Volatile collection and analysis

Volatiles were collected using a dynamic push–pull system. Air flow was maintained in the system through Teflon tubes. Charcoal filtered air was pumped into the bags at a flow of 1 L min⁻¹. A portion of the aspirated air (flow: 0.6 L min⁻¹) was withdrawn with a second pump and passed through a filter packed with 30 mg Poropak (ARS, Inc., Gainesville, FL, USA) to adsorb the volatile compounds. Volatiles were collected for 6 h during the middle of the light period of Day 2 after herbivore release (10 a.m.–4 p.m.). After the collection, the volatile compounds were desorbed by eluting the filter with 200 µL dichloromethane containing nonyl acetate as an internal standard (10 ng µL⁻¹). Samples were stored at −20°C until gas chromatography analysis. Volatile collection of P. x canescens WT trees, EV trees, and CNL knockdown plants was conducted with an air supply of 0.8 L min⁻¹ and an air withdrawal of 0.4 L min⁻¹.

Qualitative and quantitative analyses of leaf volatiles were conducted using an Agilent 6890 Series gas chromatograph coupled to an Agilent 5973 quadrupole mass selective detector (Agilent Technologies, Santa Clara, CA, USA; interface temperature, 250°C; quadrupole temperature, 150°C; source temperature, 230°C; electron energy, 70 eV) or a flame ionization detector (FID) operated at 300°C, respectively. The constituents of the volatile bouquet were separated using a ZB5 column (Phenomenex, Aschaffenburg, Germany; 30 m × 0.25 mm × 0.25 µm) and He (MS) or H₂ (FID) as carrier gas. The sample (1 µL or 2 µL) was injected without split at an initial oven temperature of 45°C. The temperature was held for 2 min and then increased to 180°C with a gradient of 6°C min⁻¹, and then further increased to 300°C with a gradient of 60°C min⁻¹ and hold of 2 min. Compounds were identified by comparison of retention times and mass spectra to those of authentic standards, or by comparison with reference spectra in the Wiley and National Institute of Standards and Technology libraries.