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.

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.

Decoding the Arsenal: Protist Effectors and Their Impact on Photosynthetic Hosts.

Interactions between various microbial pathogens including viruses, bacteria, fungi, oomycetes, and their plant hosts have traditionally been the focus of phytopathology. In recent years, a significant and growing interest in the study of eukaryotic microorganisms not classified among fungi or oomycetes has emerged. Many of these protists establish complex interactions with photosynthetic hosts, and understanding these interactions is crucial in understanding the dynamics of these parasites within traditional and emerging types of farming, including marine aquaculture. Many phytopathogenic protists are biotrophs with complex polyphasic life cycles, which makes them difficult or impossible to culture, a fact reflected in a wide gap in the availability of comprehensive genomic data when compared to fungal and oomycete plant pathogens. Furthermore, our ability to use available genomic resources for these protists is limited by the broad taxonomic distance that these organisms span, which makes comparisons with other genomic datasets difficult. The current rapid progress in genomics and computational tools for the prediction of protein functions and interactions is revolutionizing the landscape in plant pathology. This is also opening novel possibilities, specifically for a deeper understanding of protist effectors. Tools like AlphaFold2 enable structure-based function prediction of effector candidates with divergent protein sequences. In turn, this allows us to ask better biological questions and, coupled with innovative experimental strategies, will lead into a new era of effector research, especially for protists, to expand our knowledge on these elusive pathogens and their interactions with photosynthetic hosts. [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.

A New Multiplex TaqMan qPCR for Precise Detection and Quantification of Clavibacter michiganensis in Seeds and Plant Tissue.

Bacterial canker of tomato caused by Clavibacter michiganensis (Cm) is one of the most devastating bacterial diseases affecting the tomato industry worldwide. As the result of Cm colonization of the xylem, the susceptible host shows typical symptoms of wilt, marginal leaf necrosis, stem cankers, and ultimately plant death. However, is the ability of Cm to infect seeds and plants without causing symptoms what makes it an even more dangerous pathogen. Unfortunately, there are no resistant cultivars or effective chemical or biological control methods available to growers against Cm. Its control relies heavily on prevention. The implementation of a rapid and accurate detection tool is imperative to monitor the presence of Cm and prevent its spread. In this study, we developed a specific and sensitive multiplex TaqMan qPCR assay to detect Cm and distinguish it from related bacterial species that affect tomato plants. Two Cm chromosomal virulence-related genes, rhuM and tomA, were used as specific targets. The plant internal control tubulin alpha-3 was included in each of the multiplexes to improve the reliability of the assay. Specificity was evaluated with 37 bacterial strains including other Clavibacter spp. and related and unrelated bacterial pathogens from different geographic locations affecting a wide variety of hosts. Results showed that the assay is able to discriminate Cm strains from other related bacteria. The assay was validated on tissue and seed samples following artificial infection and all tested samples accurately detected the presence of Cm. The tool described here is highly specific, sensitive, and reliable for the detection of Cm and allows the quantification of Cm in seeds, roots, stems, and leaves, and roots. The diagnostic assay can also be adapted for multiple purposes such as seed certification programs, surveillance, biosafety, the effectiveness of control methods, border protection, and epidemiological studies.

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.

A Decade of Hidden Phytoplasmas Unveiled Through Citizen Science.

Climate change is impacting agriculture in many ways, and a contribution from all is required to reduce the imminent losses related to it. Recently, it has been shown that citizen science could be a way to trace the impact of climate change. However, how can citizen science be applied in plant pathology? Here, using as an example a decade of phytoplasma-related diseases reported by growers, agronomists, and citizens in general, and confirmed by a government laboratory, we explored how to better value plant pathogen monitoring data. Through this collaboration, we found that in the last decade, 34 hosts have been affected by phytoplasmas; 9, 13, and 5 of these plants were, for the first time, reported phytoplasma hosts in eastern Canada, all of Canada, and worldwide, respectively. Another finding of great impact is the first report of a 'Candidatus Phytoplasma phoenicium'-related strain in Canada, while 'Ca. P. pruni' and 'Ca. P. pyri' were reported for the first time in eastern Canada. These findings will have a great impact on the management of phytoplasmas and their insect vectors. Using these insect-vectored bacterial pathogens, we show the need for new strategies that can allow fast and accurate communication between concerned citizens and those institutions confirming their observations.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Getting to the root of a club - Understanding developmental manipulation by the clubroot pathogen.

