Novel bacteriophages for the biocontrol of Dickeya spp. in potato production Microbiological characterization and control perspectives

The bacterial pathogen Dickeya spp. is an important cause of economic losses in several agricultural and horticultural crops. Specifically in potato production, Dickeya solani is a recently emerged, more virulent Dickeya type in Western Europe. The pathogen causes blackleg (stem rot) in the field and soft rot of the tubers. As no chemical control measures are available, the search for alternatives focuses more on biocontrol using antagonists or bacteriophages. This study investigates the use of (bacterio)phages to control Dickeya solani infections in potato production. Bacteriophages or bacterial viruses specifically infect bacteria and use bacterial cells to replicate themselves. Virulent phages kill the cells they infect and are therefore ideal bacterial control agents.Four phages were isolated from soil, phages LIMElight and LIMEzero infecting the soilborne bacterium Pantoae agglomerans, and phages LIMEstone1 and LIMEstone2 infecting Dickeya solani . The infection parameters and general characteristics of these phages were examined to assess their suitability as biocontrol agents.LIMElight and LIMEzero were both found to be members of the family Podoviridae of phages, known by their short tails. Based on the microbiological characterization, LIMElight and LIMEzero were not deemed suitable candidates for further phage therapy research. Genomic analysis of LIMElight and LIMEzero was performed since they were the only podovirus phages sequenced infecting the genus Pantoea. The double-stranded DNA (dsDNA) genomes (44,546 bp and 43,032 bp, respectively) encode 55 and 57 open reading frames (ORFs). Based on the presence of a DNA dependent RNA polymerase in their genomes and their overall genome architecture, these phages could be classified in the subfamily of the Autographivirinae, within the genus of the phiKMV-like viruses. Phages LIMEstone1 and LIMEstone2 were characterized as two isolates belonging to the same species, LIMEstone. Both LIMEstone1 and LIMEstone2 infected 100% of the Dickeya solani strains tested and were also restricted to that host. From the microbiological characteristics, phages LIMEstone1 and LIMEstone2 were deemed ideal candidates for further investigation. Transmission electron microscopy of phage LIMEstone1 revealed a myovirus morphology, resembling Salmonella phage ViI. The dsDNA genome of LIMEstone1 was 152,427 bp long, comprising 201 ORFs and one tRNA. Blast and HHPred analyses of the predicted ORFs revealed a distant relation to the well-characterized E. coli phage T4, and a close relationship with Salmonella phage ViI. No integrases were found in the genome, or any other gene involved in lysogeny, confirming that LIMEstone1 is a virulent phage. The relationship between LIMEstone1 and ViI was investigated further and resulted in the creation of a new viral genus, the suggested genus Viunalikevirus . Other members belonging to this genus and sharing a substantial sequence similarity were Salmonella phages SFP10 and ΦSH19, Escherichia phages CBA120 and PhaxI, and Shigella phage phiSboM-AG3. The distinguishing features of this genus included phage morphology, the genome size and organization, gene synteny, T4-like regulation of late transcription, the use of tail spikes for host recognition rather than T4-like tail fibers, and the use of a modified form of uracil rather than thymine. The Dickeya phages LIMEstone1 and LIMEstone2 were prepared (concentrated and purified) using anion-exchange chromatography on CIM® monolithic columns. For both phages a method was optimized which included the choice of column type, buffer set and elution conditions. Combining the microbiological, molecular and purification data, the LIMEstone phages were considered suitable for phage therapy experiments. Preliminary in vivo assays were conducted on potato tubers, using D. solani strain LMG 25865 and purified suspensions of LIMEstone1 and LIMEstone2. Both phages significantly reduced soft rot incidence and severity at MOI 100; reduction was less significant at MOI 10 for LIMEstone1. In the summer of 2011, a field trial was conducted to investigate the effect of phage treatment on potato tubers artificially pre-inoculated with D. solani . In the field, all inoculated and phage treated tubers produced plants with disease symptoms. Disease severity seemed less in the phage treated plants, with 13% higher tuber yield, but this was not statistically significant. Part of the yield difference could be attributed to a larger fraction of big tubers per plant harvested from the phage treated plants, but again results were not statistically significant. The overall yield was still significantly lower than that of the non-treated (no bacterial inoculation or phage) control. This research showed that the LIMEstone phages have perspectives for controlling D. solani in potato . Further research is now essential for integration of phage therapy in a sustainable control strategy.status: publishe