Trans caryophyllene

The pure compounds of the three identified VOCs produced by HND5 were purchased from different companies: 2-methoxy-4-vinylphenol (99%) was purchased from Aladdin (Shanghai, China); and 3,4-dimethoxystyrol (technical grade) and (-)-trans-caryophyllene (98.5%) were obtained from Sigma-Aldrich (Vienna, Austria). Dual-plate experiments were used to test the antifungal activities of the compounds. petri dishes (9 cm diameter, the volume of free space was 90 mL) containing PDA medium incubated with FOC plugs were covered with petri dishes containing sterile filter papers (15 × 20 mm) containing different amounts of VOCs: 2-methoxy-4-vinylphenol: 0.5, 1, 5, 10, and 20 μL; 3,4-dimethoxystyrol: 1, 5, 10, 20, and 40 μL; and caryophyllene: 10, 20, 40, 80, and 160 μL. The control plates were covered with petri dishes containing only sterile filter papers. The plates were incubated at 28°C for 7 d, and the mycelia diameters were measured every 24 h during incubation.

For the conditioning of the adsorbents tubes, acetone of HPLC grade (Rotisolve, Carl Roth GmbH + Co KG, (Germany)) was used. The water was self-purified by a MilliQ System. For the enrichment Tenax TA (mesh 60/80) and Tenax GR (mesh 20/35) (Alltech Associates Inc.; Buchem BV (Netherlands)) and silanized glass wool (Sigma Aldrich (Germany)) were used. For tubing Tygon tubes (Carl Roth GmbH + Co KG (Germany)) were used. The Nalophan foil from Kalle GmbH (Germany) was used for wrapping.
As standards 2,4-dimethyl-1-heptene (CAS 19549-87-2, 95%) from Combi-Blocks, (+)-α-longipinene (CAS 5989-08-2, ≥99% sum of enantiomers) from Aldrich, (+)-cyclosativene (CAS 22469-52-9, 99%) from Sigma-Aldrich and (−)-trans-caryophyllene from Sigma-Aldrich (all purchased from Merck (Germany)) were used. The standards were dissolved in methanol (Rotisolve ≥ 99.98%; Carl Roth GmbH + Co KG (Germany)). A volume of 50 µl, which corresponds a mass of 100 ng of each standard, were injected on the adsorbents with a microliter syringe (Hamilton). The solvent was dried by pumping ambient air with a flow of 400 ml/min for 4 minutes over the adsorbents tube. In order to exclude contaminations, the drying procedure was carried out with blank adsorbents.

The standard compounds used for the GC analysis were thymol, trans-anethol, (+)-borneol, (−)-borneol, α-terpineol, L-carvone, (R)-(+)-limonene, eucalyptol, farnesol, neryl acetate, (±)-citronellal, citral, γ-terpinene, nerol, α-pinene, p-cymene, (−)-trans-caryophyllene, geraniol, geranyl acetate, carvacrol, eugenol, sabinene hydrate, bornyl acetate, linalyl acetate, myrcene and (±)-camphor and were purchased from Sigma-Aldrich (St. Louis, MO, USA). In addition, 1,1-Diphenyl-2-picryl-hydrazyl-hydrate (DPPH∙) was obtained from Sigma-Aldrich GmbH (Germany). All chemicals and solvents were analytical reagent grade.

For chemical analyses, EOs were diluted in cyclohexane MS SupraSolv^® for gas chromatography (Sigma-Aldrich, Steinheim, Germany). For enantiomers identification, the analytical standards of (−)-menthol, (+)-menthol, (−)-menthone, (+)-menthone, (−)-α-terpineol, (+)-α-terpineol, (−)-limonene, (+)-limonene, (−)-terpinen-4-ol, (+)-terpinen-4-ol, (−)-trans-caryophyllene, (−)-carvone, (+)-carvone, (−)-linalool, (±)-linalool, (−)-menthyl acetate, (+)-menthyl acetate, (−)-dihydrocarvone, (+)-dihydrocarvone, (−)-borneol, and undecane-2-one (all Sigma-Aldrich, Steinheim, Germany) were used.

The volatile extracts were analysed by coupled GC–MS (mass spectrometry) using a GCMSQP 2010 Ultra instrument (Shimadzu, Kyoto, Japan), fitted with a gas chromatograph (column specifications: 30 m × 0.25 mm i.d., film thickness 0.25 μm, J & W Scientific, Santa Clara, CA, USA). Helium was used as the carrier gas at a flow rate of 1 mL/min. The samples were injected into a splitless injector at the temperature of 200 °C with the injections of 1 µL being performed in splitless mode. The oven temperature was programmed to start at 50 °C for 5 min before rising to 250 °C at a rate of 5 °C/min, with a final hold time of 5 min and a 50 min total run time. Electron impact ionisation was conducted at 70 eV, with the temperature of the ion source and detector set at 200 °C. The interface temperature was 250 °C, whereas the recorded fragmentation values were scanned from 35 to 300 m/z. Plant VOCs were identified by analysing their mass spectra and comparing them with those in the NIST 08 library and then those of authentic standards (ocimene mix of isomers, trans-caryophyllene, and farnesol; Sigma-Aldrich, St. Louis, MO, USA). The compounds were quantified based on the peak area relative to the internal standard (nonacosane) provided by Sigma-Aldrich. Each treatment employed six replicates.

The following plant metabolites used in our study were ordered: (−)-caryophyllene oxide, (+)-nootkatone, zerumbone, β-amyrin, crocetin dialdehyde, saffron stigma (Sigma-Aldrich, St. Louis, MO, USA), α amyrin (Carl Roth, Karlsruhe, Germany) and taraxasterol acetate (Biorbyt, Cambridge, UK) (Figure 1). All compounds were dissolved in dimethyl sulfoxide (DMSO) in order to be used in the assays. Saffron threads were incubated in DMSO for 1 h on a rotary shaker (100 rpm); then, the supernatant was collected and used in the work. A C₇-C₃₀ saturated alkanes standard solution was also obtained from Sigma-Aldrich to calculate the GC retention indices of the sesquiterpenes formed by A. thaliana. The liquid standards used were β-elemene, ordered from Abcam plc (Cambridge, UK) and (−)-trans-caryophyllene (sum of enantiomers, purity: ≥98.0%) and (−)-α-cedrene (sum of enantiomers, purity: 96.4%) purchased from Sigma-Aldrich.