Unlocking the Potential of 46 New Bacteriophages for Biocontrol of Dickeya Solani

Abstract

Modern agriculture is expected to face an increasing global demand for food while also needing to comply with higher sustainability standards. Therefore, control of crop pathogens requires new, green alternatives to current methods. Potatoes are susceptible to several bacterial diseases, with infections by soft rot Enterobacteriaceae (SRE) being a significant contributor to the major annual losses. As there are currently no efficient ways of combating SRE, we sought to develop an approach that could easily be incorporated into the potato production pipeline. To this end, 46 phages infecting the emerging potato pathogen Dickeya solani were isolated and thoroughly characterized. The 46 isolated phages were grouped into three different groups based on DNA similarity, representing two distinct clusters and a singleton. One cluster showed similarity to phages previously used to successfully treat soft rot in potatoes, whereas the remaining phages were novel and showed only very limited similarity to previously isolated phages. We selected six diverse phages in order to create the hereto most complex phage cocktail against SRE. The cocktail was applied in a proof-of-principle experiment to treat soft rot in potatoes under simulated storage conditions. We show that the phage cocktail was able to significantly reduce the incidence of soft rot as well as disease severity after 5 days of storage post-infection with Dickeya solani. This confirms results from previous studies that phages represent promising biocontrol agents against SRE infection in potato.

Keywords: Dickeya solani; SRE; biocontrol; blackleg; phage biocontrol; phage cocktail; phage therapy; potato; soft rot; soft rot Enterobacteriaceae.

Figure 1

All vs. all nucleotide similarity analysis on all the Cluster A phages with escherichia phage P694 (KP090454) included as a reference, made using Gegenees [24].

Figure 2

Phamerator map [25]: Pairwise alignment of Mysterion, Dagda, Dagda_B1 Katbat, Luksen and Ninurta with the genome of Enterobacteria phage T7 (NC_001604) included as a reference. Each genome is represented by a vertical line with intersecting colored lines representing nucleotide similarity, red being more dissimilar and violet being more similar. Colored boxes represent genes.

Figure 3

Stability assay: Effect of 20% ethanol (red), UV radiation (orange), three freeze/thaw cycles (grey) and chloroform (blue) in vitro on the stability of six phages used in the phage cocktail. Values indicate the titer post exposure relative to the control. Error bars indicate ± SD. Asterisks indicate titers below the detection limit.

Figure 4

Potato maceration assay: Negative control (black); potatoes not exposed to neither phages nor bacteria. Phage only (grey); Potatoes washed in phage cocktail solution, but not exposed to bacteria. Positive control (red); Potatoes washed in sterile water and inoculated with bacteria. Treatment (green); Potatoes washed in phage solution and inoculated with bacteria. N = 45 for each treatment group. (A) Soft rot disease incidence (%): Positive soft rot infection was identified as the presence of >0.5 g macerated potato tissue. Numbers indicate the percentage of infected potatoes; error bars show 95% confidence interval. The treatment group showed a significant reduction compared to the untreated group (Chi-squared test, P < 0.0001). (B) Box plot showing the amount of macerated tissue (g) of potatoes after 5 days incubation.