San system

Overlying water samples were collected using a syringe on the same day and time as NH₃ volatilization samples collection. Collected water samples were filtered through a 0.45 μm membrane, then analyzed for pH using a combined reference electrode (Ф255 pH/temp/mV meter, Coulter Bechman Co., United States). A sub-sample of 50 ml filtered water was stored in clean plastic bottles at −20°C for further analysis. The concentrations of NH₄⁺-N and NO₃⁻-N in overlying water were determined by an autoanalyzer (SKALAR San⁺⁺ System, Netherlands).

At the end of the experimental period after rice harvest, three soil cores in each pot were randomly sampled at 0–20 cm depth using a soil drill (50 mm in diameter), top layer soil was sampled at selected points. Soil samples were composited, mixed manually, placed in self-sealing bags, and brought back to the laboratory in collar box with ice. Soil samples were divided into two parts: one was stored at −80°C for molecular analysis and another at −20°C for analysis of other properties. The soil pH was measured in the 1:2.5 (w/v) soil: water suspension using a combined reference electrode (Ф255 pH/temp/mV meter). Soil samples were extracted with 2.0 M KCl (1:5 soil: extractant, w/v) for NH₄⁺-N and NO₃⁻-N determination. Soil extracts were filtered through a 0.45 μm membrane filter and the NH₄⁺-N and NO₃⁻-N concentrations were determined using an autoanalyzer (SKALAR San⁺⁺ System, Netherlands). The gene copy numbers of AOA and AOB amoA, nirK, nirS, and nosZ of soil samples were determined by Shanghai Majorbio Biomedical Co., Ltd. according to the procedures detailed in Chu et al. (2020) (link) and Ye et al. (2021) (link).

The measurement methods of soil physical and chemical properties followed the protocols detailed in the study by Bao (2000 ). Soil pH was measured in 1:2.5 mixtures of soil and deionized water with a pH meter (PHS-3C, Lei-ci, Shanghai, China). SOC was determined by potassium dichromate (K₂Cr₂O₇) and sulfuric acid (H₂SO₄) oxidation and titration. Ammonium-N ( ${\text{NH}}_4^{+}$ -N) and nitrate-N ( ${\text{NO}}_3^{-}$ -N) were extracted with the ratio of 5 g fresh soil to 50 ml 2 M potassium chloride. The contents of ${\text{NO}}_3^{-}$ -N and ${\text{NH}}_4^{+}$ -N were analyzed by a continuous flow analytical system (San++ system, Skalar, Holland).

Soil pH was measured using a pH meter (FE20-FiveEasyTM pH, MettlerToledo, Germany) after shaking a soil water (1:2.5 w/v) suspension for 30 min (Guo et al., 2017 (link); Zhou et al., 2022 (link)). Soil moisture was measured gravimetrically (Guo et al., 2017 (link); Zhou et al., 2022 (link)). Total carbon (TC) and total nitrogen (TN) contents were measured using an elemental analyzer (VarioMAX, Elementar, Germany) (Guo et al., 2017 (link); Zhou et al., 2022 (link)). Ammonium (NH₄⁺-N) and nitrate (NO₃^–-N) were extracted at a ratio of 10 g fresh soil to 50 ml 2 mol L^–1 KCl. After shaking under 180 r min^–1 for 1 h, NH₄⁺-N and NO₃^–-N contents in the filtered extracts were analyzed using a continuous flow analytical system (San⁺⁺ System, Skalar, Holland) (Guo et al., 2017 (link); Zhou et al., 2022 (link)). Total phosphorus (TP) content was measured using the alkali fusion-Mo-Sb antispectrophotometric method (Guo et al., 2017 (link); Zhou et al., 2022 (link)).

Determination of soil organic carbon (SOC) by external heating of potassium dichromate [19 ]. Determination of total nitrogen (TN) by Kjeldahl digestion, the molybdate blue colorimetric method for determination of available phosphorus (AP), and atomic absorption spectrometry for determination of available potassium (AK) [19 ]. NO₃⁻-N and NH₄⁺-N were extracted at a ratio of 10 g fresh soil to 100 mL 2 M KCl. After shaking for 1 h, the extracts were filtered and analyzed by continuous flow analytical system (San++ System, Skalar, Holland) for NO₃⁻-N and NH₄⁺-N [20 (link)]. pH was measured using a pH meter (PHS-3E, INESA, China), and the soil-to-water ratio was 1:5 [21 (link)].

The plate assay is a relative efficiency test and is not a precise method to identify whether or not a strain is a P solubilizer [26 (link)]. Thus, P solubilization activity was then quantified using hydroxyapatite as a sole source of insoluble P in NBRIP broth medium [25 (link)]. The isolates were individually grown overnight in TSB broth medium and the optical density (OD_600nm) was adjusted to 0.8. The bacterial suspension (100 μL) was inoculated in 250 mL Erlenmeyer flasks containing 50 mL of NBRIP broth. The resulting media was incubated for 5 days at 30 °C under 150 rpm shaking and centrifuged at 12,000 rpm for 10 min [16 (link)]. The supernatants were filtered through 0.22 μm sterile syringe filters to remove insoluble materials [14 (link)]. The cell-free supernatants were diluted (1/50) and used to colorimetrically measure the soluble P content using a Continuous Flow Analyzer (SKALAR SAN++ SYSTEM). Uninoculated NBRIP medium served as a control. The final pH of the supernatants was also recorded. The experiment was performed in triplicate.