**1. Introduction**

Plants synthesize and release various volatile organic compounds (VOCs) [1]. VOCs play essential roles in attracting pollinators and seed-dispersers, defense against herbivores and pathogens, interplant signaling and allelopathy [2,3]. Garlic (*Allium sativum* L.), an economically important vegetable [4], contains various volatile components, including diallyl, dimethyl, and allyl methyl sulfides, disulfides, and trisulfides, as well as some other minor components, all of which are formed by the decomposition of allicin and are released upon crushing garlic. These organosulfur compounds can inhibit carcinogen activation, boost detoxifying processes, cause cell cycle arrest, stimulate the mitochondrial apoptotic pathway and increase the acetylation of histones [5]. Garlic also has potential allelopathic effects and is widely used in crop rotation and intercropping with many other crops, e.g., cucumber [6], tomato [7], eggplant [8], pepper [9]. In this kind of intercropping system, garlic has been confirmed to alleviate continuous cropping issues from the aspects of reducing plant diseases and improving the physical and chemical properties of soil [10]. Green garlic is young garlic with tender leaves that harvested at the early stage before the bulb is formed and it is consumed by people of many countries. The green garlic-cucumber intercropping system has been shown to have the benefits of improving soil fertility, promoting the activity of soil enzymes and increasing cucumber biomass [6,11]. However, there is a lack of identification of the main allelochemicals for green garlic volatiles, and knowledge about what kind of allelochemicals regulated the increased biomass of cucumber, which are the subjects in this study.

Diallyl disulfide (DADS) is a major allelochemical of the VOCs in garlic [7]. In animals, DADS has been shown having the effect of reducing cellular toxins and inhibiting the proliferation of cancer cells through several actions which include the activation of metabolizing enzymes that detoxify carcinogens, suppression of the formation of DNA adducts, antioxidant effects; regulation of cell-cycle arrest, induction of apoptosis and differentiation, histone modification and inhibition of angiogenesis and invasion [12]. Kubota et al. found that the active substances from garlic that can break the bud dormancy of grapevines are sulfur-containing compounds, among which the most effective one is DADS [13]. Previous studies found that DADS can also regulate tomato root growth by affecting cell division, endogenous phytohormone levels, expansin gene expression and the pathways of tomato root sulphate assimilation and glutathione (GSH) metabolism [7,14]. Besides, a low concentration DADS is able to promote cucumber root growth and induce main root elongation by up-regulating the expression of *CsCDKA* and *CsCDKB* genes and regulating adjusting the hormone balance of roots [15]. However, the effect of DADS on scavenging of cucumber reactive oxygen species (ROS) has not been investigated.

It is important to choose a suitable method to collect as much as possible the volatile compounds of plants in their natural state. For determination of garlic flavor components by gas chromatography-mass spectrometry (GC-MS), several sampling techniques including steam distillation (SD), simultaneous distillation and solvent extraction (SDE), microwave assisted hydrodistillation extraction (MWHD), ultrasound-assisted extraction (USE), solid-phase trapping solvent extraction (SPTE) and headspace solid-phase microextraction (HS-SPME) were applied and compared in previous studies [16,17]. Compared with SD, SDE, and SPTE, the HS-SPME method had several advantages which are rapid solvent-free extraction, no apparent thermal degradation, less laborious manipulation and less sample requirement. Five different fiber coatings were evaluated to select a suitable fiber for HS-SPME of garlic flavor components, among which the divinyl benzene/carboxen/ polydimethylsiloxane (DVB/CAR/PDMS) one was the most efficient among the investigated fibers [16]. Warren investigated the analysis of thiol compounds using a needle trap device and HS-SPME coupled to GC-MS, comparing the advantages and disadvantages of two methods [18]. Based on the above studies, the HS-SPME coupled to GC-MS method seems to be a good choice for determining the allelochemicals of green garlic. We chose a fiber-HS-SPME-GC-MS method to determine green garlic allelochemicals based on these previous studies.

