2.4 | Sample collection and determination
At the end of the culture experiments, the roots were washed with deionized water and blotted dry with absorbent paper, and the weights of the plants’ above- and below-ground parts were measured separately. Two boxes of culture substrates in each group were randomly combined into one sample, mixed and sampled by quartering. Pass through a 40-mesh sieve after grinding to determine soil enzymes and polysaccharides. Enzymatic activities of S-UE, S-NiR, S-NR, and S-CAT were evaluated using cominbio kits (Suzhou Keming Biotechnology Co., Ltd.) following the manufacturer’s guidelines, while mlbio kits (Shanghai Enzyme Biotechnology Co., Ltd.) were employed to measure the activity of S-PT enzymes. The determination of pentoses adopts the lichenol-hydrochloric acid method (Haoli et al., 2014), and the determination of hexoses adopts the anthrone-sulfuric acid method (wei-jie, 1999), and the sum of the two is calculated as the total polysaccharide content.
S-NiR activity was assessed using the cominbio kit’s method. The amount of reducing 1 μmol NO2ˉ per g soil sample per day was regarded as an enzyme activity unit. In a 2 mL centrifuge tube, 0.02 g of air-dried soil sample was mixed with 40 µL each of sodium nitrite and glucose solutions. The mixture was thoroughly combined and reacted for one hour at 25℃. Subsequently, 40 µL of aluminum potassium alum solution was added, and the content was fully agitated for thirty seconds before centrifugation was facilitated at 10,000 rpm, at 4℃, for ten minutes. At this stage, 70µL of the supernatant was extracted, along with 1:1 p-amino benzene sulfonic acid-phosphoric acid solution and 140 µL of N-1-naphthalene ethylenediamine hydrochloride solution. The components were mixed well and evaluated for absorbance at 540 nm. Control tubes were supplied with distilled water instead of sodium nitrite solution, while blank tubes had no samples. S-NR activity was assessed using the cominbio kit’s method, and the amount of 1 μmol NO2ˉ produced per gram of soil sample per day was regarded as one S-NR activity unit. Air-dried soil samples weighing 0.06 g were included in a 2 mL centrifuge tube containing 225 µL of KNO3 solution and 75 µL of Reduced Coenzyme I (NADH) solution. Samples were then subjected to a 24-hour water bath at 37℃ before centrifugation at 8000g and 25℃ for 10 min. At this stage, 130 µL of the supernatant was collected and added to 85 µL each of p-amino benzene sulfonic acid solution and α-naphthylamine solution. The resulting mixture was agitated and left for color development at 25℃ for 20 min before centrifugation at 4000g for 10 min at 25℃. 200 µL of the supernatant was transferred to a 96-well plate and monitored for absorbance at 540 nm. Distilled water replaced the KNO3 solution for the control tube, while the blank tubes omitted soil samples. The standard tube replaced the soil sample with 0.1 μmol/mL NaNO2solution. S-UE activity was assessed using the cominbio kit’s method, and 1 μg of NH3-N produced per gram of soil sample per day was defined as an enzyme activity unit. Air-dried soil samples weighing 0.06 g were placed in 2 mL centrifuge tubes after adding 20 µL of toluene and shaking, then placed at room temperature for 15 min. Afterward, 90 µL of urea and 190 µL of citric acid-potassium hydroxide solution were blended before a 24 h water bath at 37℃. Following this, centrifugation was carried out at 10,000g and 25℃ for 10 min. The supernatant was then diluted ten-fold, with 80 µL taken, and the mixture was coupled with 15 µL of phenol, methanol, acetone, absolute ethanol, and NaOH solutions. Next, 15 µL of sodium hypochlorite solution was added and mixed well before being placed at room temperature for 20 min. The absorbance of the resultant mixture was monitored at 578 nm after the inclusion of 90 µL of distilled water. Distilled water substituted urea in the control tube. S-CAT activity was assessed using the cominbio kit’s method, and the degradation of 1 μmol H2O2 catalyzed per g of air-dried soil sample per day was defined as an enzyme activity unit. Air-dried soil samples weighing 0.03 g were included in a 2 mL centrifuge tube and supplemented with 260 µL of hydrogen peroxide. Samples were subjected to a 20 min incubation at 25℃ while agitated at 500r/min. Next, 10 µL of aluminum potassium alum was added, followed by centrifugation at 8000g and 25℃ for 5 min. 180 µL of the supernatant was collected and mixed with 20 µL of sulfuric acid solution before monitoring absorptivity at 240 nm. The control tube contained distilled water instead of hydrogen peroxide, while the blank tubes contained no soil samples. S-PT activity was assessed using the mlbio kit’s method. To prepare the soil samples for testing, 0.1 g of soil was mixed with 0.9 mL of Phosphate buffer saline (PBS) before centrifugation at 4000r for 15 min. The resultant supernatant became the sample for examination. Next, 50 µL of the diluted standard and 40 µL of the sample diluent were added to the sample, which was then sealed with a sealing film and incubated at 37℃ for 30 min. Afterward, the liquid was discarded, followed by the introduction of wash solution, left for 30 s, and discarded five times. Subsequently, 50 µL of HRP enzyme-labeled reagent was included, and incubation and washing processes resumed before adding 50 µL of carbamide peroxide color reagent and 50 µL of color reagent. At this stage, color development was initiated, with 10 min allotted for completion in the dark at 37℃. Additionally, 50 µL of termination solution was infused before absorbance levels were measured at 450 nm. Blank wells that omitted samples and enzyme labeling reagents were also featured. Phosphotransferase markers (Figure. S1). The content determination method of the total polysaccharide in the soil adopts the method of pansu et al. (Pansu and Gautheyrou, 2006). Preparation of the test solution: weigh 2.5g of air-dried soil, put it in a 50mL Conical flask, then add 20 mL of 2.5M H2SO4, heat and reflux in a water bath at 100℃ for 20 min, let cool, filter, wash, and collect the filtrate. Put the residue in a 50mL Conical flask, add 1mL 13M H2SO4, and place it at room temperature for 16 h. Slowly add 25 mL of distilled water, let cool, heat at 100℃ for 5 h after airtight, let cool, filter, combine the filtrate with the above filtrate and dilute to 50mL to obtain the test solution. The total polysaccharide content was determined using the lichenol-hydrochloric acid method for pentose, while hexose was assessed using the anthrone-sulfuric acid method. Pentose and hexose markers, respectively (figure s3, figure s2). Finally, the sum of the two was calculated as the polysaccharide content.