Identification and differentiation of soft rot and blackleg bacteria from potato using nested and multiplex PCR

Abstract

The bacterial genera Pectobacterium, Dickeya and Enterobacter are plant pathogens that cause soft rot and blackleg of potato. In the present study, 25 soft rot bacteria were isolated from potato tubers and stems showing typical tuber soft rot and blackleg symptoms. The samples were obtained from different locations in Egypt. Initial identification was based on morphological, physiological and biochemical characteristics. Pathogenicity of the identified isolates was assessed on potato tuber slices. Molecular techniques (16S rRNA sequencing and PCR analysis) revealed the identification of new clades of D. solani and E. cloacae. The primers (Eca1f/Eca2r) selected for the detection of P. atrosepticum specifically amplified a 690-bp region in only two out of 25 soft rot isolates. A single 550-bp band was produced from 14 isolates of P. carotovorum subsp. carotovorum using the species-specific primer EXPCC. Out of 23 isolates, 12 isolates gave a 666-bp band using primers to detect pmrA. Moreover, D. solani isolates were confirmed by the PCR amplification of a 420-bp region using specific primers (ADE1/ADE2). Nested PCR using the primer set EXPCCF/EXPCCR followed by INPCCF/INPCCR produced a 400-bp band from all P. c. subsp. carotovorum isolates tested. Nested PCR and multiplex PCR assays provided fast and reliable detection of the major soft rot and blackleg potato pathogens using three pairs of primers (Df/Dr, Y45/46 and EXPCCF/EXPCCR) and enabled the simultaneous detection of four pectinolytic bacteria found in potato: P. atrosepticum, P. c. subsp. carotovorum, D. solani and E. cloacae.