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Appendix S1 –Experimental procedures
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Fungal strains and culture conditions
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Two strains each of two PAC species (Reininger et al., 2012) differing in virulence on spruce (Tab. S1)
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and Hebeloma crustuliniforme (strain UAMH 5460) were incubated for four weeks in 100 ml
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Erlenmeyer flasks containing 50 ml of either 20 g l-1 malt extract for PAC or liquid Pachlewski medium
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(concentrations per liter: 7.3 mM KH2PO4, 5 mM (D+)-Glucose, 2.7 mM C4H12N2O6, 7.3 mM MgSO4
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heptahydrat, 2.9 mM thiamine-HCL, 1 ml trace-element stock solution) for H. crustuliniforme on a
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shaker at 20°C and 100 rpm. Inoculation of sterile 100 ml plastic tubes containing 1:100 peat :
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vermiculite (v:v) soaked with liquid Pachlewski medium was as follows. PAC and H. crustuliniforme
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mycelia were washed with sterile high-purity water (Barnstead NANOpure DIamond™, Skan AG,
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Allschwil, Switzerland) and pureed under sterile conditions. Equal amounts of PAC and H.
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crustuliniforme mycelium were mixed together to inoculate the seedlings receiving both fungi (dual
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inoculation or ‘PAC & MYC’ treatment, see below). The concentration of mycelial inoculi was
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adjusted to 0.03 g ml-1 fresh mycelium with high-purity water in all treatments, and 2 ml of this
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suspension were used per 100 ml tube which served as experimental unit. Tubes were incubated for
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two weeks at 20°C. Picea abies (Norway spruce) seeds (Birmenstorf Tannwald, Aargau, Switzerland,
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400 m NN, year 1987) were surface sterilized for 30 minutes with 30% H2O2, rinsed in EtOH for 10 s,
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and germinated on H2O agar. Sterile seedlings were planted into tubes pre-inoculated with the
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fungus and incubated in a phytotron at a mean temperature of 19°C [16h day (120-140 µEm-2s-1) / 8h
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night (including a 2 h ramp at the start and the end of the day during which temperature and light
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increased and dropped slowly), temperature (22°C / 15°C), and 45% relative humidity (RH)].
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Experimental Setup
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The experiment was setup as a randomized block design to measure the influence of the fixed factors
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’PAC strain’ (presence/absence of one of the four tested strains), ’mycorrhization’ (presence/absence
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of mycorrhiza) and ‘block’ (to account for the heterogeneity in the phytotron two blocks including
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five replicates per treatment) on plant and PAC biomass, the degree of mycorrhization and the
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root/shoot ratio (response variables). Eight experimental units (replicates) received the same
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treatment (factor combination) and each of the two blocks comprised four replicates per treatment.
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The experiment run for 7 months in the phytotron after planting the seedlings. Plants were watered
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three times a week with deionized water as needed. Once a month each tube was fertilized with an
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equal amount (4-6 ml depending on the experimental stage) of a 1-4 ml l-1 WUXAL solution [1.4 to 5.6
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mM phosphate] (Universaldünger, Maag, Syngenta Agro AG, Dielsdorf, Switzerland).
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Sampling and data collection
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Roots of all eight replicates per treatment were carefully washed under running tap water to remove
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the peat-vermiculite. Seedlings inoculated with H. crustuliniforme were placed into large glass Petri
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dishes filled with insipid tap water to investigate the degree of mycorrhization under the binocular
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using the following classification system: 0 = no mycorrhization, 1 = 1-25% of the root tips
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mycorrhized, 2 = 26-50%, 3 = 51-75%, and 4 = 76-100%.
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Seedlings were cut into roots and shoot, dried at 50°C for 48 hours and weighed. Prior to drying, root
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segments were cut from three plants (replicates) per treatment for further analysis. Seven 5-mm-
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long segments were randomly excised from each of three main roots of each of these three plants
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for DNA extraction, and one additional segment per main root was used for reisolation. For DNA
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extraction, the segments from each plant, i.e. 21 root segments, were pooled, freeze dried and
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weighed. To estimate PAC colonization density ([g] PAC mycelium per [g] root dry weight), 3 mg of
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freeze-dried reference mycelium was added before DNA extraction (mycelium of strain C was added
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as reference to root samples containing strain A, mycelium of strain A to root samples containing
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strains B, C and D; for details see Reininger et al. (2011)) and stored at -80°C until further processing.
