OBJECTIVES:

To inoculate oilseed rape with ascospores of Leptosphaeria maculans

OPERATORS:

Staff after reading SOP who know how to inoculate oilseed rape with ascospores of Leptosphaeria maculans

PROCEDURE:

(1) Obtain ascospores from naturally infected oilseed rape stem debris or defined crosses in vitro

(2) Inoculate oilseed rape plants using ascospore ‘shower' method

General Method

Inoculation with ascospores produced on naturally affected oilseed rape stems

1. Get oilseed rape stems with mature pseudothecia and cut the stems into pieces (1-2 cm in length). Attach the stem pieces to the underside of tray cover and spray the stem pieces with distilled water until run off.

2. Place the tray cover over the tray with plants to let ascospore ‘rain down' onto the plants for 2 h at 15°C.

Warning: Don't let ascospores ‘shower' last too long; otherwise the inoculum is too heavy, it will be difficult to do the assessment and the too heavy infection will kill the whole leaf.

3. Seal the tray cover to maintain required wetness treatment.

4. Symptoms will appear 5-19 days after inoculation at 5-25°C.

Inoculation with ascospores produced by cross in vitro

1. Get the plates with mature pseudothecia and unseal the plate. Peel the layer of water agar on the top of the plate.

2. Cut the mature pseudothecia with pieces of agar and stick to the inside of a tray cover. Place the tray cover over the tray with plants to let ascospore ‘rain down' onto the plants for 24 h at 15°C.

Warning: Don't seal the tray cover; otherwise there is no flow air get into the tray and ascospores can not be evenly ‘rain' down onto plants. Usually, less ascospores are produced on agar in vitro than on affected stem debris under natural conditions, let the ascospore ‘shower' last long to get enough inoculum.

3. Seal the tray cover to maintain required wetness treatment.

4. Symptoms will appear 8-24 days after inoculation at 5-25°C.