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).