Use of phage ϕ6 to inactivate Pseudomonas syringae pv. actinidiae in kiwifruit plants: in vitro and ex vivo experiments.

Over the last years, the global production and trade of kiwifruit has been severely impacted by Pseudomonas syringae pv. actinidiae (Psa), a phytopathogen that causes a disease in kiwifruit plants known as bacterial canker. The available treatments for this disease are still scarce, with the most common involving frequently spraying the orchards with disinfectants, copper-based bactericides and/or antibiotics. Moreover, these treatments should be avoided due to their high toxicity to the environment and promotion of bacterial resistance. Phage therapy may be an alternative approach to inactivate Psa. The present study investigated the potential application of the already commercially available bacteriophage (or phage) ϕ6 to control Psa infections. The inactivation of Psa was assessed in vitro, using liquid culture medium, and ex vivo, using artificially contaminated kiwifruit leaves with two biovar 3 (a highly aggressive pathogen) strains (Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10). In the in vitro experiments, the phage ϕ6 was effective against both strains (maximum reduction of 2.2 and 1.9 CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). In the ex vivo tests, the decrease was lower (maximum reduction 1.1 log and 1.8 CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). The results of this study suggest that the commercially available phage ϕ6 can be an effective alternative to control Psa infections in kiwifruit orchards.

Efficiency of Phage φ6 for Biocontrol of Pseudomonas syringae pv. syringae: An in Vitro Preliminary Study.

Pseudomonas syringae is a plant-associated bacterial species that has been divided into more than 60 pathovars, with the Pseudomonas syringae pv. syringae being the main causative agent of diseases in a wide variety of fruit trees. The most common treatments for biocontrol of P. syringae pv. syringae infections has involved copper derivatives and/or antibiotics. However, these treatments should be avoided due to their high toxicity to the environment and promotion of bacterial resistance. Therefore, it is essential to search for new approaches for controlling P. syringae pv. syringae. Phage therapy can be a useful alternative tool to the conventional treatments to control P. syringae pv. syringae infections in plants. In the present study, the efficacy of bacteriophage (or phage) φ6 (a commercially available phage) was evaluated in the control of P. syringae pv. syringae. As the plants are exposed to the natural variability of physical and chemical parameters, the influence of pH, temperature, solar radiation and UV-B irradiation on phage φ6 viability was also evaluated in order to develop an effective phage therapy protocol. The host range analysis revealed that the phage, besides its host (P. syringae pv. syringae), also infects the Pseudomonas syringae pv. actinidiae CRA-FRU 12.54 and P. syringae pv. actinidiae CRA-FRU 14.10 strains, not infecting strains from the other tested species. Both multiplicities of infection (MOIs) tested, 1 and 100, were effective to inactivate the bacterium, but the MOI 1 (maximum reduction of 3.9 log CFU/mL) was more effective than MOI 100 (maximum reduction of 2.6 log CFU/mL). The viability of phage φ6 was mostly affected by exposure to UV-B irradiation (decrease of 7.3 log PFU/mL after 8 h), exposure to solar radiation (maximum reduction of 2.1 PFU/mL after 6 h), and high temperatures (decrease of 8.5 PFU/mL after 6 days at 37 °C, but a decrease of only 2.0 log PFU/mL after 67 days at 15 °C and 25 °C). The host range, high bacterial control and low rates of development of phage-resistant bacterial clones (1.20 × 10-3) suggest that this phage can be used to control P. syringae pv. syringae infections in plants, but also to control infections by P. syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit. Although the stability of phage φ6 was affected by UV-B and solar radiation, this can be overcome by the application of phage suspensions at the end of the day or at night.