Development of a species-specific PCR assay for identification and detection of Dickeya solani

Abstract

Dickeya solani associated with other members of Pectobacterium spp. and Dickeya spp. (Pectobacteria) causes the destructive potato blackleg in the Iranian province of Isfahan. The seed tuber dispersal ability and better adaptation of D. solani to warm climates raise major concerns about the spread and establishment of the pathogen across the country. Therefore, monitoring of the target pathogen on potato seeds and diseased plants is necessary to make effective decisions to limit the spread of the pathogen. However, the similarity in symptom development and phenotypic characteristics of Pectobacteria makes it difficult to distinguish D. solani in the collected samples. The aim of the present study was to develop a reliable PCR-based method for the specific detection of D. solani in naturally infected samples. In ERIC-PCR genotyping of different species and subspecies of Pectobacteria, a distinct PCR product around 950 bp was amplified only in D. solani strains. The amplicon was cloned, sequenced, analyzed and found to be highly homologous to the ugpC-1 gene sequence. Based on the sequenced fragment, a primer pair (DSF1/DSR2) was designed that allowed the specific amplification of a 895 bp band from the isolates of D. solani, but not from non-target Pectobacteria and other bacterial species tested in the present study. The approximate limit of detection of the PCR assay has been estimated to be approximately 2.7 × 10⁴ CFU/ml. The developed D. solani specific PCR assay can likely serve as a valuable method for various purposes.