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, what makes Cm an even more dangerous pathogen is its ability to infect seeds and plants without causing symptoms. 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. 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.[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 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.

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.

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.