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

Monika Kałużna,Joanna Puławska,Piotr Sobiczewski,Fernando Tavares,Pedro Albuquerque

doi:10.1007/s00253-016-7295-0

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 ~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.

Highlights

Bacterial canker of fruit trees occurs in stone fruit growing areas all over the world (Agrios 2005)
Bacteria that cause bacterial canker on stone fruit trees belong to three genomospecies: gs 1—P. syringae pv. syringae (Pss); gs 2—P. syringae pv. morsprunorum race 1 (Psm1); and gs 3—P. syringae pv. morsprunorum race 2 (Psm2), Appl Microbiol Biotechnol (2016) 100:3693–3711
GATTa and L-lactate utilisation tests allowed further discrimination of pathovars and races: 49 isolates were identified as P. syringae pv. morsprunorum race 1 (Psm1), 10 as race 2 of this pathovar, 53 as pathovar syringae (Pss) and 56 as belonging to atypical taxa, having most of the features of Pss without, the ability of esculine hydrolysis (Table 1)

Summary

Introduction

Bacterial canker of fruit trees occurs in stone fruit growing areas all over the world (Agrios 2005). In Poland, the disease incidence on stone fruit trees orchards is observed every year with different intensity and is becoming more economically significant. In the last vegetative seasons, bacterial canker was dangerous to stone fruit trees, and to apple and pear trees. The causal agents of the disease belong to the polyphagous Pseudomonas syringae species, able to infect more than 180 plant species, both annual and perennial, including fruit trees, ornamental plants and vegetables. Bacteria that cause bacterial canker on stone fruit trees belong to three genomospecies (gs): gs 1—P. syringae pv. Morsprunorum race 1 (Psm1); and gs 3—P. syringae pv. Morsprunorum race 2 (Psm2), Appl Microbiol Biotechnol (2016) 100:3693–3711 Bacteria that cause bacterial canker on stone fruit trees belong to three genomospecies (gs): gs 1—P. syringae pv. syringae (Pss); gs 2—P. syringae pv. morsprunorum race 1 (Psm1); and gs 3—P. syringae pv. morsprunorum race 2 (Psm2), Appl Microbiol Biotechnol (2016) 100:3693–3711

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