# VOCs Are Relevant Biomarkers of Elicitor-Induced Defences in Grapevine
## 2 Results and Discussion
### SBSE-GC-MS Analysis of VOC Emission Elicited by Bastid(r)  
We first assessed VOC production induced by the marketed Bastid(r) elicitor. Bastid(r) is a mixture of chitol oligosaccharidesaccharides (COS) and oligogalacturonides (OGA) as active components, in which OGA dimers formed in the presence of calcium ions are stabilised by COS. This complex exhibits higher eliciting activity in Arabidopsis cell suspensions than the individual components do [23]. It triggers plant defence in tomato [24] and potato [25], and controls diseases of a number of crops. It is marketed for control of grape powdery mildew and downy mildew. In order to assess a basal blend of the experimental system (containment atmosphere + Bastid(r)), preliminary analyses of the VOCs collected in a tent where Bastid(r) or water were sprayed in the absence of plants were performed (data not shown). The molecules detected following water treatment were 2-aziridinylethyl amine; acetic acid, methyl ester; cyclic octaatomic sulphur; oxime-, methoxy-phenyl-, and toluene. The  
Figure 1: Comparison of VOC signal responses from elicitor-treated grapevine leaves, detected by SPME fibre and SBSE stir bar in bag containment. Amounts (\(\mu\)g/L) of eight targeted VOCs collected for 4 h on D3 after sulphated laminarin (PS3, 2.5 g/L) treatment of four plants (A to D) individually placed in a bag, using SPME fibres and SBSE stir bars (TwistersTM) as sensors. molecules detected following Bastid(r) treatment were glycolid; toluene; 2-propenoic acid, 3-(3,4-dimethoxyyphenyl)-, (_E_)-fumaronitrile, and nonane, 2,2,4,4,6,8,8-heptamethyl-. Toluene was detected following both treatments; it may have been present in the ambient atmosphere of the greenhouse. Others may have come from water contaminants, Bastid(r) degradation products, and pollutants such as sprayer components. Next, we analysed the VOCs emitted by Bastid(r)-treated plants, collected on D1, D3, and D5. PCA analysis of the data showed time-dependent VOC emission (Figure S1). The data obtained from the scan mode analysis of the samples collected at the three time points from three independent experiments were filtered according to ANOVA significance and to the fold change (FC) relative to H\({}_{2}\)O control criteria (significant for \(p\) < 0.05, induced above 1.5-fold at least twice in the three experiments). As a result, seven VOCs were selected. Two of them were sesquiterpenes: \(\alpha\)-farnesene and \(\beta\)-caryophyllene (Table 1). The other ones were decanal; toluene; 2-oxo-4-phenyl-6-(4-chlorophenylphenyl)-1,2-dihydropyrimidine; 4,4'-bi-4H-pyran, 2,2',6,6'-tetrakis(1,1-dimethylethyl)-4,4'-dimethyl- and 3,4-dihydrosoquinoline-7-ol, 1-[4-hydroxybenzyl]-6-methoxy-. \(\alpha\)-farnesene, \(\beta\)-caryophyllene, decanal, and toluene can be produced and emitted by plants [26,27]. However, 2-oxo-4-phenyl-6-(4-chlorophenylphenyl)-1,2-dihydropyrimidine; 4,4'-bi-4H-pyran, 2,2',6,6'-tetrakis(1,1-dimethylethyl)-4,4'-dimethyl- and 3,4-dihydrosoquinoline-7-ol, 1-[4-hydroxybenzyl]-6-methoxy- probably did not originate from the plants despite their significant accumulation after elicitor treatment. It is unlikely that these compounds were present in the elicitor formulation as they were not systematically detected following Bastid(r) treatment (Table 1), or in the plant-less tents sprayed with Bastid(r). Their origin remains unexplained. Figure 2: Chemical formulas detected in the bag or the tent containments. VOCs were collected for 4 h from four plants treated with water and individually enclosed in bags (bag) and from six plants gathered in a tent (tent). Radar plot illustrations correspond to the combined lists of VOCs detected on D3 and D5 after treatment. Chemical formulas were determined by GC-MS analysis and NIST identification. The release of these VOCs was time-dependent, with a variable time of significant emission depending on the VOC and the experiment (Table 1). The profile of significant VOC emission in response to Bastid(r) was also variable among experiments (Table 1).