title: Development of SCAR markers for rapid and specific detection of Pseudomonas syringae pv. morsprunorum races 1 and 2, using conventional and real-time PCR.
abstract: Specific primers were developed to detect the causal agent of stone fruit bacterial canker using conventional and real-time polymerase chain reaction (PCR) methods. PCR melting profile (PCR MP) used for analysis of diversity of Pseudomonas syringae strains, allowed to pinpoint the amplified fragments specific for P. syringae pv. morsprunorum race 1 (Psm1) and race 2 (Psm2), which were sequenced. Using obtained data, specific sequence characterised amplified region (SCAR) primers were designed. Conventional and real-time PCRs, using genomic DNA isolated from different bacterial strains belonging to the Pseudomonas genus, confirmed the specificity of selected primers. Additionally, the specificity of the selected DNA regions for Psm1 and Psm2 was confirmed by dot blot hybridisation. Conventional and real-time PCR assays enabled accurate detection of Psm1 and Psm2 in pure cultures and in plant material. For conventional PCR, the detection limits were the order of magnitude ~10(0) cfu/reaction for Psm1 and 10(1) cfu/reaction for Psm2 in pure cultures, while in plant material were 10(0)-10(1) cfu/reaction using primers for Psm1 and 3 × 10(2) cfu/reaction using primers for Psm2. Real-time PCR assays with SYBR Green I showed a higher limit of detection (LOD) - 10(0) cfu/reaction in both pure culture and in plant material for each primer pairs designed, which corresponds to 30-100 and 10-50 fg of DNA of Psm1 and Psm2, respectively. To our knowledge, this is the first PCR-based method for detection of the causal agents of bacterial canker of stone fruit trees.
authors.affiliation: Research Institute of Horticulture, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland. monika.kaluzna@inhort.pl.
authors.forename: Monika
authors.initials: M
authors.lastname: Kałużna
authors.affiliation: Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO), InBIO, Laboratório Associado, Universidade do Porto, Campus Agrário de Vairão, 4485-661, Vairão, Portugal.|Faculdade de Ciencias, Departamento de Biologia, Universidade do Porto, Edifício FC4, Via Panoramica No. 36, 4150-564, Porto, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Albuquerque
authors.affiliation: Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO), InBIO, Laboratório Associado, Universidade do Porto, Campus Agrário de Vairão, 4485-661, Vairão, Portugal.|Faculdade de Ciencias, Departamento de Biologia, Universidade do Porto, Edifício FC4, Via Panoramica No. 36, 4150-564, Porto, Portugal.
authors.forename: Fernando
authors.initials: F
authors.lastname: Tavares
authors.affiliation: Research Institute of Horticulture, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Sobiczewski
authors.affiliation: Research Institute of Horticulture, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Puławska
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques; D017931:DNA Primers / Q000235:genetics*; D005819:Genetic Markers; D010935:Plant Diseases / Q000382:microbiology; D044224:Pseudomonas syringae / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D005819:Genetic Markers
keywords: Dot blot hybridisation; PCR MP; Real-time PCR; SCAR primers; Stone fruit tree pathogens
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