Similar to other biological active factors, allelochemicals derived from plants can target some specific biological processes, which include destroying membrane permeability, influencing photosynthetic and respiratory chain electron transport, influencing cell division and ultrastructure, changing enzyme activity, altering reactive oxygen species (ROS) levels and effecting expression of related genes [19]. ROS are involved in many biological processes, such as growth, development, response of biotic and environmental stresses and programmed cell death [20,21]. It has been reported that higher ROS levels might cause oxidative damage to plants. In order to reduce this damage, plants have developed efficient antioxidant defense systems to scavenging ROS, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbic acid (ASA), GSH, monodehydroascorbate (MDA), the expressions of their synthetic genes are related to oxidation reactions [22,23].

In this study, we will explore the regulation mechanism of VOCs derived from green garlic in scavenging ROS of cucumber from the following three aspects. Firstly, the major effective allelochemicals of VOCs that collected from green garlic will be identified; secondly, the different ROS scavengers induced by VOCs of green garlic and the ROS levels will be investigated among treated cucumber seedlings; thirdly, the expression levels of six genes (*CsPOD, CsCAT, CscAPX, CsMDAR, CsGPX, CsSOD*) related to oxidation reactions will be checked following the treatment of cucumber seedlings

with VOCs and DADS. This study will provide more information for understanding the mechanism of green garlic VOCs in scavenging ROS of vegetables.

#### **2. Results**

#### *2.1. Identification of Volatile Allelochemicals in Green Garlic*

Identification of the effective components of VOCs collected from green garlic is the basis to reveal its mechanism in scavenging ROS. For the VOCs collected from the green garlic segments that were contained in the SPME vial, only two compounds including DADS and diallyl sulfide (DAS) were identified (Figure 1A and Table 1). The retention time and relative peak area of DADS were 13.83 min and 99.52%, respectively, while, DAS had a retention time of 7.04 min and a relative peak area of 0.48%. Among the VOCs collected using green garlic segments that were put in a sealed desiccator, 17 compounds were detected (Table S1), in which there were three sulfur compounds including DADS, methyl propenyl disulfide and DAS, with relative peak areas of 86.33%, 1.53% and 0.35%, respectively (Figure 1B and Table 1). All the other compounds were heterocyclic compounds and long chain hydrocarbon compounds, which are impurities in air. There were 42 compounds identified in the VOCs obtained from the whole green garlics that were placed in a sealed desiccator (Table S2). Among the 42 detected compounds there was only one sulfur compound, which was DADS with a relative peak area of 15.3% (Figure 1C and Table 1), while the others were also heterocyclic compounds and long chain hydrocarbon compounds. The qualitative identification result showed that DADS is the main volatile compound of green garlic. Compared with the whole green garlic, the cut green garlic segments produced more DADS with about a 5-fold difference.

**Figure 1.** Identification of the collected volatiles from green garlic volatiles by GC-MS. (**A**) Cut (SPME vial); (**B**) Cut (sealed desiccator); (**C**) Whole (sealed desiccator); (**D**) MS spectrum and compound structure of DADS. *n* = 3, three biological replicates.


**Table 1.** Total sulfur compounds of green garlic volatile compounds among different collecting methods.

The result of an external standard method for quantitative analysis of volatiles showed that the linear relation between DADS concentration (mmol/L) (y) and peak area (x) fits a simple linear regression equation (Equation (1)):

$$\mathbf{y} = \mathbf{5E} + \mathbf{0}\mathbf{9x} + \mathbf{3E} + \mathbf{07}, \mathbf{R}^2 = \mathbf{0}.9903\tag{1}$$

Based on this equation, the DADS that released by green garlics has a concentration of about 0.08 mg/g.

Our biological tests showed that the green garlic treatment had a consistent effect with the DADS treatment (Figure 2). When the concentration of DADS was higher than 1 mmol/L, cucumber leaves turned yellowish (Figure 2). Inversely, when the green garlic treatment was less than 2 g or the concentration of DADS was lower than 1 mmol/L, the cucumber leaves had no morphology changes (Figure 2). The amount of DADS volatilized by 2 g of green garlic approached 1 mmol/L DADS treatment based on Equation (1). In addition, higher concentrations of green garlic volatiles and DADS can cause cucumber leaves turn to soft and rot (Figure 2). These results confirmed that DADS is the main allelochemical of green garlic volatiles.

