A sensitive real-time PCR assay for the detection of the two Melampsora medusae formae speciales on infected poplar leaves

Abstract

Melampsora medusae is a quarantine fungus in the European Union (EU) that causes a damaging leaf rust disease on poplars. Two formae speciales of the pathogen can be distinguished, M. medusae f. sp. deltoidae and M. medusae f. sp. tremuloidae, but the EU plant health directive 2000/29/EC currently in force does not make the distinction between them. EU countries must have the ability to detect and identify rapidly the introduction of these quarantine fungi and to conduct extensive surveys in case of outbreaks. Efficient detection tools are thus needed. In this study, a sensitive real-time PCR assay was developed to detect the presence of M. medusae in poplar leaf samples. A unique primer/hydrolysis probe combination targeting both formae speciales was designed using species-specific polymorphisms observed within the internal transcribed spacer region. An additional primer/hydrolysis probe combination was designed from a region of the 28S rDNA that is highly conserved in the genus Melampsora and used in a separate real-time PCR assay in order to check the quality of the DNA extracted from Melampsora urediniospores. The test developed demonstrated a high sensitivity since it enables the reproducible detection of two M. medusae urediniospore in a mixture of 2 mg of urediniospores (ca 800 000 urediniospores) of other Melampsora species. This new real-time PCR tool should be useful for laboratories in charge of official analyses since it has many advantages over the techniques currently used to monitor this quarantine pathogen in Europe

Use of QuantitativePCRDetection Methods to Study Biocontrol Agents and Phytopathogenic Fungi and Oomycetes in Environmental Samples

“…Particularly relevant is the detection of quarantine pathogens, because molecular analyses are likely to impact on large-scale eradication schemes or plant trade (Schena et al 2006;Montes-Borrego et al 2011). Due to its high specificity and sensitivity, qPCR is increasingly included in official protocols of the European Plant Protection Organization (http:// archives.eppo.org/index.htm) for the certification, production and assessment of healthy plant materials (Blanco-Meneses and Ristaino 2011; Boutigny et al 2013). Given the high importance of an accurate detection of quarantine pathogens and the risk of false positive/negative results, a statistical procedure has been proposed to determine the cycle cut-off and the corresponding limit of detection in qPCR (Chandelier et al 2010).…”

Molecular studies of rust on European aspen suggest an autochthonous relationship shaped by genotype

“…Fungal communities in tree foliage are not uniformly distributed or stationary ( Toju et al., 2019 ); their composition is influenced by several factors including host genotype ( Albrectsen et al., 2010b ), growth site ( Siddique et al., 2021 ), seasonal factors ( Albrectsen et al., 2010b ), ontogeny ( Bourassa et al., 2005 ; Boutigny et al., 2013a ), tissue type ( Siddique et al., 2021 ), and host-associated consumers ( Arnold et al., 2003 ; Albrectsen et al., 2010b ; Siddique et al., 2021 ). The mechanisms underpinning these relationships are not fully understood but it has been suggested that both the host’s chemical defense responses ( Ullah et al., 2017 ; Witzell et al., 2022 ) and competition between members of the mycobiome may help shape the microbial community ( Wemheuer et al., 2019 ; Siddique et al., 2021 ).…”

“…Additionally, molecular detection and quantification techniques are increasingly used to analyze individual trees or small groups of trees ( Feau et al., 2009 ; Ioos et al., 2010 ; Tan et al., 2010 ). Fungi share a conserved ITS rDNA sequence that can be amplified using specific primers, enabling PCR-based molecular verification and quantification of fungal infestations ( Bourassa et al., 2005 ; Husson et al., 2013 ; Boutigny et al., 2013a ; Boutigny et al., 2013b ; Bergeron et al., 2019 ; Siddique et al., 2022 ). The Melampsora genus includes several species that use P. tremula as summer host including M. medusae, M. larici-populina , which are exotic in Sweden, and M. pinitorqua , which is native to Sweden.…”

Genome-enhanced detection and identification of fungal pathogens responsible for pine and poplar rust diseases

“…Real-time PCR assays were also developed for the simultaneous detection of both formae speciales of M . medusae , based on polymorphisms found within the ITS region, and also for the detection of the forma specialis deltoidae only, based on the large ribosomal RNA subunit [18, 19]. Regions other than ribosomal DNA were also targeted.…”