2.2 Artificial root exudate experiment
The sieved and air-dried soil samples (3 kg dry weight) were
respectively placed in a 50-L plastic bucket and flooded with deionized
water to a depth of 2–3 cm. The soils were pre-incubated for two weeks
in the dark at 25 °C. The experiment consisted of five treatments with
four replicates in a completely randomized design. Afterwards, the soil
was mixed well, and subsamples of 50 g (equivalent to 30 g dry soil)
were placed in 500-mL incubation serum bottles. Deionized water was
added to each bottle to a level of 2–3 cm above the soil surface, and
the bottles were sealed using a rubber stopper to ensure anoxic
conditions. To imitate root exudates, which are mainly dominated by
sugars, amino acids, and organic acids (Lopez‐Sangil et al., 2017; Xiong
et al., 2019), we chose different proportions of glucose
(C6H12O6), oxalic acid
(H2C2O4), and alanine
(C3H7NO2). We adjusted
three different C/N ratios (CN6-, CN10-, and CN80-treatments) by adding
different amounts of alanine, along with a treatment without alanine
(C-only). Treatment with only distilled H2O was used as
a control. The total amount of added C was maintained at 5% SOC, about
0.6 mg C g-1 soil dry weight. The relative
contributions of sugar, amino acids, and organic acid-derived C were
within the ranges reported in the literature (Qiao et al., 2016; Liu et
al., 2020).
The solutions were prepared every five days by dissolving the
abovementioned proportions of glucose, oxalic acid, and alanine in 50 mL
of deionized water. The details are presented in Table 1 and Table S1.
To mimic a pattern of continuous exudate excretion, we manually injected
0.5 mL of the solution into the soil samples using a 10-mL syringe
daily. All samples were incubated at 25 °C in the dark for 45 days. Four
bottle samples were chosen randomly and destructively harvested at days
3, 12, and 45 after initial exudate addition. Before harvesting the soil
samples, four leachate water samples were collected in a 50-mL
centrifuge tube and the soil samples were then mixed thoroughly; both
leachate water and soil samples were stored at 4 °C for further
measurements.