responses. In the Brassica napus genome, we identified six novel EDS1 genes, among which four were responsive to clubroot infection, a major rapeseed disease resistant to chemical control. Developing resistant cultivars is a potent and economically viable strategy to control clubroot infection. Bioinformatics analysis revealed conserved domains and structural uniformity in Bna-EDS1 homologs. Bna-EDS1 promoters harbored elements associated with diverse phytohormones and stress responses, highlighting their crucial roles in plant defense. A functional analysis was performed with Bna-EDS1 overexpression and RNAi transgenic lines. Bna-EDS1 overexpression boosted resistance to clubroot and upregulated defense-associated genes (PR1, PR2, ICS1, and CBP60), while Bna-EDS1 RNAi increased plant susceptibility, indicating suppression of the defense signaling pathway downstream of NBS-LRRs. RNA-Seq analysis identified key transcripts associated with clubroot resistance, including phenylpropanoid biosynthesis. Activation of SA regulator NPR1, defense signaling markers PR1 and PR2, and upregulation of MYC-TFs suggested that EDS1-mediated clubroot resistance potentially involves the SA pathway. Our findings underscore the pivotal role of Bna-EDS1-dependent mechanisms in resistance of B. napus to clubroot disease, and provide valuable insights for fortifying resistance against Plasmodiophora brassicae infection in rapeseed.

Development of a rapid and efficient system for CR genes identification based on hairy root transformation in Brassicaceae

Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection. Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease. The traditional way of R gene functional validation requires stable transformation that is both time- and labor-consuming. In this study, a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed. The transformation positive rate was over 80% in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation. The system was applicable to different B. napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea. In particular, two known CR genes, CRA3.7.1 and CRA8.2.4 were used respectively, as example to show that the system works well for CR gene study combined with subsequent P. brassicae infection in B. napus. Most importantly, it works both in over-expression that led to disease resistance, as well as in RNAi which led to disease susceptible phenotype. Therefore, this system can be used in batch-wise identification of CR genes, and also offered the possibility of manipulating key genes within the P. brassicae genome that could improve our knowledge on host–pathogen interaction.

Systematic analysis of Heat Shock Protein 70 (HSP70) gene family in radish and potential roles in stress tolerance

The 70 kD heat shock proteins (HSP70s) represent a class of molecular chaperones that are widely distributed in all kingdoms of life, which play important biological roles in plant growth, development, and stress resistance. However, this family has not been systematically characterized in radish (Raphanus sativus L.). In this study, we identified 34 RsHSP70 genes unevenly distributed within nine chromosomes of R. sativus. Phylogenetic and multiple sequence alignment analyses classified the RsHSP70 proteins into six distinct groups (Group A–F). The characteristics of gene structures, motif distributions, and corresponding cellular compartments were more similar in closely linked groups. Duplication analysis revealed that segmental duplication was the major driving force for the expansion of RsHSP70s in radish, particularly in Group C. Synteny analysis identified eight paralogs (Rs-Rs) in the radish genome and 19 orthologs (Rs-At) between radish and Arabidopsis, and 23 orthologs (Rs-Br) between radish and Chinese cabbage. RNA-seq analysis showed that the expression change of some RsHSP70s were related to responses to heat, drought, cadmium, chilling, and salt stresses and Plasmodiophora brassicae infection, and the expression patterns of these RsHSP70s were significantly different among 14 tissues. Furthermore, we targeted a candidate gene, RsHSP70–23, the product of which is localized in the cytoplasm and involved in the responses to certain abiotic stresses and P. brassicae infection. These findings provide a reference for further molecular studies to improve yield and stress tolerance of radish.

Identification of transcriptional modules linked to the drought response of Brassica napus during seed development and their mitigation by early biotic stress.

