Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae

Boutigny, Anne-Laure; Guinet, Cécile; Vialle, Agathe; Hamelin, Richard; Andrieux, Axelle; Frey, Pascal; Husson, Claude; Ioos, Renaud

doi:10.1016/j.funbio.2013.04.001

Cited by 11 publications

(11 citation statements)

References 26 publications

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“…In turn, the decision to rate a Ct value as a positive or indeterminate result may be influenced by internal rules, which are not the same between laboratories. In addition, slight variations induced by the operator or the equipment, such as pipetting errors, temperature drift or thermic heterogeneity of the thermal cycler block may have an effect on the stringency of the PCR reaction and thus, in turn may affect the analytical specificity and sensitivity 33,37–39 .…”

Section: Discussionmentioning

confidence: 99%

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum

Ioos¹,

Aloi

Piškur

et al. 2019

Sci Rep

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Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii , causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F . circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F . circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F . circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

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Section: Discussionmentioning

confidence: 99%

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum

Ioos¹,

Aloi

Piškur

et al. 2019

Sci Rep

Self Cite

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“… Mean C t values ± standard deviation using the Mel28S real‐time PCR test, DNA extracts were normalized at 0·5 ng μ L −1 prior to test (Boutigny et al ., ). …”

Section: Methodsmentioning

confidence: 97%

“…The quality of all DNA extracts was assessed according to Boutigny et al . (), and using the Mel PCR primers/probe, designed from a region of the 28S rDNA that is highly conserved in the genus Melampsora .…”

Section: Methodsmentioning

confidence: 99%

Simultaneous monitoring and quantification of Melampsora allii‐populina and Melampsora larici‐populina on infected poplar leaves using a duplex real‐time PCR assay

et al. 2015

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The rust fungi Melampsora larici-populina (Mlp) and Melampsora allii-populina (Map) are the main phytosanitary constraints for commercial poplar cultivation in Europe. Although Mlp is more aggressive and prevalent than Map, the two species may co-infect the same poplar tree or even the same poplar leaf, making the epidemiological surveys of each species difficult to achieve. In this study, a new duplex real-time PCR assay targeting each species was developed, based on single-copy genes. This test proved to be specific, inclusive and was successfully used to detect and quantify each species, starting from urediniospore samples or directly from infected poplar leaves, with or without visible symptoms. This new molecular tool was also assessed for comparative studies of time-course infection experiments on artificially inoculated poplar leaf discs. These studies showed that the growth dynamics of Map were significantly slower when the two species were co-inoculated on the same leaf disc, confirming that Map is less aggressive than Mlp.

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“…Populus deltoides ) or Populus (e.g. Populus tremuloides ), respectively (Shain, ; Boutigny et al., ).…”

Section: Pest Categorisationmentioning

confidence: 99%

Pest categorisation of Melampsora medusae

Jeger¹,

Bragard²,

Caffier³

et al. 2018

EFS2

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Following a request from the European Commission, the EFSA Plant Health Panel performed a pest categorisation of Melampsora medusae, a well-defined and distinguishable fungal species of the family Melampsoraceae. The pathogen is regulated in Annex IAI of Council Directive 2000/29/EC as a harmful organism whose introduction into the EU is banned. M. medusae is a heteroecious rust fungus with Populus spp. as primary telial hosts and various conifers (Larix, Pinus, Pseudotsuga, Abies, Picea and Tsuga spp.) as secondary aecial hosts. M. medusae is native to North America and has spread to South America, Africa, Asia, Oceania, as well as the EU, where M. medusae f. sp. deltoidae has been reported with a restricted distribution and low impacts from Belgium, south-west France and southern Portugal. The pest could spread to other EU countries, via dissemination of spores, movement of host plants for planting and cut branches. Climate is assumed not to be a limiting factor for the establishment of the pathogen in the EU. M. medusae is the most widespread and important Melampsora rust in North America. In western Canada, extensive damage has been reported to conifers and Populus spp. in nurseries and plantations as well as in woodlands. M. medusae is damaging in both Australia and New Zealand. The pest could have economic and environmental impacts in the EU if aggressive isolates of M. medusae were introduced into the EU. Import prohibition of host plants for planting is an available measure to reduce the risk of further introductions. Some resistant Populus cultivars are available. Moreover, increasing the genetic diversity of poplar plantations can prevent disease impacts. The main uncertainty concerns the factors explaining the low pathogenicity of the populations of M. medusae present in the EU. The criteria assessed by the Panel for consideration as a potential quarantine pest are met (the pest is present, but with a restricted distribution, and is officially under control). Given that plants for planting are not the main pathway of spread, not all criteria for consideration as a regulated non-quarantine pest are met.

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Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae

Cited by 11 publications

References 26 publications

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum

Simultaneous monitoring and quantification of Melampsora allii‐populina and Melampsora larici‐populina on infected poplar leaves using a duplex real‐time PCR assay

Pest categorisation of Melampsora medusae

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