<p>Previous studies have shown that some polyphenols have anti-ice nucleation activity (anti-INA) against ice-nucleating bacteria that contribute to frost damage. In the present study, leaf disk freezing assay, a test of <i>in vitro</i> application to plant leaves, was performed for the screening of anti-INA, which inhibits the ice nucleation activity of an ice-nucleating bacterium <i>Erwinia ananas</i> in water droplets on the leaf surfaces. The application of polyphenols with anti-INA, kaempferol 7-<i>O</i>-β-glucoside and (–)-epigallocatechin gallate, to the leaf disk freezing assay by cooling at −4–−6 °C for 3 h, revealed that both the compounds showed anti-INAs against <i>E. ananas</i> in water droplets on the leaf surfaces. Further, this assay also revealed that the extracts of five plant leaves showed high anti-INA against <i>E. ananas</i> in water droplets on leaf surfaces, indicating that they are the candidate resources to protect crops from frost damage.</p> <p>Water droplets with ice-nucleating bacteria on leaf disks were hardly frozen by addition of leaf crude extracts. Unfrozen leaf disks survived freezing injury.</p
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