Protocol for inoculation with Leptosphaeria maculans

Inoculation with ascospores

Three inoculation methods can be used: dry ascospores directly ‘rain down' onto plants; spray with ascospore suspension in water; pinpoint inoculation by placing a 10 µL drop of ascospore suspension on leaf surface. After inoculation, plants need to be maintained at least 24h wetness. One or 2 weeks after inoculation, depending on the temperature, disease development on the plants can be assessed.

Inoculation with conidia

Three inoculation methods can be used: spray with conidia suspension; pinpoint inoculation by placing a 10 µL drop of conidia suspension on leaf surface without wounding the leaf; pinpoint inoculation by placing a 10 µL drop of conidia suspension on the wounded site of leaf surface. After inoculation, plants need to be maintained at least 72h wetness. Two or 3 weeks after inoculation, depending on the temperature, disease development on the plants can be assessed.

Ascospore is more infective than conidia, it is easy to get infection with inoculation of ascospores regardless of the inoculation methods. However, for limited inoculum, for example ascospores of isogenic isolates from crosses in vitro , it is difficult to get enough ascospores to make ascospore suspension; the ‘ascospore shower' method is more efficient. Pinpoint inoculation with conidia could not infect plants without wounding the plants before inoculation. Spray with conidia suspension could not always get infection. Therefore, for inoculation with conidia, wounding and pinpoint inoculation is an efficient method.

Protocol for production of inoculum ( conidia and ascospores)

Production of conidia

For genetics study, it usually needs quite a lot of conidia to make conidia suspension. Three methods of producing conidia can be used:

(1) culture isolates onto V8 and seal with Parafilm, incubate in darkness at 20°C for one week and then move to UV light;

(2) culture isolates onto V8 and seal with Parafilm, incubate in darkness at 20°C for one week, then overlay the plate with 1.5% water agar, seal the plates with Parafilm and put under UV light;

(3) culture isolates onto V8 without sealing the plates with Parafilm, incubate the plates at 20°C with 12h light/12h darkness for one to two weeks.

It was found out that few conidia were obtained with method (1), plenty of conidia were obtained with methods (2) and (3). Therefore, methods (2) and (3) are good for production of conidia for genetic study.

Production of ascospores from debris

For production of ascospores from naturally infected debris, collect oilseed rape stems ( c . 30-50 cm in length, including tap roots) showing stem base canker or phoma stem lesions after harvest. Incubate these stem pieces outdoors in labelled freely draining plastic trays. Regularly check the maturation of pseudothecia, when most of the pseudothecia on these stems are mature, dry the stems at room temperature and store at –15ºC until required.

Production of ascospores from cross in vitro

The two isolates to be paired are cultivated on V8-agar (in 5cm diameter Petri dishes) for one week at 25°C with a 12h photoperiod (mixture of white and near-UV light). Petri dishes are NOT sealed with parafilm. One agar plug (diameter around 5 mm) of each isolate is deposited on a 50 cm diameter Petri dish containing V8-agar (Mycelium upside down). The two plugs are placed in a 5 to 10 mm distance in the centre of the plate as follows:

After one week (conditions described above), the cultures are covered with 1.5% water agar (cooled at around 45 °C). 5 to 6 ml of water agar for each agar plate. 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). The plates are sealed again and the cultures are maintained at 11 – 12°C under blacklight (OSRAM 38W tubes) with a 12h-photoperiod (distance between the plates and the light: around 15 cm). 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 (Mengistu et al 1993).

Protocol for isolation of Leptosphaeria maculans from leaf lesions or stem lesions

Sterilise the leaves or small piece of stem with lesions with a sodium hypochlorite solution ( c . 1% available chlorine) for 3 min. cut a small piece of lesion (0.3 × 0.3 cm) from the surface sterilised leaves and place in a Petri dish on distilled water agar amended with 20 units/ml penicillin and 40 units/ml streptomycin. Incubate the cultures at 20°C in darkness. After 5 days, transfer a small piece of mycelium to Petri dishes containing 4% w/v potato-dextrose agar (PDA) with 20 units/mL penicillin and 40 units/ml streptomycin and incubate at 20°C for 10 days. Isolates taken from leaf lesions can be identified as L. maculans or L .biglobosa , on the basis of colony morphology and absence/presence of a yellow pigment in the PDA medium. Colonies which produce a yellow pigment in the colourless PDA, have white, fluffy mycelium, occasionally with yellow or brown drops of liquid, and produce pycnidia mainly in central, older parts of the colony, can be classified as L. biglobosa . Colonies which do not produce an intense pigment, have flat white mycelium, less aerial hyphae, relatively few pycnidia, and sometimes patches of beige-brown mycelium, can be classified as L. maculans .

Protocol for single ascospore isolation from mature pseudothecia of Leptosphaeria maculans

Attach pieces of oilseed rape stem (2 ´0.5 cm) bearing mature L. maculans pseudothecia well to the undersides of 9 cm diameter Petri dish lids with petroleum jelly or Vaseline. Spray the pieces of stem with distilled water using a laboratory sprayer to induce ascospore release. Then place the lid above a 9 cm diameter Petri dish containing distilled water agar (DWA) and incubate at room temperature ( c. 20°C). Monitor the dish every hour for ascospore release under a binocular stereo-microscope at ´60 magnification. When ascospores have been released onto the surface of DWA (after 1-3 h), cut out small blocks of DWA containing single ascospores with a sterile scalpel under a binocular stereo-microscope at ´60 magnification and transfer to 9 cm diameter Petri dishes containing PDA. Seal the plate containing the single ascospore with Parafilm. Incubate the plates with single ascospores at 20°C for 10 days to produce a colony. The identity of the resulting colonies can be assessed by colony morphology and pigment production. Colonies which produce a yellow pigment in the colourless PDA, have white, fluffy mycelium, occasionally with yellow or brown drops of liquid, and produce pycnidia mainly in central, older parts of the colony, can be classified as L. biglobosa . Colonies which do not produce an intense pigment, have flat white mycelium, less aerial hyphae, relatively few pycnidia, and sometimes patches of beige-brown mycelium, can be classified as L. maculans .