In order to capture the drought impacts on seed quality acquisition in Brassica napus and its potential interaction with early biotic stress, seeds of the 'Express' genotype of oilseed rape were characterized from late embryogenesis to full maturity from plants submitted to reduced watering (WS) with or without pre-occurring inoculation by the telluric pathogen Plasmodiophora brassicae (Pb + WS or Pb, respectively), and compared to control conditions (C). Drought as a single constraint led to significantly lower accumulation of lipids, higher protein content and reduced longevity of the WS-treated seeds. In contrast, when water shortage was preceded by clubroot infection, these phenotypic differences were completely abolished despite the upregulation of the drought sensor RD20. A weighted gene co-expression network of seed development in oilseed rape was generated using 72 transcriptomes from developing seeds from the four treatments and identified 33 modules. Module 29 was highly enriched in heat shock proteins and chaperones that showed a stronger upregulation in Pb + WS compared to the WS condition, pointing to a possible priming effect by the early P. brassicae infection on seed quality acquisition. Module 13 was enriched with genes encoding 12S and 2S seed storage proteins, with the latter being strongly upregulated under WS conditions. Cis-element promotor enrichment identified PEI1/TZF6, FUS3 and bZIP68 as putative regulators significantly upregulated upon WS compared to Pb + WS. Our results provide a temporal co-expression atlas of seed development in oilseed rape and will serve as a resource to characterize the plant response towards combinations of biotic and abiotic stresses.

Proteome- and metabolome-level changes during early stages of clubroot infection in Brassica napus canola.

Clubroot is a destructive root disease of canola (Brassica napus L.) caused by Plasmodiophora brassicae Woronin. Despite extensive research into the molecular responses of B. napus to P. brassicae, there is limited information on proteome- and metabolome-level changes in response to the pathogen, especially during the initial stages of infection. In this study, we have investigated the proteome- and metabolome- level changes in the roots of clubroot-resistant (CR) and -susceptible (CS) doubled-haploid (DH) B. napus lines, in response to P. brassicae pathotype 3H at 1-, 4-, and 7-days post-inoculation (DPI). Root proteomes were analyzed using nanoflow liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS). Comparisons of pathogen-inoculated and uninoculated root proteomes revealed 2515 and 1556 differentially abundant proteins at one or more time points (1-, 4-, and 7-DPI) in the CR and CS genotypes, respectively. Several proteins related to primary metabolites (e.g., amino acids, fatty acids, and lipids), secondary metabolites (e.g., glucosinolates), and cell wall reinforcement-related proteins [e.g., laccase, peroxidases, and plant invertase/pectin methylesterase inhibitors (PInv/PMEI)] were identified. Eleven nucleotides and nucleoside-related metabolites, and eight fatty acids and sphingolipid-related metabolites were identified in the metabolomics study. To our knowledge, this is the first report of root proteome-level changes and associated alterations in metabolites during the early stages of P. brassicae infection in B. napus.

WeiTsing: a new face of Ca2+-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 Ca2+ spikes, despite in different spatiotemporal patterns, to activate downstream Ca2+-dependent immune signaling cascades. While multiple families of Ca2+-permeable channels at the plasma membrane have been uncovered, the counterparts responsible for Ca2+ 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 Ca2+-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 Ca2+ release from intracellular repertoire in promoting plant disease resistance.

Evaluation of genetic variation of morphological and clubroot-resistance traits of radish and metabonomic analysis of clubroot-resistant cultivar

Global commercial production of radish (Raphanus sativus) is highly threatened by Plamodiophora brassicae. Germplasm evaluation and the deep analysis of variants with clubroot-resistant trait are needed to produce quality cultivars. The genetic diversity was analyzed for 97 radish germplasms based on 20 agronomic traits and the response to clubroot. Additionally, differentially regulated metabolites (DRMs) between healthy and P. brassicae-infected resistant cultivars were measured. The genetic diversity index values of quality traits varied from 0.396 to 1.350, and that of quantitative traits varied from 1.65–2.15. The variation of radish quantitative traits ranged from 19.18% to 54.44%. The principal component analysis indicated 8 comprehensive indicators, allowing classification of germplasms into 4 classes, with 71 in class I, 20 in class II, 4 in class III, and 2 in class IV. The results of classification of germplasms by the response to clubroot infection only or by comprehensive agronomic traits including response of plants to clubroot disease infection were similar. Among all traits assessed in the present study, the clubroot severity and the depth of in-soil root were closely linked. Metabolomic analysis revealed that the metabolites changes of clubroot resistant cultivar “DHBC” pre- and post- P. brassicae infection was relatively stable. P. brassicae infection resulted in limited DRMs, significant down-regulation was identified on ginsenoside, dihydro-2(3H)-thiophenone, proline, 1-pyrroline, while apiosylglucosyl 4-hydroxybenzoate, chrycolide, glucomalcomiin, and phosphocholine were significantly up-regulated. We revealed more extensive genetic variation in quantitative traits than in quality indexes in radish and we summarized that the response of radish to clubroot disease may be a dominate indicator in evaluation of radish varieties. Finally, untargeted metabolomic study confirmed the clubroot resistance of “DHBC” and revealed a sophisticated metabolites (including some precious pharmaceutical ingredients) variation in response to clubroot infection, offers new directions in the studies of clubroot resistance mechanism of radish.

