Telomere-to-telomere Genome Assembly of the Clubroot Pathogen Plasmodiophora Brassicae.

Plasmodiophora brassicae (Woronin, 1877), a biotrophic, obligate parasite, is the causal agent of clubroot disease in brassicas. The clubroot pathogen has been reported in more than 80 countries worldwide, causing economic losses of hundreds of millions every year. Despite its widespread impact, very little is known about the molecular strategies it employs to induce the characteristic clubs in the roots of susceptible hosts during infection, nor about the mechanisms it uses to overcome genetic resistance. Here, we provide the first telomere-to-telomere complete genome of P. brassicae. We generated ∼27 Gb of Illumina, Oxford Nanopore, and PacBio HiFi data from resting spores of strain Pb3A and produced a 25.3 Mb assembly comprising 20 chromosomes, with an N50 of 1.37 Mb. The BUSCO score, the highest reported for any member of the group Rhizaria (Eukaryota: 88.2%), highlights the limitations within the Eukaryota database for members of this lineage. Using available transcriptomic data and protein evidence, we annotated the Pb3A genome, identifying 10,521 protein-coding gene models. This high-quality, complete genome of P. brassicae will serve as a crucial resource for the plant pathology community to advance the much-needed understanding of the evolution of the clubroot pathogen.

HO-CR and HOLL-CR: new forms of winter oilseed rape (Brassica napus L.) with altered fatty acid composition and resistance to selected pathotypes of Plasmodiophora brassicae (clubroot).

The priority in oilseed rape (Brassica napus L.) research and breeding programs worldwide is to combine different features to develop cultivars tailored to specific applications of this crop. In this study, forms with a modified fatty acid composition of seed oil were successfully combined with a source of resistance to Plasmodiophora brassicae Wor., a harmful protist-causing clubroot. Three HO-type recombinants in F6-F12 generations with oleic acid content of 80.2-82.1% and one HOLL-type F6 inbred mutant recombinant (HOmut × LLmut), with a high oleic acid content (80.9%) and reduced linolenic acid content (2.3%), were crossed with the cultivar Tosca, resistant to several pathotypes of P. brassicae. The work involved genotyping with the use of DNA markers specific for allelic variants of desaturase genes responsible for the synthesis of oleic and linolenic fatty acids, CAPS (FAD2 desaturase, C18:1), and SNaPshot (FAD3 desaturase, C18:3), respectively. Of 350 progenies in the F3 generation, 192 (55%) were selected for further studies. Among them, 80 HO (≥ 72%) lines were identified, 10 of which showed resistance to at least one up to four P. brassicae pathotypes. Thirty lines in the selected progeny contained high oleic acid and less than 5% linolenic acid; eight of them belonged to the HOLL type conferring resistance to at least one pathotype. Two HO lines and two HOLL lines were resistant to four pathotypes. The resulting HO-CR and HOLL-CR inbred lines with altered seed oil fatty acid composition and resistance to P. brassicae represent unique oilseed rape material with the desired combination of valuable traits.

A Basic Guide to the Propagation and Manipulation of the Clubroot Pathogen, Plasmodiophora brassicae.

Clubroot caused by the obligate parasite Plasmodiophora brassicae is a devastating disease affecting the canola industry worldwide. The socio-economic impact of clubroot can be significant, particularly in regions where Brassica crops are a major agricultural commodity. The disease can cause significant crop losses, leading to reduced yield and income for farmers. Extensive studies have been conducted to understand the biology and genetics of the pathogens and develop more effective management strategies. However, the basic procedures used for pathogen storage and virulence analysis have not been assembled or discussed in detail. As a result, there are discrepancies among the different protocols used today. The aim of this article is to provide a comprehensive and easily accessible resource for researchers who are interested in replicating or building upon the methods used in the study of the clubroot pathogen. Here, we discuss in detail the methods used for P. brassicae spore isolation, inoculation, quantification, propagation, and molecular techniques such as DNA extraction and PCR. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of Plasmodiophora brassicae resting spores and propagation Support Protocol 1: Evans blue staining to identify resting spore viability Support Protocol 2: Storage of Plasmodiophora brassicae Basic Protocol 2: Generation of single spore isolates from P. brassicae field isolates Basic Protocol 3: Phenotyping of Plasmodiophora brassicae isolates Basic Protocol 4: Genomic DNA extraction from Plasmodiophora brassicae resting spores Basic Protocol 5: Molecular detection of Plasmodiophora brassicae.

Identification of Clubroot (Plasmodiophora brassicae) Resistance Loci in Chinese Cabbage (Brassica rapa ssp. pekinensis) with Recessive Character.

