Section III, Note 5:

Syringomycin bioassay (5)

1. Inoculate 5 ml of NBY cultures with P. syringae pv. syringae mutant strains using a sterile loop and incubate for 16 h at 25°C with shaking.

2. Harvest 1 ml of the overnight culture by centrifugation (14,000 rpm for 1 min).

3. Resuspend the cells in 1 ml of sterile distilled water (SDW) and spin the cells down by centrifugation (14,000 rpm for 1 minute).

4. Resuspend the cells in 1 ml of SDW and then dilute the sample to an O.D.₄₂₀=0.3 (~2 x 10⁸ cfu/ml).

5. Inoculate a 5 µl droplet of the cell suspension to potato dextrose agar (20 ml/plate) supplemented with 0.4% casamino acids and 1.5% glucose and incubate at 25°C for 3-5 days.

6. Overspray the plates lightly with an arthrospore suspension of the bioassay fungus, Geotrichum candidum strain F-260, which is sensitive to syringomycin, and incubate for 16 h at 25°C.

7. A zone of inhibition around the bacterial colony is indicative of the production of syringomycin.