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.

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Plasmodiophora brassicae (club root).

10.1079/cpc.41865.20210102194 ◽

2021 ◽

Author(s):

Graham McGrann

Keyword(s):

Crop Rotation ◽

Plasmodiophora Brassicae ◽

Brassica Species ◽

Infested Soil ◽

Yield Losses ◽

Varietal Resistance ◽

Weed Species ◽

Clubroot Disease ◽

Local Spread ◽

Resting Spores

Abstract Plasmodiophora brassicae is a root-infecting protist pathogen that causes clubroot disease in brassica species. The organism is soil-borne and has long-lived resting spores that can survive in soil for more than 15 years. Local spread of motile zoospores can be facilitated by wet conditions but most dispersal of the pathogen is through the movement of infested soil. P. brassicae has a wide host range in the brassica family including numerous weed species. Control of the disease is difficult but clubroot can be managed by a combination of crop rotation, varietal resistance, improved agronomic practice such as improved drainage and the application of lime of related products to raise pH which can limit the effects of the disease. There are currently no effective fungicides for the widespread control of clubroot. Yield losses range from 10 to 15% but can exceed 50% under disease conducive environmental conditions.

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First Report of Clubroot on Capsella bursa-pastoris Caused by Plasmodiophora brassicae in Sichuan Province of China

Plant Disease ◽

10.1094/pdis-04-13-0395-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 687-687 ◽

Cited By ~ 2

Author(s):

L. Ren ◽

X. P. Fang ◽

C. C. Sun ◽

K. R. Chen ◽

F. Liu ◽

...

Keyword(s):

Plasmodiophora Brassicae ◽

Tap Water ◽

Disease Incidence ◽

Morphological Characteristics ◽

Pcr Amplification ◽

Sichuan Province ◽

Clubroot Disease ◽

Cruciferous Vegetable ◽

First Report ◽

Resting Spores

Shepherd's purse (Capsella bursa-pastoris (L.) Medicus) is an edible and wild medicinal plant widely distributed in China. This plant has been cultivated in Shanghai, China, since the end of the 19th century. Infection of C. bursa-pastoris by Plasmodiophora brassicae, the causal agent of clubroot disease on Brassica spp. has been reported in Korea (2), but is not known to occur in China. In February of 2011, stunted and wilted shepherd's purse (SP) plants were observed in a field planted to oilseed rapes (B. napus) in Sichuan Province of China. Symptomatic SP plants also exhibited root galls. Disease incidence was 6.2% and 100% for SP and B. napus, respectively. Root galls on diseased SP plants were collected for pathogen identification. Many resting spores were observed when the root galls were examined under a light microscope. The resting spores were circular in shape, measuring 2.0 to 3.1 μm in diameter (average 2.6 μm). PCR amplification was conducted to confirm the pathogen. DNA was extracted from root galls and healthy roots (control) of SP. Two primers, TC2F (5′-AAACAACGAGTCAGCTTGAATGCTAGTGTG-3′) and TC2R (5′-CTTTAGTTGTGTTTCGGCTAGGATGGTTCG-3′) were used to detect P. brassicae (1). No PCR amplifications were observed with the control DNA as template. A fragment of the expected size (approximately 520 bp) was obtained when DNA was amplified from diseased roots of SP. These results suggest that the pathogen in the galled roots of SP is P. brassicae. Pathogenicity of P. brassicae in SP was tested on plants of both SP and Chinese cabbage (CC) (B. campestris ssp. pekinensis). A resting spore suspension prepared from naturally infected SP roots was mixed with a sterilized soil in two plastic pots, resulting in a final concentration of 5 × 106 spores/g soil. Soil treated with the same volume of sterile water was used as a control. Seeds of SP and CC were pre-germinated on moist filter paper for 2 days (20°C) and seeded into the infested and control pots, one seed per pot for planted for CC and four seeds per pot for SP. The pots were placed in a chamber at 15 to 25°C under 12 h light and 12 h dark. Plants in each pot were uprooted after 4 weeks and the roots of each plant were washed under tap water and rated for clubroot disease. No disease symptoms were observed in the control treatments of SP or CC. Plants of both species showed symptoms of clubroot, with the disease incidence of 62.5% and 100% on SP and CC, respectively. The pathogen was isolated from diseased roots of each plant and confirmed as P. brassicae based on morphological characteristics and PCR detection. To our knowledge, this is the first report of clubroot disease on C. bursa-pastoris in Sichuan Province of China. This finding suggests that it may be necessary to manage C. bursa-pastoris in cruciferous vegetable (cabbage, turnip) and oilseed rape production fields. References: (1) T. Cao et al. Plant Dis. 91:80, 2007. (2) W. G. Kim et al. Microbiology 39:233, 2011.

