Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

Lamarche, Josyanne; Potvin, Amélie; Pelletier, G.; Stewart, Don; Feau, Nicolas; Alayon, Dario Isidro Ojeda; Dale, Angela; Coelho, Aaron; Uzunovic, Adnan; Bilodeau, Guillaume J.; Brière, S. C.; Hamelin, Richard; Tanguay, Philippe

doi:10.1371/journal.pone.0134265

Cited by 53 publications

(51 citation statements)

References 74 publications

(56 reference statements)

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“…The sensitivity of this assay allowed detection of 1–3 copies of the ITS target region of pure O. c‐j DNA 95% of the time. Similar results were reported by researchers working on trace detection of fungal DNA, where LOD values varying between 1 and 25 copies were obtained (Johnson, Bibby, Wong, Agrawal, & Bustin, ; Lamarche et al., ; Liu et al., ). We obtained positive O. c‐j detection signal in 100% of DNA samples extracted from the equivalent to one conidium per PCR.…”

Section: Discussionsupporting

confidence: 86%

qPCR quantification of Ophiognomonia clavigignenti‐juglandacearum from infected butternut trees under different release treatments

et al. 2018

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Summary The use of a molecular assay for quantifying conidia of Ophiognomonia clavigignenti‐juglandacearum, the fungal pathogen responsible of butternut canker, was investigated. Purified DNA from conidia collected on glass fibre filters of a passive rain collectors was quantified using a TaqMan real‐time quantitative polymerase chain reaction (qPCR) assay. The qPCR assay could specifically discriminate the target species from all other North American known species of Ophiognomonia, and it was sensitive enough to repeatedly detect one conidium. A linear relationship between numbers of conidia and qPCR Ct values was determined, and used to assess the sporulation of the pathogen under trees that were released to promote their vigour. In total, 977 samples of field‐captured conidia from 49 trees, at two locations, and from two successive growing seasons were analysed. No significant difference of sporulation was observed under control and release treatments. However, our results demonstrated that qPCR assay was reliable for detecting and quantifying O. clavigignenti‐juglandacearum from environmental samples, which will be useful to assess further control methods for this disease.

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Section: Discussionsupporting

confidence: 86%

qPCR quantification of Ophiognomonia clavigignenti‐juglandacearum from infected butternut trees under different release treatments

et al. 2018

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“…One of the challenges of developing assays with primers and probes targeting conserved gene regions is the paucity of variable regions among closely related species. This limitation has been reported in Pseudogymnoascus (Shuey et al ., ) and Phytophthora kernoviae (Lamarche et al ., ), where the genes and genome regions commonly used for assay development did not possess sufficient variation to discriminate between the target species and its close relative. Discriminating among lineages of P. ramorum was even more challenging as polymorphisms were rare in the ITS (Kroon et al ., ), β‐tubulin and CBEL (Bilodeau et al ., ) and the cytochrome c oxidase subunit 1 ( Cox 1 ) gene (Kroon et al ., ).…”

Section: Discussionmentioning

confidence: 99%

“…The DNA concentration of this dilution was translated to the equivalent number of genome copies as described by Lamarche et al . ().…”

Section: Methodsmentioning

confidence: 97%

Improved detection and identification of the sudden oak death pathogen Phytophthora ramorum and the Port Orford cedar root pathogen Phytophthora lateralis

et al. 2019

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Early detection provides the best way to prevent introduction and establishment of alien plant pathogens. Amplification of DNA by PCR has revolutionized the detection and monitoring of plant pathogens. Most of those assays rely on the amplification of a fraction of the genome of the targeted species. With the availability of whole genomes for a growing number of fungi and oomycetes it is becoming possible to compare genomes and discover regions that are unique to a target organism. This study has applied this pipeline to develop a set of hierarchical TaqMan real‐time PCR detection assays targeting DNA of all four Phytophthora ramorum lineages, and a closely related species, P. lateralis. Nine assays were generated: three targeting DNA of all P. ramorum lineages, one for each lineage of P. ramorum, one for P. lateralis and one targeting DNA of P. ramorum and P. lateralis. These assays were very accurate and sensitive, ranging from 98.7% to 100% detection accuracy of 2–10 gene copies of the targeted taxa from pure cultures or inoculated tissues. This level of sensitivity is within the lowest theoretical limit of detection of DNA. It is expected that these assays will be useful because of their high level of specificity and the ease with which they can be multiplexed because of the inherent flexibility in primer and probe design afforded by their lack of conservation in non‐target species.

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“…For example, as there are far fewer described oomycetes compared to plants and fungi, this limitation may subsequently influence the diversity of genera detected (Blackwell, 2011;Hawksworth & Rossman, 1997;Rossman & Palm, 2006). Plus, any genic region, including ITS and ATP9-NAD9, has its pros and cons for identification; these include copy number, sufficient variability for species resolution, gene marker universality, and availability of a large variety of reference sequences (Edger et al, 2014;Lamarche et al, 2015Lamarche et al, , 2014. Any given bioinformatics pipeline also introduce biases in the final taxonomic assignments and abundance used throughout the process (Majaneva, Hyytiäinen, Varvio, Nagai, & Blomster, 2015).…”

Section: Discussionmentioning

confidence: 99%

High‐resolution biomonitoring of plant pathogens and plant species using metabarcoding of pollen pellet contents collected from a honey bee hive

et al. 2019

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The Canadian beekeeping industry is spread across the country, with the greatest proportion of managed honey bee colonies occurring in the Prairie Provinces. Nationally, the number of beekeepers has recently been trending upwards. Simultaneously, agronomic and environmental plant pest incidents are increasing due to a number of factors, including the introduction of exotic organisms through international trade, which is a major pathway for the introduction of potentially invasive alien species and quarantine pests. Therefore, regulatory agencies are interested in developing high‐throughput tools to achieve earlier detection of unwanted species in order to expedite application of mitigating measures to limit the impacts of their introduction. This study evaluates the potential of pollen pellet contents collected by honey bees to monitor plant pests using metabarcoding, a high‐throughput sequencing (HTS) approach for monitoring complex environmental samples. The study used the ITS1 intergenic region to target oomycetes and fungi, the ATP9‐NAD9 spacer to specifically target Phytophthora species, and the ITS2 region to target plant species. From the HTS results, a number of plants that were detected corresponded to known hosts of certain pathogens or species closely related to potentially invasive plant species. Genera including phytopathogenic species found in the pollen samples comprised Fusarium sp., Ophiostoma sp., Peronospora sp., Phytophthora sp., and Pythium sp. Correlations, high entropy, and co‐occurrences between certain plants and oomycetes or fungi were observed. The potential for using honey bee‐collected pollen pellets to study phytopathogens in a given environment is demonstrated here, and this concept could represent a promising complementary tool for the surveillance of phytopathogens or unwanted plants with previously described air and insect sampling methods if the protocol was applied with additional genetic markers.

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Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

Cited by 53 publications

References 74 publications

qPCR quantification of Ophiognomonia clavigignenti‐juglandacearum from infected butternut trees under different release treatments

qPCR quantification of Ophiognomonia clavigignenti‐juglandacearum from infected butternut trees under different release treatments

Improved detection and identification of the sudden oak death pathogen Phytophthora ramorum and the Port Orford cedar root pathogen Phytophthora lateralis

High‐resolution biomonitoring of plant pathogens and plant species using metabarcoding of pollen pellet contents collected from a honey bee hive

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