Integrative Transcriptome, miRNAs, Degradome, and Phytohormone Analysis of Brassica rapa L. in Response to Plasmodiophora brassicae.

Clubroot is an infectious root disease caused by Plasmodiophora brassicae in Brassica crops, which can cause immeasurable losses. We analyzed integrative transcriptome, small RNAs, degradome, and phytohormone comprehensively to explore the infection mechanism of P. brassicae. In this study, root samples of Brassica rapa resistant line material BrT24 (R-line) and susceptible line material Y510-9 (S-line) were collected at four different time points for cytological, transcriptome, miRNA, and degradome analyses. We found the critical period of disease resistance and infection were at 0-3 DAI (days after inoculation) and 9-20 DAI, respectively. Based on our finding, we further analyzed the data of 9 DAI vs. 20 DAI of S-line and predicted the key genes ARF8, NAC1, NAC4, TCP10, SPL14, REV, and AtHB, which were related to clubroot disease development and regulating disease resistance mechanisms. These genes are mainly related to auxin, cytokinin, jasmonic acid, and ethylene cycles. We proposed a regulatory model of plant hormones under the mRNA-miRNA regulation in the critical period of P. brassicae infection by using the present data of the integrative transcriptome, small RNAs, degradome, and phytohormone with our previously published results. Our integrative analysis provided new insights into the regulation relationship of miRNAs and plant hormones during the process of disease infection with P. brassicae.

Genome-Wide Identification and Functional Characterization of Stress Related Glyoxalase Genes in Brassica napus L.

Rapeseed (Brassica napus L.) is not only one of the most important oil crops in the world, but it is also an important vegetable crop with a high value nutrients and metabolites. However, rapeseed is often severely damaged by adverse stresses, such as low temperature, pathogen infection and so on. Glyoxalase I (GLYI) and glyoxalase II (GLYII) are two enzymes responsible for the detoxification of a cytotoxic metabolite methylglyoxal (MG) into the nontoxic S-D-lactoylglutathione, which plays crucial roles in stress tolerance in plants. Considering the important roles of glyoxalases, the GLY gene families have been analyzed in higher plans, such as rice, soybean and Chinese cabbage; however, little is known about the presence, distribution, localizations and expression of glyoxalase genes in rapeseed, a young allotetraploid. In this study, a total of 35 BnaGLYI and 30 BnaGLYII genes were identified in the B. napus genome and were clustered into six and eight subfamilies, respectively. The classification, chromosomal distribution, gene structure and conserved motif were identified or predicted. BnaGLYI and BnaGLYII proteins were mainly localized in chloroplast and cytoplasm. By using publicly available RNA-seq data and a quantitative real-time PCR analysis (qRT-PCR), the expression profiling of these genes of different tissues was demonstrated in different developmental stages as well as under stresses. The results indicated that their expression profiles varied among different tissues. Some members are highly expressed in specific tissues, BnaGLYI11 and BnaGLYI27 expressed in flowers and germinating seed. At the same time, the two genes were significantly up-regulated under heat, cold and freezing stresses. Notably, a number of BnaGLY genes showed responses to Plasmodiophora brassicae infection. Overexpression of BnGLYI11 gene in Arabidopsis thaliana seedlings confirmed that this gene conferred freezing tolerance. This study provides insight of the BnaGLYI and BnaGLYII gene families in allotetraploid B. napus and their roles in stress resistance, and important information and gene resources for developing stress resistant vegetable and rapeseed oil.

