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).