Canadian Forest Service Publications

Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusa f. sp. deltoidae. 2013. Boutigny, A.-L.; Guinet, C.; Vialle, A.; Hamelin, R.C.; Andrieux, A.; Frey, P.; Husson, C.; Ioos, R. Fungal Biol. 117:389-398.

Year: 2013

Available from: Laurentian Forestry Centre

Catalog ID: 35284

Language: English

CFS Availability: PDF (request by e-mail)

Abstract

Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S ). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe.

Plain Language Summary

The fungus Melampsora medusae is the pathogen responsible for poplar rust, considered one of the most damaging poplar diseases in the world. In Europe, this rust species is considered a quarantine species by the European and Mediterranean Plant Protection Organization and its presence is closely monitored. There are two types of this rust fungus: f. sp. deltoidae (the host of which is the eastern cottonwood) and f. sp. tremuloidae (the host of which is the trembling aspen).

The objective of this study conducted in Europe was to develop a quick and reliable test for detecting the presence of f.sp. deltoidae, the variety causing the greatest financial losses for nurseries. The test makes use of a technique based on the detection of the pathogen’s DNA molecules. This technique, called quantitative polymerase chain reaction (or in real time, qPCR), involves the use of a DNA extraction (e.g., obtained from leaves) to quickly detect the presence of the pathogen responsible for poplar rust. This test can be used for phytosanitary control purposes in order to prevent the introduction and spread of this rust in Europe.

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