Plasmodiophora brassicae Wor., the clubroot pathogen, is the perfect example of an "atypical" plant pathogen. This soil-borne protist and obligate biotrophic parasite infects the roots of cruciferous crops, inducing galls or clubs that lead to wilting, loss of productivity, and plant death. Unlike many other agriculturally relevant pathosystems, research into the molecular mechanisms that underlie clubroot disease and Plasmodiophora-host interactions is limited. After release of the first P. brassicae genome sequence and subsequent availability of transcriptomic data, the clubroot research community have implicated the involvement of phytohormones during the clubroot pathogen's manipulation of host development. Herein we review the main events leading to the formation of root galls and describe how modulation of select phytohormones may be key to modulating development of the plant host to the benefit of the pathogen. Effector-host interactions are at the base of different strategies employed by pathogens to hijack plant cellular processes. This is how we suspect the clubroot pathogen hijacks host plant metabolism and development to induce nutrient-sink roots galls, emphasizing a need to deepen our understanding of this master manipulator.

The clubroot pathogen Plasmodiophora brassicae: A profile update.

BACKGROUND: Plasmodiophora brassicae is the causal agent of clubroot disease of cruciferous plants and one of the biggest threats to the rapeseed (Brassica napus) and brassica vegetable industry worldwide. DISEASE SYMPTOMS: In the advanced stages of clubroot disease wilting, stunting, yellowing, and redness are visible in the shoots. However, the typical symptoms of the disease are the presence of club-shaped galls in the roots of susceptible hosts that block the absorption of water and nutrients. HOST RANGE: Members of the family Brassicaceae are the primary host of the pathogen, although some members of the family, such as Bunias orientalis, Coronopus squamatus, and Raphanus sativus, have been identified as being consistently resistant to P. brassicae isolates with variable virulence profile. TAXONOMY: Class: Phytomyxea; Order: Plasmodiophorales; Family: Plasmodiophoraceae; Genus: Plasmodiophora; Species: Plasmodiophora brassicae (Woronin, 1877). DISTRIBUTION: Clubroot disease is spread worldwide, with reports from all continents except Antarctica. To date, clubroot disease has been reported in more than 80 countries. PATHOTYPING: Based on its virulence on different hosts, P. brassicae is classified into pathotypes or races. Five main pathotyping systems have been developed to understand the relationship between P. brassicae and its hosts. Nowadays, the Canadian clubroot differential is extensively used in Canada and has so far identified 36 different pathotypes based on the response of a set of 13 hosts. EFFECTORS AND RESISTANCE: After the identification and characterization of the clubroot pathogen SABATH-type methyltransferase PbBSMT, several other effectors have been characterized. However, no avirulence gene is known, hindering the functional characterization of the five intercellular nucleotide-binding (NB) site leucine-rich-repeat (LRR) receptors (NLRs) clubroot resistance genes validated to date. IMPORTANT LINK: Canola Council of Canada is constantly updating information about clubroot and P. brassicae as part of their Canola Encyclopedia: https://www.canolacouncil.org/canola-encyclopedia/diseases/clubroot/. PHYTOSANITARY CATEGORIZATION: PLADBR: EPPO A2 list; Annex designation 9E.

Multilocus sequence typing of diverse phytoplasmas using hybridization probe-based sequence capture provides high resolution strain differentiation.

