**4. Materials and Methods**

#### *4.1. Materials and Equipment*

Cucumber seeds of Jinyou 40, a North China fresh market type variety, were obtained from Tianjin Kernel Cucumber Research Institute (Tianjin, China). Garlic bulbs of Gailiang were provided by the College of Horticulture, Northwest A&F University (Yangling, Shaanxi Province, China). To prepare the DADS stock solutions, laboratory grade DADS (purity 80%) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and was firstly dissolved in Tween-80 with a ratio of 1:2 (*w*/*w*), then distilled water was added to get a 10 mmol/L stock solution which was stored at 4 ◦C for further use [35]. SPME fibers (50/30 μm DVB/CAR/PDMS, Sigma-Aldrich Co.) were used to absorb the VOCs and GC-MS (ISQ, Thermo Fisher, Waltham, MA, USA) was used to identify the collected VOCs. A ultraviolet spectrophotometer (UNIC 3200, UNIC, Shanghai, China) was used to measure the enzyme activities and ROS content. A microscope (BX63, Olympus, Japan) was used for histological observation of H2O2 effects.

#### *4.2. Plant Growth and Experimental Design*

Garlic bulbs of uniform size (approximately diameter = 4 cm, height = 4 cm and weight = 35 g) were selected and sown in plastic pots (diameter = 21 cm) filled with sterilized culture substrate. The green garlic plants that grew to 25 cm in plant height (about 30 days after sowing) were used for collecting VOCs and identification of green garlic allelopathic compounds and co-culturing with cucumber seedlings.

Germinated cucumber seeds were planted in plastic pots filled with sterilized culture substrate and cultivated in growth chamber with a day/night temperature of 25/18 ◦C, a day/night regime of 16/8 h and a relative humidity of 80%. The cucumber seedlings at two-true-leaf stage were used for following experiments.

To verify whether DADS is the main allelochemical of green garlic VOCs, bioassay analysis was performed. The detached cucumber leaves were treated with different weights of green garlic (0.0, 1.0, 2.0, 3.0, 5.0 g) that was cut into 1 cm segments and different concentrations of DADS (0, 0.5, 1, 2, 3, 5, and 10 mmol/L) in Petri dishes (see details in Figure 2). To ensure the treatment effect, the green garlic pieces were replaced with fresh ones and DADS was added again after 3 days of treatment. The morphology changes of these detached cucumber leaves were observed at the end of the sixth day.

To explore the effect of VOCs that derived from green garlic on scavenging ROS of cucumber, green garlic plants and cucumber seedlings were co-cultured in a greenhouse of Northwest A&F University, Yangling (N 34◦16 , E 108◦4 ), China. Green garlic plants and cucumbers were co-cultured for 10 days in a plastic mini-greenhouse (60 × 60 × 58 cm), in which 12 cucumber seedlings were surrounded with green garlic seedlings. Four treatments including different number of green garlic seedlings (derived from 0, 6, 12 and 18 garlic bulbs, respectively) were used in this experiment (see details in Figure S1). Each treatment was replicated three times. To investigate the effect of DADS on ROS scavenging in cucumber, 12 cucumber seedlings were also planted in the plastic mini- greenhouse, and 5 mL of DADS solution with a concentration of 1 mmol/L were sprayed on each cucumber seedling, while an equal volume of distilled water was sprayed as a control (see details in Figure S1). During the 10-day cultivation of these cucumber seedlings, DADS and distilled water were sprayed twice, on the first and sixth day. For all treatments, the second true leaves of cucumber were sampled. One part of the leaves were put in a stationary liquid for histological observation of H2O2, and the other parts of the leaves were stored at −80 ◦C for ROS and genes assay.

### *4.3. Identification of Green Garlic Allelochemicals by Fiber-HS-SPME-GC-MS*

The volatiles derived from green garlic samples were collected by three methods. For the first one, we used the headspace solid phase microextraction (HS-SPME) method, in which the green garlic plants were firstly cut into approximately 1 cm segments and 2 g samples were weighed and put into a 40 mL SPME vial (see details in Figure S2) which was pre-incubated for 10 min at 45 ◦C, and then the SPME fiber was exposed for 45 min at the same temperature to adsorb the volatiles. For the second and the third method, a hermetic glass container with 8 L in volume was used to collect the volatiles, the difference being that the green garlic seedlings were cut into segments or not (see details in Figure S2). Eight uniform green garlic seedlings were selected and put into these containers for three hours, then the SPME fibers were used to absorb the produced volatiles. All the collected volatile compounds were directly measured by a GC-MS system. Moreover, an external standard method was used for quantitative analysis of these volatiles, in which 2 mL DADS standard solutions with concentrations of

0.1, 0.5, 1.0, 1.5 and 2.0 mmol/L were prepared and added into a 20 mL SPME vial. Therefore, the final concentrations of DADS in SPME vials that used for the external standard method were 0.01, 0.05, 0.10, 0.15, 0.20 mmol/L, respectively.

The GC was equipped with a DB-5MS column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, Santa Clara, CA, USA). UHP helium was used as the carrier gas at a flow rate of 1 mL min−1. The desorption time was 2.5 min. The injector temperature was set at 230 ◦C and injection was performed in a split mode and split ratio of 30:1. The gas flow was set at 0.8 mL/min. The temperature was programed as follows: 40 ◦C for 2 min, then rising at 6 ◦C /min to 170 ◦C and held for 5 min, where it then was increased at 10 ◦C /min to 250 ◦C, where it was held for 1 min, running for 40 min. For MS, the electron multiplier was operating at 70 eV and all samples were analyzed in electron impact mode. Mass ranges of *m*/*z* 40–450 u for green garlic analysis were acquired. The temperatures of the transfer line and ion trap were both held at 250 ◦C. All the treatments were done in triplicates.

The compound TIC was used to identify the allelochemicals of VOCs. The Xcalibar workstation NIST standard spectrum database was used to search for each detected component, and the related literatures and standard spectra were used to check and confirm the inspection results. Only components with a matching degree greater than 800 were reported, and the relative content of each component was calculated based on the area normalization method.
