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+assays/3_destructive_sampling/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/3_destructive_sampling/dataset/pheno_data.csv filter=lfs diff=lfs merge=lfs -text

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README.md

+# **2025_mycorrhization_experiment**
+
+## This is the git repository for the publication:
+
+**An Ectomycorrhizal Fungus Alters Development Stages Within Pedunculate Oak’s Endogenous Growth Rhythm**
+
+
+
+## Authors & Affiliations:
+
+**Felix Zimmermann** (*Corresponding Author*; Research Unit Biodiversity and Conservation Biology, Swiss Federal Research Institute WSL, Birmensdorf, Switzerland; felix.zimmermann@wsl.ch; [ORCID: 0009-0002-0762-2454](https://orcid.org/0009-0002-0762-2454))
+
+**Marie-Lara Bouffaud** (Department of Soil Ecology, Helmholtz Centre for Environmental Research - UFZ, Halle/Saale, Germany; [ORCID: 0000-0001-6112-2982](https://orcid.org/0000-0001-6112-2982))
+
+**Sylvie Herrmann** (Department of Soil Ecology, Helmholtz Centre for Environmental Research - UFZ, Halle/Saale, Germany; [ORCID: 0000-0001-8220-3853](https://orcid.org/0000-0001-8220-3853))
+
+**Marco Göttig** (Institute of Biology, Philipps University Marburg, Marburg, Germany)
+
+**René Graf** (Research Unit Biodiversity and Conservation Biology, Swiss Federal Research Institute WSL, Birmensdorf, Switzerland; [ORCID: 0009-0002-9567-9089](https://orcid.org/0009-0002-9567-9089))
+
+**Mika Tarkka** (Department of Soil Ecology, Helmholtz Centre for Environmental Research - UFZ, Halle/Saale, Germany; [ORCID: 0000-0003-4630-351X](https://orcid.org/0000-0003-4630-351X))
+
+**Lars Opgenoorth** (Laboratory of Plant Ecology and Geobotany, Institute of Biology, Philipps University Marburg, Marburg, Germany; [ORCID: 0000-0003-0737-047X](https://orcid.org/0000-0003-0737-047X))
+
+**Daniel Croll** (Laboratory of Evolutionary Genetics, Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland; [ORCID: 0000-0002-2072-380X](https://orcid.org/0000-0002-2072-380X))
+
+**Martina Peter** (Research Unit Biodiversity and Conservation Biology, Swiss Federal Research Institute WSL, Birmensdorf, Switzerland; [ORCID: 0000-0002-6365-6889](https://orcid.org/0000-0002-6365-6889))
+
+**Benjamin Dauphin** (*Corresponding Author*; Research Unit Biodiversity and Conservation Biology, Swiss Federal Research Institute WSL, Birmensdorf, Switzerland; benjamin.dauphin@wsl.ch; [ORCID: 0000-0003-0982-4252](https://orcid.org/0000-0003-0982-4252))
+
+
+## Content:
+
+### Data:
+
+* Here you can find the [Phenotypic Data File](https://git.nfdi4plants.org/felix.zimmermann/2025_mycorrhization_experiment/-/blob/main/assays/2_destructive_sampling/dataset/pheno_data.csv?ref_type=heads).
+* Here you can find the [Timeseries Data File](https://git.nfdi4plants.org/felix.zimmermann/2025_mycorrhization_experiment/-/blob/main/assays/1_growth_monitoring/dataset/time_series.csv?ref_type=heads).
+* Here you can find the [Experimental Setup Data File](https://git.nfdi4plants.org/felix.zimmermann/2025_mycorrhization_experiment/-/blob/main/assays/3_experimental_setup/dataset/randomization.csv?ref_type=heads).
+
+### Scripts:
+
+* Here you can find scripts for [Figure 1 / Phenotypic Data Analysis](https://git.nfdi4plants.org/felix.zimmermann/2025_mycorrhization_experiment/-/blob/main/workflows/1_phenotypic_data_figure_1/figure1_phenotypicdata.R?ref_type=heads).
+* Here you can find scripts for [Figure 2 / Timeseries Data Analysis](https://git.nfdi4plants.org/felix.zimmermann/2025_mycorrhization_experiment/-/blob/main/workflows/2_timeseries_data_figure2/figure2_timeseries.R?ref_type=heads).
+* Here you can find scripts for the [Supplementary Information Analysis](https://git.nfdi4plants.org/felix.zimmermann/2025_mycorrhization_experiment/-/blob/main/workflows/3_additional_analyses_SI/SI.R?ref_type=heads).
+
+### Results:
+
+* Here you can find results of [Figure 1 / Phenotypic Data Analysis](https://git.nfdi4plants.org/felix.zimmermann/2025_mycorrhization_experiment/-/tree/main/runs/1_phenotypic_data_figure1?ref_type=heads).
+* Here you can find results of the [Figure 2 / Timeseries Data Analysis](https://git.nfdi4plants.org/felix.zimmermann/2025_mycorrhization_experiment/-/tree/main/runs/2_timeseries_data_figure2?ref_type=heads).
+* Here you can find results of the [Supplementary Information Analysis](https://git.nfdi4plants.org/felix.zimmermann/2025_mycorrhization_experiment/-/tree/main/runs/3_additional_analyses_SI?ref_type=heads).
+   
+
+## To use our code:
+
+1. Download/clone repository.
+
+2. Open the "2025_Myco_Experiment.Rproj" file with RStudio.
+
+3. Run scripts under "workflows" (you can find "workflows" under "Files" in the "Files/Plots/Packages etc." window).
+
+
+## Citation:
+
+
+
+## Link to publication:
+
+
+
+## ARC DOI:
+
+

