T4-related bacteriophage LIMEstone isolates for the control of soft rot on potato caused by 'Dickeya solani'

Abstract

The bacterium 'Dickeya solani', an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage ViI. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with 'Dickeya solani', the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics.

Figure 1. LIMEstone isolates.

A) EM picture of phage LIMEstone1. Phage negatively stained with 2% phosphotungstate. Scale bar represents 100 nm. B) HindII restriction digestion of 0.5 and 1.0 µg DNA of LIMEstone1 and LIMEstone2, respectively.

Figure 2. The genome of phage LIMEstone1 (152,427 bp).

The inner ring represents ORFs on the forward strand, the outer ring the reverse strand. Proteins in italics show no sequence similarity with T4. ORFs in blue are confirmed as structural proteins, putative homing endonucleases are depicted in green. Promoters are indicated with arrows, factor-independent terminators with stem-loop structures.

Figure 3. Phage therapy assay on potato tubers cv. Bintje and Kondor.

Tubers treated with ‘Dickeya solani’ strain LMG 25865 were compared with phage treated tubers and with a water-treated control. Error bars indicate standard deviation. Significant differences were tested with the Kruskal-Wallis multiple comparison tests at p<0.05 en the Mann-Whitney U test for comparison of two samples. Letters indicate significant differences, capitals between treatments, small letters within cultivars.

Figure 4. Field trial results.

A) Total yield of the tubers in mass. B) Tuber size distribution in percentages of the total number of tubers. In the bars of fraction >55 mm, letters indicate statistical significance (p<0.05) as determined with the Kruskal-Wallis non-parametric test. Other fractions are not significantly different from each other.