The soil-borne pathogen Plasmodiophora brassicae is the causal agent of clubroot, a major disease in Chinese cabbage (Brassica rapa ssp. pekinensis). The host's resistance genes often confer immunity to only specific pathotypes and may be rapidly overcome. Identification of novel clubroot resistance (CR) from germplasm sources is necessary. In this study, Bap246 was tested by being crossed with different highly susceptible B. rapa materials and showed recessive resistance to clubroot. An F2 population derived from Bap246 × Bac1344 was used to locate the resistance Quantitative Trait Loci (QTL) by Bulk Segregant Analysis Sequencing (BSA-Seq) and QTL mapping methods. Two QTL on chromosomes A01 (4.67-6.06 Mb) and A08 (10.42-11.43 Mb) were found and named Cr4Ba1.1 and Cr4Ba8.1, respectively. Fifteen and eleven SNP/InDel markers were used to narrow the target regions in the larger F2 population to 4.67-5.17 Mb (A01) and 10.70-10.84 Mb (A08), with 85 and 19 candidate genes, respectively. The phenotypic variation explained (PVE) of the two QTL were 30.97% and 8.65%, respectively. Combined with gene annotation, mutation site analysis, and real-time quantitative polymerase chain reaction (qRT-PCR) analysis, one candidate gene in A08 was identified, namely Bra020861. And an insertion and deletion (InDel) marker (co-segregated) named Crr1-196 was developed based on the gene sequence. Bra013275, Bra013299, Bra013336, Bra013339, Bra013341, and Bra013357 in A01 were the candidate genes that may confer clubroot resistance in Chinese cabbage. The resistance resource and the developed marker will be helpful in Brassica breeding programs.

Map-based cloning and functional analysis of a major quantitative trait locus, BolC.Pb9.1, controlling clubroot resistance in a wild Brassica relative (Brassica macrocarpa).

KEY MESSAGE: A causal gene BoUGT76C2, conferring clubroot resistance in wild Brassica oleracea, was identified and functionally characterized. Clubroot is a devastating soil-borne disease caused by the obligate biotrophic pathogen Plasmodiophora brassica (P. brassicae), which poses a great threat to Brassica oleracea (B. oleracea) production. Although several QTLs associated with clubroot resistance (CR) have been mapped in cultivated B. oleracea, none have been cloned in B. oleracea. Previously, we found that the wild B. oleracea B2013 showed high resistance to clubroot. In this study, we constructed populations using B2013 and broccoli line 90196. CR in B2013 is quantitatively inherited, and a major QTL, BolC.Pb9.1, was identified on C09 using QTL-seq and linkage analysis. The BolC.Pb9.1 was finely mapped to a 56 kb genomic region using F2:3 populations. From the target region, the candidate BoUGT76C2 showed nucleotide variations between the parents, and was inducible in response to P. brassicae infection. We generated BoUGT76C2 overexpression lines in the 90196 background, which showed significantly enhanced resistance to P. brassicae compared to the WT line, suggesting that BoUGT76C2 corresponds to the resistance gene BolC.Pb.9.1. This is the first report on the CR gene map-based cloning and functional analysis from wild relatives, which provides a theoretical basis to the understanding of the molecular mechanism of CR, and lays a foundation to improve the CR of cultivated B. oleracea.

Role of Brassica rapa SWEET genes in the defense response to Plasmodiophora brassicae.

BACKGROUND: Interactions of plants with biotic stress factors including bacteria, fungi, and viruses have been extensively investigated to date. Plasmodiophora brassicae, a protist pathogen, causes clubroot disease in Cruciferae plants. Infection of Chinese cabbage (Brassica rapa) plants with P. brassica results in the formation of root galls, which inhibits the roots from absorbing soil nutrients and water. Sugar, the major source of carbon for all living organisms including pathogens and host plants, plays an important role in plant growth and development. OBJECTIVE: To explore the roles of BrSWEET2, BrSWEET13, and BrSWEET14 in P. brassicae resistance, Arabidopsis thaliana T-DNA knockout mutants sweet2, sweet13, and sweet14 were employed. METHODS: To isolate total RNA from the collected root nodules, the root tissues washed several times with running water and frozen tissues with liquid nitrogen. Total RNA was extracted using the Spectrum™ Plant Total RNA Kit (SIGMA) and cDNA was synthesized in a 20 µl reaction volume using the ReverTra Ace-α-® kit (TOYOBO). Real-time PCR was performed in a 10 µl reaction volume containing 1 µl of template DNA, 1 µl of forward primer, 1 µl of reverse primer, 5 µl of 2× iQTM SYBR® Green Supermix (BioRad), and 2 µl of sterile distilled water. The SWEET genes were genotyped using BioFACT™ 2× TaqBasic PCR Master Mix 2. RESULTS: Both sweet2 and sweet14 showed strong resistance to P. brassicae compared with wild-type Arabidopsis and Chinese cabbage plants and sweet13 mutant plants. Pathogenicity assays indicated that the SWEET2 gene plays an important role in clubroot disease resistance in higher plants.

