Development of SCAR markers for rapid and specific detection of Pseudomonas syringae pv. morsprunorum races 1 and 2, using conventional and real-time PCR

• Published in: Applied microbiology and biotechnology Vol. 100; no. 8; pp. 3693 - 3711
• Main Authors: Kałużna, Monika, Albuquerque, Pedro, Tavares, Fernando, Sobiczewski, Piotr, Puławska, Joanna
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-04-2016; Springer; Springer Nature B.V
• Subjects: Analysis; Bacteria; bacterial canker; Bacterial infections; Bacterial Proteins - genetics; Bacterial Typing Techniques; Biological markers; Biomedical and Life Sciences; Biotechnology; Deoxyribonucleic acid; detection limit; Detection limits; Diseases and pests; DNA; DNA Primers - genetics; Fruit trees; Fruits; Genes; Genetic Markers; Genomics; Hybridization; Identification; Identification and classification; Life Sciences; melting; Methods and Protocols; Microbial Genetics and Genomics; Microbiology; nucleic acid hybridization; Pathogens; Physiological aspects; Physiology; Plant Diseases - microbiology; Polymerase chain reaction; Pseudomonas syringae; Pseudomonas syringae - genetics; Pseudomonas syringae - isolation & purification; Pseudomonas syringae pv. morsprunorum; quantitative polymerase chain reaction; races; Real-Time Polymerase Chain Reaction - methods; Serology; Species Specificity; Stone fruits; Studies; Trees; Stone fruit tree pathogens; Real-time PCR; PCR MP; SCAR primers; Dot blot hybridisation
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-016-7295-0

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 ~10⁰ cfu/reaction for Psm1 and 10¹ cfu/reaction for Psm2 in pure cultures, while in plant material were 10⁰–10¹ cfu/reaction using primers for Psm1 and 3 × 10² cfu/reaction using primers for Psm2. Real-time PCR assays with SYBR Green I showed a higher limit of detection (LOD) − 10⁰ 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.