Most Plasmodiophora brassicae Populations in Single Canola Root Galls from Alberta Fields are Mixtures of Multiple Strains

Clubroot, caused by Plasmodiophora brassicae, is an important disease on canola in Alberta, Canada. The pathogen is grouped into pathotypes according to their virulence reaction on differential hosts. Multiple pathotypes or strains are known exist in one field, one plant, or even one gall. This study was conducted with the objective of testing the prevalence of the coexistence of multiple strains in a single gall. In all, 79 canola clubroot galls were collected from 22 fields across Northern Alberta in 2018. Genomic DNA extracted from these single galls was analyzed using RNase H-dependent PCR (rhPCR). The rhPCR primers were designed to amplify a partial sequence of a dimorphic gene, with one primer pair specific to one sequence and the other primer pair specific to the alternative sequence. The amplification of both sequences from DNA obtained from a single gall would indicate that it contains two different P. brassicae strains. The rhPCR analyses indicated that the P. brassicae populations in 50 of the 79 galls consisted of more than one strain. This result emphasizes the need for cautious interpretation of results when a single-gall population is subject to pathotyping or being used as inoculum in plant pathology research. It also confirms that the maintenance of pathotype diversity within single root galls is a common occurrence which has implications for the durability, and stewardship, of single-gene host resistance.

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Characterization of Five Molecular Markers for Pathotype Identification of the Clubroot Pathogen Plasmodiophora brassicae

Phytopathology ◽

10.1094/phyto-11-17-0362-r ◽

2018 ◽

Vol 108 (12) ◽

pp. 1486-1492 ◽

Cited By ~ 3

Author(s):

Jing Zheng ◽

Xuliang Wang ◽

Qian Li ◽

Shu Yuan ◽

Shiqing Wei ◽

...

Keyword(s):

Molecular Markers ◽

Plasmodiophora Brassicae ◽

Expression Patterns ◽

Chain Reaction ◽

Clubroot Disease ◽

Polymerase Chain ◽

Differential Hosts ◽

Reaction Cycles ◽

Important Disease

Clubroot disease is an important disease on cruciferous crops caused by Plasmodiophora brassicae infections. The pathotypes have been classified based on the reactions of differential hosts. However, molecular markers of particular pathotypes for P. brassicae are limited. In this study, we found five genetic markers in association with different pathotypes. Different gene expression patterns among different pathotypes (P4, P7, P9, and P11) were assayed according to the transcriptome data. The assay indicated that molecular markers PBRA_007750 and PBRA_009348 could be used to distinguish P11 from P4, P7, and P9; PBRA_009348 and Novel342 could distinguish P9 from P4, P7, and P11; and PBRA_008439 and Novel342 could represent a kind of P4. Polymerase chain reaction cycles ranging from 25 to 30 were able to identify the predominant pathotype in general. Therefore, these molecular markers would be a valuable tool to identify and discriminate pathotypes in P. brassicae population.

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Specific Genes Identified in Pathotype 4 of the Clubroot Pathogen Plasmodiophora brassicae

Plant Disease ◽

10.1094/pdis-05-18-0912-re ◽

2019 ◽

Vol 103 (3) ◽

pp. 495-503 ◽

Cited By ~ 2

Author(s):

Jing Zheng ◽

Xuliang Wang ◽

Yang Xiao ◽

Shiqing Wei ◽

Die Wang ◽

...

Keyword(s):

Molecular Markers ◽

Plasmodiophora Brassicae ◽

Identification System ◽

Transcriptome Data ◽

Expression Of Genes ◽

Different Types ◽

Differential Hosts ◽

Differential Expression Of Genes ◽

Important Disease ◽

Six Genes

Clubroot is an important disease of cruciferous crops caused by Plasmodiophora brassicae, and pathotypes are classified based on the response of differential hosts. This study was conducted to identify genetic markers able to differentiate pathotypes. Differential expression of genes between pathotype 4 (P4) and pathotype 7 (P7) was assessed according to transcriptome data of molecular marker screening. Among the pathotypes (P2, P4, P5, P7, P9, P10, and P11) tested, six genes were exclusive to P4, dividing the isolates into three types: PBRA_003263 and PBRA_003268 were present in all P4 isolates, PBRA_000003/Novel512 were found in a type of P4 (P4-1), and Novel137/PBRA_005772 were found in another P4 type, P4-2. Amplicons for all six genes were produced for only one isolate, which we named P4-3. This study is the first to establish a molecular identification system for P4 the, predominant pathotype in China. The genes identified might serve as molecular markers for differentiation of P4 from other pathotypes and may also distinguish different types of P4.