The root meristem growth factor BrRGF6 positively regulates Chinese cabbage to infection of clubroot disease caused by Plasmodiophora Brassicae.

Chinese cabbage has a high annual demand in China. However, clubroot disease caused by the infection of Plasmodiophora brassicae seriously affects its yield. Transcriptome analysis identified a root meristem growth factor 6 (BrRGF6) as significantly up-regulated in Chinese cabbage roots infected with Plasmodiophora brassicae. Quantitative reverse-transcription polymerase chain reaction and in situ hybridization analysis showed higher BrRGF6 expression in susceptible materials than in resistant materials. After Plasmodiophora brassicae infection, BrRGF6 expression was significantly up-regulated, especially in susceptible materials. Gene function analysis showed that the roots of Arabidopsis mutant rgf6 grew faster than the wild-type, and delayed the infection progress of Plasmodiophora brassicae. A Protein, nuclear transcription factor Y subunit C (BrNF-YC), was screened from yeast two-hybrid library of Chinese cabbage induced by Plasmodiophora brassicae, and verified to interact with BrRGF6 by yeast two-hybrid co-transfer. Yeast one-hybrid and β-Glucuronidase activity analysis showed that BrNF-YC could directly bind to and strongly activate the promoter of BrRGF6. Transgenic verification showed that BrRGF6 or BrNF-YC silenced Chinese cabbage significantly decreased the expression of BrRGF6, accelerated root development, and reduced incidence of clubroot disease. However, after overexpression of BrRGF6 or BrNF-YC, the phenotype showed a reverse trend. Therefore, BrRGF6 silencing accelerated root growth and enhanced resistance to clubroot disease, which was regulated by BrNF-YC.

High-throughput sequencing reveals tuber mustard genes responsive to Plasmodiophora brassicae in the early stage of infection

Tuber mustard is a kind of Brassica crop, swollen stem of this crop is the material of mustard pickle, which has high economic value. Clubroot is a serious disease of brassicaceae plants, causing huge losses every year. Although some clubroot resistant cultivars of tuber mustard were bred, the mechanism by which tuber mustard responds to the pathogen Plasmodiophora brassicae needs to be elucidated. Morphologic and cytological changes of tuber mustard roots were observed during the pathogenetic process, and 72 h after inoculation was found to be the key stage of the interaction between the root of tuber mustard and the pathogen. A transcriptome sequencing was performed using the roots inoculated and mock-inoculated by P. brassicae at 72 h. A total of 2294 up-regulated and 6533 down-regulated genes were identified, and the expression levels of 11 differentially expressed genes were verified by qPCR. Genes related to MAPK signaling pathway-plant, plant hormone signal transduction, phenylpropanoid biosynthesis, nitrogen metabolism, ABC transporters, plant-pathogen interaction were involved in tuber mustard response to infection by P. brassicae. After the pathogen inoculation, among the differentially expressed genes, most of the PR1 like genes were upregulated, while most of BAK1 like genes, MAPK like genes, SUMM2 like genes, JAZ like genes, and NPR1 like genes were downregulated. The miRNA sequencing results showed that miR160a-5p, miR319a, miR395i, miR400 and their relative targets were negatively co-expressed, which might be involved in the regulation of tuber mustard response to the pathogen. This study is helpful for further studying the functions of tuber mustard genes during P. brassicae infection.

