National Seed Health System
Sb 4.2 Pseudomonas syringae pv glycinea – Ground bulk seed
|PATHOGEN: Pseudomonas syringae pv. glycinea (syn: Pseudomonas amygdali pv. glycinea)|
|HOST: Soybean (Glycine max)|
|COMMON NAME: bacterial blight|
|METHOD: Sb 4.2 Ground bulk seed–Serological and pathogenicity confirmation (Alvarez et al.,1995)(formerly Sb2.2)|
|METHOD CLASS: STANDARD (A)|
|SAMPLE: : 5000 seeds|
1. Five subsamples of 1000 dry soybean seeds are grinded in a Stein Mill for 1 min, then added to 600 ml of sterile saline (0.85% NaCl) and the suspension placed on a rotary shaker for 2 hr at 25°C at 220 rpm.
2. Threefold serial dilutions are made from the suspension and 0.1 ml aliquots plated on King’s B medium amended with cephalexin.
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. Presumptive colonies of each subsample are confirmed as P. s. glycinea by the following pathogenicity and slide agglutination tests.
1. Pathogenicity is determined by inoculating 15-day-old, greenhouse-grown soybean seedlings (cvs. Oakland, Beeson, Acme, and Flambeau) by rubbing leaves with a sterile cotton swab dipped in an aqueous suspension of the presumptive colony (approximately 105 cfu/ml).
2. The seedlings are incubated in light for 48 hr at 90% relative humidity in a mist chamber at 25°C, then transferred to the greenhouse and observed for necrotic lesions on leaves 4-7 days after inoculation.
1. Ten microliters of bacterial suspension of each colony (105 cfu/ml) was mixed in polystyrene Micro ELISA plates (Dynatech Corp.) with 10 µl of a 1:1,000 aqueous dilution of the antiserum obtained from A. Calzolari (Osservatorio Regionale per le Malattie delle Plante, Bologna, Italy).
2. The plates are agitated for 1 hr at 25°C on a rotary shaker at 220 rpm, and agglutination is determined under a stereoscopic microscope.
King’s B Medium
DI water 1 liter
Proteose peptone #3 20g
Glycerol 15ml MgSO4 * 7H2O 6g
*4ml cephalexin from the stock solution per liter applied after autoclaving. (Stock = 1g per 100ml water)
Alvarez, E., Braun. E. J., and McGee, D.C. 1995. New assays for detection of Pseudomonas syringae pv. glycinea in soybean seed. Plant Dis. 79:12-14.