Be 1.1 Pseudomonas syringae pv phaseolicola 2017-05-08T13:18:28+00:00

National Seed Health System

 Be 1.1 – Pseudomonas syringae pv. phaseolicola (syn: P. savastanoi pv phaseolicola)

VERSION: 1.0
DATE: 12/2012
PATHOGEN: Pseudomonas syringae pv. phaseolicola (syn: P. savastanoi pv phaseolicola)
HOST: common bean (Phaseolus vulgaris)
COMMON NAME: halo blight
METHOD: Be 1.1 Plate Test (STA Laboratories, Longmont, CO)
METHOD CLASS: STANDARD (A)
SAMPLE: 5000 seeds

PROCEDURE:

1. 5 subsamples of 1,000 seeds are placed in 0.85% NaCl buffer in a ratio of 1:3.
2. Samples are shaken vigorously for 3 to 4 hours, or refrigerated overnight.
3. Part of the extract is centrifuged at 10,000 rpm for 10 minutes. Most of the supernatant liquid is discarded, and pellet is resuspended.
4. Two more dilutions are made, one directly from the bean extract, and a 1:10 dilution from this original solution.
5. 0.1ml of each of the three dilutionsis plated in duplicate onto KBBC and MSP media.
6. Liquid is spread evenly over each plate, and plates are incubated at 27-30ºC for 5 to 7 days.
7. Plates are evaluated for suspected P.s. pv. phaseolicola colonies. Suspect colonies are confirmed by fluorescence, oxidase, reaction on BBD media, and pathogenicity testing.
8. Pathogenicity is shown by mixing a slightly turbid solution of the bacteriumin sterile water, and inoculating the undersides of susceptible bean leaves with a cotton swab. Plants are incubated at high humidity for 7 to 10 days, then checked for lesions surrounded by chlorotic halos.

MEDIA PREPARATION:

KBBC:

King’s B media 900ml

After autoclaving filter sterilize the following adding to media just prior to pouring:

Boric acid 100ml of 1.5% aqueous solution
Cephalexin 80mg
Cycloheximide 20mg

MSP:

Sucrose 20g
Peptone 5g
K2HPO4/td> 0.5g
MgSO4 * 7H2O 0.25g
Agar 20g
H2O 1 liter

After autoclaving filter sterilize the following adding to media just prior to pouring:

Cycloheximide 200mg
Cephalexin 80mg
Vancomycin 10mg
Bromthymol blue 15mg

BBD:

Yeast extract 1g
Glycerol 5g
Bacto peptone 5g
Bacto agar 15g
H2O 750ml

Autoclave separately for no more than 15 minutes:

Powdered milk 15g
H2O 250ml

REFERENCES:

Saettler, A. W. 1991. Halo blight. Page 30 in: Compendium of Bean Disease. Robert Hall (edit.) American Phytopathological Society, St. Paul, MN.

Van Vuurde, J. W. L. and Van den Bovenkamp, G. W. 1989. Detection of Pseudomonas syringae pv. phaseolicola in bean. Pages 30-40 in: Detection of bacteria in seed and other planting material. Saettler, A. W., Schaad N. W. and Roth, D. A. (Eds.) American Phytopathological Society. St. Paul, MN. 122 pp.

Mohan, S. K. and Schaad,N. W. 1987. An improved agar plating assay for detecting Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. phaseolicola in contaminated bean seed. Phytopathology. 77: 1390-1395.