2.3.2. Collection of Plant Volatiles by Dynamic Headspace Method

Volatiles emitted from (1) phloem from healthy and infected trees, (2) annual branches, and (3) foliage from seedlings were sampled from the field and collected by dynamic headspace method at the Forestry Bureau in Lingwu city. In the laboratory, each plant part was placed inside the collection bag (Oven Bags Turkey Size, Reynolds Consumer Products, Inc., Lake Forest, IL, USA) and tied tightly. After the air in the bag was drained by the atmospheric sampler (QC-1S, Beijing Municipal Institute of Labour Protection), the filtered air was pumped into the bag through the glass tube with activated charcoal using the atmospheric sampler. The other corner of the bag was attached to a glass tube with 70 mg of Porapak Q (80–100 mesh), the adsorbents were sandwiched between glass wool in the glass tube. The atmospheric sampler pumped clean air at a flow rate of 150 mL/min. Volatiles were collected by the adsorbents for 24 h and were then eluted with 800 μL of n-hexane. The eluent was kept at −20 °C and brought back to Beijing for analysis. Volatiles of empty collection bags were collected as a control.

Volatiles collected by HS-SPME and dynamic headspace method were analyzed by GC-MS-QP2010 SE (Shimadzu, Kyoto, Japan) following the same procedure. The GC was equipped with a Restek Rtx-5MS capillary column (30 m × 0.25 mm × 0.25 μm). Helium was used as the carrier gas at a constant flow rate of 1 mL/min, the injection volume was 1.0 μL in the split mode with a 40:1 split. The oven temperature started at 50 °C, and was increased at 6 °C/min to 180 °C and held for 6 min, then increased at 10 °C/min to 280 °C and held for 10 min. The mass spectra were recorded in the electron impact mode at 70 eV (source at 220 °C, scanned mass range: 29–500 m/z). Data analyses were performed using GCMS solution 4.1.1 (Shimadzu, Japan) with the National Institute of Standards and Technology (NIST) database. The relative content of each volatile compound was identified by peak area calculated by the normalized method [21].