A technique for the extraction and purification of viable <i>Plasmodiophora brassicae</i> resting spores from host root tissue

Castlebury, Lisa A.; Maddox, Joseph V.; Glawe, Dean A.

doi:10.1080/00275514.1994.12026435

Cited by 38 publications

(26 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The resting spores were extracted and purified according to the method described by T. Asano44. We purified the resting spores under 50% (w/v) sucrose solution by centrifugation and further purified by using continuous gradient of HS-40 colloidal silica (LUDOX, 40 wt% suspension in water; Sigma-Aldrich) as the protocol45. Resting spores were suspended in freshly prepared 2% chloramine-T solution (wt/vol) at room temperature for 20 min and then washed twice in sterile water by centrifugation at 1,700 g for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Integrated omics study of lipid droplets from Plasmodiophora brassicae

Gao

et al. 2016

Sci Rep

View full text Add to dashboard Cite

Plasmodiophora brassicae causes clubroot disease in cruciferous. In this report, lipid droplets were observed in the resting spores of P. brassicae. 295 lipid droplet-associated proteins were identified and categorized into nine groups. Transcriptome analysis of these proteins during three different zoosporic stages revealed differences in gene expression pattern. GO enrichment analysis revealed that these proteins associated with lipid droplets were mainly linked to biosynthesis and metabolism. GC-MS analysis revealed that lipid droplets contain seven types of free fatty acids: saturated fatty acids C16:0 and C18:0, and unsaturated fatty acids C18:1Δ9, C18:1Δ11, C18:2, C20:4 and C20:5. P. brassicae accumulated a large amount of triacylglycerols (TAGs). We systematically analyzed the putative proteins involved in TAG biosynthesis and its metabolic pathway. KEGG pathway analysis defined 3390 genes, including 167 genes involved in lipid metabolism. Transcriptome analysis revealed that 162 candidate enzymes involved in lipid metabolism were differential expressed. Our omics studies are the first to investigate the lipid droplet organelles in P. brassicae, providing a reference resource to study protist lipid droplets.

show abstract

Section: Methodsmentioning

confidence: 99%

Integrated omics study of lipid droplets from Plasmodiophora brassicae

Gao

et al. 2016

Sci Rep

View full text Add to dashboard Cite

show abstract

“…P. brassicae resting spores were isolated according to the procedure of Castlebury et al (4). P. brassicae resting spores were isolated according to the procedure of Castlebury et al (4).…”

Section: Methodsmentioning

confidence: 99%

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

2007

View full text Add to dashboard Cite

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.

show abstract

“…The sucrose method used by Castlebyry et al (1994) was conducted in a side-by-side fashion to eliminate the impurities, except for resting spores originating from plant tissue. First, the root galls were washed several times with running water to eliminate soil particles and foreign materials, after which the resting spores of P. brassicae were separated after grinding the samples in sterilized water with a homogenizer (13,500 rpm/5minutes).…”

Section: Methodsmentioning

confidence: 99%

Chinese Cabbage Clubroot Pathogen,Plasmodiophora brassicae, Is Genetically Stable

Heo¹,

Jang²,

Choi

et al. 2009

Mycobiology

View full text Add to dashboard Cite

Single spore isolates of Plasmodiophora brassicae e4 and e9 obtained from diseased Chinese cabbage were identified as race 4 and race 9, respectively, by the Williams' differential variety set. To confirm the possibility of variation in same generation and progeny of a single spore isolate of P. brassicae, random amplified polymorphic DNA (RAPD) analysis was conducted using the URP 3, 6 and OPA 7 primers. There was no difference in band type at each part of the gall of Chinese cabbage obtained by inoculation of e4 and e9 and amplification using the URP 3 and 6 primers when the same generation was analyzed. In addition, the progeny analysis, which was expanded to the third generation and conducted using the URP 3 and OPA 7 primers, revealed no differences in the band type of the e4 isolate. Based on these results, the single spore isolate of P. brassicae was genetically stable.

show abstract

A technique for the extraction and purification of viable Plasmodiophora brassicae resting spores from host root tissue

Cited by 38 publications

References 10 publications

Integrated omics study of lipid droplets from Plasmodiophora brassicae

Integrated omics study of lipid droplets from Plasmodiophora brassicae

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

Chinese Cabbage Clubroot Pathogen,Plasmodiophora brassicae, Is Genetically Stable

Contact Info

Product

Resources

About