Flood inoculation of seedlings on culture medium to study interactions between Pseudomonas syringae pv. actinidiae and kiwifruit.

Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is a serious threat to kiwifruit production. Highly virulent strains of Psa biovar3 (Psa3) have spread rapidly to kiwifruit production areas worldwide. Therefore, there is an urgent need to develop critical management strategies for bacterial canker based on dissecting the interactions between Psa and kiwifruit. Here, we developed a rapid and reliable flood-inoculation method using kiwifruit seedlings grown on Murashige and Skoog medium. This method has several advantages over inoculation of conventional soil-grown plants. We demonstrated the utility of a kiwifruit seedling assay to study the virulence of Psa biovars and Psa3 virulence factors, including the type III secretion system (T3SS). Kiwifruit seedlings inoculated with Psa3 developed severe necrosis within 1 week, whereas those inoculated with a T3SS-deficient hrcN mutant of Psa3 did not. This method was also useful for analyzing expression profiles of genes involved in Psa3 virulence during infection, and revealed that the expression of genes encoding the T3SS and type III secreted effectors were strongly induced in planta. Our results indicate that the T3SS has an important role in Psa3 virulence, and the flood-inoculation assay using kiwifruit seedling is suitable for analyzing Psa and kiwifruit interactions.