National Seed Health System
Be 2.1 Pseudomonas syringae pv syringae
|PATHOGEN: Pseudomonas syringae pv. syringae|
|HOST: common bean (Phaseolus vulgaris)|
|COMMON NAME: bacterial brown spot|
|METHOD: Be 2.1 Agar Plate Assay (Mohan and Schaad 1987)|
|METHOD CLASS: TEMPORARY STANDARD (B)|
|SAMPLE: 1 kg of seeds|
1. Three one kg of seed are soaked in 3L of sterile saline solution with 0.01% Tween 20 for 20 hours at 5°C.
2. Each suspension is mixed with a sterile glass rod and a 150ml sample of the extract is drawn.
3. A 100ml potion of the extract is concentrated 10-fold by centrifugation at 12,000g for 10 min. The pellet is re-suspended in 10ml sterile saline (0.85% NaCl).
4. Aliquots of 0.1ml of 10-fold diluted, undiluted, and 10-fold concentrated extracts are plated onto selective media KBC and MSP in triplicate.
5. Plates are incubated at room temperature (23±2°C) for 3 to 4 days.
6. The 10-fold concentrate is stored at 3-5°C until plates are read. This is re-centrifuged 10-fold more and plated as above if plates at the 10-fold concentrated sample only show a few saprophytic colonies.
7. Presumptive positive colonies are confirmed by biochemical and pathogenicity testing (Sands et al., 1980). Colonies on KBC are 3-3.5mm in diameter, flat, circular, translucent, creamy white, and showed blue fluorescence under UV light. Colonies on MSP are 3mm in diameter after 3 days at room temperature. They are circular, raised, globose, glistening, and light yellow with a less dense center. After 3 days the medium around the colony turned light yellow.
Mohan S. K. and Schaad N. W. 1987. An improved agar plating assay for detecting Pseudomonas syringae pv. syringae and P. s. pv. phaseolicola in contaminated bean seed. Phytopathology. 77(10):1390-1395 Sands D. C., Schroth, M. N. and Hildebrand, D. C. 1980. Pseudomonas. Pages 36-44 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. NW Schaad (ed.) American Phytopathological Society, St. Paul MN. 72p.