Sb 4.1 Pseudomonas syringae pv glycinea – Soaked bulk seed 2017-05-19T16:47:42+00:00

National Seed Health System

Sb 4.1  Pseudomonas syringae pv glycinea – Soaked bulk seed

VERSION: 1.0
DATE: 2001
PATHOGEN: Pseudomonas syringae pv. glycinea (syn: Pseudomonas amygdali pv. glycinea)
HOST: Soybean (Glycine max)
COMMON NAME: bacterial blight
METHOD: Sb 4.1 Soaked bulk seed – Biochemical confirmation (Chauveau, 1988) (formerly Sb 2.1)
METHOD CLASS: STANDARD (A)
SAMPLE: : 5000 seeds

PROCEDURE:

1. Five subsamples of 1000 soybean seeds are soaked for 24 hr at 4-5°C in 600 ml of sterile tap water adjusted to pH 6.5 with a phosphate buffer solution.

2. Threefold serial dilutions are made from the soaking solution and 0.1ml aliquots plated on King’s B medium amended with cephalexin (KBC).

3. After incubation at 25°C for 2-3 days, presumptive colonies of P. s. glycinea, exhibiting a blue fluorescence under UV light (370 nm), are re-isolated onto KBC.

4. Five presumptive colonies of each subsample are subculture onto King’s B medium.

5. These subcultures are then confirmed as P. s. glycinea by a positive reaction for levan production and negative reactions in oxidase and esculin hydrolysis tests.

Media:

King’s B Medium
DI water 1 liter
Proteose peptone #3 20g
K2HPO4 2.5g
Glycerol 15ml MgSO4 * 7H2O 6g
Agar 20g
*4ml cephalexin from the stock solution per liter applied after autoclaving. (Stock = 1g per 100ml water)
Esculin Hydrolysis Agar
DI Water 1 liter
NH4H2PO4 0.5g
K2HPO4 0.5g
MgSO4 * 7H2O 0.2g
NaCl 5g
Yeast extract 5g
Ferric ammonium citrate 0.5g
Esculin 1g
Agar 12g
*pH should be about 6.8.
Levan Agar
DI Water 1 liter
Sucrose 50g
Nutrient agar 23g

REFERENCES:

Chauveau, J. F. 1988, personal communication