Phytoplasmas are insect-vectored, difficult-to-culture bacterial pathogens that infect a wide variety of crop and non-crop plants, and are associated with diseases that can lead to significant yield losses in agricultural production worldwide. Phytoplasmas are currently grouped in the provisional genus 'Candidatus Phytoplasma', which includes 49 'Candidatus' species. Further differentiation of phytoplasmas into ribosomal groups is based on the restriction fragment length polymorphism (RFLP) pattern of the 16S rRNA-encoding operon, with more than 36 ribosomal groups (16Sr) and over 100 subgroups reported. Since disease symptoms on plants are not associated with phytoplasma identity, accurate diagnostics is of critical importance to manage disease associated with these microorganisms. Phytoplasmas are typically detected from plant and insect tissue using PCR-based methods targeting universal taxonomic markers. Although these methods are relatively sensitive, specific and are widely used, they have limitations, since they provide limited resolution of phytoplasma strains, thus necessitating further assessment of biological properties and delaying implementation of mitigation measures. Moreover, the design of PCR primers that can target multiple loci from phytoplasmas that differ at the sequence level can be a significant challenge. To overcome these limitations, a PCR-independent, multilocus sequence typing (MLST) assay to characterize an array of phytoplasmas was developed. Hybridization probe s targeting cpn60, tuf, secA, secY, and nusA genes, as well as 16S and rp operons, were designed and used to enrich DNA extracts from phytoplasma-infected samples for DNA fragments corresponding to these markers prior to Illumina sequencing. This method was tested using different phytoplasmas including 'Ca. P. asteris' (16SrI-B), 'Ca. P. pruni' (16SrIII-A),'Ca. P. prunorum' (16SrX-B), 'Ca. P. pyri' (16SrX-C), 'Ca. P. mali' (16SrX-A), and 'Ca. P. solani' (16SrXII-A). Thousands of reads were obtained for each gene with multiple overlapping fragments, which were assembled to generate full-length (typically >2 kb), high-quality sequences. Phytoplasma groups and subgroups were accurately determined based on 16S ribosomal RNA and cpn60 gene sequences. Hybridization-based MLST facilitates the enrichment of target genes of phytoplasmas and allows the simultaneous determination of sequences corresponding to seven different markers. In this proof-of-concept study, hybridization-based MLST was demonstrated to be an efficient way to generate data regarding 'Ca. Phytoplasma' species/strain differentiation.

First report of potato (Solanum tuberosum L.) blackleg disease caused by Dickeya solani in Mayabeque, Cuba.

Blackleg, caused by Pectobacterium spp. and Dickeya spp., is an important disease of potatoes. During the period from November 20 and March 2021, stems of potato plants showing necrosis and rot symptoms, and chlorotic leaves, were collected from commercial production areas of the Mayabeque province of Cuba (Fig. 1A). After disinfestation of affected stems, small fragments of the stem were cut and macerated in a sterile 0.85% NaCl solution. Serial dilutions of bacterial suspension were prepared and streaked onto nutrient agar in Petri plates. Two colonies per sample showing the characteristic of "fried egg" were selected for further investigation, and an isolated was selected and named D7. The isolated bacterium was rod shaped, gram-negative, motile, oxidase and indole production negative, with anaerobic growth, and able to use lactose as carbon source in Mac Conkey Agar medium. One colony of the isolate D7 was selected and multiplied. Total DNA of the bacteria cells was extracted and used to amplify the genes pelADE (Nassar et al., 1996) and gapA (Cigna et al., 2017), to differentiate Dickeya from Pectobacterium. The sequence obtained showed 99.75% and 99.88% nucleotide identity with Dickeya solani for pelADE (Genbank accession number ON644347) and gapA (Genbank accession number ON644346), respectively. To confirm the pathogenicity of the isolate D7, four 15-day-old potato plants, including two plants of each 'Otolia' and 'Naima' potatoes were inoculated with a bacterial suspension of the isolate D7 (108 CFU/ml) in sterile water by stabbing. Control plants were stabbed with sterile water. Inoculated plants were maintained at 28°C, relative humidity of about 90%, and at 12 h light/12 h dark, as described by (Chen et al. 2014). After 3 to 5 days, typical blackleg disease symptoms (water-soaked lesions and necrosis) developed at the inoculated areas of plants (Fig. 1B). No symptom was observed in the control plants. Bacterium was re-isolated from symptomatic plants and the isolates had the same cultural, physiological, and biochemical characteristics to the isolate D7. To our knowledge, this is the first report of D. solani causing blackleg in potato fields in Cuba. Further studies to determine the spread of this pathogen in potato producing areas in Cuba is underway.

Rapid Molecular Diagnostics in the Field and Laboratory to Detect Plant Pathogen DNA in Potential Insect Vectors.

A variety of sensitive and specific molecular diagnostic assays has been described for detecting nucleic acids in biological samples that may harbor pathogens of interest. These methods include very rapid, isothermal nucleic acid amplification methods that can be deployed outside of the laboratory environment, such as loop-mediated isothermal DNA amplification (LAMP) and recombinase-polymerase amplification (RPA). However, all molecular diagnostic assays must be preceded by nucleic acid extraction from the biological samples of interest, which provides suitable template molecules for the assays. To exploit the features of the amplification assays and be utilized outside of the lab, these methods must be rapid and avoid the need for typical laboratory chemicals and equipment. We describe a protocol for the extraction of DNA from field-collected insects that can be implemented at the point of collection and used to detect the presence of DNA sequences from potential plant pathogens that may be vectored by the insects. This protocol provides template DNA that is suitable for PCR, LAMP, and RPA. The FTA PlantSaver card-based DNA extraction product was also confirmed to amplify the mitochondrial cytochrome oxidase 1 (CO1) universal barcode that could later be sequenced to identify any insect. Lastly, we provide an example using field-collected insects, Neokolla (Graphocephala) heiroglyphica, and demonstrate the detection of the plant pathogen Xylella fastidiosa in carrier insects using PCR, RPA, and LAMP.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Cannabis sativa L. (marijuana) in Quebec, Canada.

