A method for the extraction and enumeration of resting spores of Plasmodiophora brassicae from infested soil

North Dakota leads the United States in canola (Brassica napus L.) production (4). A canola field with a distinct patch of dead plants spreading over an area of approximately 0.4 ha was detected in Cavalier County, North Dakota, in early September 2013. Numerous spots within the patch had plant mortalities >80%. Dead plants pulled from the soil had roots with severe galling and clubbing. Clubbed roots were brittle and disintegrated easily when pressed between fingers. Root and soil samples collected at several locations within and outside the affected patch were pooled in separate groups. All plants collected in the patch were symptomatic but those collected outside were not. In the lab, total genomic DNA from three symptomatic and two healthy root samples was extracted using standard procedures and freehand slices were prepared for observation with a compound microscope. Also, DNA from pooled soil samples was extracted using FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH). Round resting structures ranging from 2.2 to 4.2 μm in diameter were observed by microscopic examination of symptomatic root tissues. These structures resembled those typically produced by Plasmodiophora brassicae Woronin. This initial identification was later confirmed through PCR analysis using the species specific primers TC1F/R and TC2F/R (1). PCR products of 548 bp (TC1F/R) and 519 bp (TC2F/R) were produced in the three symptomatic and two infested soil samples, confirming the presence of P. brassicae. PCR amplicons were not detected in healthy root and soil samples. Pathogenicity tests were conducted in greenhouse to fulfill Koch's postulates. Briefly, five square plastic pots (10 × 10 × 13 cm) were filled with a 10-cm layer of Sunshine Mix #1 potting mix (Fison Horticulture, Vancouver, BC, Canada) and then 1 g of ground root galls (approximately 5 × 105 resting spores) was spread evenly on its surface and covered with 2 cm of soilless mix. A similar number of pots were filled only with soilless mix and used as controls. All pots were planted with two seeds of canola cv. Westar and incubated in greenhouse conditions at 21°C and 16 h light daily. The experiment was conducted twice. Four weeks after planting, all plants in the inoculated pots had developed galls while plants in control pots were symptomless. Presence of P. brassicae resting spores in the newly developed galls was confirmed by microscopic observations and PCR. Based on the symptoms, morphology of resting spores, PCR reactions, and pathogenicity tests, we confirm the presence of P. brassicae on canola. While P. brassicae has been reported as widespread in North America (2), to our knowledge, this is the first report of clubroot on canola in North Dakota and the United States. Clubroot became the most important disease affecting canola production in central Alberta, Canada, within 5 years of its discovery in 2003 (3); since then, the disease has been detected in Saskatchewan and Manitoba (3), Canadian provinces that share borders with North Dakota. Considering the difficulties in management of clubroot, measures should be initiated to limit the spread of the disease before it could pose a threat to United States canola production. References: (1) T. Cao et al. Plant Dis. 91:80, 2007. (2) G. Dixon J. Plant Growth Regul. 28:194, 2009. (3) S. Strelkov and S. Hwang. Can. J. Plant Pathol. 36(S1):27, 2014. (4) USDA-NASS, Ag. Statistics No. 81, 2012.

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Seedling age and inoculum density affect clubroot severity and seed yield in canola

Canadian Journal of Plant Science ◽

10.4141/cjps10066 ◽

2011 ◽

Vol 91 (1) ◽

pp. 183-190 ◽

Cited By ~ 34

Author(s):

S. F. Hwang ◽

H. U. Ahmed ◽

S. E. Strelkov ◽

B. D. Gossen ◽

G. D. Turnbull ◽

...

Keyword(s):

Plant Height ◽

Seed Yield ◽

Negative Impact ◽

Plasmodiophora Brassicae ◽

Inoculum Density ◽

Infested Soil ◽

Seedling Age ◽

Control Seed ◽

Resting Spores ◽

Infested Field

Hwang, S. F., Ahmed, H. U., Strelkov, S. E., Gossen, B. D., Turnbull, G. D., Peng, G. and Howard, R. J. 2011. Seedling age and inoculum density affect clubroot severity and seed yield in canola. Can. J. Plant Sci. 91: 183–190. Clubroot, caused by Plasmodiophora brassicae, is a serious threat to canola (Brassica napus, B. rapa) production in western Canada because of its long-lived resting spores, high spore production potential, and negative impact on seed yield when inoculum pressure is high. The effect of inoculum density was studied by diluting heavily infested field soil with pathogen-free soil-less potting mix at seven increments, ranging from completely pathogen-free to 100% infested soil, and also by incorporating resting spores into the soil-less mix at concentrations of 1×105 to 1×108 spores cm−3, along with a non-inoculated control. Seed of the susceptible canola cultivar 34 SS 65 was planted in soil of each treatment, grown to maturity, and rated for plant height, seed yield, and clubroot severity (0–3 scale) at harvest. Clubroot severity increased and plant height and seed yield decreased with increasing inoculum density. To assess the effect of seedling age on reaction to clubroot, resting spores of P. brassicae were inoculated onto roots of 1-, 2-, 3- and 4-wk-old seedlings of 34 SS 65. In addition, seed (i.e., 0-wk-old seedlings) was sown into infested soil. Inoculation of young seedlings resulted in higher clubroot severity, shorter plants and lower yield than inoculation of older seedlings. These results indicate that seed treatment fungicides with a long residual period (4 wk or more) may be useful for the management of clubroot.

