Protocol for the production of mature apothecia of Pyrenopeziza brassicae (light leaf spot of oilseed rape) from post-harvest crop debris.

OBJECTIVE:

To define the procedures for producing mature apothecia (sexual fruiting bodies) of Pyrenopeziza brassicae (light leaf spot of oilseed rape) from crop debris prior to the production of ascospores (sexual airborne spores) for use in further experiments. This protocol developed from two papers by Gilles et al :

Gilles T, Ashby AM, Fitt BDL, Cole T (2001) Development of Pyrenopeziza brassicae apothecia on agar and oilseed rape debris. Mycological Research 105: 705-714

Gilles T, Fitt BDL, Jeger MJ (2001) Effects of environmental factors on development of Pyrenopeziza brassicae (light leaf spot) apothecia on oilseed rape debris. Phytopathology 91: 392-398

OPERATORS OF THE PROCEDURE:

Experienced or trained plant pathologists or other biologists.

PROCEDURE:

1. Separate upper stem debris into pod casings (preferably showing signs of infection [dried brown lesions]) and stem material.

2. Discard stem material and store pods casings in plastic bags under laboratory conditions.

3. Soak required number of pod casings for 16 hours in a beaker with rain water.

4. Place 10 wetted pod casings in a plastic box (12.5 x 8 cm) on three layers of filter paper (Whatman No.1) which has been wetted with 7 ml of rain water.

5. Place plastic box in an incubator or controlled environment cabinet at 15°C (light or dark, as it doesn't matter).

6. Monitor apothecial development from day 8 onwards using a stereo binocular microscope. Apothecia are considered “mature” when the fully expanded hymenium can be observed (Gilles et al ., 2001, Mycol. Res. 105, 705-714).

RECORD KEEPING:

Record all details (soak date, start date, monitor dates, etc) in laboratory notebook with accompanying unique experiment identification code for experiment series/project.

PROCEDURE REVIEW:

The procedure will be reviewed annually.

SAFETY:

No safety issues when implementing GLP.