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The three root segments for PAC reisolation resulting from each seedling were surface-sterilized in
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30% H2O2 for 30 s and 10 s in EtOH and incubated on terramycin-malt agar (20 g l-1 malt extract, 15 g
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l-1 agar, 50 mg l-1 terramycin®) at room temperature.
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DNA extraction, microsatellite PCR and microsatellite fragment analysis
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Frozen root-reference-mixtures were homogenized in 2 ml safe-lock tubes, using a Retsch machine
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MM 200, adding a small metal ball and a few grains of sand. DNA extraction followed the
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manufacturers protocol of the DNeasy plant mini kit (Qiagen, Hilden, Germany) except for the lysis
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buffer which was replaced by hexadecyltrimethylammonium bromide (CTAB) according to Rogers et
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al. (1989) and Rogers and Bendich (1994). Microsatellite PCR was performed in 15 µl volumes
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containing 2 µl 1:40 diluted DNA, 50 mM KCl, 10 mM Tris-HCl, 1,5 mM MgCl2, 200 µM dNTPs
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(Amersham Pharmacia Biotech), 0,4 µM of each primer and 0,3 U Taq polymerase (Amersham
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Pharmacia Biotech). The running conditions were 2 min at 94°C followed by 33 cycles of denaturation
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for 30 s at 94°C, annealing for 30 s at 53°C and extension for 30 s at 72°C (followed by a final
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extension step of 10 min at 72°C) (Queloz et al., 2008). For the microsatellite fragment analysis 15-
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fold diluted amplicons of the PCRs were prepared and 4 µL of the dilutions were mixed with 9.05 µL
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Hi-DiTM formamide and 0.25 µL GeneScanTM 500 LIZTM (Applied Biosystems). Fragment lengths and
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the area under the light emission curve (AUC) of each fragment were measured using an ABI 3730xl
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DNA Analyzer (Applied Biosystems) and analyzed using the GeneMapper v. 4.0 software (Applied
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Biosystems) (Queloz et al., 2010). Biomass of PAC mycelia in and on roots was estimated using the
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method described in Reininger et al. (2011).
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Statistical analysis
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All statistical analyses were done with the statistical software R (R Development Core Team, 2010).
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Due to very high correlation of root and shoot biomass (data not shown), analyses were done with
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total plant biomass except for the root/shoot ratio analysis. Six seedlings had to be excluded from
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data analysis since mycorrhization failed. ANOVA models [1]-[4] including interactions between
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factors were examined (µ = overall mean):
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Plant biomass = µ + PAC strain + mycorrhization + block
[1]
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Root/shoot ratio = µ + PAC strain + mycorrhization + block
[2]
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PAC biomass = µ + PAC strain + mycorrhization + block
[3]
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Degree of mycorrhization = µ + PAC strain + block
[4]
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Reductions of the full models [1]-[3] were sought using the ‘stepAIC’ (Akaike Information Criterion)
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function implemented in ‘R’. The factor ’mycorrhization’ in models [1]-[3] is binary with 1 = H.
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crustuliniforme added and 0 = no H. crustuliniforme added. Letters above the bars in bar charts were
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calculated with the function ‘pairwise.t.test’ to find significant differences between treatments.
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Correlation between plant dry weight and endophytic PAC biomass amount were examined for
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plants inoculated with PAC only (single inoculation) and for dual inoculated plants with PAC and H.
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crustuliniforme using linear regression. Best transformations of the models were sought comparing
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residual analyses (Tukey-Anscombe plot, Q-Q plot, leverage plot).
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References
Queloz, V., Duo, A., and Grünig, C.R. (2008) Isolation and characterization of microsatellite markers
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Resour 8: 1322-1325.
Queloz, V., Duo, A., Sieber, T.N., and Grünig, C.R. (2010) Microsatellite size homoplasies and null
alleles do not affect species diagnosis and population genetic analysis in a fungal species
complex. Mol Ecol Resour 10: 348-367.
R Development Core Team (2010) R: A language and environment for statistical computing. Vienna,
Austria: R Foundation for Statistical Computing.
Reininger, V., Grünig, C.R., and Sieber, T.N. (2011) Microsatellite-based quantification method to
estimate biomass of endophytic Phialocephala species in strain mixtures. Microb Ecol 61:
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Reininger, V., Grünig, C.R., and Sieber, T.N. (2012) Host species and strain combination determine
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Rogers, S.O., and Bendich, A.J. (1994) Extraction of total cellular DNA from plants, algae and fungi.
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