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.