Extraction and analysis of volatile organic compounds

The extraction and analysis of volatile organic compounds (VOCs) were conducted in leaf samples collected from both treatments (n = 8).

The headspace solid-phase microextraction (HS-SPME) technique was used to extract the volatile compound. For capture of the volatile constituents, 0.45 g of leaves were placed in clear screw cap vials with 15 mL of deionized water. The vial was placed in water bath at 95 °C for 1 h. The volatile phase was exposed to CAR/PDMS fibre (Carboxen/Polydimethylsiloxane, film thickness 75 μm, Supelco, Bellefonte, PA, USA) for 15 min.

The qualitative and quantitative (area normalization method) analyses of the volatile components were performed on a gas chromatograph coupled to a mass spectrometer (CG-MS, Shimadzu, QP-5000), with an OV-5 fused silica capillary column (30 m × 0.25 mm × 0.25 μm, Ohio Valley Specialty Chemical, Inc.), operating at an MS ionization voltage of 70 eV, with helium as the carrier gas (1.0 mL min⁻¹). The following chromatography conditions were used: injector at 240 °C, detector at 230 °C, split 1/20, and the temperature programme: 50 °C, 2 min; 50–180 °C, 3 °C min⁻¹; 180–230 °C, 10 °C min⁻¹. The compounds were identified by comparison of the acquired mass spectra with those stored in the GC/MS database of the system (NIST 62 lib.) and retention indices (RI; Adams 2007). The RI were obtained from the injection of a mixture of n-alcanes (Sigma-Aldrich, C9-C24), employing the same temperature programme conditions described above for GC/MS, applying the equation of Van den Dool and Kratz (1963). For each sample the fibre was conditioned in GC-FID (Shimadzu, GC-2010/AOC-20i) with the temperature programme: 50–240 °C, 7 °C min⁻¹.