Comprehensive analysis of the full-length transcripts and alternative splicing involved in clubroot resistance in Chinese cabbage

Chinese cabbage is an economically important Brassica vegetable worldwide, and clubroot, which is caused by the soil-borne protist plant pathogen Plasmodiophora brassicae is regarded as a destructive disease to Brassica crops. Previous studies on the gene transcripts related to Chinese cabbage resistance to clubroot mainly employed RNA-seq technology, although it cannot provide accurate transcript assembly and structural information. In this study, PacBio RS II SMRT sequencing was used to generate full-length transcriptomes of mixed roots at 0, 2, 5, 8, 13, and 22 days after P. brassicae infection in the clubroot-resistant line DH40R. Overall, 39 376 high-quality isoforms and 26 270 open reading frames (ORFs) were identified from the SMRT sequencing data. Additionally, 426 annotated long noncoding RNAs (lncRNAs), 56 transcription factor (TF) families, 1 883 genes with poly(A) sites and 1 691 alternative splicing (AS) events were identified. Furthermore, 1 201 of the genes had at least one AS event in DH40R. A comparison with RNA-seq data revealed six differentially expressed AS genes (one for disease resistance and five for defensive response) that are potentially involved in P. brassicae resistance. The results of this study provide valuable resources for basic research on clubroot resistance in Chinese cabbage.

Ethylene Plays a Dual Role during Infection by Plasmodiophora brassicae of Arabidopsis thaliana.

Plasmodiophora brassicae infection leads to hypertrophy of host roots and subsequent formation of galls, causing huge economic losses to agricultural producers of Cruciferae plants. Ethylene (ET) has been reported to play a vital role against necrotrophic pathogens in the classic immunity system. More clues suggested that the defense to pathogens in roots may be different from the acrial. The ET pathway may play a positive role in the infection of P. brassicae, as shown by recent transcriptome profiling. However, the molecular basis of ET remains poorly understood. In this study, we investigated the potential role of ethylene against P. brassicae infection in an ein3/eil1 double-mutant of Arabidopsis thaliana (A. thaliana). After infection, ein3/eil1 (Disease Index/DI: 93) showed more susceptibility compared with wild type (DI: 75). Then, we inoculated A. thaliana Columbia-0 (Col-0) with P. brassicae by 1-aminocyclopropane-1-carboxylic acid (ACC) and pyrazinamide (PZA), respectively. It was found that the symptoms of infected roots with ACC were more serious than those with PZA at 20 dpi (day post infection). However, the DI were almost the same in different treatments at 30 dpi. WRKY75 can be directly regulated by ET and was upregulated at 7 dpi with ACC, as shown by qRT-PCR. The wrky75-c mutant of A. thaliana (DI: 93.75) was more susceptible than the wild type in Arabidopsis. Thus, our work reveals the dual roles of ET in infection of P. brassicae and provides evidence of ET in root defense against pathogens.

Starch content changes and metabolism-related gene regulation of Chinese cabbage synergistically induced by Plasmodiophora brassicae infection.

Clubroot is one of the major diseases adversely affecting Chinese cabbage (Brassica rapa) yield and quality. To precisely characterize the Plasmodiophora brassicae infection of Chinese cabbage, we developed a dual fluorescent staining method for simultaneously examining the pathogen, cell structures, and starch grains. The number of starch (amylopectin) grains increased in B. rapa roots infected by P. brassicae, especially from 14 to 21 days after inoculation. Therefore, the expression levels of 38 core starch metabolism genes were investigated by quantitative real-time PCR. Most genes related to starch synthesis were up-regulated at 7 days after P. brassicae inoculation, whereas the expression levels of starch degradation-related genes were increased at 14 days after inoculation. Then, genes encoding the core enzymes involved in starch metabolism were investigated by assessing their chromosomal distributions, structures, duplication events, and synteny among Brassica species. Genome comparisons indicated that 38 non-redundant genes belonging to six core gene families related to starch metabolism are highly conserved among Arabidopsis thaliana, B. rapa, Brassica nigra, and Brassica oleracea. Previous genome sequencing projects have revealed that P. brassicae obtained host nutrients by manipulating plant metabolism. Starch may serve as a carbon source for P. brassicae colonization, as indicated by histological observations and transcriptomic analysis. Results of this study may elucidate the evolution and expression of core starch metabolism genes and provide researchers with novel insights into the pathogenesis of clubroot in B. rapa.

