Protocol for the inoculation of oilseed rape plants with ascospores of Pyrenopeziza brassicae (light leaf spot) using the ascospore shower method.

OBJECTIVE:

To define the procedures for inoculating oilseed rape plants with ascospores (sexual wind-dispersed spores) of Pyrenopeziza brassicae (light leaf spot) using an ascospore shower. This protocol was developed from a paper by Gilles et al :

Gilles T, Fitt BDL, McCartney HA, Papastamati K, Steed JM (2001) The roles of ascospores and conidia of Pyrenopeziza brassicae in light leaf spot epidemics on winter oilseed rape (Brassica napus) in the UK. Annals of Applied Biology 138: 141-152.

OPERATORS OF THE PROCEDURE:

Experienced or trained plant pathologists or other biologists.

PROCEDURE:

1. Produce mature apothecia (sexual fruiting bodies) from pod debris or agar plates using respective SOP.

2. Arrange pots containing plants in seed trays (6 pots/plants per tray) and place clean microscope slides adjacent to plants at same level as the target leaves to be inoculated (e.g. true leaves 2 and 3, or true leaves4 and 5, etc).

3. Attach the stem pieces or pieces of agar containing mature apothecia to the underside of plant tray cover using petroleum jelly.

4. Place the tray cover over the tray with plants ensuring there is a 2 cm gap between the lid and the plant tray. This ensures that relative humidity is reduced, encouraging ascospore release.

5. Let ascospores ‘rain down' onto plants for 24 h.

6. Remove lids, remove debris/agar and remove microscope slides.

7. Spray plants with distilled water and refit lids tightly to maintain leaf wetness for a period of 48 h. Incubate at 15°C or temperature appropriate for particular experiment.

8. Stain microscope slides with Trypan blue, cover and count number of ascospores deposited on an area of c .350mm 2 and calculate spore numbers per cm 2 deposited.

9. Assess plants for visual infection symptoms (presence of acervuli [asexual conidial fruiting bodies]) depending on incubation temperature.

RECORD KEEPING:

Record all details (inoculation 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.