2.4 Quantification of rice allelochemicals
Quantification of allelochemicals from focal rice cultivars ‘Huagan-3’
and ‘Lingyou-6173’ was performed with a liquid extraction-solid phase
extraction followed by High Performance Liquid Chromatography (HPLC).
The rice samples collected from the experiment 2 were freeze-dried and
ground, and 20 mg was extracted with 10 mL MeOH for 12 h and filtered.
The filtrates were evaporated to dryness individually under nitrogen
gas. Dry residues were dissolved in 2 mL 50% aqueous methanol then
filtered through a 0.22 μm filter. The filtered samples were
subsequently subjected to HPLC for quantitative analysis.
Quantitative analysis was carried out by injecting 10 μL into an HPLC
(HPLC-1525; Waters, Worcester, MA, USA) with a UV detector set at 320
nm. Elution was performed at a constant flow rate of 1.0 mL
min-1 at 40 ℃ with acetonitrile and a mixture of 0.5%
formic acid in a gradient: initial mobile phase of 10% acetonitrile for
4 min, increased to 35% for 4 min, then decreased to 10% for 4 min,
for a total run time of 12 min. The allelochemicals
tricin and momilactone B were
quantified by interpolating the peak area on the HPLC chromatogram to a
standard curve constructed from the peak area of authentic tricin and
momilactone B (Yang & Kong, 2017).
2.5
PLFA analysis
The soil samples collected from the experiment 3 were subjected to PLFA
analysis that was conducted following method of Sun et al. (2014) with
minor modifications. Briefly, freeze-dried soil was extracted with a
mixture of CHCl3-MeOH-citrate butter (1:2:0.8, v/v/v),
and the phospholipids were separated from other lipids on a
silica-gelfilled solid-phase extraction cartridge (0.50 g Si; Supelco,
Inc., Bellefonte, PA, USA). The resulting fatty acid methyl esters
(FAMEs) were identified and quantified by GC-MS. The identification of
the FAMEs was based on retention time comparisons with FAMEs controls
(Supelco, Inc., Bellefonte, PA, USA). Quantification was performed by
calibration against standard solutions of nonadecanoate methylester
(C19:0), which were also considered as internal standards.
A total of 16 PLFAs were identified and quantified for the analysis.
Twelve fatty acids (i-15:0, a-15:0, i-16:0, i-17:0, 16:1 ω7c, 16:1ω9c,
cy-17:0, 14:0, 15:0, 16:0, 17:0 and 18:0) were considered as bacterial.
Among them, four fatty acids (i-15:0, a-15:0, i-16:0 and i-17:0) were
identified as Gram-positive bacteria (Gram+) and 3
fatty acid (16:1 ω7c, 16:1ω9c and cy-17:0) were identified as
Gram-negative bacteria (Gram-). Fungi were assessed by
18:1ω7c and 18:1ω9c. Actinomycetes were assessed by 10Me17:0 and
10Me18:0.