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.