Archived 10.20.2017 Be 1.1 – Pseudomonas syringae pv. phaseolicola (syn: P. savastanoi pv phaseolicola)
| Version | 1.0 |
|---|---|
| Date | 12/2012 [Archived 10.20.2017] |
| 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:
- 5 subsamples of 1,000 seeds are placed in 0.85% NaCl buffer in a ratio of 1:3.
- Samples are shaken vigorously for 3 to 4 hours, or refrigerated overnight.
- Part of the extract is centrifuged at 10,000 rpm for 10 minutes. Most of the supernatant liquid is discarded, and pellet is resuspended.
- Two more dilutions are made, one directly from the bean extract, and a 1:10 dilution from this original solution.
- 0.1ml of each of the three dilutions is plated in duplicate onto KBBC and MSP media.
- Liquid is spread evenly over each plate, and plates are incubated at 27-30ºC for 5 to 7 days.
- Plates are evaluated for suspected P.s. pv. phaseolicola colonies. Suspect colonies are confirmed by fluorescence, oxidase, reaction on BBD media, and pathogenicity testing.
- Pathogenicity is shown by mixing a slightly turbid solution of the bacterium in 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:
| Reagent | Quantity |
|---|---|
| King’s B media | 900ml |
After autoclaving filter, sterilize the following adding to media just prior to pouring:
| Reagent | Quantity |
|---|---|
| Boric acid | 100ml of 1.5% aqueous solution |
| Cephalexin | 80mg |
| Cycloheximide | 20mg |
MSP:
| Reagent | Quantity |
|---|---|
| Sucrose | 20g |
| Peptone | 5g |
| K2HPO4 | 0.5g |
| MgSO4 * 7H2O | 0.25g |
| Agar | 20g |
| H2O | 1 liter |
After autoclaving filter, sterilize the following adding to media just prior to pouring:
| Reagent | Quantity |
|---|---|
| Cycloheximide | 200mg |
| Cephalexin | 80mg |
| Vancomycin | 10mg |
| Bromthymol blue | 15mg |
BBD:
| Reagent | Quantity |
|---|---|
| Yeast extract | 1g |
| Glycerol | 5g |
| Bacto peptone | 5g |
| Bacto agar | 15g |
| H2O | 750ml |
Autoclave separately for no more than 15 minutes:
| Reagent | Quantity |
|---|---|
| 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.