CVP plating |
Isolation of pure bacterial colonies and viable bacterial cells |
Depending on the sample source and presence of other microorganisms, recovery of bacteria on the medium up to 100% |
Very rarely Pectobacterium and Dickeya spp. isolates may not grow on the medium or do not produce cavities; incubation up to 5 days at optimal temperature is required to assess the cavity formation; pectin source can affect the quality of the medium |
Cuppels & Kelman (1974) and Helias et al. (2011) |
Enrichment in PEB medium |
Enrichment of low bacterial populations of Pectobacterium spp. and Dickeya spp., useful for isolation of bacteria from environmental samples |
Frequently even 1–10 bacterial cell are enriched to the detectable densities: usually 102–103 cells mL−1 bacteria should be present in starting material |
Despite medium selectivity, other bacterial species antagonistic to Pectobacterium and/or Dickeya spp. maybe also enriched, hence false-negative results |
Pérombelon & van der Wolf (2002) |
PCR with species-specific primers |
Differentiation of Pectobacterium and Dickeya spp. from other bacteria, differentiation of Pectobacterium from Dickeya spp. Can be applied in a singlex and multiplex setting for detection of several pathogens |
From 1 cfu mL−1 to 105 cells mL−1 depending on assay, primer sets, conditions; an average detection limit is 104 cells mL−1 in plant extracts |
In general PCR works better on pure bacterial colonies or purified genomic DNA, false-positive reactions possible, the results should be confirmed with other methods, the assays detect also nonviable bacterial cells and DNA |
Please see the Table 3 for reference |
Real-time PCR (TaqMan™, SYBR green) |
Quantitative and qualitative detection of bacteria, for TaqMan™, additionally assays with higher specificity due to the presence of probe complementary to the target DNA. TaqMan assays be applied in a singlex and multiplex setting for detection of several pathogens |
Pure genomic DNA of good quality is prerequisite |
Relatively expensive for routine use, samples need to be investigated in duplicates or triplicates together with positive and negative controls |
Please see the Table 3 for reference |
Repetitive Sequence-Based PCR (REP-PCR) |
Differentiation of Pectobacterium and Dickeya spp. isolates |
High concentration of pure genomic DNA of good quality is prerequisite, viable bacterial cells used for DNA isolation are necessary |
High resolution, even closely related species can be differentiated from each other, results on two different gels cannot be directly compared, the assay should include control designated strains |
Versalovic et al. (1994) |
Pulsed field gel electrophoresis (PFGE) |
Differentiation of Pectobacterium and Dickeya spp. isolates |
High concentration of pure genomic DNA of good quality is prerequisite, |
High resolution, even strains can be differentiated from each other, results on two different gels cannot be directly compared, the assay should include control designated strains |
Lee et al. (2006) and Kim et al. (2009) |
Restriction Fragment Length Polymorphism- PCR (PCR-RFLP) |
Differentiation of Pectobacterium and Dickeya spp. isolates |
High concentration of pure genomic DNA of good quality is prerequisite, viable bacterial cells used for DNA isolation are necessary |
Results on two different gels cannot be directly compared, the assay should include control designated strains, relatively good resolution |
Boccara et al. (1991) and Waleron et al. (2002) |
Multi Locus Sequence Tagging (MLST) |
Differentiation of Pectobacterium and Dickeya spp. isolates |
Qualitative detection |
Selection of good target genes may be difficult |
Pitman et al. (2010), De Boer et al. (2012) and Waleron et al. (2013) |