Harvest and plant measurements
Before destructively harvesting the plants at advanced tillering stage
on 25th August 2021, we measured leaf chlorophyll
content using a chlorophyll content meter (CCM-330; Opti-Sciences,
Hudson, NY). For each plant, the top three fully expanded leaves were
measured and for each leaf, three measurements were taken and averaged
to account for spatial heterogeneity. Afterward, leaves were collected
and dried at 70 °C for 48 h to determine leaf N content, and the rest of
the aboveground plant material was also harvested and dried at 70 °C for
48 h. Total shoot biomass was measured on a dry weight basis.
Roots were excavated from rhizoboxes and rhizosphere soil was collected
after vigorous shaking to remove most of the loosely bound soil. One
subsample of rhizosphere soil was immediately frozen with dry ice for
microbial analyses whereas the other subsample was stored at 4 °C for
substrate-induced respiration measurements. Thereafter, the rest of the
root material was stored at -20 °C before thoroughly washing the roots.
Representative samples from washed roots were scanned at 1200 dpi
resolution using a flatbed scanner (Epson Perfection V800 Photo, 8-bit
grayscale images). Scanned root images were analyzed with RhizoVision
Explorer (Seethepalli et al. 2021) with the following setting:
Image thresholding level: 180, enable edge smoothing: true, edge
smoothing threshold: 2, root pruning threshold: 15, dpi: 1200. We used
each root diameter class to determine the following root traits:
specific root length (root length divided by root dry biomass, SRL),
specific root area (root area divided by root dry biomass, SRA), root
tissue density (root dry biomass divided by root volume, RTD), and root
length weighted average fine root diameter (FRD) as recommended by
others (Rose 2017; Rose & Lobet 2019).