Spore based infection assay on Pinus sylvestris seedlings with Diplodia sapinea

Use 2 year old container seedlings of Scots pine ( Pinus sylvestris ).

Ensure seedlings have no visible symptoms and have undergone two annual cycles.

Apply the last fungicide treatment 8 weeks before the first inoculation.

Replant seedlings in plastic pots filled with potting soil

Maintain plants at 20–25°C, 16 h of light per day, and high humidity (>90% RH) for 4 d after inoculation, then switch to moderate humidity (60% RH).

Grow Diplodia sapinea on VMM (Vogels Minimal Medium) for 21 d at approximately 27°C under constant light (5000–6500 lx) to induce sporulation.

Harvest the spores from the plate by adding approximately 2 ml of 0.01% (v/v) Tween to the plate and rinse the surface of the colony several times by pipetting. Repeat the procedure for a higher yield.

Dilute the spore suspension to 2x10⁶ spores/ml for inoculation.

Assess spore viability by spreading 600 µl of the spore suspension onto three VMM plates. Incubate for 7 h at 27°C and record the germination status of 200 spores per plate. Aim for an average germination rate of 88%.

Confirm the absence of hyphal fragments microscopically.

Wounded or not wounded plants can be inoculated. Wounding is likely to influence symptom development.

Wounding: Use a sterile scalpel to make a 5 mm long cut down to the cambium on dormant seedlings and on the last year’s growth segments of actively growing seedlings. Be careful that the cut does not become too deep.

Inoculation with Spores:

Pipetting: Apply 2 µl of spore suspension (approx. 4000 spores) directly onto the wound or unwounded stem.

Spraying: Spray approximately 360 µl (approx. 720,000 spores) from a distance of 10 cm onto the wound or unwounded stem.

Control Treatments: Use sterile 0.01% Tween 20 for mock inoculation.

Evaluate the symptoms 4- and 6-weeks post-inoculation based on the classification into previously determined symptom classes, for example:

1. No symptoms

2. Necrotic needles, no symptoms on stem

3. Upper third of shoot necrotic

4. Upper two thirds necrotic

5. Seedling dead

After 6 weeks, cut off and discard the shoot tip.

Remove an approx. 1.5 cm long piece of the stem above the wound (if applicable), remove needles, and use for reisolation.

Sterilize stem pieces:

Immerse in 70% ethanol for 30 s.

Immerse in 3% NaOCl for 60 s.

Rinse twice in sterile water for 30 s each.

Dry on sterile filter paper.

Cut four approx. 2 mm long pieces from the center of the stem samples and place on MYP agar. Incubate for 27 d at room temperature and ambient daylight.

Sort isolates into morphological groups from day 4 of cultivation.

Extract fungal DNA of a representational number of isolates for molecular identification.

Use species-specific primers to identify D. sapinea (for example as described in Adamson et al. 2021).