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Expression and Role of Biosynthetic, Transporter, Receptor, and Responsive Genes for Auxin Signaling during Clubroot Disease Development

International Journal of Molecular Sciences ◽

10.3390/ijms21155554 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5554

Author(s):

Arif Hasan Khan Robin ◽

Gopal Saha ◽

Rawnak Laila ◽

Jong-In Park ◽

Hoy-Taek Kim ◽

...

Keyword(s):

Plasmodiophora Brassicae ◽

Fold Increase ◽

Auxin Signaling ◽

Post Inoculation ◽

Disease Development ◽

Secondary Phases ◽

Auxin Receptor ◽

Clubroot Disease ◽

Root Tissues

Auxins play a pivotal role in clubroot development caused by the obligate biotroph Plasmodiophora brassicae. In this study, we investigated the pattern of expression of 23 genes related to auxin biosynthesis, reception, and transport in Chinese cabbage (Brassica rapa) after inoculation with P. brassicae. The predicted proteins identified, based on the 23 selected auxin-related genes, were from protein kinase, receptor kinase, auxin responsive, auxin efflux carrier, transcriptional regulator, and the auxin-repressed protein family. These proteins differed in amino acids residue, molecular weights, isoelectric points, chromosomal location, and subcellular localization. Leaf and root tissues showed dynamic and organ-specific variation in expression of auxin-related genes. The BrGH3.3 gene, involved in auxin signaling, exhibited 84.4-fold increase in expression in root tissues compared to leaf tissues as an average of all samples. This gene accounted for 4.8-, 2.6-, and 5.1-fold higher expression at 3, 14, and 28 days post inoculation (dpi) in the inoculated root tissues compared to mock-treated roots. BrNIT1, an auxin signaling gene, and BrPIN1, an auxin transporter, were remarkably induced during both cortex infection at 14 dpi and gall formation at 28 dpi. BrDCK1, an auxin receptor, was upregulated during cortex infection at 14 dpi. The BrLAX1 gene, associated with root hair development, was induced at 1 dpi in infected roots, indicating its importance in primary infection. More interestingly, a significantly higher expression of BrARP1, an auxin-repressed gene, at both the primary and secondary phases of infection indicated a dynamic response of the host plant towards its resistance against P. brassicae. The results of this study improve our current understanding of the role of auxin-related genes in clubroot disease development.

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Endomembrane-Targeting Plasmodiophora brassicae Effectors Modulate PAMP Triggered Immune Responses in Plants

Frontiers in Microbiology ◽

10.3389/fmicb.2021.651279 ◽

2021 ◽

Vol 12 ◽

Author(s):

Md Musharaf Hossain ◽

Edel Pérez-López ◽

Christopher D. Todd ◽

Yangdou Wei ◽

Peta C. Bonham-Smith

Keyword(s):

Plasmodiophora Brassicae ◽

Plant Immunity ◽

Cell Communication ◽

Inhibitory Effect ◽

Effector Proteins ◽

Post Inoculation ◽

Endomembrane System ◽

Cell Secretion ◽

Clubroot Disease ◽

Golgi Bodies

Plasmodiophora brassicae is a devastating obligate, intracellular, biotrophic pathogen that causes clubroot disease in crucifer plants. Disease progression is regulated by effector proteins secreted by P. brassicae. Twelve P. brassicae putative effectors (PbPEs), expressed at various stages of disease development [0, 2, 5, 7, 14, 21, and 28 days post inoculation (DPI)] in Arabidopsis and localizing to the plant endomembrane system, were studied for their roles in pathogenesis. Of the 12 PbPEs, seven showed an inhibitory effect on programmed cell death (PCD) as triggered by the PCD inducers, PiINF1 (Phytophthora infestans Infestin 1) and PiNPP1 (P. infestans necrosis causing protein). Showing the strongest level of PCD suppression, PbPE15, a member of the 2-oxoglutarate (2OG) and Fe (II)-dependent oxygenase superfamily and with gene expression during later stages of infection, appears to have a role in tumorigenesis as well as defense signaling in plants. PbPE13 produced an enhanced PiINF1-induced PCD response. Transient expression, in Nicotiana benthamiana leaves of these PbPEs minus the signal peptide (SP) (ΔspPbPEGFPs), showed localization to the endomembrane system, targeting the endoplasmic reticulum (ER), Golgi bodies and nucleo-cytoplasm, suggesting roles in manipulating plant cell secretion and vesicle trafficking. ΔspPbPE13GFP localized to plasma membrane (PM) lipid rafts with an association to plasmodesmata, suggesting a role at the cell-to-cell communication junction. Membrane relocalization of ΔspPbPE13GFP, triggered by flagellin N-terminus of Pseudomonas aeruginosa (flg22 – known to elicit a PAMP triggered immune response in plants), supports its involvement in raft-mediated immune signaling. This study is an important step in deciphering P. brassicae effector roles in the disruption of plant immunity to clubroot disease.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.823051 ◽

