Br 2.1 Phoma lingam 2017-06-14T17:50:48+00:00

National Seed Health System

Br 2.1 Phoma lingam

DATE: 2001
PATHOGEN: Phoma lingam (sexual: Leptosphaeria maculans)
HOST: Brassica sp.
COMMON NAME: black leg; canker; dry rot
METHOD: Br 2.1 Freezing Blotter Method (STA)
SAMPLE: 1000 – 10,000 seeds

REVISION HISTORY: Version 1.1: Procedure #4. Botran solution correction


1. Prepare a solution of 0.05% Botran.

2. Place 1 sterilized 10×15 inch brown blotter paper on each 11×16 inch styrofoam tray.

3. On top of the brown paper, place two sterilized 11×9 inch white blotter papers side by side, slightly overlapping.

4. Add enough of the Botran (0.05%) solution to each tray to thoroughly wet the blotters.

5. Distribute the seed evenly over each of the trays by hand or with a vacuum planter.

6. Punch holes in each plastic ziploc bag.

7. Place each tray into a 14×20 inch plastic ziploc bag.

8. Incubate the trays at room temperature for a minimum of 8 hours.

9. Freeze the sample for 8-24 hours.

10. Remove the trays from the freezer and incubate under NUV/Fluorescent light with a 12 hour photoperiod at 20-28 C° for 10 days.


Tegli, S., Sereni, A. and Surico, G. 2002. PCR-based assay for the detection of Curtobacterium flaccumfaciens pv. flaccumfaciens in bean seeds. Letters in Applied Microbiology. 35(4):331-337. Versalovic, J., Schneider, M., de Bruijn, F. J. and Lupski, J. R. 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods in Molecular and Cellular Biology. 5:472–489.

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.