Methods for Assessment of Viability and Germination of Plasmodiophora brassicae Resting Spores

Clubroot caused by the obligate biotrophic parasite Plasmodiophora brassicae is a destructive soil borne disease of cruciferous crops. Resting spores of P. brassicae can survive in the soil for a long period without hosts or external stimulants. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. In this study, we evaluated several methods for the assessment of viability and germination rate of P. brassicae resting spores. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Due to DNA degradation of heat treatments, acetone was selected to evaluate the efficiency of propidium monoazide (PMA)–quantitative PCR (qPCR) used for the quantification of DNA from viable cells. The staining with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) and the use of differential interference contrast microscopy were suitable for the determination of resting spore germination rates. The latter method also allowed recording individual germination states of spores. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. brassicae resting spores. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control.

Severity of clubroot in kale related to management practices and soil attributes

ABSTRACT: Clubroot disease, caused by Plasmodiophora brassicae, limits the production of Brassica spp. worldwide. Little is known about the factors related to the development of the disease in kale (Brassica oleracea var. acephala) plants and in crops in mountainous areas under tropical conditions. This study examined the severity of clubroot in kale crops as well as identify potential flaws in management and the soil and relief factors related to its occurrence. The study was conducted in 24 kale fields in the mountainous region of Rio de Janeiro (Brazil). Soil and kale growth management practices adopted in the region were identified and samples of soil and plants were collected. Subsequently, soil and relief attributes, disease severity, biomass and nutrient and Al contents and accumulation in the plants were determined. There was a high spread of the pathogen in the areas. Inappropriate and recurrent practices in the region were detected, e.g., sequential cultivation of host species, low adoption of soil fertility analysis and liming and conservation practices, and community use of agricultural machinery and implements without prior cleaning. The disease was associated with more acidic soils, subject to greater water accumulation and with high levels of Al3+ as well as with higher Al contents and accumulation in the roots. Management practices must be adopted in the region to reduce the potential inoculum of P. brassicae and to increase soil fertility.

A Novel Target (Oxidation Resistant 2) in Arabidopsis thaliana to Reduce Clubroot Disease Symptoms via the Salicylic Acid Pathway without Growth Penalties

The clubroot disease (Plasmodiophora brassicae) is one of the most damaging diseases worldwide among brassica crops. Its control often relies on resistant cultivars, since the manipulation of the disease hormones, such as salicylic acid (SA) alters plant growth negatively. Alternatively, the SA pathway can be increased by the addition of beneficial microorganisms for biocontrol. However, this potential has not been exhaustively used. In this study, a recently characterized protein Oxidation Resistant 2 (OXR2) from Arabidopsis thaliana is shown to increase the constitutive pathway of SA defense without decreasing plant growth. Plants overexpressing AtOXR2 (OXR2-OE) show strongly reduced clubroot symptoms with improved plant growth performance, in comparison to wild type plants during the course of infection. Consequently, oxr2 mutants are more susceptible to clubroot disease. P. brassicae itself was reduced in these galls as determined by quantitative real-time PCR. Furthermore, we provide evidence for the transcriptional downregulation of the gene encoding a SA-methyltransferase from the pathogen in OXR2-OE plants that could contribute to the phenotype.

Root Transcriptome and Metabolome Profiling Reveal Key Phytohormone-Related Genes and Pathways Involved Clubroot Resistance in Brassica rapa L.