**Figure 2.** The biological test and its results of green garlic volatiles and DADS on cucumber leaf. (**A**) effect of green garlic (0.0, 1.0, 2.0, 3.0, 5.0 g) volatiles on cucumber leaf color and structure. (**B**) effect of DADS (0, 0.5, 1, 2, 3, 5, and 10 mmol/L) on cucumber leaf color and structure. Five leaves from 5 plants were used for each treatment.

*2.2. E*ff*ect of Green Garlic Volatiles and DADS on Cucumber ROS and Antioxidant Activity*

2.2.1. Effect of Green Garlic Volatiles and DADS on Cucumber ROS (O2 •− and H2O2)

Figure 3A showed the results of histological observation of H2O2 in cucumber leaves stained by DAB dye. Compared with the control groups, the colors of dyed cucumber leaves gradually turned much darker with the increasing concentrations of green garlic volatiles and DADS (Figure 3A). This suggested that green garlic volatiles and DADS led to an increasing H2O2 content at histological levels. After 10-days of co-culture with green garlic, the O2 •− contents of treated cucumber leaves were significantly lower that of control (*p* < 0.05). Similarly, the O2 •− contents of cucumber leaves under 1 mmol/L DADS treatment was also lower than that of control group (Figure 3B). The H2O2 content in cucumber leaves exhibited a rising trend with the increase of the concentration of green garlic volatiles. Besides, the H2O2 contents in cucumber leaves of all green garlic treatments and DADS were significantly higher than that of control (*p* < 0.05), which were consistent with the histological observations of H2O2 in cucumber leaves (Figure 3A).

**Figure 3.** Effect of green garlic volatiles and DADS on cucumber ROS, antioxidant substance and antioxidant enzymes activity. (**A**) Histological observation of H2O2 in cucumber leaves that stained by DAB dye. (**B**,**C**), effect of green garlic volatiles and DADS on cucumber ROS (O2 •− and H2O2); (**D**–**F**), effect of green garlic volatiles and DADS on cucumber antioxidant substance content (MDA, GSH and ASA); (**G**–**I**), effect of green garlic volatiles and DADS on cucumber antioxidant enzyme activity (SOD, CAT and POD). \* *p* < 0.05; \*\* *p* < 0.01; ANOVA, followed by Tukey test and *t*-test. Data are means ± standard errors (*n* = 3 for (**B**,**C**), three biological replicates).

2.2.2. Effect of Green Garlic Volatiles and DADS on Cucumber Antioxidant Substances (MDA, GSH and ASA)

The MDA content of cucumber leaves co-cultured with 6-bulbs of green garlic was significantly lower than that of control (*p* < 0.05) (Figure 3D), while, that for co-cultures with 12- and 18-bulbs of green garlic the MDA content was significantly higher than the control group (*p* < 0.05) (Figure 3D). When cucumber seedlings were treated with DADS, the MDA content of cucumber leaves was also significantly lower than that of control (*p* < 0.01) which was consistent with the treatment co-cultured

with 6-bulbs of green garlic (Figure 3D). The GSH content had an increasing trend with the increasing number of bulbs of green garlic. The GSH contents of 6-bulb green garlic and DADS treatment were both significantly lower than that of control (*p* < 0.05), but the treatments with 12- and 18-bulbs of green garlic were higher than that of control (Figure 3E). The ASA content in cucumbers treated with green garlic bulbs and DADS were consistently lower than that of control (Figure 3F).

2.2.3. Effect of Green Garlic Volatiles and DADS on Cucumber Antioxidant Enzymes (SOD, CAT and POD)

In this study, the SOD activity of cucumber that was co-cultured with 18-bulbs of green garlic was significantly higher than other treatments (*p* < 0.05) (Figure 3G). There were no significant difference among the treatments co-cultured with 6- and 12-bulbs of green garlic, the treatment of DADS and the control groups (*p* < 0.05) (Figure 3G). All the green garlic volatiles treatments had lower CAT activity than the control (*p* < 0.01). For the CAT activity, the treatments of DADS exhibited similar results to the treatments of green garlic volatiles (Figure 3H). Both the green garlic volatiles and DADS treatments had higher POD activity than that of control (*p* < 0.01) (Figure 3I).
