An Efficient Triplex TaqMan Quantitative PCR to Detect a Blackleg-Causing Lineage of Pectobacterium brasiliense in Potato Based on a Pangenome Analysis.

is an important bacterial pathogen causing blackleg (BL) in potatoes. Nevertheless, is often detected in seed lots that do not develop any of the typical blackleg symptoms in the potato crop when planted. Field bioassays identified that strains can be categorized into two distinct classes, some able to cause blackleg symptoms and some unable to do it. A comparative pangenomic approach was performed on 116 strains, of which 15 were characterized as BL-causing strains and 25 as non-causative. In a genetically homogeneous clade comprising all BL-causing strains, two genes only present in the BL-causing strains were identified, one encoding a predicted lysozyme inhibitor Lprl (LZI) and one encoding a putative Toll/interleukin-1 receptor (TIR) domain-containing protein. TaqMan assays for the specific detection of BL-causing were developed and integrated with the previously developed generic assay into a triplex TaqMan assay. This simultaneous detection makes the scoring more efficient as only a single tube is needed, and it is more robust as BL-causing strains of should be positive for all three assays. Individual strains were found to be either positive for all three assays or only for the assay. In potato samples, the mixed presence of BL-causing and not BL-causing strains was observed as shown by the difference in Ct value of the TaqMan assays. However, upon extension of the number of strains, it became clear that in recent years additional BL-causing lineages of were detected for which additional assays must be developed.