2.4. Root measurements
The roots were sampled in 2018 using the soil core method (Bohm, 1979). Four samples per plot were collected using a 50 mm diameter core. The samples were collected from four soil layers: 0‑5, 5‑10, 10‑15 and 15‑20 cm. The roots were washed using a hydropneumatic elutriation system (Smucker et al., 1982) to remove mineral particles from the samples. Organic contamination was removed manually, and digital images were obtained with an Epson Perfection 4870 photo scanner (Seico Epson Corp., Japan) at a resolution of 1200 dpi. The images were analysed using Aphelion Dev image analysis software (ADCIS S.A. and Amerinex Applied Imaging, Herouville, Saint-Clair, France), and the root characteristics were then determined. The procedure for the image analysis was described by Bauhus and Messier (1999) and comprised four main steps: filtering, segmentation, preparation of root skeletons and morphometric measurements. The obtained root lengths were divided into eight diameter classes (<0.02, 0.02‑0.05, 0.05‑0.1, 0.1‑0.2, 0.2‑0.5, 0.5‑1.0, 1.0‑2.0, and >2.0 mm), and the rot morphometric parameters were calculated. The root length density (RLD) was calculated by dividing the total root length (L ) by the volume of the soil sample (V ) (Eq. 1).
(1)
\begin{equation} \text{RLD}=\frac{L}{V}\nonumber \\ \end{equation}
The mean root diameter (MRD) was calculated as the weighted mean of the root length (li ) with the particular diameter classes (di ) as the weights (Eq. 2).
(2)
\begin{equation} \text{MRD}=\frac{\sum_{i=1}^{n}{l_{i}d_{i}}}{\sum_{i=1}^{n}d_{i}}\nonumber \\ \end{equation}
The root surface area (RSA) was calculated as the product of the root segment lengths (li ) and their diameters (di ) (Eq. 3).
(3)
\begin{equation} \text{RSA}=\sum_{i=1}^{n}{\pi d_{i}l_{i}}\nonumber \\ \end{equation}
The root volume density (RVD) was calculated as the product of the root segment lengths (li ) and their diameters (di ) (Eq. 4).
(4)
\begin{equation} \text{RVD}=\sum_{i=1}^{n}{\pi\left(\frac{d_{i}}{2}\right)^{2}l_{i}}\nonumber \\ \end{equation}
After scanning, the roots were dried at a temperature of 70 °C to determine the root dry matter density (RDMD). Then, the specific root length (SRL) was calculated as a ratio of the RLD to the RDMD (Eq. 5).
(5)
\begin{equation} \text{SRL}=\frac{\text{RLD}}{\text{RDMD}}\nonumber \\ \end{equation}
Based on the RDMD and the DM of the aboveground yields, the root:shoot ratio (RSR) was determined (Eq. 7) as follows
(7)
\begin{equation} \text{RSR}=\frac{\text{RDMD}\times V_{s}}{\text{SDM}}\nonumber \\ \end{equation}
where Vs is the volume of the upper soil layer and SDM is the mean annual shoot dry matter.