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.