assays/1_experimental_setup/protocols/experimental_setup_protocol.md

+**Experimental Setup Protocol**
+
+We micropropagated and rooted the pedunculate oak (*Quercus robur* L.) clone DF159 according to Herrmann et al. (1998). In brief, we alternatively used micro-cuttings of axillary and apical buds to maintain the endogenous growth rhythm of oak plants (Herrmann et al., 2016). After micropropagation under sterile conditions in glass vessels, we transferred individual cuttings to sterile glass tubes containing active charcoal medium for in vitro rooting. We performed both the micropropagation and in vitro rooting at 24°C, 50% air humidity, and under long-day conditions (16h of light per day) with a photosynthetic photon flux density of 90-100 µmol m-2⋅s-1.
+After plant production, we prepared the experimental culture system according to Tarkka et al. (2013). In short, soil from an oak forest in the Harz mountains, Saxony Anhalt, Germany (51.400972, 11.125389) from the A0 (humus, 0-10 cm) and A1/A2 (organic, 10–30 cm) horizons was sieved at 2 mm and sterilized at Synergy Health Däniken AG (Däniken, Switzerland) using γ-irradiation with a dosage of 70–90 kGy. We cultivated *P. croceum* strain F1598 (ATCC MYA-4870; Herrmann et al., 1998, 2015) and *C. geophilum strain* 1.58 (CBS 143616; Peter et al., 2016) on Melin Norkrans Modified (MMN; Marx, 1969) agar medium in darkness at 20°C (Herrmann et al., 1998). From the plate cultures, we prepared MMN liquid cultures. Finally, we transferred the liquid cultures onto 10:1 (v/v) mixtures of vermiculite and peat (autoclaved three times) and incubated them in darkness at 20°C for four weeks.
+Next, we applied four treatments on the clone after plant randomization by development stage, rooting date, and stem length; the control treatment, the *C. geophilum* treatment, the *P. croceum* treatment, and the co-inoculation treatment. Finally, we planted the oaks in 12×12 cm2 squared Petri dishes (roots grown inside, shoots grown outside the Petri dishes), assessed the initial weight and sealed the dishes with parafilm. In total, we prepared two sets of 48 plants each (with 12 plants per treatment, Fig. 1a), with the second set being produced three weeks after the first set. We then grew the plants in a growth chamber at 20–22°C, 80–90% air humidity, and with 16h of light as previously described.
+
+
+**References:**
+
+Herrmann, S., Grams, T. E. E., Tarkka, M. T., Angay, O., Bacht, M., Bönn, M., Feldhahn, L., Graf, M., Kurth, F., Maboreke, H., Mailander, S., Recht, S., Fleischmann, F., Ruess, L., Schädler, M., Scheu, S., Schrey, S., & Buscot, F. (2016). Endogenous rhythmic growth, a trait suitable for the study of interplays between multitrophic interactions and tree development. Perspectives in Plant Ecology, Evolution and Systematics, 19, 40–48. https://doi.org/10.1016/j.ppees.2016.02.003
