TITLE: Spore based infection assay on Pinus sylvestris seedlings with Diplodia sapinea
AUTHORS: Anne Oostlander, Laura Brodde, Miriam von Bargen, Rasmus Enderle, Marco Leiterholt, Dagmar Trautmann, Malin Elfstrand, Jan Stenlid, André Fleißner

[DESCRIPTION]
This protocol describes a spore based method for assessing Diplodia sapinea pathogenicity in Scots pine seedlings (Pinus sylvestris), including inoculation, symptom evaluation, and reisolation.

[STEPS]
SECTION: Plant Material and Greenhouse Conditions
1. Use 2 year old container seedlings of Scots pine (Pinus sylvestris).
SECTION: Plant Material and Greenhouse Conditions
2. Ensure seedlings have no visible symptoms and have undergone two annual cycles.
SECTION: Plant Material and Greenhouse Conditions
3. Apply the last fungicide treatment 8 weeks before the first inoculation.
SECTION: Plant Material and Greenhouse Conditions
4. Replant seedlings in plastic pots filled with potting soil
SECTION: Plant Material and Greenhouse Conditions
5. 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).
SECTION: Fungal Inoculum Preparation
6. Grow Diplodia sapinea on VMM (Vogels Minimal Medium) for 21 d at approximately 27°C under constant light (5000–6500 lx) to induce sporulation.
SECTION: Fungal Inoculum Preparation
8. Dilute the spore suspension to 2x106 spores/ml for inoculation.
SECTION: Fungal Inoculum Preparation
9. 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%.
SECTION: Fungal Inoculum Preparation
10. Confirm the absence of hyphal fragments microscopically.
SECTION: Fungal Inoculum Preparation
7. 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.
SECTION: Inoculation
11. Wounded or not wounded plants can be inoculated. Wounding is likely to influence symptom development.
SECTION: Inoculation
12. 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.
SECTION: Inoculation
13. Inoculation with Spores:
SECTION: Inoculation
13.1. Pipetting: Apply 2 µl of spore suspension (approx. 4000 spores) directly onto the wound or unwounded stem.
SECTION: Inoculation
13.2. Spraying: Spray approximately 360 µl (approx. 720,000 spores) from a distance of 10 cm onto the wound or unwounded stem.
SECTION: Inoculation
13.3. Control Treatments: Use sterile 0.01% Tween 20 for mock inoculation.
SECTION: Symptom Assessment
14. 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

SECTION: Sampling for reisolation
15. After 6 weeks, cut off and discard the shoot tip.
SECTION: Sampling for reisolation
16. Remove an approx. 1.5 cm long piece of the stem above the wound (if applicable), remove needles, and use for reisolation.
SECTION: Surface Sterilization
17. Sterilize stem pieces: 
SECTION: Surface Sterilization
17.2.  Immerse in 3% NaOCl for 60 s.
SECTION: Surface Sterilization
17.3. Rinse twice in sterile water for 30 s each.
SECTION: Surface Sterilization
17.4. Dry on sterile filter paper.
SECTION: Isolation and Identification
18. 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.
SECTION: Isolation and Identification
19. Sort isolates into morphological groups from day 4 of cultivation.
SECTION: Isolation and Identification
20. Extract fungal DNA of a representational number of isolates for molecular identification.
SECTION: Isolation and Identification
21. Use species-specific primers to identify D. sapinea (for example as described in Adamson et al. 2021).
SECTION: Surface Sterilization
17.1. Immerse in 70% ethanol for 30 s.