Plasmodiophora brassicae, an obligate biotrophic pathogen-causing clubroot disease, can seriously affect Brassica crops worldwide, especially Chinese cabbage. Understanding the transcriptome and metabolome profiling changes during the infection of P. brassicae will provide key insights in understanding the defense mechanism in Brassica crops. In this study, we estimated the phytohormones using targeted metabolome assays and transcriptomic changes using RNA sequencing (RNA-seq) in the roots of resistant (BrT24) and susceptible (Y510-9) plants at 0, 3, 9, and 20 days after inoculation (DAI) with P. brassicae. Differentially expressed genes (DEGs) in resistant vs. susceptible lines across different time points were identified. The weighted gene co-expression network analysis of the DEGs revealed six pathways including “Plant–pathogen interaction” and “Plant hormone signal transduction” and 15 hub genes including pathogenic type III effector avirulence factor gene (RIN4) and auxin-responsive protein (IAA16) to be involved in plants immune response. Inhibition of Indoleacetic acid, cytokinin, jasmonate acid, and salicylic acid contents and changes in related gene expression in R-line may play important roles in regulation of clubroot resistance (CR). Based on the combined metabolome profiling and hormone-related transcriptomic responses, we propose a general model of hormone-mediated defense mechanism. This study definitely enhances our current understanding and paves the way for improving CR in Brassica rapa.

Plasmodiophora brassicae in Mexico, from anecdote to fact

For years, the presence of clubroot disease and its causal agent, Plasmodiophora brassicae, in Mexico has been given by granted. However, after a long search in the scientific literature in English and Spanish, as well as grey literature including thesis and government reports, we were not able to find any information regarding the actual detection of the pathogen, hosts affected, areas with the disease, or any real information about clubroot ('hernia de la col', in Mexico). To confirm if P. brassicae was indeed in Mexico, we started a true detective adventure. First, we identified agricultural communities in south-east Mexico known to grow cruciferous crops. Second, we asked to the growers if they have ever seen clubroot symptoms, showing them during the inquires pictures of the characteristic galls that might have been present in their crops. Third, we collected soil from two of the communities with positive response and grew an array of cruciferous in the soil as baits to 'fish' the clubroot pathogen. We detected the presence of galls in the roots of 32 plants and observed the presence of resting spores. Through a P. brassicae specific PCR assay, we were able to confirm the presence of the clubroot pathogen in the samples and in Mexico for the very first time. This study is the first report and identification of P. brassicae in Mexico, opening the doors to understand the genetic diversity of this elusive and devastating plant pathogen.

Unraveling the Association between Metabolic Changes in Inter-Genus and Intra-Genus Bacteria to Mitigate Clubroot Disease of Chinese Cabbage

Clubroot disease caused by the obligate parasite Plasmodiophora brassicae is a serious threat to cabbage production worldwide. Current clubroot control primarily relies on a fungicide, but this has a negative impact on the environment and the use of a single biocontrol agent cannot efficiently control the disease. Thus, the combined application of different biocontrol agents has been proposed as a promising alternative. In this study, we used bacterial biocontrol agents as a co-culture (inter-genus and intra-genus) and mono-culture to mitigate the clubroot disease of Chinese cabbage. We evaluated their biocontrol effect and plant growth promoter (PGP) traits in in vitro and in vivo experiments. This study revealed that the inter-genus bacterial co-culture significantly suppresses the incidence of clubroot disease and enhances plant growth compared with intra-genus and mono-culture. In pairwise interaction, we observed that Bacillus cereus BT-23 promotes the growth of Lysobacter antibioticus 13-6 (inter-genus bacterial co-culture), whereas L. capsici ZST1-2 and L. antibioticus 13-6 (intra-genus microbial co-culture) are antagonists to each other. Furthermore, a total of 5575 metabolites, 732 differentially expressed metabolites (DEMs), and 510 unique metabolites were detected through the LC-MS/MS technique in the bacterial co-culture. The number of unique metabolites in inter-genus bacterial co-culture (393 metabolites) was significantly higher than in the intra-genus bacterial co-culture (117 metabolites). Further analysis of DEMs showed that the DEMs were mainly involved in four kinds of metabolism pathways, i.e., carbohydrate metabolism, amino metabolism, nucleotide metabolism, and metabolism of cofactors and vitamins. The contents of some secondary metabolites with biocontrol activity and plant growth-promoting functions were increased in inter-genus bacterial co-culture, indicating that inter-genus bacterial co-culture has a solid potential to suppress clubroot disease. We conclude that the inter-genus bacterial interaction changes the community metabolism and improves several secondary metabolites functions with respect to disease control and PGP ability.