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Current and Future Pathotyping Platforms for Plasmodiophora brassicae in Canada

Plants ◽

10.3390/plants10071446 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1446

Author(s):

Heather H. Tso ◽

Leonardo Galindo-González ◽

Stephen E. Strelkov

Keyword(s):

Crop Production ◽

Plasmodiophora Brassicae ◽

Cost Effective ◽

Turnaround Time ◽

Molecular Techniques ◽

Differential Set ◽

Management Plans ◽

Increased Sensitivity ◽

Phenotypic Methods ◽

Important Disease

Clubroot, caused by Plasmodiophora brassicae, is one of the most detrimental threats to crucifers worldwide and has emerged as an important disease of canola (Brassica napus) in Canada. At present, pathotypes are distinguished phenotypically by their virulence patterns on host differential sets, including the systems of Williams, Somé et al., the European Clubroot Differential set, and most recently the Canadian Clubroot Differential set and the Sinitic Clubroot Differential set. Although these are frequently used because of their simplicity of application, they are time-consuming, labor-intensive, and can lack sensitivity. Early, preventative pathotype detection is imperative to maximize productivity and promote sustainable crop production. The decreased turnaround time and increased sensitivity and specificity of genotypic pathotyping will be valuable for the development of integrated clubroot management plans, and interest in molecular techniques to complement phenotypic methods is increasing. This review provides a synopsis of current and future molecular pathotyping platforms for P. brassicae and aims to provide information on techniques that may be most suitable for the development of rapid, reliable, and cost-effective pathotyping assays.

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Genetic structure of Plasmodiophora brassicae populations virulent on clubroot resistant canola (Brassica napus)

Plant Disease ◽

10.1094/pdis-09-20-1980-re ◽

2021 ◽

Author(s):

Homa Askarian ◽

Alireza Akhavan ◽

Leonardo Galindo González ◽

Sheau-Fang Hwang ◽

Stephen Ernest Strelkov

Keyword(s):

Brassica Napus ◽

Genetic Structure ◽

Plasmodiophora Brassicae ◽

Single Spore ◽

Specific Primers ◽

Brassica Napus L ◽

Before And After ◽

Differential Hosts ◽

And Migration ◽

Simple Sequence

Clubroot, caused by Plasmodiophora brassicae Woronin, is a significant threat to the canola (Brassica napus L.) industry in Canada. Clubroot resistance has been overcome in more than 200 fields since 2013, representing one of the biggest challenges to sustainable canola production. The genetic structure of 36 single-spore isolates derived from 12 field isolates of P. brassicae collected before and after the introduction of clubroot resistant (CR) canola cultivars (2005-2014) was evaluated by simple sequence repeat (SSR) marker analysis. Polymorphisms were detected in 32 loci with the identification of 93 distinct alleles. A low level of genetic diversity was found among the single-spore isolates. Haploid linkage disequilibrium and number of migrants suggested that recombination and migration were rare or almost absent in the tested P. brassicae population. A relatively clear relationship was found between the genetic structure and virulence phenotypes of the pathogen as defined on the differential hosts of Somé et al., Williams and the Canadian Clubroot Differential (CCD) set. Although genetic variability within each pathotype group, as classified on each differential system, was low, significant genetic differentiation was observed among the pathotypes. The highest correlation between genetic structure and virulence was found among matrices produced with genetic data and the hosts of the CCD set, with a threshold index of disease of 50% to distinguish susceptible from resistant reactions. Genetically homogeneous single-spore isolates provided a more complete and clearer picture of the population genetic structure of P. brassicae, and the results suggest some promise for the development of pathotype-specific primers.

