Development of qPCR Detection Assay for Potato Pathogen Pectobacterium atrosepticum Based on a Unique Target Sequence

Abstract

The recent taxonomic diversification of bacterial genera Pectobacterium and Dickeya, which cause soft rot in plants, focuses attention on the need for improvement of existing methods for the detection and differentiation of these phytopathogens. This research presents a whole genome-based approach to the selection of marker sequences unique to particular species of Pectobacterium. The quantitative real-time PCR assay developed is selective in the context of all tested Pectobacterium atrosepticum strains and is able to detect fewer than 102 copies of target DNA per reaction. The presence of plant DNA extract did not affect the sensitivity of the assay.

Quantitative Real-Time PCR Assay for the Detection of Pectobacterium parmentieri, a Causal Agent of Potato Soft Rot

“…The search for species-specific sequences was carried out using the workflow described in a previous study [ 20 ]. Briefly, this workflow splits the genome of the type Ppa strain into short sections, then each section is compared with a negative database of “non-target” genomes and a positive database of “target genomes” and, as a result, regions are identified that occur in all Ppa genomes and are not found in genomes of other species.…”

“…To search for species-specific sequences, custom databases were constructed using BLAST ( (accessed on 25 February 2021)). The search for species-specific regions for amplification was carried out using the workflow presented in the previous study [ 20 ].…”

“…For the experiment, potato tubers of the most widespread variety, “Gala”, were obtained from a market. They were washed and soaked in a bacterial suspension to infect the tubers, following the same protocol as in a previous study [ 20 ]. Then, the tubers were incubated at 28 °C.…”

“…However, it still does not allow for species-specific detection and is somewhat outdated, due to the changed understanding of the taxonomy of the group. Recently, the authors developed a pipeline for searching unique sequences for genomic groups and tested it in the context of P. atrosepticum , a genetically distinct species of SRP [ 20 ]. This paper describes how this workflow can be used to design a quantitative real-time PCR assay to discriminate closely related species.…”

Curtobacterium spp. and Curtobacterium flaccumfaciens: Phylogeny, Genomics-Based Taxonomy, Pathogenicity, and Diagnostics

“…Another 23 strains were isolated from the affected plants using a semi-selective medium for the isolation of Curtobacterium [54]. The remaining strains were taken from the collection of phytopathogenic bacteria of the Laboratory of Molecular Bioengineering of the Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Moscow, Russia), used in previous research [55,56].…”