Marijuana (Cannabis sativa L.) is legal in Canada for medical and recreational purposes and is currently a multi-million-dollar industry. The province of Quebec follows British Columbia and Ontario in production acreage (Government of Canada 2018). During the growing season 2020-2021, five greenhouse growers throughout Quebec reported the presence of signs and symptoms reminiscent of powdery mildew including the presence of white powdery patches on the adaxial sides of leaves of several C. sativa cultivars. From one commercial facility, infected leaves of three cannabis cultivars (Sour Diesel, Orange Krush, and Lemon Sour) were photographed and the fungal mycelium was collected for identification in the laboratory. Fungal mycelium on leaf tissue was white and amphigenous and displayed unbranched hyaline conidiophores ranging from 130 to 275 µm in height (n = 50). Conidiophores arose from the upper surface of hyphal mother cells ranging from 35-70 × 8-13 µm in diameter (n = 25) and formed catenescent conidia. Conidia were broad ellipsoid-ovoid and measured, 24 to 35 × 12 to 19 µm (n = 50), and hyphae ranged from 3-8 µm in diameter (n = 30). Based on previous description (Qiu et al. 2020), the fungus was placed within the Golovinomyces genus. The species identification was confirmed through multi-locus phylogenetic using internal transcribed spacer (ITS), 28S large ribosomal subunit, and chitin synthase I (CHS1) genes amplified as recommended (Qiu et al. 2020), and directly sequenced with amplification primers (Centre Hospitalier de l'Université Laval de Quebec, CA). The three marker sequences shared 100% similarity for all the samples analyzed and were deposited in Genbank under accession numbers: OM131434 (28S), OM131448 (ITS), and OM141118 (CHS1). The phylogenetic analysis of the multi-locus sequences amplified grouped all three Quebec marijuana isolates in the G. ambrosiae accessions, confirming their identification. Pathogenicity was confirmed by transferring conidia onto detached healthy leaves of hop plants (Humulus lupulus) cultivar Northern Brewer kept under greenhouse conditions (28C, 50-60% relative humidity, and 14 h light) via paint brush inoculation. Hop leaves were used as surrogate due to the restricted availability of marijuana leaves. Inoculated leaves were placed in the growth chamber set at 20C, 50-60% relative humidity, and long days conditions as previously suggested (Weldon et al. 2020). The leaves developed powdery mildew colonies after 21 days, and the fungus was confirmed to be G. ambrosiae following morphological characterization and amplification of CHS1. Powdery mildew caused by G. ambrosiae (previous Golovinomyces cichoracearum) has been reported affecting hemp (Cannabis sativa) in New York and Oregon, United Sates (Weldon et al. 2020; Wiseman et al. 2021), and in British Columbia, Canada (Pépin et al. 2018; Punja et al. 2021), and this is the first report of G. ambrosiae causing powdery mildew on marijuana in Quebec. REFERENCES Government of Canada 2018. Online, retrieved January 7, 2021 https://www150.statcan.gc.ca/n1/daily-quotidien/180430/dq180430b-eng.htm Pépin N, Punja ZK, Joly DL. 2018. First report of powdery mildew caused by Golovinomyces cichoracearum sensu lato on Cannabis satia in Canada. Plant Disease. 102(12):2644. Doi: https://doi.org/10.1094/PDIS-04-18-0586-PDN Punja, Z. P. (2021). First report of the powdery mildew pathogen of hops, Podosphaeria macularis, naturally infecting cannabis (Cannabis sativa L., marijuana) plants under field conditions, Canadian Journal of Plant Pathology, Doi: https://doi.org/10.1080/07060661.2021.1960424. Qiu, P.-L., Liu, S.-Y., Bradshaw, M., Rooney-Latham, S., Takamatsu, S., Bulgakov, T. S., Tang, S.-R., Feng, J., Jin, D.-N., Aroge, T., Li, Y., Wang, L.-L., and Braun, U. 2020. Multi-locus phylogeny and taxonomy of an unresolved, heterogeneous species complex within the genus Golovinomyces (Ascomycota, Erysiphales), including G. ambrosiae, G. circumfusus and G. spadiceus. BMC Microbiology. 20:51. Doi : https://doi.org/10.1186/s12866-020-01731-9. Weldon WA, Ullrich MR, Smart LB, Smart CD, Gadoury DM. 2020. Cross-infectivity of powdery mildew isolates originating from hemp (Cannabis sativa) and Japanese hop (Humulus japonicus) in New York. Plant Health Progress. 21(1):47-53. Doi: https://doi.org/10.1094/PHP-09-19-0067-RS Wiseman, M. S., Bates, T. A., Garfinkel, A. R., Ocamb, C. M., and Gent, D. H. 2021. First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Cannabis sativa in Oregon. Plant Disease 105(9):2733. Doi: https://doi.org/10.1094/PDIS-11-20-2455-PDN.

