Collection of root exudates and analyses
On 21st August 2021, we installed the root exudation
traps with modifications from Phillips, Erlitz, Bier & Bernhardt 2008,
where we removed the transparent window of the rhizobox, and the most
distal part of the roots was carefully excavated from the soil and
washed with distilled water. Then roots were put in the exudation traps
(20 ml syringes) filled with glass beads and carbon (C)-free nutrient
solution and allowed to acclimatize for 2 days (Fig. 1b). On
23rd August 2021, the nutrient solution from the
exudation traps was sucked out and replaced with a new C-free nutrient
solution. After 48 hours (25th August 2021), the
solution from the exudation traps was collected. The trapped solution
was immediately passed through a membrane filter (Captive Agilent
Premium syringe filter with 0.7µm glass fiber membrane) and stored in
dry ice to transport to the lab and, thereafter, stored at -20 °C. Two
exudation traps were installed per rhizobox and pooed together to make
one composite sample to obtain enough solution for subsequent lab
analyses. Exuded organic C (OCEXU) in trapped solution
was measured with a multi N/C analyzer (multi N/C analyzer 2100S,
Analytik Jena). Roots in the trap solutions were cut and scanned to
determine the root surface area by using RhizoVision Explorer
(Seethepalli et al., 2021).
We also measured the potential activity of the phosphomonoesterase
(PHOEXU) enzyme in the exudation solution by using a
fluorogenic artificial substrate (Marx, Wood & Jarvis 2001; Kumar &
Pausch 2022). For this, frozen exudate solutions were allowed to thaw
first at 4 °C and later at room temperature. Exudate solution aliquots
were used to fluorogenically measure PHO activity. Fluorogenic
methylumbelliferone (MUB)-based artificial substrate was used to measure
the potential activity of PHOEXU enzyme and each field
replicate was measured in analytic triplicates and the potential
activity was expressed in units of nmol MUB cleaved
ml-1 h-1.