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Virulence Spectrum of Single-Spore and Field Isolates of Plasmodiophora brassicae Able to Overcome Resistance in Canola (Brassica napus)

Plant Disease ◽

10.1094/pdis-03-20-0471-re ◽

2021 ◽

Vol 105 (1) ◽

pp. 43-52 ◽

Cited By ~ 2

Author(s):

Homa Askarian ◽

Alireza Akhavan ◽

Victor P. Manolii ◽

Tiesen Cao ◽

Sheau-Fang Hwang ◽

...

Keyword(s):

Brassica Napus ◽

Confidence Interval ◽

Plasmodiophora Brassicae ◽

Large Collection ◽

Single Spore ◽

Brassica Napus L ◽

Clubroot Resistance ◽

Field Isolates ◽

Virulence Spectrum ◽

Important Disease

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important disease of canola (Brassica napus L.) that is managed mainly by planting clubroot-resistant (CR) cultivars. Field isolates of P. brassicae can be heterogeneous mixtures of various pathotypes, making assessments of the genetics of host–pathogen interactions challenging. Thirty-four single-spore isolates were obtained from nine field isolates of the pathogen collected from CR canola cultivars. The virulence patterns of the single-spore and field isolates were assessed on the 13 host genotypes of the Canadian Clubroot Differential (CCD) set, which includes the differentials of Williams and Somé et al. Indices of disease (IDs) severity of 25, 33, and 50% (±95% confidence interval) were compared as potential thresholds to distinguish between resistant and susceptible reactions, with an ID of 50% giving the most consistent responses for pathotype classification purposes. With this threshold, 13 pathotypes could be distinguished based on the CCD system, 7 on the differentials of Williams, and 3 on the hosts of Somé et al. The highest correlations were observed among virulence matrices generated using the three threshold IDs on the CCD set. Genetically homogeneous single-spore isolates gave a clearer profile of the P. brassicae pathotype structure. Novel pathotypes, not reported in Canada previously, were identified among the isolates. This large collection of single-spore isolates can serve as a reference in screening and breeding for clubroot resistance.

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Identification of the single gene for calmodulin in Dictyostelium discoideum.

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1851 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1851-1854 ◽

Cited By ~ 28

Author(s):

H Goldhagen ◽

M Clarke

Keyword(s):

Dictyostelium Discoideum ◽

Genomic Dna ◽

Single Gene ◽

Sequence Determination ◽

Coding Sequence ◽

Calmodulin Gene ◽

Cloned Cdna

We report the isolation and sequence determination of a cDNA containing most of the coding sequence for Dictyostelium discoideum calmodulin. The cloned cDNA was used as a probe to examine the complexity of D. discoideum genomic DNA. These studies indicated that D. discoideum cells possess a single calmodulin gene.

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Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.97.1.222 ◽

2000 ◽

Vol 97 (1) ◽

pp. 222-227 ◽

Cited By ~ 24

Author(s):

J. Herrick ◽

X. Michalet ◽

C. Conti ◽

C. Schurra ◽

A. Bensimon

Keyword(s):

Fluorescent Probe ◽

Copy Number ◽

Genomic Dna ◽

Single Gene ◽

Gene Copy Number ◽

Gene Copy

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Inactivation of DFR (Dihydroflavonol 4-reductase) Gene Transcription Results in Blockage of Anthocyanin Production in Yellow Onions (Allium cepa)

HortScience ◽

10.21273/hortsci.39.4.889c ◽

2004 ◽

Vol 39 (4) ◽

pp. 889C-889 ◽

Cited By ~ 1

Author(s):

Sunggil Kim* ◽

Marla Binzel ◽

Sunghun Park ◽

Kil-Sun Yoo ◽

Leonard Pike

Keyword(s):