Identification of Micro Ribonucleic Acids and Their Targets in Response to Plasmodiophora brassicae Infection in Brassica napus

Clubroot disease, which is caused by the soil-borne pathogen Plasmodiophora brassicae War (P. brassicae), is one of the oldest and most destructive diseases of Brassica and cruciferous crops in the world. Plant microRNAs [micro ribonucleic acids (miRNAs)] play important regulatory roles in several developmental processes. Although the role of plant miRNAs in plant-microbe interaction has been extensively studied, there are only few reports on the specific functions of miRNAs in response to P. brassicae. This study investigated the roles of miRNAs and their targets during P. brassicae infection in a pair of Brassica napus near-isogenic lines (NILs), namely clubroot-resistant line 409R and clubroot-susceptible line 409S. Small RNA sequencing (sRNA-seq) and degradome-seq were performed on root samples of 409R and 409S with or without P. brassicae inoculation. sRNA-seq identified a total of 48 conserved and 72 novel miRNAs, among which 18 had a significant differential expression in the root of 409R, while only one miRNA was differentially expressed in the root of 409S after P. brassicae inoculation. The degradome-seq analysis identified 938 miRNA target transcripts, which are transcription factors, enzymes, and proteins involved in multiple biological processes and most significantly enriched in the plant hormone signal transduction pathway. Between 409R and 409S, we found eight different degradation pathways in response to P. brassicae infection, such as those related to fatty acids. By combining published transcriptome data, we identified a total of six antagonistic miRNA-target pairs in 409R that are responsive to P. brassicae infection and involved in pathways associated with root development, hypersensitive cell death, and chloroplast metabolic synthesis. Our results reveal that P. brassicae infection leads to great changes in miRNA pool and target transcripts. More interestingly, these changes are different between 409R and 409S. Clarification of the crosstalk between miRNAs and their targets may shed new light on the possible mechanisms underlying the pathogen resistance against P. brassicae.

Comparison of the effects of three fungicides on clubroot disease of tumorous stem mustard and soil bacterial community

PurposeThe application of fungicides is one of the main strategies to prevent clubroot disease. Currently, numerous studies focus on changes in the soil microbial community at different levels of clubroot disease severity. However, the effects of fungicides on the soil microbial community and causative pathogen, Plasmodiophora brassicae, while preventing clubroot disease remain unclear.MethodsIn this study, we evaluated the control efficacy of three fungicides (fluazinam, metalaxyl-mancozeb, and carbendazim) on clubroot disease of tumorous stem mustard in greenhouse experiment. Uninoculated and Water treatments after inoculation were performed as controls. At three (3 W) and six weeks (6 W) post-inoculation of P. brassicae, soil properties, bacterial composition (sequencing of 16S rRNA genes), and effector gene expression of the pathogen were analyzed. The correlation of these factors with disease index (DI) was explored.ResultsFluazinam was the most effective in controlling clubroot disease of tumorous stem mustard with a controlled efficacy of 59.81%, and the abundance of P. brassicae in the soil decreased 21.29% after 3 weeks of treatment. Compared with other treatments, twelve out of twenty effector genes showed higher expression in fluazinam 3 W samples. Different fungicides had different effects on soil properties. EC (electrical conductivity), the main factor that positively associated with DI, was significantly lower in fluazinam treatment than the other two fungicide treatments. The application of fungicides, especially carbendazim, significantly reduced bacterial α-diversity and the composition of soil bacteria. Pseudomonas, Microbacterium, and Sphingobacterium (positively correlated with DI) were enriched in Water, metalaxyl-mancozeb, and carbendazim treatments, but were less abundant in fluazinam treatment. Among the three fungicide treatments, DI was significantly negatively correlated with Shannon and Chao 1 indices. Soil properties and the top bacterial genera that positively correlated with DI were influenced to a lesser degree in the fluazinam treatment.ConclusionAmong three fungicides, fluazinam was the most effective agent with the highest control effects against clubroot disease. The strong virulence of fluazinam against P. brassicae was one of the main reasons for the prevention of clubroot disease, and in addition the alteration of rhizosphere bacterial community by fluazinam to the detriment of P. brassicae infection. Based on our results, EC could be an indicator of the severity of clubroot disease.