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

Purpose The 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. Methods In 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. Results Fluazinam 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. Conclusion Among 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.

Effect of pathogen virulence on pathogenicity, host range and reproduction of Plasmodiophora brassicae, the causal agent of clubroot disease

A series of greenhouse experiments was conducted to evaluate the effect of Plasmodiophora brassicae virulence on clubroot development and propagation of resting spores in 86 plant species from 19 botanical families. Plants were artificially inoculated with two isolates of P. brassicae, which were either virulent on clubroot-resistant oilseed rape cv. Mendel (P1 (+)) or avirulent on this cultivar (P1). Clubroot severity and the number of resting spores inside the roots were assessed 35 days post inoculation. Typical clubroot symptoms were observed only in the Brassicaceae family. P1 (+)-inoculated species exhibited more severe symptoms (2 to 10–fold more severe), bigger galls (1.1 to 5.8 fold heavier) and higher number of resting spores than the P1-inoculated plants. Among all Brassica species, Bunias orientalis, Coronopus squamatus and Raphanus sativus were fully resistant against both isolates, while Camelina sativa, Capsella bursa-pastoris, Coincya momensis, Descurainia sophia, Diplotaxis muralis, Erucastrum gallicum, Neslia paniculata, Sinapis alba, S. arvensis, Sisymbrium altissimum, S. loeselii and Thlaspi arvense were highly susceptible. Conringia orientalis, Diplotaxis tenuifolia, Hirschfeldia incana, Iberis amara, Lepidium campestre and Neslia paniculata were completely or partially resistant to P1-isolate but highly susceptible to P1 (+). These results propose that the basis for resistance in these species may be similar to that found in some commercial cultivars, and that these species could contribute to the build-up of inoculum of virulent pathotypes. Furthermore, the pathogen DNA was detected in Alopecurus myosuroides, Phacelia tanacatifolia, Papaver rhoeas and Pisum sativum. It can concluded that the number and diversity of hosts for P. brassicae are greater than previously reported.

Identification and Characterization of Circular RNAs in Brassica rapa in Response to Plasmodiophora brassicae

BackgroundPlasmodiophora brassicae is a soil-borne pathogen that attacks the roots of cruciferous plants, causing clubroot disease. CircRNAs are non-coding RNAs widely exist in plant and animal species which can acting as “microRNA (miRNA) sponges” and “competing endogenous RNAs (ceRNAs)”. Knowledge of circRNAs has been updated continuously and rapidly. However, the information about circRNAs in the regulation of clubroot-disease resistance is limited in Brassica rapa. ResultsHere, the Chinese cabbage (BJN 222) containing clubroot resistance gene (CRa) resistant to the Pb4 was susceptible to the PbE of P. brassicae. To investigate the mechanism of cicRNAs responsible for clubroot-disease resistance in Brassica rapa, the circRNA-seq was performed roots of BJN 222 at 0 d, 8 d, and 23 d after inoculated with Pb4 and PbE. A total of 1636 circRNAs were detected distributed on 10 chromosomes. Furthermore, total 231 differentially expressed circRNAs between groups were screened. Parental genes of circRNAs functions analysis results indicated that the expression of circRNAs was affected not only by inoculation time but also by the pathogenicity of P. brassicae. However, the “Phenylalanine, tyrosine, and tryptophan biosynthesis” pathway was significant enriched between the two pathotypes at different inoculation times. All the expression of target genes annotated with “receptor-like protein kinase,” “zinc finger protein,” “LRR-repeat protein,” and “hormone-related” identified from the circRNA-miRNA-mRNA network were analyzed. 5 target genes were consistent with the expression pattern of novel_circ_000495 at 8 dpi, but only Bra026508 was significantly up-regulated. ConclusionThe up-regulated novel_circ_000495 might suppressed the expression of miR5656-y, leading to the up-regulation of Bra026508. Our results provided new insights to clubroot resistance mechanisms of B.rapa and laid a foundation for further research on the function of circRNAs responsible for the pathogen infection.