Isolation, detection and characterization of Pectobacterium and Dickeya species

This chapter outlines isolation, detection and characterization methods for soft rot Pectobacteriaceae (SRP) and finishes with recommendations for diagnostics of SRP and perspectives for improved detection using metagenomic and pan-genomic approaches. For dilution plating and isolation of SRP, crystal violet pectate is still the medium of preference, although it is poorly selective. To improve the diagnostic sensitivity of detection methods, enrichment methods are used in which selective growth of the pathogen is enhanced by incubation in a pectate broth under low oxygen conditions. For molecular characterization, various finger printing techniques are described, but today analysis based on phylogenetic markers are preferred, in particular multi-locus sequence typing of housekeeping genes and comparative genetics using whole-genome sequences. For phenotypic characterization, methods are used based on serological, biochemical and physiological features. Currently the most precise phenotyping method is protein mass fingerprinting using a MALDI-TOF Mass Spectrometry. For detection of the pathogen, DNA-based amplification methods are generally used, including conventional PCR, real time (TaqMan) PCR assays and LAMP assays. They can detect the pathogen at a low density and allow recognition of the pathogens at different taxonomic levels. An inventory has been included of recently developed primer and probe combinations.