The potential of bacteriophages to control Xanthomonas campestris pv. campestris at different stages of disease development.

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that invades the xylem of Brassica crops. Current chemical and antibiotics-based control measures for this bacterium are unsustainable and inefficient. After establishing a representative collection of Xcc strains, we isolated and characterized bacteriophages from two clades of phages to assess their potential in phage-based biocontrol. The most promising phages, FoX2 and FoX6, specifically recognize (lipo) polysaccharides, associated with the wxc gene cluster, on the surface of the bacterial cell wall. Next, we determined and optimized the applicability of FoX2 and FoX6 in an array of complementary bioassays, ranging from seed decontamination to irrigation- and spray-based applications. Here, an irrigation-based application showed promising results. In a final proof-of-concept, a CaCl2 -formulated phage cocktail was shown to control the outbreak of Xcc in the open field. This comprehensive approach illustrates the potential of phage biocontrol of black rot disease in Brassica and serves as a reference for the broader implementation of phage biocontrol in integrated pest management strategies.

Purification of Bacteriophages Using Anion-Exchange Chromatography.

In bacteriophage research and therapy, most applications ask for highly purified phage suspensions. The standard technique for this is ultracentrifugation using cesium chloride gradients. This technique is cumbersome, elaborate and expensive. Moreover, it is unsuitable for the purification of large quantities of phage suspensions.The protocol described here, uses anion-exchange chromatography to bind phages to a stationary phase. This is done using an FLPC system, combined with Convective Interaction Media (CIM®) monoliths. Afterward, the column is washed to remove impurities from the CIM® disk. By using a buffer solution with a high ionic strength, the phages are subsequently eluted from the column and collected. In this way phages can be efficiently purified and concentrated.This protocol can be used to determine the optimal buffers, stationary phase chemistry and elution conditions, as well as the maximal capacity and recovery of the columns.

Characterization of Novel Bacteriophages for Biocontrol of Bacterial Blight in Leek Caused by Pseudomonas syringae pv. porri.

Pseudomonas syringae pv. porri, the causative agent of bacterial blight in leek (Allium porrum), is increasingly frequent causing problems in leek cultivation. Because of the current lack of control measures, novel bacteriophages were isolated to control this pathogen using phage therapy. Five novel phages were isolated from infected fields in Flanders (vB_PsyM_KIL1, vB_PsyM_KIL2, vB_PsyM_KIL3, vB_PsyM_KIL4, and vB_PsyM_KIL5), and were complemented with one selected host range mutant phage (vB_PsyM_KIL3b). Genome analysis of the phages revealed genome sizes between 90 and 94 kb and an average GC-content of 44.8%. Phylogenomic networking classified them into a novel clade, named the "KIL-like viruses," related to the Felixounalikevirus genus, together with phage phiPsa374 from P. syringae pv. actinidiae. In vitro characterization demonstrated the stability and lytic potential of these phages. Host range analysis confirmed heterogeneity within P. syringae pv. porri, leading to the development of a phage cocktail with a range that covers the entire set of 41 strains tested. Specific bio-assays demonstrated the in planta efficacy of phages vB_PsyM_KIL1, vB_PsyM_KIL2, vB_PsyM_KIL3, and vB_PsyM_KIL3b. In addition, two parallel field trial experiments on three locations using a phage cocktail of the six phages showed variable results. In one trial, symptom development was attenuated. These data suggest some potential for phage therapy in controlling bacterial blight of leek, pending optimization of formulation and application methods.