PCR‐based specific and sensitive detection ofPectobacterium carotovorumssp.carotovorumby primers generated from a URP‐PCR fingerprinting‐derived polymorphic band

Abstract

A 24-mer primer pair was generated by sequencing a URP-PCR fingerprinting-derived polymorphic band that is uniquely shared in Pectobacterium carotovorum ssp . carotovorum strains (Pcc). The primer set (EXPCCF/EXPCCR) amplified a single band of expected size (0·55 kb) from genomic DNA obtained from 29 Pcc strains and three Pectobacterium carotovorum ssp. wasabiae (Pcw) strains, but not from other P. carotovorum subspecies atrosepticum , betavasculorum or odoriferum , or from other Erwinia spp. or bacterial genera. The Rsa I digestion profile of the amplified bands divided Pcc strains into five groups with a unique profile from Pcw strains. First-round PCR detected between 5 × 10 2 and 1 × 10 3 colony forming units (CFU) mL − 1 and detection sensitivity was increased to as few as 2-4 CFU mL − 1 after second-round (nested) PCR. This PCR protocol was used directly to detect Pcc strains in infected plant tissues.

Detection of Pectobacteria Causal Agents of Potato Soft Rot in North Western Provinces of Iran

“…Detection of bacteria with primers EXPCCF and EXPCCR, specific for P. carotovororum subsp carotovorom, P. carotovorum sub sp carotovorum was amplified with a set of primers EXPCCF and EXPCCR as described by [12], synthesized by Bioneer (England). Amplification was performed in a final volume of 50 µL PCR mixture containing 5 µL 10X PCR buffer, 0.5 µL each of desoxy nucleotides dATP, dCTP, dGTP, dTTP, 0.5 µmol (2.5 U)Taq polymerase and 1 µL (100 ng) primers.…”

“…Amplification was performed in a final volume of 50 µL PCR mixture containing 5 µL 10X PCR buffer, 0.5 µL each of desoxy nucleotides dATP, dCTP, dGTP, dTTP, 0.5 µmol (2.5 U)Taq polymerase and 1 µL (100 ng) primers. PCR amplification was performed in an Eppendorf thermal cycler (Germany), using the following program [12] one cycle of 4 min at 94°C as initial denaturation step, 30 cycles of 1 min at 94°C, 1 min at 60°C, 2 min at 72°C and one cycle as final extension for 7 min at 72°C.…”

“…However, diversity of isolates in serological properties does not allow for reliable and accurate identification and there are high serologically heterogeneity and cross-reactions in this technique [11]. As an alternative, PCRbased methods have been used for specific and rapid detection and identification of pathogens isolates, it is notable that PCR techniques greatly enhance detection sensitivity, simplicity and rapidity compared with other methods of identification and are based on specific amplification of a target DNA sequence that is unique to a bacterial genome [9,12] (Table 1). Also, since Pectobacterium carotovorum subsp.…”

Pectobacterium carotovorum subsp. carotovorum can cause potato blackleg in temperate climates

“…to cause blackleg is undisputed (Lumb et al 1986;Helias et al 2000;Toth et al 2003) but the role of Pcc in the blackleg syndrome is controversial. Pcc is widely distributed in the world and has a broad host range (De Boer et al 1987;Smith and Bartz 1990;Kang et al 2003). Studies under high temperature conditions in North America Harrison 1977, 1980) and Australia (Peltzer and Sivasithamparam 1985) showed that Pcc isolates, inoculated into tubers or stems, could cause typical blackleg symptoms in the field.…”

Identification and genetic diversity of Jordanian potato soft rot isolates, Pectobacterium carotovorum subspecies carotovorum (DYE 1969)

“…Within the past 30 years, there has been a shift from microbiological and serological methods to molecular approaches (PalacioBielsa et al, 2009;Czajkowiski et al, 2014). DNA markers have been used for the rapid detection of different strains of P. carotovorum subspecies carotovorum (Pcc) (Kang et al, 2003;Zhu et al, 2010;Rahmanifer et al, 2012;Azadmanesh et al, 2013). DNA sequence analysis of gene pmrA, which is linked to pathogenicity (response regulator), was used to evaluate the relationship among Pcc isolates collected from different regions of Morocco and results were equivalent to enterobacterial repetitive intergenic consensuspolymerase chain reaction (ERIC-PCR) and 16S rDNA sequencing (Kettani-Halabi et al, 2013).…”