Interactions in the Brassica napus–Pyrenopeziza brassicae pathosystem and sources of resistance to P. brassicae (light leaf spot)

AbstractPyrenopeziza brassicae, cause of light leaf spot (LLS), is an important pathogen of oilseed rape and vegetable brassicas and has a wide geographic distribution. Exploitation of host resistance remains the most sustainable and economically viable solution for disease management. This study evaluated 18 oilseed rape cultivars or breeding lines for host resistance against P. brassicae in glasshouse experiments. Selected cultivars/lines were inoculated with eight single‐spore isolates of the pathogen obtained from three different regions in England. Analysis of P. brassicae infection‐related changes on host plants identified leaf deformation as a characteristic feature associated with P. brassicae infection, this showed poor correlation to LLS severity measured as the amount of pathogen sporulation on infected plants. Resistant host phenotypes were identified by limitation of P. brassicae sporulation, with or without the presence of a necrotic response (black flecking phenotype). Investigation of this pathosystem revealed significant differences between cultivars/lines, between isolates, and significant cultivar/line‐by‐isolate interactions. In total, 37 resistant and 16 moderately resistant interactions were identified from 144 cultivar/line‐by‐isolate interactions using statistical methods. Most of the resistant/moderately resistant interactions identified in this study appeared to be nonspecific towards the isolates tested. Our results suggested the presence of isolate‐specific resistant interactions for some cultivars. Several sources of resistance have been identified that are valuable for oilseed rape breeding programmes.

Mitogen-Activated Protein Kinase MAPKKK7 from Plasmodiophora brassicae Regulates Low-Light-Dependent Nicotiana benthamiana Immunity.

MAPKKK is the largest family of mitogen-activated protein kinase (MAPK) cascades and is known to play important roles in plant pathogen interaction by regulating fungal cell proliferation, growth, and pathogenicity. Thus far, only a few have been characterized because of the functional redundancy of MAPKKKs. In this study, it is interesting that Plasmodiophora brassicae (Pb)MAPKKK7 was clustered into the A3 subgroup of plant MAPKKKs by a phylogenetic analysis and also with the BCK1 and STE groups of fungal MAPKKKs. PbMAPKKK7 function in reactive oxygen species accumulation and cell death in Nicotiana benthamiana was characterized. Agroinfiltration with the PbMAPKKK7 mutated protein kinase domain relieved these changes. Interestingly, the induction of cell death was dependent on light intensity. Transcriptional profiling analysis demonstrated that PbMAPKKK7 was highly expressed during cortex infection stages, indicating its important role in P. brassicae infection. These functional analyses of PbMAPKKK7 build knowledge of new roles of the MAPK cascade pathway in N. benthamiana and P. brassicae interactions.

In silico analysis and overexpression of chitinase class IV gene in Brassica juncea improves resistance against Alternaria brassicae

Indian mustard (Brassica juncea), an elite oilseed crop in the Brassicae family is challenged by various biotic stresses. Alternaria leaf blight is the most devastating disease in B. juncea caused by Alternaria brassicae. Hence engineering B. juncea plants for improved Alternaria disease resistance becomes imperative. Plant chitinases are key players in the plant immune system that offers successful protection to fungal infections over a broad spectrum of diseases. Chitinase class IV gene was isolated and functionally characterized in Indian mustard (Brassica juncea). Based on phylogenetic analysis, Bj chitinase class IV gene was closely related to Brassica napus followed by other Brassica species. To understand the defensive role of this gene, it was cloned in (Pet28a+) vector and expressed in the prokaryotic expression host Escherichia coli BL21. The purified protein showed antifungal activity against A. brassicae. To further discern its role in disease resistance, B. juncea transgenic lines were raised by overexpressing Bj chitinase IV gene and further confirmed by PCR and southern blotting. In addition, independent B. juncea transgenic lines with high transcript levels of chitinase class IV gene were selected for disease assays against A. brassicae infection. Pathological studies showed that the rate of A. brassicae lesions development and size was significantly reduced in the Bj chitinase transgenic lines compared with non transgenic plants. Interestingly, Bj chitinase transgenic lines showed increased activity of ROS scavenging enzymes when compared to control plants. Collectively our results suggest that overexpression of Bj chitinase IV gene in B. juncea improves resistance against Alternaria leaf blight.