Pseudomonas syringae pv. actinidiae (Psa) is the causative agent of the bacterial canker of kiwifruit (Actinidia spp.). Phage therapy has been suggested as a viable alternative approach to controlling this disease, but its efficacy is limited by the emergence of phage-resistant mutants. Carvacrol is an essential oil that may be useful for the control of Psa. Combination therapies can be used to overcome resistance development. Here, the combination of phages (single phage suspensions of phages PN05 and PN09, and a cocktail of both phages) and carvacrol was investigated in controlling Psa planktonic and biofilm forms in vitro. The phage therapy alone (with phages PN05 and PN09), and the carvacrol alone (minimum inhibitory concentration 2.0 mg/mL), inhibited Psa growth, but the combined effect of both therapies was more effective. The phages alone effectively inhibited Psa growth for 24 h, but Psa regrowth was observed after this time. The carvacrol (2.0 mg/mL) alone prevented the biofilm formation for 48 h, but did not destroy the pre-formed biofilms. The combined treatment, phages and carvacrol (2.0 mg/mL), showed a higher efficacy, preventing Psa regrowth for more than 40 h. In conclusion, the combined treatment with phages and carvacrol may be a promising, environment-friendly and cost-effective approach to controlling Psa in the kiwifruit industry.

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes canker in kiwifruit. Few conventional control methods are effective against this bacterium. Therefore, alternative approaches, such as phage therapy are warranted. In this study, a lytic bacteriophage (PN09) of Psa was isolated from surface water collected from a river in Hangzhou, China in 2019. Morphologically, PN09 was classified into the Myoviridae family, and could lyse all 29 Psa biovar 3 strains. The optimal temperature and pH ranges for PN09 activity were determined as 25 to 35 °C and 6.0 to 9.0, respectively. The complete genome of PN09 was found to be composed of a linear 99,229 bp double-stranded DNA genome with a GC content of 48.16%. The PN09 endolysin (LysPN09) was expressed in vitro and characterized. LysPN09 was predicted to belong to the Muraidase superfamily domain and showed lytic activity against the outer-membrane-permeabilized Psa strains. The lytic activity of LysPN09 was optimal over temperature and pH ranges of 25 to 40 °C and 6.0 to 8.0, respectively. When recombinant endolysin LysPN09 was combined with EDTA, Psa strains were effectively damaged. All these characteristics demonstrate that the phage PN09 and its endolysin, LysPN09, are potential candidates for biocontrol of Psa in the kiwifruit industry.

Lambda Red recombineering is a DNA cloning and engineering technique involving recombination between homologous regions. The homologous recombination is mediated by the lambda Red genes consisting of red alpha, red beta and gam. Three lambda Red recombineering systems are currently available; the first is the plasmid-based system, in which lambda Red genes were cloned into temperature-sensitive plasmids; the second is the prophage-based system, in which lambda Red genes containing prophage were integrated into the Escherichia coli genome; the third is the integrative form system, characterized by the integration of lambda Red genes (or their counterparts) into the E. coli genome. In this study, a novel integrative form recombineering host, E. coli LS-GR, was constructed through the integration of functional recombineering elements including lambda Red genes, recA, araC and aacC1 into the E. coli DH10B genome. LS-GR shows high recombination efficiency for medium copy number vector and single copy number BAC vector modifications. The results indicate that LS-GR could be used as a general recombineering host strain.

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