Phage Cocktail Therapy for Biocontrol of Kiwifruit Bacterial Canker Caused by <i>Pseudomonas syringae</i> pv. <i>Actinidiae</i>

Kiwifruit bacterial canker (KBC), caused by Pseudomonas syringae pv. actinidiae (Psa), threatens global kiwifruit production. Traditional control methods face challenges like bacterial resistance and environmental issues. In this study, four lytic phages (pSM43, pGZ41, pWA51, and pSO21) were isolated and characterized using Psa M228 as the host bacterium. Notably, the phage pGZ41 represents a novel phylogenetic lineage, and the four-phage cocktail demonstrates significant advantages over previously reported single phages or mixtures in terms of lytic spectrum and resistance management. These phages exhibit distinct structural features and biological properties. Genomic sequencing classified them as double-stranded DNA viruses with genome sizes spanning 38,130 - 100,813 base pairs (bp), encoding 91 to 309 putative opening frames. The complete genome sequences have been deposited in GenBank under accession numbers PX673947 (pSM43), PX673946 (pGZ41), PX673948 (pWA51), and PX673949 (pSO21). The phage cocktail has a significant inhibitory effect on the growth of Psa M228 in vitro, in Arabidopsis thaliana and kiwifruit leaf discs. In vitro tests demonstrated that phage cocktails could effectively suppress bacterial growth and delay the emergence of resistant strains within a short period. In A. thaliana, phages exhibited strong antibacterial capabilities, reducing bacterial load by 4.92 log CFU/g in the treatment group and 4.33 log CFU/g in the prevention group compared to the infected control group. In kiwifruit leaf discs assays, the prevention group treated with phage cocktails exhibited superior efficacy, reducing lesion areas by 3-5 times compared to the treatment group. The significant efficacy in plant models, combined with the high environmental stability of the phages (particularly pGZ41), underscores the strong potential of this phage cocktail as a practical, sustainable, and environmentally friendly biocontrol agent for integrated management of kiwifruit canker in orchard settings. This multi-level validation suggests these phages as promising biocontrol agents for controlling kiwifruit canker caused by Psa.