# VOCs Are Relevant Biomarkers of Elicitor-Induced Defences in Grapevine
## 2 Results and Discussion  
### VOC Sampling Method  
We first focused on the method of VOC collection with a view to transferring it to the vineyard in the future. In previous studies [21], VOCs were collected from vines individually enclosed in bags using fibres coated with divinylbenzene (DVB), carboxen (CAR), and polydimethylsiloxane (PDMS). Solid-phase microextraction (SPME) has proved to be a useful tool to collect plant VOCs [22]. It uses a fused silica fibre coated with a polymeric material to adsorb and pre-concentrate volatile compounds. However, SPME lacks sensitivity because of the low amount of polymeric sorbent available on fibre. Moreover, fibres are fragile and can break easily, so they are difficult to use in the field. Therefore, we also assessed Twister(tm), another sensor of the Stir Bar Sportive Extraction (SBSE) type. Twisters(tm) are magnetic stir rods coated with a polymeric material. They have a more significant sorbent volume than SPME fibre has. Like SPME fibres, SBSE can be used to pre-concentrate and adsorb volatile compounds in gas samples. In the present study, an SPME fibre and a stir bar were both placed in the foliage of a same plant in order to compare the nature and the intensity of the collected VOCs. The experiment was performed using six plants individually wrapped in a bag and elicited with sulphated laminarin (PS3, 2.5 g/L), as previously described by [PERSON], et al. [21]. VOCs were collected 3 days (D3) after elicitation.  
The concentration (\(\upmu\)g/L) of detected volatiles was determined using external calibration with monoterpene, sesquiterpene, and green leaf volatile (GLV). These compoundswere identified against the NIST-14 library and the retention times of the corresponding standards. As previously reported, PS3 induced VOC emission in grapevine [21]. Targeted analysis of eight VOCS showed that SBSE led to a better signal response for hexenyl acetate, \(\beta\)-caryophyllene, \(\beta\)-ocimene, and \(\alpha\)-farnesene, while \(\alpha\)-pinene was better detected by SPME (Figure 1). SPME fibre lacks sensitivity for oxygenated and linear terpenes (linal, \(\beta\)-ocimene), sesquiterpenes (\(\beta\)-caryophyllene, \(\alpha\)-farnesene) and GLVs (hexenyl acetate, methyl salicate). Therefore, SBSE stir bars, even coated with a non-polar adsorbent (PDMS), could be considered a more appropriate sensor for the sampling of VOCs of large polarity and chemical nature.  
We also assessed another mode of bagging plants for VOC trapping. In previous experiments [21], grapevines were individually enclosed in bags. However, the inner side of the bag was quickly covered with mist due to plant transpiration. The level of the analysed VOC was also quite variable, maybe due to physical stress related to the bag and/or variability of the response among plants (Figure 1). For these reasons, we analysed VOC emission by plants grouped under a tent and compared it with that of plants enclosed individually, following water treatment. Sampling was performed on day 1 (D1), day 3 (D3), and day 5 (D5) after water treatment. The comparison of VOC emission showed huge differences between the two collection systems, supporting our hypothesis of a stress related to individual bagging. We indeed observed a higher number and diversity of water-induced VOCs in the bag containment mode (Figure 2). In view of these results, the tent mode was retained for the next experiments because it appeared less stressful for grapevine.