At the first day of harvest (475 d after planting), the shoot length and root collar diameter of each sapling were measured. The roots were carefully excavated from the soil, washed and cleaned from adherent soil particles. Where possible, three representative root branches per species and soil compartment were isolated in all six soil layers of the rhizotrons and digitalised on a flat-bed scanner for image analysis to determine specific fine root area (SRA, cm2 g-1 dry matter), specific fine root length (SRL, cm g-1 dry matter) and total fine root surface area using WinRhizo 2005c software (Régent Instruments Inc., Québec, QC, Canada). All biomass samples were oven-dried (70 °C, 48 h) and weighed for dry weight determination.
For quantifying the vertical distribution of root biomass in the rhizotrons, we calculated the relative cumulative root biomass in the six soil depth layers of the boxes and described the depth distribution by the exponential function y = 1 – βd given by Gale and Grigal (1987) which expresses the cumulative proportion of root biomass y as a function of soil depth d and a specific factor β. The relative growth rate of the roots (RGR, mg d-1 g-1 root mass) was estimated by subtracting the initial root mass (determined in five saplings per species at the day of planting) from the root mass of the harvested saplings divided by the duration of the experiment and relating to initial root mass. The plant material was grounded with a disc mill and the C and N concentrations detected in a mass spectrometer (Delta plus, Finnigan MAT, Bremen, Germany). The colonisation with AM and EM was determined as described previously (Lang et al., 2011a), and differed between ash (85%) and beech (44%), but not between mono or mixed systems. Compared to field observations, both ectomycorrhizal and arbuscular mycorrhizal colonisation rates were within typical ranges (Pena et al., 2010; Lang et al., 2011a).
For estimating the root-induced respiratory activity in immediate vicinity of the roots, we estimated root respiration by calculating root growth respiration from the expression Rg = (RGRroot+73.7)/4.31 given by Reich et al. (1998) with RGRroot being the relative growth rate of the roots, and on the assumption that root maintenance respiration is approximated by Rm = 0.106 x N following Ryan (1991) with N being the nitrogen concentration of root dry mass. By subtracting the calculated root respiration (growth plus maintenance respiration) and the pure soil respiration, measured in the root-free rhizotrons, from the measured total net CO2 efflux, we obtained an estimate of the root-induced additional soil respiration in the rhizosphere. The root respiration rates of ash calculated from root RGR and root N content were checked against independent in situ measurements conducted with miniature root cuvettes (1.5 mL microcentrifuge tubes) placed around root segments of ash saplings (n = 5) using miniature planar CO2 optodes (Presens, Regensburg, Germany) for online optoanalytical measurement of net CO2 release over 180 min at one minute intervals..
During the harvest soil samples from the upper 20 cm-layer located below the gas flux sampling area were extracted for chemical analysis. To exclude an effect of soil depth on soil properties additional samples were taken in each soil layer. The soil pH was analysed in a suspension with 10 g soil and mixed with 25 mL H2O using a Vario pH meter (WTW GmbH, Weilheim, Germany). The gravimetric soil water content was determined by weighing the soil samples before and after drying at 105
°C for 24 h. The nitrate (mg N-NO3
kg-1 dw) and ammonium (mg N-NH4+
kg-1dw) concentrations were estimated by extracting soil samples in 0.5 M K2SO4 solution (1:3 wet soil mass to solution ratio) directly after collection. The samples were shaken for 1 h and passed through folded filters (150 mm in diameter, 65 g m-2, Sartorius Stedim, Aubagne, France). The NO3
and NH4+
concentrations of the filtered extracts were analysed using continuous flow injection colorimetry (SAN+ Continuous Flow Analyzer, Skalar Instruments, Breda, The Netherlands). Nitrate was determined by the copper cadmium reduction method (ISO method 13395) and NH4+
by the Berthelot reaction method (ISO method 11732). The contents of Corg
and Ntotal were determined in a mass spectrometer (Delta plus, Finnigan MAT, Bremen, Germany) after grounding the dry soil in a disc mill. The bulk density of the
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material was determined in 5 cm soil depth under the gas flux sampling area using plastic cores with a defined volume of 10.8 cm3 after Schlichting et al. (1995). The particle size distribution of the soil material in the rhizotrons was analysed in five replicate samples using the sieving and pipette method (Schlichting et al., 1995).
5.3.6 Data analysis
All statistical analyses were carried out with SAS 9.1 software (Statistical Analysis System, SAS Institute Inc., Cary, NC, USA). Cumulative gas fluxes were calculated by summing up all measurements done in a rhizotron considering the number of measurements taken and the length of the entire measuring period (324 d). The gas fluxes varied considerably between the different measurement days as it is common for GHG fluxes from soil, so that we refrained from showing the time course. All data were tested for normal distribution using the Shapiro-Wilk test and for homogeneity of variances applying the Levene test. The block effect of the two climate chambers was tested with a two-factorial ANOVA considering the two factors ―treatment‖ and ―bloc‖ and an interaction term (―treatment x block‖). For the various soil chemical properties, the gas fluxes and the biological parameters, no bloc (chamber) effect was detected. To investigate the effects of beech and ash roots on various parameters, one-way ANOVA with a post hoc Tukey-Kramer test was used to locate significant differences among treatment means for data showing normal distribution. If the data were not normally distributed or variances were not homogeneous, the non-parametric Kruskal-Wallis test was used to test for significant differences between means. The significance of differences between two treatments was subsequently investigated with the Wilcoxon U-test. A paired t test was used to test for significant differences in normally distributed soil parameters between the soil state at harvest and the experiment‘s beginning. Linear regression analysis was conducted to relate the chemical soil properties of the uppermost 20 cm to various biological parameters (listed in Tables 5.1 and 5.2), and to relate gas fluxes to soil chemical properties and biological parameters. In all analyses, significance was determined at P < 0.05.
5.4 RESULTS