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Molecular Detection of Plasmodiophora brassicae, Causal Agent of Clubroot of Crucifers, in Plant and Soil

Plant Disease ◽

10.1094/pd-91-0080 ◽

2007 ◽

Vol 91 (1) ◽

pp. 80-87 ◽

Cited By ~ 48

Author(s):

Tiesen Cao ◽

Jalpa Tewari ◽

Stephen E. Strelkov

Keyword(s):

Ribosomal Rna ◽

Plasmodiophora Brassicae ◽

Rrna Gene ◽

Infested Soil ◽

Resting Spores ◽

One Step ◽

Dna Fragment ◽

Polymerase Chain ◽

Total P ◽

Step Polymerase Chain Reaction

Clubroot of crucifers, caused by Plasmodiophora brassicae, recently has been identified in canola (Brassica napus) fields in Alberta, Canada. An effective strategy for managing the disease is to avoid planting cruciferous crops in P. brassicae-infested soil, because the pathogen produces resting spores that can remain infectious for many years. A simple, one-step polymerase chain reaction (PCR) protocol was developed to detect the pathogen in plant and soil samples. The primers TC1F and TC1R, based on a P. brassicae partial 18S ribosomal RNA (rRNA) gene sequence from GenBank, yielded a 548-bp product in the optimized PCR. A second pair of primers, TC2F and TC2R, which amplified a fragment of the 18S and internal transcribed spacer (ITS) 1 regions of the rDNA repeat, also was tested and produced a 519-bp product. Neither set of primers amplified any DNA fragment from noninfected plant hosts, noninfested soil, or common soil fungi and bacteria tested in this study. Quantities of 100 fg or less of total P. brassicae DNA, or 1 × 103 resting spores per gram of soil, could be detected consistently using these primers and PCR protocol, corresponding to an index of disease of 11% or lower when the soil was bioassayed. The protocol also enabled detection of P. brassicae in symptomless root tissue 3 days after inoculation with the pathogen. Therefore, the PCR assay described in this study could provide a reliable diagnosis for routine detection of P. brassicae in plant and soil materials in a specific and rapid manner.

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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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Propidium Monoazide Improves Quantification of Resting Spores of Plasmodiophora brassicae with qPCR

Plant Disease ◽

10.1094/pdis-05-16-0715-re ◽

2017 ◽

Vol 101 (3) ◽

pp. 442-447 ◽

Cited By ~ 15

Author(s):

Fadi Al-Daoud ◽

Bruce D. Gossen ◽

Justin Robson ◽

Mary Ruth McDonald

Keyword(s):

Heat Treatment ◽

Plasmodiophora Brassicae ◽

Quantitative Polymerase Chain Reaction ◽

Infested Soil ◽

Propidium Monoazide ◽

Spore Viability ◽

Heat Treated ◽

Resting Spores ◽

Polymerase Chain ◽

The Impact

Plasmodiophora brassicae, which causes clubroot of Brassica crops, persists in soil as long-lived resting spores. Quantitative polymerase chain reaction (qPCR) analysis is often used to quantify resting spores but does not distinguish between DNA of viable and nonviable spores. The impact of pretreating spores with propidium monoazide (PMA), which inhibits amplification of DNA from nonviable microorganisms, was assessed in several experiments. Spore suspensions from immature and mature clubs were heat treated; then, PMA-PCR analyses and bioassays were performed to assess spore viability. Prior to heat treatment, assessments comparing PMA-PCR to qPCR for mature spores were similar, indicating that most of these spores were viable. However, only a small proportion (<26%) of immature spores were amplified in PMA-PCR. Bioassays demonstrated that clubroot severity was much higher in plants inoculated with mature spores than with immature spores. Heat treatment produced little or no change in estimates of mature spores from qPCR but spore estimates from PMA-PCR and clubroot severity in bioassays were both substantially reduced. Estimates of spore concentration with PMA-PCR were less consistent for immature spores. To facilitate use of PMA-PCR on infested soil, a protocol for extracting spores from soil was developed that provided higher extraction efficiency than the standard methods.

