Aug 01, 2024
Version 1
Spore based infection assay on Pinus sylvestris seedlings with Diplodia sapinea V.1
- Anne Oostlander1,
- Laura Brodde2,
- Miriam von Bargen1,
- Rasmus Enderle3,
- Marco Leiterholt1,
- Dagmar Trautmann3,
- Malin Elfstrand2,
- Jan Stenlid2,
- André Fleißner1
- 1Institute of Genetics, Technical University Braunschweig, Braunschweig, Germany;
- 2Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden;
- 3Institute of Forest Protection, Julius Kuehn Institute (JKI), Braunschweig
Protocol Citation: Anne Oostlander, Laura Brodde, Miriam von Bargen, Rasmus Enderle, Marco Leiterholt, Dagmar Trautmann, Malin Elfstrand, Jan Stenlid, André Fleißner 2024. Spore based infection assay on Pinus sylvestris seedlings with Diplodia sapinea. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyw9revx9/v1
Manuscript citation:
Oostlander AG, Brodde L, Bargen M von, Leiterholt M, Trautmann D, Enderle R et al. A reliable and simple method for the production of viable pycnidiospores of the Pine pathogen Diplodia sapinea and a spore-based infection assay on Scots Pine. Plant Dis 2023; 107(11):3370–7.
doi.org/10.1094/PDIS-01-23-0107-RE
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 26, 2024
Last Modified: August 01, 2024
Protocol Integer ID: 104147
Keywords: Diplodia sapinea, Diplodia tip blight, infection assay, Pinus sylvestris infection, pycnidiospores, Sphaeropsis sapinea
Abstract
This protocol describes a spore based method for assessing Diplodia sapinea pathogenicity in Scots pine seedlings (Pinus sylvestris), including inoculation, symptom evaluation, and reisolation.
Materials
Material:
Petri dishes
D. sapinea (e.g. ex-type strain CBS 138184)
Light shelf with cold white daylight (e.g. Osram Lumilux 18W/865), intensity from 5000 to 6500 lux
Cleanbench
Centrifuge
Sterile scalpel
Ethanol (70%)
Sodium hypochlorite (3% NaOCl)
Sterile water
Plant Material:
Scots pine (P. sylvestris) container seedlings
Potting soil
Plastic pots
Minimal medium (VMM):
as described in Vogel, 1964
20 ml Vogel’s solution
20 g sucrose
15 g agar
add 1 l water autoclave
Trace element solution:
50 g citric acid
50 g zinc sulfate (J.T. Baker)
10 g ammonium iron(II) sulfate
2,5 g copper sulfate
0,5 g manganese sulfate
0,5 g boric acid
0,5 g sodium molybdate
add 1 l water
Chloroform (1 ml) is added as a preservative, store at room temperature.
Vogel’s solution:
125 g sodium citrate
250 g potassium dihydrogen phosphate
100 g ammonium nitrate
10 g magnesium sulfate
5 g calcium chloride
5 ml trace element solution
2,5 ml biotin solution
add 1 l water
Chloroform (2 ml) is added as a preservative, store at room temperature.
Biotin solution:
0,1 g biotin
add 1 l water
Store at -20°C
0,01 % (v/v) Tween:
0,1 ml Tween
add 1 l water
aliquod and autoclave
Plant Material and Greenhouse Conditions
Plant Material and Greenhouse Conditions
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).
Fungal Inoculum Preparation
Fungal Inoculum Preparation
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.
Inoculation
Inoculation
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.
Symptom Assessment
Symptom Assessment
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
Sampling for reisolation
Sampling for reisolation
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.
Surface Sterilization
Surface Sterilization
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.
Isolation and Identification
Isolation and Identification
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).
Protocol references
Adamson, Kalev/Laas, Marili/Blumenstein, Kathrin/Busskamp, Johanna/Langer, Gitta J./Klavina, Darta/Kaur, Anu/Maaten, Tiit/Mullett, Martin S./Müller, Michael M./Ondrušková, Emília/Padari, Allar/Pilt, Enn/Riit, Taavi/Solheim, Halvor/Soonvald, Liina/Tedersoo, Leho/Terhonen, Eeva/Drenkhan, Rein (2021). Highly clonal structure and abundance of one haplotype characterise the Diplodia sapinea populations in Europe and Western Asia. Journal of fungi (Basel, Switzerland)
https://doi.org/10.3390/jof7080634.