OBJECTIVES:

To produce inoculum (ascospores) of Leptosphaeria maculans

OPERATORS:

Staff after reading this SOP who have had appropriate training in recognising pseudothecia maturation who know how to get ascospores of Leptosphaeria maculans from defined cross or from naturally infected oilseed rape stem debris

PROCEDURE:

(1) Obtain ascospores from naturally infected oilseed rape stem debris

(2) Obtain ascospores from cross in vitro

General Method

Obtain ascospores from naturally infected oilseed rape stem debris

1. Collect winter oilseed rape stems (30-60 cm long including tap roots) with stem base canker 2 weeks after harvest from untreated plots of field experiments at Rothamsted.

2. Place the stems in freely draining plastic trays (45 75 cm) lined with hessian sacking material.

3. Put the tray with stems outdoors to allow pseudothecia and ascospores to mature in natural conditions.

4. Sample the stems from the trays weekly to monitor the maturation of ascospores. Sample ten stems randomly. Excise ten pseudothecia from each stem and check the maturation stage of each pseudothecia.

5. Once ascospores are mature (pseudothecia reach maturation stage class D), bring the stems into the laboratory and dry at room temperature. Store the dry stems with mature pseudothecia at -20° until wanted.

Warning: According to the purpose of experiment, sometimes it is necessary to do single ascospore isolation from the stem to confirm that L. maculans ascospores are produced on these stems.

6. To harvest ascospores, the stems with mature pseudothecia are cut into pieces (2-3 cm in length) and attached to the underside of 9 cm Petri dish lids with Vaseline. Spray distilled water until run off to induce ascospore release, and then place the lids over Petri dish bases.

7. After about 3 h at room temperature (20°C), a large number of ascospores will be released into the bases of the dishes. Combine ascospore suspensions from several dishes and adjust the concentration to the required amount using a haemocytometer slide.

Warning: Ascospores germinate within 2 h at room temperature, so it is necessary to make the ascospore suspension as soon as possible.

Obtain ascospores from cross in vitro

1. Culture the two opposite mating type isolates on V8-agar (in 5cm diameter Petri dishes) for one week at 25°C with a 12h photoperiod. Petri dishes are NOT sealed with parafilm.

2. Put an agar plug (about 5 mm in diameter) of each isolate centrally on a 5 cm diameter Petri dish containing V8-agar (mycelium upside down). The two plugs should be separated by a distance of 5 to 10 mm.

3. After one week, pour 5 to 6 ml of 1.5% water agar cover the cultures.

Warning: It is important to perfectly recover the whole surface of the plate (if not, mycelium will grow on the surface of the agar overlay and pseudothecia will not be visible).

4. Seal plates with Parafilm and maintain cultures at 11 – 12°C under black light (OSRAM 38W tubes) with a 12h-photoperiod (distance between the plates and the light: around 15 cm).

5. The pseudothecia are produced under the water-agar layer within 4 to 6 weeks. They are usually, but not always, developed at the confrontation line between the two isolates.