The tested plant volatile compounds were selected on the basis of the previous studies, which identified the compounds that convey chemical information of host location to tea leafhopper (Empoasca onukii) and are attractive to tea leafhopper (Cai et al. 2017; Xu et al. 2017). They are representatives of various classes of plant volatile compounds, including green leaf compounds [(Z)-3-hexenol, (Z)-3-hexenyl acetate and (Z)-3-hexenyl butyrate], phenylpropanoids/benzenoids (benzaldehyde and ethyl benzoate), and terpenoids [limonene, ocimene (mixture of isomers), and (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT)]. These compounds and an internal standard (IS, ethyl decanoate) were high-purity grade.
The tested compounds were mixed at equal volumes. Forty microliters of the mixture was loaded onto a rubber septum, which was then placed in a refrigerator at 4°C. After the mixture was completely absorbed (about 10 h), the rubber septum was used for tests in the wind tunnel. Before each test, the rubber septum was placed in a fume cupboard at 20 ± 2 ℃ for 3 h.
The wind tunnel had a polycarbonate flight section (length × width × height) of 200 cm × 60 cm × 60 cm (Fig. 1). Air was blown into the tunnel by a fan through an activated carbon filter and honeycomb-structured plastic, which were located inside a filter housing. The air exiting the tunnel was passed through a 100-mesh metal screen and a box filled with activated charcoal before being extracted from the room containing the wind tunnel via an exhaust system. Smooth airflow in the wind tunnel was confirmed by burning a mosquito coil at the site of odor source before each test. Hot-film anemometers (AR866, Dongguan Science & Technology Co. Ltd., Dongguan, China) were used to measure the wind speed at the five points near the exit of the wind tunnel.
The rubber septum was threaded onto a string with paper clips. The string was hung in the center of the flight section of the wind tunnel at the upwind end. There was a 20-cm distance between the rubber septum and the honeycomb-structured plastic, and a 30-cm distance between the rubber septum and the top of the wind tunnel. The wind tunnel was lit diffusely from above at about 200 lux. The room was kept at 20 ± 2 ℃, 70–80% relative humidity (R.H.). Before each test, the flight sections were cleaned with ethyl alcohol and maintained in a ventilated environment for 8 h.
Sampling was performed at 12:00 h. Samples were collected at 20 cm, 60 cm, 120 cm and 180 cm away from the odor source in the middle of the wind tunnel (four samples). Another eight samples were collected on the left and right sides of the middle four samples (at a distance of 20 cm from the middle sample). In total, samples were collected from 12 sites in the wind tunnel (see Fig. 1). The air inlet of stainless steel adsorbent tubes (Markes, UK; packed with 200 mg of Tenax™, 60–80 mesh) was at about the same height as the odor source. Air was collected at a flow rate of 100 mL min− 1 for 100 min using a microprocessor-controlled air sampling pump (Mini-pump Σ30; Shibata, Japan).
The trials included three treatments: low wind speed (0.09 m s− 1), high wind speed (0.39 m s− 1), and a blank control. In the low and high wind speed treatments, the odor sources were the same (rubber septa loaded with 40 µL volatile compound mixture). In the blank control, nothing was loaded on the rubber septum. Each treatment was replicated four times. Different treatments were tested on three consecutive days, and all of the wind tunnel tests were completed within 20 days.
To estimate the emission from the rubber septum, the volatiles emitted from the rubber septum were collected in a push/pull system at the same time of the wind tunnel test. The rubber septum was maintained in a 30-mL glass holding chamber (2.1 cm i.d., 8 cm length). Charcoal-purified air entered the holding chamber at a rate of 100 ml min− 1. After passing over the rubber septum, the air was pulled through the same stainless steel adsorbent tubes as those used in the wind tunnel test. Volatiles were collected at the 4th, 22nd, 40th, 58th, 76th, and 94th minute of the wind tunnel test, and each collection lasted for 2 minutes. Between collections, the flowing gas was retained in the chamber containing the rubber septum. The conditions of the collection room were the same as those used in the wind tunnel test.
After collection, samples were analyzed immediately as described previously (Cai et al. 2015). All samples were spiked with 5 ng IS, and were analyzed by coupled thermal desorption (TD; TD100, Marks, UK) and GC-MS (GCMS-QP2010, Shimadzu, Japan) with a DB-5 MS capillary column (60 m × 0.25 mm i.d., 0.25 µm film thickness; J&W Scientific, USA). The adsorbent tubes were heated at 275°C for 5 min while the desorbed volatile compounds from the tube were focused into the cold trap at 4°C with high-purity helium. Following sample transfer, the cold trap was rapidly heated to 290°C. Then, the desorbed compounds were injected into the GC. The oven temperature of the GC was initially set to 45°C for 2 min, then increased by 5°C min− 1 to 70°C and held for 15 min, increased by 2°C min− 1 to 160°C, and then increased by 30°C min− 1 to 260°C and maintained for 10 min. Ionization was achieved via electron impact at 70 eV and 250°C, and compounds were analyzed in the SIM mode. The calibration curve was established as described previously (Cai et al. 2015) by plotting the abundance ratio of analyte to IS against the mass ratio of analyte to IS. The calibration curve was updated immediately before analyzing each batch of samples.
The collection ratio and relative collection ratio were used to describe the dispersal behavior of slow-release volatiles in the wind tunnel. The formulae used to these ratios were as follows: Collection ratio = (amount collected at a point/amount released from the source) × ‰; Relative collection ratio = (collection ratio at a point/total collection ratio of the three points at the same horizontal distance from the odor source) × %. The amount released from the odor source was estimated as follows: 50 × average amounts detected at the 4th, 22nd, 40th, 58th, 76th, and 94th minute.
All statistical tests were carried out using SAS V8.2 (SAS Institute, Cary, NC, USA). Differences in the collection ratios or relative collection ratios at a collection point between high and low wind speed were determined using two-sample t-test for means. Principal component analysis (PCA) was used to compare the profiles of volatiles released at the odor source and those collected at different points, and to compare the collection ratios of nine compounds at different collection points (Mumm et al. 2004; Hare and Sun 2011). For volatile profiles, the percentages of compounds in the collected or released volatiles were log10 (X + 0.00001)-transformed, mean centered, and represented as a covariance matrix before PCA. For collection ratios of compounds, the data were normalized, log10 (X + 0.00001)-transformed, mean centered, and represented as a covariance matrix before PCA. A mixed-model ANOVA of the factor scores for PCA was used to detect significant variations in the profiles of volatiles and the collection ratios of nine compounds. The ANOVA for the PCA based on the profiles included the horizontal position of the points (right, middle, and left), the vertical distance of the points from the source (0 cm, 20 cm, 60 cm, 120 cm, and 180 cm), and their interaction as fixed effects, and replicate as a random effect. The horizontal position and vertical distance of the source odor was, respectively, middle and 0 cm. For the PCA based on the collection ratios of nine compounds, compounds were considered as fixed effects and replicate as a random effect.