2. Materials and methods

2.1. Study area

The field experiment was conducted in Xuwei New District (34°37′N, 119°29′E), Lianyungang City, Jiangsu Province, where the annual average rainfall and evaporation were 901 mm and 855 mm, respectively. The experimental site was divided into three parts: low salinity (< 3 g/kg), medium salinity (5~8 g/kg), and high salinity (> 10 g/kg). BeforeM. azedarach was cultivated, the experimental site was plowed to a depth of 40 cm, and the row spacing was 2.0 × 3.0 m.

2.2. Sample collection

In August 2020 (after 40 months of M. azedarach growth), the samples of soil and roots ofM. azedarach were collected from each plot [high (H), medium (M) and low (L) salinity]. Samples of rhizosphere soil (soil attached to M. azedarach roots) and bulk soil (soil away from M. azedarach roots) were collected in each plot. Three replicates were collected in each plot, with each replicate consisting of six sampling points. All soil samples were air-dried and then sieved through a 0.147 mm sieve for soil chemical and enzymatic activities analysis.
Six replicates of roots were sampled in each plot, and each replicate comprised three sampling points. The samples were stored at -80 ℃ for analysis of root metabolome. Root samples (R) collected in different salinities were labelled: RH, RM and RL for high, medium and low salinity, respectively.

2.3. Measurement of soil properties

Soil salinity was determined by the conductivity method (Yue et al., 2020). Soil pH was assessed by a calibrated pH meter (FE28-Standard, Mettler Toledo, Greifensee, Switzerland) (Zhang & Pang, 1999). Soil water-soluble Na+, K+, Ca2+, and Mg2+were measured by ICP-OES (Optima 2100DV, Perkin-Elmer, Waltham, Massachusetts, USA) (Yang et al., 2016). Soil water-soluble Cl- content was determined by the silver nitrate titration method (Asakai, 2018).
Soil organic matter was determined using the potassium dichromate method (Osman et al., 2013), available phosphorus by the molybdenum-antimony colorimetric method (Wang et al., 2011) and available potassium by flame photometry (Bilias & Barbayiannis, 2019).
Soil enzyme activities were measured using aprpriate kits (Solarbio, Beijing, China). Activity of soil alkaline phosphatase (S-AP) was measured by the determination of phenol produced during the hydrolysis of the substrate (Li et al., 2021b). Activity of soil urease activity (S-UE) was obtained by measuring NH3-N produced by the urease hydrolysis of urea based on the indophenol blue colorimetric method (Huang et al., 2014).

2.4. Characterizations of M. azedarach metabolome

2.4.1. Metabolite extraction

An amount of 50 mg of fresh M. azedarach roots was weighed accurately ground in 1000 μL of extractant (methanol-acetonitrile-water, 2:2:1, v/v) containing internal standard (l,2-chlorophenylalanine, 2 mg) at 45 Hz for 10 min in a Tissuelyser-32; the suspension was allowed to stand at -20 ℃ for 1 hour, followed by centrifugation at 16,114 g for 15 min (Sun et al., 2022). The supernatant was collected and dried in a vacuum concentrator. The dried metabolites were reconstructed in the extractant (acetonitrile-water, 1:1, v/v), sonicated in an ice-water bath for 10 min, and centrifuged at 16,114 g for 15 minutes; the supernatant was used for metabolomic analysis (Li et al., 2022).

2.4.2. UPLC-Q-TOF-MS for metabolite analysis

Extracts were analyzed by UPLC (Waters UPLC Acquity I-Class PLUS) and QTOF system (Waters UPLC Xevo G2-XS QTof) with a chromatographic column Acquity UPLC HSS T3, mobile phase A (0.1% formic acid aqueous solution), and mobile phase B (0.1% formic acid acetonitrile) (Fan et al., 2022). Metabolites need to be ionized before they can be detected by mass spectrometry. The ESI source operation parameters were as follows: capillary voltage at 2000 V (positive ion mode) or -1500 V (negative ion mode), a cone voltage at 30 V, a source temperature of 150 °C, desolvation to 500 °C and a desolvation gas flow to 800 L·h-1 (Liu et al., 2022).

2.4.3. Metabolome data processing

The raw data were collected by MassLynx (v4.2) and processed by Progenesis QI software (Huang et al., 2022), before identified based on Progenesis QI software, the online METLIN database, and Bemec’s proprietary library. The criteria for differential metabolites (DEM) were FC≥2.0 or FC≤0.5,p -value<0.05, and VIP>1.0.

2.5. Statistical analyses

An independent sample t -test was done by IBM SPSS Statistics 20 (IBM, Armonk, NY, USA), with p < 0.05 considered statistically significant. Graphs were drawn by Graphpad prism v8.0.1 and R package ggplot 2 (v3.2.0). The Venn diagrams were constructed by the R package Venn diagram (v1.6.20). The network diagram was produced by Cytoscape (v3.6.1).