Table 2.

Methods used for the isolation, detection and differentiation of soft rot Pectobacterium and Dickeya spp. in practical applications and research

Method	Application	Sensitivity, Detection Level	Remarks	Reference
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)