A Hydroponic-Based Bioassay to Facilitate Plasmodiophora brassicae Phenotyping.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most devastating diseases affecting the canola/oilseed rape (Brassica napus) industry worldwide. Currently, the planting of clubroot-resistant (CR) cultivars is the most effective strategy used to restrict the spread and the economic losses linked to the disease. However, virulent P. brassicae isolates have been able to infect many of the currently available CR cultivars, and the options to manage the disease are becoming limited. Another challenge has been achieving consistency in evaluating host reactions to P. brassicae infection, with most bioassays conducted in soil and/or potting medium, which requires significant space and can be labor intensive. Visual scoring of clubroot symptom development can also be influenced by user bias. Here, we have developed a hydroponic bioassay using well-characterized P. brassicae single-spore isolates representative of clubroot virulence in Canada, as well as field isolates from three Canadian provinces in combination with canola inbred homozygous lines carrying resistance genetics representative of CR cultivars available to growers in Canada. To improve the efficiency and consistency of disease assessment, symptom severity scores were compared with clubroot evaluations based on the scanned root area. According to the results, this bioassay offers a reliable, less expensive, and reproducible option to evaluate P. brassicae virulence, as well as to identify which canola resistance profile(s) may be effective against particular isolates. This bioassay will contribute to the breeding of new CR canola cultivars and the identification of virulence genes in P. brassicae that could trigger resistance and that have been very elusive to this day.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Starch Plays a Key Role in Sporosorus Formation by the Powdery Scab Pathogen Spongospora subterranea.

Powdery scab disease, caused by the soilborne protist Spongospora subterranea f. sp. subterranea, poses a major constraint to potato production worldwide. Disease symptoms include damage to the tuber skin and the formation of root galls. This study aimed to investigate the potential mechanism behind the formation of sporosori, which are aggregates of resting spores, within root galls. Scanning electron microscopy analysis revealed that the early stage of gall formation, characterized by a white color, involved the accumulation of starch grains, which later disappeared as the gall matured and turned brown. The mature brown galls were found to contain fully formed sporosori. Light microscopy examination of ultramicrotome sections of the root galls showed that the high-amylopectin starches were surrounded by a plasmodium, a precursor to sporosorus. These findings suggest that starch grains contribute to the formation of a sponge-like structure within the sporosori. A significant reduction in total starch levels in both the root galls and their associated roots was observed compared with healthy roots. These findings indicate starch consumption by sporosori during the maturation of root galls. Interestingly, analysis of the transcript levels of starch-related genes showed downregulation of genes encoding starch degrading enzymes and an amylopectin-debranching enzyme, whereas genes encoding a starch synthase and a protein facilitating starch synthesis were upregulated in the infected roots. Overall, our results demonstrate that starch is consumed during sporosorus formation, and the pathogen likely manipulates starch homeostasis to its advantage for sporosorus development within the root galls.

Changes in Diversity and Composition of Rhizosphere Bacterial and Fungal Community between Resistant and Susceptible Pakchoi under Plasmodiophora brassicae.

Plasmodiophora brassicae (P. brassicae) is a soil-born pathogen worldwide and can infect most cruciferous plants, which causes great yield decline and economic losses. It is not well known how microbial diversity and community composition change during P. brassicae infecting plant roots. Here, we employed a resistant and a susceptible pakchoi cultivar with and without inoculation with P. brassicae to analyze bacterial and fungal diversity using 16S rRNA V3-V4 and ITS_V1 regions, respectively. 16S rRNA V3-V4 and ITS_V1 regions were amplified and sequenced separately. Results revealed that both fungal and bacterial diversity increased, and composition was changed in the rhizosphere soil of the susceptible pakchoi compared with the resistant cultivar. In the four groups of R_mock, S_mock, R_10d, and S_10d, the most relatively abundant bacterium and fungus was Proteobacteria, accounting for 61.92%, 58.17%, 48.64%, and 50.00%, respectively, and Ascomycota, accounting for 75.11%, 63.69%, 72.10%, and 90.31%, respectively. A total of 9488 and 11,914 bacteria were observed uniquely in the rhizosphere soil of resistant and susceptible pakchoi, respectively, while only 80 and 103 fungi were observed uniquely in the correlated soil. LefSe analysis showed that 107 and 49 differentially abundant taxa were observed in bacteria and fungi. Overall, we concluded that different pakchoi cultivars affect microbial diversity and community composition, and microorganisms prefer to gather around the rhizosphere of susceptible pakchoi. These findings provide a new insight into plant-microorganism interactions.

Characterization of a virulence factor in Plasmodiophora brassicae, with molecular markers for identification.

Symptom severity on differential host lines is currently used to characterize and identify pathotypes of Plasmodiophora brassicae, which is an obligate, soil-borne chromist pathogen that causes clubroot disease on canola (Brassica napus) and other brassica crops. This process is slow, variable and resource intensive; development of molecular markers could make identification of important pathotypes faster and more consistent for deployment of cultivars with pathotype-specific resistance. In the current study, a variant of gene 9171 was identified in the whole-genome sequences of only the highly virulent pathotypes of P. brassicae from around the world, including the new cohort of virulent pathotypes in Canada; its presence was confirmed using three KASP marker pairs. The gene was not present in the initial cohort of pathotypes identified in Canada. The putative structure, domains, and gene ontogeny of the protein product of gene 9171 were assessed using on-line software resources. Structural analysis of the putative protein produced by gene 9171 indicated that it was localized in the cytosol, and likely involved in cellular processes and catalytic activity. Identification of gene 9171 represents a potentially useful step toward molecular identification of the pathotypes of P. brassicae.