2022 ◽

Vol 12 ◽

Author(s):

Yao Wang ◽

Birger Koopmann ◽

Andreas von Tiedemann

Keyword(s):

Evans Blue ◽

Plasmodiophora Brassicae ◽

Germination Rate ◽

Inoculum Potential ◽

Differential Interference Contrast Microscopy ◽

Calcofluor White ◽

Clubroot Disease ◽

Resting Spores ◽

Dual Staining

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.

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MAPKK Inhibitor U0126 Inhibits Plasmodiophora brassicae Development

Phytopathology ◽

10.1094/phyto-07-17-0240-r ◽

2018 ◽

Vol 108 (6) ◽

pp. 711-720 ◽

Cited By ~ 2

Author(s):

Tao Chen ◽

Kai Bi ◽

Yanli Zhao ◽

Xueliang Lyu ◽

Zhixiao Gao ◽

...

Keyword(s):

Plasmodiophora Brassicae ◽

Transcriptional Profiling ◽

Specific Inhibitor ◽

Mapk Signaling ◽

Cellular Growth ◽

Mitogen Activated Protein Kinase ◽

Clubroot Disease ◽

Mapk Kinase ◽

Mapk Cascades ◽

Resting Spores

Mitogen-activated protein kinase (MAPK) cascades play a central role in cellular growth, proliferation, and survival. MAPK cascade genes have been extensively investigated in model plants, mammals, yeast, and fungi but are not characterized in Plasmodiophora brassicae, which causes clubroot disease in cruciferous plants. Here, we identified 7 PbMAPK, 3 PbMAPKK, and 9 PbMAPKKK genes in the P. brassicae genome. Transcriptional profiling analysis demonstrated that several MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK) genes were preferentially expressed in three different zoosporic stages. Based on yeast two-hybrid assays, PbMAKKK7 interacted with PbMAKK3 and PbMAKK3 interacted with PbMAK1/PbMAK3. The PbMAKKK7-PbMAKK3-PbMAK1/PbMAK3 cascade may be present in P. brassicae. U0126, a potent and specific inhibitor of MAPKK, could inhibit the germination of P. brassicae resting spores. U0126 was used to treat the resting spores of P. brassicae and coinoculate rapeseed, and was proven to significantly relieve the severity of clubroot symptoms in the host plant and delay the life cycle of P. brassicae. These results suggest that MAPK signaling pathways may play important roles in P. brassicae growth, development, and pathogenicity.

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Plasmodiophora brassicae infection threshold – How many resting spores are required for infection of canola (Brassica napus)

10.1101/2021.05.21.445124 ◽

2021 ◽

Author(s):

Kher Zahr ◽

Yalong Yang ◽

Snezana Dijanovic ◽

Heting Fu ◽

Michael Harding ◽

...

Keyword(s):

Plasmodiophora Brassicae ◽

Management Strategies ◽

Detection Methods ◽

Single Spore ◽

Clubroot Disease ◽

Highly Sensitive ◽

Resting Spores ◽

Low Probability ◽

Highly Sensitive Detection ◽

Important Disease

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other brassica crops. Improved understanding of host and pathogen biology is frequently useful in guiding management strategies. In order to better understand infection thresholds, seven-day old seedlings of canola cultivar Westar were inoculated with serially diluted concentrations of P. brassicae resting spores. Controlled soil and plant inoculation assays were performed and the plants maintained in a greenhouse for 42 days and clubroot disease severity evaluated visually. Clubroot symptoms were observed in soils containing as low as one spore/mL soil and on plants inoculated with as few as ≤ 100 resting spores. These thresholds were lower than any previously reported. The results indicated the importance of highly sensitive detection methods for P. brassicae diagnosis and quantification methods for clubroot risk prediction in soils. Furthermore, these results highlighted the low probability of obtaining P. brassicae single spore isolates.