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Wybrane zagadnienia z biologii grzyba Plasmodiophora brassicae Wor. [Some problems in the life-cycle of fungus Plasmodiophora brassicae, Wor.]

Acta Agrobotanica ◽

10.5586/aa.1973.012 ◽

2015 ◽

Vol 26 (1) ◽

pp. 147-160 ◽

Cited By ~ 1

Author(s):

B. Nowicki

Keyword(s):

Life Cycle ◽

Soil Ph ◽

Plasmodiophora Brassicae ◽

Acid Soil ◽

Infested Soil ◽

Alkaline Soils ◽

Resting Spores ◽

Ph Range

The quickest loss of infectivity of <i>Plasmodiophora brassicae</i> Wor. resting spores was observed in acid soil. Jnlectivity was ratained longer in neutral and alkaline soils. The infection of cabbage seedlings took place in a broad pH range from 3.3 to 8.1, the optimum soil pH for infection being at 5.3 - 5.7. When the number of spores in the soil increased the infection took place in the infection took place in the broader pH range. The plants which were planted as seedlings in infested soil were infected in a broader pH range than plants which were grown from seeds in infested soil.

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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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The life history of Plasmodiophora brassicae Woron

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1972.0008 ◽

1972 ◽

Vol 180 (1058) ◽

pp. 103-112 ◽

Cited By ~ 109

Keyword(s):

Life History ◽

Root Hair ◽

Plasmodiophora Brassicae ◽

Root Hairs ◽

Secondary Phase ◽

Tissue Cultures ◽

Resting Spore ◽

Resting Spores ◽

Seedling Roots ◽

History Of

Resting spore germination and the root hair stages of the life history of Plasmodiophora brassicae were studied in stained preparations of infected Brassica rapa seedling roots. Naked protoplasts, usually possessing two unequal flagella, were released from resting spores through a small circular pore. They penetrated the root hairs of B. rapa and there developed into plasmodia which, after becoming multinucleate, cleaved to form zoosporangia containing incipient zoospores. Biflagellate zoospores were released from root hair zoosporangia and fused in pairs, although karyogamy did not occur. The resulting binucleate zoospores infected the cortical dells of B. rapa to form binucleate plasmodia, the earliest stages of the secondary phase of the life history. These findings are discussed in relation to previous studies on the life history of P. brassicae in Brassica plants and in Brassica tissue cultures, and a new complete life history, including nuclear fusion in the secondary plasmodium, is suggested for the organism.

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

Plant Disease ◽

10.1094/pdis-02-21-0410-re ◽

2021 ◽

Author(s):

Nazanin Zamani-Noor ◽

Sinja Brand ◽

Hans-Peter Soechting

Keyword(s):

Plasmodiophora Brassicae ◽

Brassica Species ◽

Post Inoculation ◽

Clubroot Disease ◽

Alopecurus Myosuroides ◽

Pathogen Virulence ◽

Iberis Amara ◽

Thlaspi Arvense ◽

Resting Spores ◽

Pathogen Dna

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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Genetic Transformation of the Obligate Parasite Plasmodiophora brassicae

Phytopathology ◽

10.1094/phyto-01-13-0010-r ◽

2013 ◽

Vol 103 (10) ◽

pp. 1052-1057 ◽

Cited By ~ 12

Author(s):

J. Feng ◽

Sheau-Fang Hwang ◽

S. E. Strelkov

Keyword(s):

Genetic Transformation ◽

Fluorescent Protein ◽

Plasmodiophora Brassicae ◽

Expression Vectors ◽

Obligate Parasite ◽

Putative Transformants ◽

Resting Spores ◽

Selection Step ◽

Protoplast Preparation ◽

Green Fluorescent

A protocol for genetic transformation of the obligate parasite Plasmodiophora brassicae, causal agent of clubroot of crucifers, was developed. In this protocol, protoplast preparation was superseded with lithium acetate treatment and the selection step was omitted. In two independent experiments, germinating resting spores of P. brassicae were transformed by two fungal expression vectors containing either a green fluorescent protein (gfp) gene or a hygromycin resistance (hph) gene. Putative transformants were produced from both transformations, with ≈50% of the obtained galls containing resting spores from which transforming DNA could be detected by polymerase chain reaction (PCR). PCR, quantitative PCR (qPCR), and genome walking conducted on selected transformants indicated that the transforming DNA was intergraded into the P. brassicae genome. Transcript of hph but not gfp was detected by reverse-transcription qPCR from selected transformants. From all galls produced by transformants, no GFP activity could be identified. Verified transformants were inoculated on canola and new galls were generated. PCR and qPCR analyses based on these galls indicated that transforming DNA was still resident in P. brassicae. This is the first report on genetic transformation of P. brassicae. The information and data generated from this study will facilitate research in multiple areas of the clubroot pathosystem.

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