Genomic Dna ◽

Single Gene ◽

Color Difference ◽

Gene Transcript ◽

Red Color ◽

Reductase Gene ◽

Pcr Rflp ◽

Primary Determinant ◽

Rapid Amplification ◽

Anthocyanin Production

Anthocyanin, one of the flavonoids, is a primary determinant of red color in onions. Inheritance studies indicate that a single gene determines the color difference between yellow and red onions. In order to establish which gene might be responsible for this color difference, full-length cDNAs of five structural genes: chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS) were cloned using degenerate PCR and RACE (Rapid Amplification of cDNA Ends). RT-PCR was carried out for these five genes to examine differential expression between yellow and red colored bulbs. Accumulation of the DFR gene transcript only occurred in red onions. In F3 populations which originated from the cross between yellow and red parents, DFR transcript was detected only in red F3 lines, not in yellow F3 lines. To design molecular markers for selection of yellow and red DFR alleles, the DFR gene was sequenced from genomic DNA isolated from both types of onions. The genomic DNA sequence revealed the DFR gene consists of six exons and five introns. A PCR-RFLP marker was designed based on 2% polymorphic nucleotide sequence of the DFR gene between yellow and red onions. The co-segregation of markers and red color were observed in F2 segregating populations, supporting the conclusion that color difference in red and yellow onions is likely to be due to the lack of an active DFR gene.

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Seed treatment of canola (Brassica napus) with the endomycorrhizal fungus Piriformospora indica does not reduce clubroot

Canadian Journal of Plant Science ◽

10.1139/cjps-2020-0126 ◽

2020 ◽

Author(s):

Afsaneh Sedaghatkish ◽

Bruce D. Gossen ◽

Mary Ruth McDonald

Keyword(s):

Brassica Napus ◽

Microscopic Observation ◽

Seed Treatment ◽

Plasmodiophora Brassicae ◽

Piriformospora Indica ◽

Brassica Crops ◽

Growth Room ◽

Treated Seed ◽

Important Disease

A Basidiomycete endomycorrhizal fungus, Piriformospora indica, colonizes and promotes the growth of canola and other Brassica crops, and can reduce diseases of other crops. Clubroot is an important disease of Bbrassica crops caused by the obligate, soil-borne pathogen Plasmodiophora brassicae. The effect of P. indica on clubroot severity in canola was assessed in replicated growth room studies. Seed was treated with P. indica using a proprietary process. Microscopic observation confirmed that canola roots grown from treated seed were colonized by P. indica. However, P. indica did not consistently reduce clubroot severity and did not promote the growth of canola.

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The adenosine2 gene of Drosophila melanogaster encodes a formylglycineamide ribotide amidotransferase

Genome ◽

10.1139/g93-122 ◽

1993 ◽

Vol 36 (5) ◽

pp. 924-934 ◽

Cited By ~ 3

Author(s):

S. Y. K. Tiong ◽

D. Nash

Keyword(s):

Drosophila Melanogaster ◽

Amino Acid ◽

Genomic Dna ◽

Gene Rearrangement ◽

Single Gene ◽

Genetic Material ◽

Biosynthetic Enzyme ◽

E Coli ◽

Enzymatic Function ◽

Rearrangement Breakpoint

Drosophila melanogaster genomic DNA spanning an adenosine2 gene rearrangement breakpoint (in cytological map region 26B1-2) was cloned and a composite ade2 base sequence was derived from this DNA and from a corresponding cDNA. Based on genetic evidence, the ade2 gene is thought to encode the purine biosynthetic enzyme formylglycineamide ribotide amidotransferase (FGARAT). The cDNA hybridizes to a 4.8-kb message that encodes a 1354 amino acid polypeptide with extensive similarity to Escherichia coli FGARAT. The D. melanogaster FGARAT amino acid sequence is considerably more like that of the E. coli enzyme than is the FGARAT of B. subtilis, which has two polypeptides with, collectively, 969 amino acids. It is suggested that the taxonomically anomalous similarity between the eukaryotic FGARAT and that from E. coli may indicate horizontal exchange of genetic material. On the basis of substantially greater conservation of sequences snared by all three species compared with those present only in E. coli and D. melanogaster, we suggest that no radical alteration of enzymatic function accompanied the transition between the single-gene and the two-gene state.Key words: Drosophila melanogaster, purine biosynthesis genes, formylglycineamide ribotide amidotransferase, formylglycineamidine ribotide synthase, adenosine2 locus, horizontal gene transfer.

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