Plasmodiophora brassicae in Mexico: From Anecdote to Fact.

For years, the presence of clubroot disease and its causal agent, Plasmodiophora brassicae, in Mexico has been stated as a fact. However, an intensive search of the scientific literature in English and Spanish, as well as gray literature including theses and government reports, did not reveal any information about the actual detection of the pathogen, affected hosts, or areas with clubroot presence, or any information about clubroot (hernia de la col in Mexico). We followed a multistep process to confirm whether P. brassicae was indeed in Mexico. First, we identified agricultural communities with a history of cruciferous crop cultivation. Second, we asked growers if they had seen clubroot on their crops, using pictures of the characteristic root galls. Third, we collected soil from the locations where clubroot was reported and looked for clubroot/P. brassicae in the soil using several cruciferous bait plants. For the first time we confirm the presence of the clubroot pathogen P. brassicae in Mexico, through a bioassay, the presence of resting spores, and a P. brassicae-specific PCR assay. The identification of P. brassicae in Mexico will contribute to our understanding of the genetic diversity of this elusive and devastating plant pathogen in future studies.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Detection of blueberry stunt phytoplasma in Eastern Canada using cpn60-based molecular diagnostic assays.

Blueberry stunt phytoplasma (BBSP; 'Candidatus Phytoplasma asteris') is an insect-vectored plant pathogen that causes severe yield losses in blueberry (Vaccinium corymbosum), which is the most valuable fruit crop in Canada. Rapid, field-based diagnostic assays are desirable tools for the control of BBSP, as part of an integrated, proactive approach to production management termed biovigilance. We designed and validated a chaperonin-60 (cpn60)-targeted LAMP assay for detection of BBSP, providing a rapid, low cost, field-deployable diagnostic option. Our validation demonstrates that the assay is reproducible, with high analytical specificity and improved sensitivity when compared with 16S rRNA nested PCR. We applied the validated LAMP assay to nearly 2000 blueberry samples from Québec and Nova Scotia over three growing seasons (2016-2018). Our surveys revealed that BBSP is present in most sites across both provinces, though detection of the pathogen in individual plants varied in different tissues across sampling dates and across years, and evidence of spread between plants was limited. To quantify pathogen load in select plants, we designed additional qPCR and ddPCR assays, also based on cpn60. We found that pathogen load fluctuates in individual plants, both within and between growing seasons. Finally, we designed an interactive map to visualize the results of our surveys. These results provide a validated diagnostic assay that can be used as part of a biovigilance strategy for detecting and controlling infections caused by BBSP.

A clubroot pathogen effector targets cruciferous cysteine proteases to suppress plant immunity.

Plant pathogen effector proteins are key to pathogen virulence. In susceptible host Brassicas, the clubroot pathogen, Plasmodiophora brassicae, induces the production of nutrient-sink root galls, at the site of infection. Among a list of 32 P. brassiae effector candidates previously reported by our group, we identified SSPbP53 as a putative apoplastic cystatin-like protein highly expressed during the secondary infection. Here we found that SSPbP53 encoding gene is conserved among several P. brassicae pathotypes and that SSPbP53 is an apoplastic protein able to directly interact with and inhibit cruciferous papain-like cysteine proteases (PLCPs), specifically Arabidopsis XYLEM CYSTEINE PEPTIDASE 1 (AtXCP1). The severity of clubroot disease is greatly reduced in the Arabidopsis xcp1 null mutant (AtΔxcp1) after infection with P. brassicae resting spores, indicating that the interaction of P. brassicae SSPbP53 with XCP1 is important to clubroot susceptibility. SSPbP53 is the first cystatin-like effector identified and characterized for a plant pathogenic protist.

