Live cell imaging of <i>Plasmodiophora brassicae</i>—host plant interactions based on a two‐step axenic culture system

Tu, J. C.; Bush, J.T.; Bonham‐Smith, Peta C.; Wei, Yangdou

doi:10.1002/mbo3.765

Cited by 10 publications

(9 citation statements)

References 35 publications

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“…Clear images of P. brassicae nuclei stained with DAPI were obtained ( Figure 10 ). The dual staining of CFW combined with the lipophilic fluorescent dye Nile red has been employed for examining the structures and development of P. brassicae in B. napus callus tissues ( Tu et al, 2019 ). Nile red has also been used to stain intracellular lipid droplets abundant in resting spores of P. brassicae ( Bi et al, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

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

2022

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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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Section: Discussionmentioning

confidence: 99%

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

2022

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“…While secondary infection by P. brassicae was detected in the CR B. rapa hosts in this study, there was no change in the root structure, with the vascular system developing normally. In contrast, biotrophic life history of P. brassicae renders significant changes to elucidating morphological and cellular characteristics of P. brassicae development in host tissues during clubroot disease initiation and development ( Tu et al, 2019 ). The vascular system of the susceptible host was severely distorted.…”

Section: Discussionmentioning

confidence: 99%

Histopathology of the Plasmodiophora brassicae-Chinese Cabbage Interaction in Hosts Carrying Different Sources of Resistance

Liu

Strelkov²,

Sun³

et al. 2022

Front. Plant Sci.

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Clubroot is a serious soil-borne disease of crucifers caused by the obligate parasite Plasmodiophora brassicae. The genetic basis and histopathology of clubroot resistance in two Chinese cabbage (Brassica rapa ssp. pekinensis) inbred lines Bap055 and Bap246, challenged with pathotype 4 of P. brassicae, was evaluated. The Chinese cabbage cultivar “Juxin” served as a susceptible check. The resistance in Bap055 was found to be controlled by the CRa gene, while resistance in Bap246 fit a model of control by unknown recessive gene. Infection of the roots by P. brassicae was examined by inverted microscopy. Despite their resistance, primary and secondary infection were observed to occur in Bap055 and Bap246. Primary infection was detected at 2 days post-inoculation (DPI) in “Juxin,” at 4 DPI in Bap055, and at 6 DPI in Bap246. Infection occurred most quickly on “Juxin,” with 60% of the root hairs infected at 10 DPI, followed by Bap055 (31% of the root hairs infected at 12 DPI) and Bap246 (20% of the root hairs infected at 14 DPI). Secondary infection of “Juxin” was first observed at 8 DPI, while in Bap055 and Bap246, secondary infection was first observed at 10 DPI. At 14 DPI, the percentage of cortical infection in “Juxin,” Bap055 and Bap246 was 93.3, 20.0, and 11.1%, respectively. Although cortical infection was more widespread in Bap055 than in Bap246, secondary infection in both of these hosts was restricted relative to the susceptible check, and the vascular system remained intact. A large number of binucleate secondary plasmodia were observed in “Juxin” and the vascular system was disrupted at 16 DPI; in Bap055 and Bap246, only a few secondary plasmodia were visible, with no binucleate secondary plasmodia. The defense mechanisms and expression of resistance appears to differ between Chinese cabbage cultivars carrying different sources of resistance.

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“…Resting spores can survive in soil for up to 20 years and germinate to release primary zoospores upon stimulation by root exudates, followed by primary infection of root hairs (Al‐Daoud et al, 2017; Dixon, 2009). Resting spores of P. brassicae are the target of studies defining pathogen life cycle (Liu et al, 2020), P. brassicae ‐plant interactions (Tu, Bush, Bonham‐Smith, & Wei, 2019), pathotype classification (Holtz, Hwang, & Strelkov, 2018; Strelkov et al, 2006), germplasm screening and resistance evaluation (Deora, Gossen, & McDonald, 2013; Fei et al, 2016; Gludovacz, Deora, McDonald, & Gossen, 2014), nucleic acid or protein isolation (Fu et al, 2020; Schwelm et al, 2015) and chemical treatments (Mitani et al, 2003). Crude resting spore homogenate was previously prepared by gall homogenisation (Wen et al, 2020) while purified resting spore suspension was obtained by multiple centrifugation in sucrose solution (Feng, Hwang, & Strelkov, 2013).…”

Section: Discussionmentioning

confidence: 99%

A high‐throughput turbidimetric method for quantitative preparation of Plasmodiophora brassicae inoculum for bioassays

Yang

Wang

Hong

et al. 2022

Annals of Applied Biology

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Clubroot caused by Plasmodiophora brassicae is one of the major diseases on cruciferous crops. This disease has been a problem all over the world and led to serious economic losses in cruciferous crop production. The improvement of clubroot management is dependent on the effectiveness of bioassays on pathogenicity or plant resistance.P. brassicae resting spore inoculum prepared from clubroot tissues was widely used in bioassays. Traditionally, resting spore concentration was measured by visual counting with a haemocytometer; however, this method is time-consuming, labour intensive and with poor repeatability due to the tiny size of resting spores. In this study, we established a turbidimetric method to measure P. brassicae resting spore concentration of the inoculum. A regression curve was generated by plotting optical density of gradiently diluted resting spore suspension against the corresponding resting spore concentrations, which resulted in a logarithmic regression equation. This method was validated and proved to be robust and effective in determining resting spore concentration of different samples, and was further confirmed by bioassay of infection and clubroot development. In conclusion, we established a standard protocol to prepare P. brassicae inoculum and provided an effective method for resting spore concentration determination. The results of this study can be widely used in research activities on clubroot sustainable management when an effective assessment of P. brassicae biomass is needed.

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Live cell imaging of Plasmodiophora brassicae—host plant interactions based on a two‐step axenic culture system

Cited by 10 publications

References 35 publications

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

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

Histopathology of the Plasmodiophora brassicae-Chinese Cabbage Interaction in Hosts Carrying Different Sources of Resistance

A high‐throughput turbidimetric method for quantitative preparation of Plasmodiophora brassicae inoculum for bioassays

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