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A PCR method that combines biological and enzymatic amplification of PCR targets was developed for the detection of Pseudomonas syringae pv. actinidiae on kiwifruit leaves. A nested PCR was performed with primers designed from the coding sequence of the cfl gene, which is involved in production of the phytotoxin coronatine. The first and second primer sets efficiently amplified expected 665 and 310-bp fragments, respectively. With two successive amplifications, as few as 20 CFU/ml of P. syringae pv. actinidiae could be detected on ethidium bromide-stained agarose gel. Leaf samples were collected from 4 kiwifruit trees showing yellow halo spots on leaves and incubated in pepton-sucrose broth for 12 h at 16oC before PCR amplification. Positive detection was obtained with one sample, which was proved as a diseased plant in the next spring.