Endomembrane-Targeting Plasmodiophora brassicae Effectors Modulate PAMP Triggered Immune Responses in Plants.

Plasmodiophora brassicae is a devastating obligate, intracellular, biotrophic pathogen that causes clubroot disease in crucifer plants. Disease progression is regulated by effector proteins secreted by P. brassicae. Twelve P. brassicae putative effectors (PbPEs), expressed at various stages of disease development [0, 2, 5, 7, 14, 21, and 28 days post inoculation (DPI)] in Arabidopsis and localizing to the plant endomembrane system, were studied for their roles in pathogenesis. Of the 12 PbPEs, seven showed an inhibitory effect on programmed cell death (PCD) as triggered by the PCD inducers, PiINF1 (Phytophthora infestans Infestin 1) and PiNPP1 (P. infestans necrosis causing protein). Showing the strongest level of PCD suppression, PbPE15, a member of the 2-oxoglutarate (2OG) and Fe (II)-dependent oxygenase superfamily and with gene expression during later stages of infection, appears to have a role in tumorigenesis as well as defense signaling in plants. PbPE13 produced an enhanced PiINF1-induced PCD response. Transient expression, in Nicotiana benthamiana leaves of these PbPEs minus the signal peptide (SP) (Δsp PbPEGFPs), showed localization to the endomembrane system, targeting the endoplasmic reticulum (ER), Golgi bodies and nucleo-cytoplasm, suggesting roles in manipulating plant cell secretion and vesicle trafficking. Δsp PbPE13GFP localized to plasma membrane (PM) lipid rafts with an association to plasmodesmata, suggesting a role at the cell-to-cell communication junction. Membrane relocalization of Δsp PbPE13GFP, triggered by flagellin N-terminus of Pseudomonas aeruginosa (flg22 - known to elicit a PAMP triggered immune response in plants), supports its involvement in raft-mediated immune signaling. This study is an important step in deciphering P. brassicae effector roles in the disruption of plant immunity to clubroot disease.

Transcriptome Analysis Identifies Plasmodiophora brassicae Secondary Infection Effector Candidates.

Plasmodiophora brassicae (Wor.) is an obligate intracellular plant pathogen affecting Brassicas worldwide. Identification of effector proteins is key to understanding the interaction between P. brassicae and its susceptible host plants. To date, there is very little information available on putative effector proteins secreted by P. brassicae during a secondary infection of susceptible host plants, resulting in root gall production. A bioinformatics pipeline approach to RNA-Seq data from Arabidopsis thaliana (L.) Heynh. root tissues at 17, 20, and 24 d postinoculation (dpi) identified 32 small secreted P. brassicae proteins (SSPbPs) that were highly expressed over this secondary infection time frame. Functional signal peptides were confirmed for 31 of the SSPbPs, supporting the accuracy of the pipeline designed to identify secreted proteins. Expression profiles at 0, 2, 5, 7, 14, 21, and 28 dpi verified the involvement of some of the SSPbPs in secondary infection. For seven of the SSPbPs, a functional domain was identified using Blast2GO and 3D structure analysis and domain functionality was confirmed for SSPbP22, a kinase localized to the cytoplasm and nucleus.

The CpnClassiPhyR Is a Resource for cpn60 Universal Target-Based Classification of Phytoplasmas.

Phytoplasmas are plant-pathogenic bacteria that are associated with yield losses in many crop plants worldwide. Phytoplasma strain differentiation is accomplished using in silico restriction fragment length polymorphism (RFLP) analysis of 16S ribosomal RNA-encoding gene sequences, which has resulted in the definition of ribosomal groups and subgroups of phytoplasmas. Due to limitations associated with this approach, a complementary classification scheme was recently developed based on RFLP analysis of the single-copy, protein-encoding gene chaperonin-60 (cpn60). We present the CpnClassiPhyR, software that facilitates phytoplasma strain classification using both RFLP and automated phylogenetic analysis of cpn60 sequences. This software is available through a web interface at http://cpnclassiphyr.ca.