Adapting the inoculation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from branch microbiome

Pseudomonas syringae pv. actinidiae (Psa), the bacterium that causes kiwifruit bacterial canker, is a common field occurrence that is difficult to control globally. Currently, exploring the resources for efficient biocontrol bacteria is a hot spot in the field. The common strategy for isolating biocontrol bacteria is to directly isolate biocontrol bacteria that can secrete diffusible antibacterial substances, most of which are members of Bacillus, Pseudomonas and Streptomycetaceae, from disease samples or soil. Here, we report a new approach by adapting the typical isolation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from the branch microbiome. Using this unique approach, we isolated a group of kiwifruit biocontrol agents (KBAs) from the branch microbiome of Psa-resistant varieties. Thirteen of these showed no antagonistic activity in vitro, which depends on the secretion of antibacterial compounds. However, they exhibited antibacterial activity via cell-to-cell contacts mimicked by co-culture on agar plates. Through biocontrol tests on plants, two isolates, KBA13 and KBA19, demonstrated their effectiveness by protecting kiwifruit branches from Psa infection. Using KBA19, identified as Pantoea endophytica, as a representative, we found that this bacterium uses the type VI secretion system (T6SS) as the main contact-dependent antibacterial weapon that acts via translocating toxic effector proteins into Psa cells to induce cell death, and that this capacity expressed by KBA19 is common to various Psa strains from different countries. Our findings highlight a new strategy to identify efficient biocontrol agents that use the T6SS to function in an antibacterial metabolite-independent manner to control wood diseases.