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ClubrootTracker: A Resource to Plan a Clubroot-Free Farm

Plant Health Progress ◽

10.1094/php-01-20-0003-rs ◽

2020 ◽

Vol 21 (3) ◽

pp. 185-187 ◽

Cited By ~ 1

Author(s):

Kevin Muirhead ◽

Christopher D. Todd ◽

Yangdou Wei ◽

Peta Bonham-Smith ◽

Edel Pérez-López

Keyword(s):

Plasmodiophora Brassicae ◽

Intracellular Parasite ◽

13Th Century ◽

Clubroot Disease ◽

Online Tool ◽

Obligate Intracellular ◽

Obligate Intracellular Parasite ◽

The World ◽

Resting Spores ◽

Ready Access

Clubroot is a devastating disease affecting cruciferous crops worldwide. Clubroot was first described in the 13th century in Russia and from that moment has been affecting European, Asian, and American brassica production. Plasmodiophora brassicae is the clubroot causal agent, and it is an obligate intracellular parasite that, as soil-borne resting spores, can remain viable in soil for many years. This persistence in the soil is a major negative contributing factor to the management of clubroot disease and highlights the importance for brassica growers to have ready access to current information on the distribution of the pathogen. The interactive online tool ClubrootTracker ( http://clubroottracker.ca ) has been developed to enable users to view pathogen and disease presence in geographic locations across the world. ClubrootTracker, as described in this manuscript, has been developed to provide brassica farmers a tool that will contribute to clubroot management and aid in planning a clubroot-free farm. This tool is an open resource that has the main goal of acquisition of GPS information in reporting the pathogen or the disease by the researchers working with it around the world.

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Germination of Plasmodiophora brassicae resting spores stimulated by a non-host plant

European Journal of Plant Pathology ◽

10.1007/s10658-005-2797-0 ◽

2005 ◽

Vol 113 (3) ◽

pp. 275-281 ◽

Cited By ~ 25

Author(s):

Hanna Friberg ◽

Jan Lagerlöf ◽

Birgitta Rämert

Keyword(s):

Host Plant ◽

Plasmodiophora Brassicae ◽

Resting Spores

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A New Identification Method Reveals the Resistance of an Extensive-Source Radish Collection to Plasmodiophora brassicae Race 4

Agronomy ◽

10.3390/agronomy11040792 ◽

2021 ◽

Vol 11 (4) ◽

pp. 792

Author(s):

Haohui Yang ◽

Yuxiang Yuan ◽

Xiaochun Wei ◽

Xiaohui Zhang ◽

Haiping Wang ◽

...

Keyword(s):

Plasmodiophora Brassicae ◽

Central China ◽

Clubroot Disease ◽

Cruciferous Vegetable ◽

Inoculation Methods ◽

Identification Method ◽

South Central ◽

Disease Occurrence ◽

Resistant Varieties ◽

The Stability

Raphanus sativus, an important cruciferous vegetable, has been increasingly affected by clubroot disease. Establishing a stable and accurate resistance identification method for screening resistant germplasms is urgently needed in radish. In this study, the influence of inoculum concentration, inoculation methods, and pH of the substrate on disease occurrence was studied. The result showed that the disease index (DI) was highest at 2 × 108 spores/mL, the efficiency of two-stage combined inoculation methods was higher than others, and pH 6.5 was favorable for the infection of P. brassicae. By using this new method, DIs of 349 radish germplasms varying from 0.00 to 97.04, presented significantly different levels of resistance. Analysis showed that 85.06% germplasms from China were susceptible to P. brassicae, whilst 28 accessions were resistant and mainly distributed in east, southwest, northwest, and south-central China. Most of the exotic germplasms were resistant. Repeated experiments verified the stability and reliability of the method and the identity of germplasm resistance. In total, 13 immune, 5 highly resistant and 21 resistant radish accessions were identified. This study provides an original clubroot-tolerance evaluation technology and valuable materials for the development of broad-spectrum resistant varieties for sustainable clubroot management in radish and other cruciferous crops.

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