+
+Herrmann, S., Munch, J. ‐C., & Buscot, F. (1998). A gnotobiotic culture system with oak microcuttings to study specific effects of mycobionts on plant morphology before, and in the early phase of, ectomycorrhiza formation by Paxillus involutus and Piloderma croceum. New Phytologist, 138(2), 203–212. https://doi.org/10.1046/j.1469-8137.1998.00105.x
+
+Marx, D. H. (1969). The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. II. Production, identification, and biological activity of antibiotics produced by Leucopaxillus cerealis var. Piceina. Phytopathology, 59(4), 411–417.
+
+Peter, M., Kohler, A., Ohm, R. A., Kuo, A., Krützmann, J., Morin, E., Arend, M., Barry, K. W., Binder, M., Choi, C., Clum, A., Copeland, A., Grisel, N., Haridas, S., Kipfer, T., LaButti, K., Lindquist, E., Lipzen, A., Maire, R., … Martin, F. M. (2016). Ectomycorrhizal ecology is imprinted in the genome of the dominant symbiotic fungus Cenococcum geophilum. Nature Communications, 7(1), 12662. https://doi.org/10.1038/ncomms12662
+
+Tarkka, M. T., Herrmann, S., Wubet, T., Feldhahn, L., Recht, S., Kurth, F., Mailänder, S., Bönn, M., Neef, M., Angay, O., Bacht, M., Graf, M., Maboreke, H., Fleischmann, F., Grams, T. E. E., Ruess, L., Schädler, M., Brandl, R., Scheu, S., … Buscot, F. (2013). OakContig DF 159.1, a reference library for studying differential gene expression in Quercus robur during controlled biotic interactions: Use for quantitative transcriptomic profiling of oak roots in ectomycorrhizal symbiosis. New Phytologist, 199(2), 529–540. https://doi.org/10.1111/nph.12317
+

assays/2_growth_monitoring/protocols/growth_monitoring_protocol.md

+**Growth Monitoring Protocol**
+
+Day 1 of the experiment is defined as the first day after a four-week acclimatization period in protective plastic bags.  Throughout eight weeks of the experiment, we assessed the development stages of the plants twice a week according to Herrmann et al. (2015). In short, *Q. robur* growth is characterized by the succession of developmental stages A (bud rest) and B (bud swelling), corresponding to the root flush of a growth cycle, followed by stages C (shoot elongation) and D (leaf expansion), corresponding to the shoot flush. We weighed the plants weekly and replenished any weight loss with sterilized tap water to maintain optimal growth conditions at a soil water content of 12-15%.
+
+**References:**
+
+Herrmann, S., Recht, S., Boenn, M., Feldhahn, L., Angay, O., Fleischmann, F., Tarkka, M. T., Grams, T. E. E., & Buscot, F. (2015). Endogenous rhythmic growth in oak trees is regulated by internal clocks rather than resource availability. Journal of Experimental Botany, 66(22), 7113–7127. https://doi.org/10.1093/jxb/erv408
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