Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of a bacterial canker in kiwifruit plants and has caused economic losses worldwide. Currently, the primary strategies to control this pathogen include the use of copper-based compounds and even antibiotics. However, the emergence of isolates of Psa that are resistant to these agrochemicals has raised the need for new alternatives to control this pathogen. Bacteriophages have been proposed as an alternative to control bacterial infections in agriculture, including Psa. Here, we show the isolation and characterization of 13 phages with the potential to control Psa infections in kiwifruit plants. The phages were characterized according to their host range and restriction fragment length polymorphism (RFLP) pattern. Four phages were selected according to their lytic effect on the bacteria and their tolerance to different environmental conditions of pH (4–7), temperature (4–37 °C), and solar radiation exposure (30 and 60 min). The selected phages (CHF1, CHF7, CHF19, and CHF21) were sequenced, revealing a high identity with the podophage of Psa phiPSA2. In vitro assays with kiwifruit leaf samples demonstrated that the mixture of phages reduced the Psa bacterial load within three hours post-application and was able to reduce the damage index in 50% of cases. Similarly, assays with kiwifruit plants maintained in greenhouse conditions showed that these phages were able to reduce the Psa bacterial load in more than 50% of cases and produced a significant decrease in the damage index of treated plants after 30 days. Finally, none of the selected phages were able to infect the other bacteria present in the natural microbiota of kiwifruit plants. These results show that bacteriophages are an attractive alternative to control Psa infections in kiwifruit plants.

Bacteriophages have been proposed as biological controllers to protect plants against different bacterial pathogens. In this scenario, one of the main challenges is the low viability of phages in plants and under adverse environmental conditions. This work explores the use of 12 compounds and 14 different formulations to increase the viability of a phage mixture that demonstrated biocontrol capacity against Pseudomonas syringae pv. actinidiae (Psa) in kiwi plants. The results showed that the viability of the phage mixture decreases at 44 °C, at a pH lower than 4, and under UV radiation. However, using excipients such as skim milk, casein, and glutamic acid can prevent the viability loss of the phages under these conditions. Likewise, it was demonstrated that the use of these compounds prolongs the presence of phages in kiwi plants from 48 h to at least 96 h. In addition, it was observed that phages remained stable for seven weeks when stored in powder with skim milk, casein, or sucrose after lyophilization and at 4 °C. Finally, the phages with glutamic acid, sucrose, or skim milk maintained their antimicrobial activity against Psa on kiwi leaves and persisted within kiwi plants when added through roots. This study contributes to overcoming the challenges associated with the use of phages as biological controllers in agriculture.

Background: Since 2008, the kiwifruit industry has been significantly impacted by Pseudomonas syringae pv. actinidiae (Psa), the agent responsible for bacterial canker in kiwifruit. Existing treatments, such as copper-based compounds and antibiotics, have faced challenges related to resistance and soil contamination. Phage therapy is a promising and safe alternative for controlling this pathogen. This study aimed to evaluate the use of a mixture of four isolated and characterized bacteriophages as potential biocontrol agents against Psa. Methods: Trials were conducted at two locations in Chile, where Psa presence was reported during the 2019/2020 and 2020/2021 seasons, with a focus on the spring stages. Different formulations were tested each season to evaluate possible improvements in effectiveness. Pseudomonas spp. isolates obtained from epiphyte populations were characterized using morphological, biochemical (LOPAT), and molecular techniques. Results: Field trials demonstrated that the phage mixture effectively reduced the damage associated with Psa on kiwi leaves, resulting in a decrease in the Pseudomonas spp. bacterial load (42.9% for Peumo and 25% for Linares) at both locations during the first season trials. This decrease is associated with a reduction in the incidence and severity of the disease in kiwi plants in the Peumo orchard. In both seasons, bacteriophages reduce Psa symptoms in treated kiwi plants compared to untreated controls, at least at one location and evaluation. In both orchards during the first season, bacteriophages also outperformed copper- and antibiotic-based treatments used by farmers. Bacteriophage therapy is eco-friendly and safe for both applicators and consumers.