First Report of Dickeya dianthicola Causing Blackleg Disease on Potato in Morocco

Pectinolytic bacteria belonging to the genera Pectobacterium and Dickeya cause blackleg and soft rot diseases on several plants and crops including Solanum tuberosum (Pérombelon 2002). In Morocco, Pectobacterium carotovorum is described as the main causal agent of these diseases (Terta et al. 2010). In March 2016, blackleg symptoms were observed in commercial potato fields in the north of Morocco. Using molecular microbiology tools, these pathogenic isolates on potato plants have been characterized as belonging to Dickeya dianthicola. The isolation of pectinolytic bacteria (including species of the genera Dickeya and Pectobacterium) was performed on the standard medium crystal violet pectate (CVP) agar (Hélias et al. 2012). Identification of pectinolytic bacteria on CVP plates was carried out by observing the formation of a depression around bacterial colonies. All cultures were kept at 28 to 30°C for at least 3 days. Routine subcultures were performed on Luria-Bertani (LB) agar plates. To further determine the identity of the potential pectinobacteria, PCR was used with primer sets ADE1/ADE2 and Y1/Y2 targeting the pelADE genes (Nasser et al. 1996). Results from PCR screening showed that 69 isolates were identified as Pectobacterium spp. and 20 as Dickeya spp. Ten out of 20 Dickeya isolates were subjected to PCR amplification targeting the dnaX (Sławiak et al. 2009) and rrs 16S rDNA (with universal primers PA and 518r) genes. Nucleotide sequences of the PCR products were determined. The sequences were deposited under accession numbers KX985805–814 for dnaX and KX985795–804 for rrs. Both dnaX and rrs sequences showed 100 and 97% identity with published D. dianthicola sequences, respectively. A multilocus sequence analysis (MLSA) of the concatenated 16S rDNA and dnaX genes fragments of the Moroccan isolates in comparison with complete genomes from D. chrysanthemi NCPPB 516, D. dadantii 3937, D. dianthicola RNS049, D. solani 3337, D. zeae 1591, and P. atrosepticum CFBP6276 showed that all Moroccan Dickeya strains were clustered in the D. dianthicola clade. Koch’s postulates were completed on potato plants cv. Lusa in order to verify the pathogenicity of the 10 Moroccan D. dianthicola isolates. Potato tubers were planted in 2-liter pots and incubated at 25 to 28°C with regular watering in a greenhouse. Three weeks after stem emergence, 10 µl of a cell suspension at 107 cfu/ml of each Dickeya strain was injected into the lower part of stem with a sterile syringe. Six plants were infected by each of the 10 D. dianthicola isolates. Additional plants infiltrated by a sterile NaCl 0.85% solution were used as a control. Fourteen days after inoculation, all inoculated stems by D. dianthicola showed black and rotten stems, but control plants remained symptomless. Bacteria were isolated from the lesions and confirmed to be Dickeya sp. pathogens. In conclusion, combination of molecular and symptomatic analysis demonstrated the occurrence of D. dianthicola species from some potato fields in Morocco, and showed that D. dianthicola strains are virulent on potato plants. These findings will increase awareness and offer new technologies to producers and phytosanitary authorities in prophylactic measures to avoid the spread of Dickeya in potatoes. © 2017, American Phytopathological Society. All rights reserved.