3 octanone

Sodium chloride and hexane (≥95%) were obtained from Fisher Scientific (Pittsburgh, PA, USA). Sodium sulphate (anhydrous) was obtained from ECP-Analytical (Auckland, New Zealand). 4-Decanone* (≥97.0%) was obtained from Tokyo Chemical Company (Tokyo, Japan). 4-Methyl-2-pentanone* (≥98%) was obtained from BDH Chemicals Ltd. (Poole, UK). Dichloromethane (≥99.9%), methanol (≥99.8%), sodium hydroxide solution (1N), 3-octanone (≥98%), δ-octalactone (98%), γ-decalactone (≥98%), δ-decalactone (≥98%), γ-dodecalactone (≥97%), δ-dodecalactone (≥97%), δ-tetradecalactone (98%) were obtained from Merck (Darmstadt, Germany). A C₇-C₃₀ Saturated Alkane Standard was obtained from Supelco (Bellefonte, PA, USA). Liquid nitrogen, liquid carbon dioxide, hydrogen carrier gas (instrument grade, >99.98%), and nitrogen (instrument grade, >99.99%) were obtained from BOC Ltd. (Auckland, New Zealand).

Identities of all VOCs considered as potential marker substances were confirmed through analysis of pure reference substances.
Acetone, 2-butanone, hexanal, nonanal, isoprene and benzene were acquired from Ionimed Analytik GmbH (Innsbruck, Austria). Butane, pentane and hexane were bought from Supelco (Bellefonte, USA). Methyl-isobutyl-ketone, 1-propanol, styrene, 3-octanone, 2-heptanone, furan, heptane, methylacetate, 2-methylfuran, 2-ethylfuran, 2-pentylfuran, 2-pentanone, 2-hexanone, 3-hexanone, 3-methyl-2-butanone, 3-methyl-2-pentanone, 2-propanethiol and dimethylsulfide were purchased from Fluka/Sigma-Aldrich (Steinheim, Germany). 2-Methyl-butanal and dimethyldisulfide were bought from Abbott GmbH & Co.KG (Wiesbaden, Germany). Ethylbenzene was acquired from Merck (Darmstadt, Germany). 3-methylfuran and 3-methylbutanal was purchased from TCI Europe N.V. (Zwijndrecht, Belgium).

The two VOCs 1-Octen-3-ol (CAS number 3391-86-4) and 3-Octanone (CAS number 106–68-3) purchased from Sigma-Aldrich (≥98% purity), have a concentration of 784 g/L. 1-Octen-3-ol, known as mushroom alcohol, is a secondary alcohol derived from 1-Octene. It is approved by the U.S. Food and Drug Administration as a food additive. It has 128.21 molecular weight; insoluble in water, soluble in oils and miscible at room temperature in ethanol, solubility of 1.836 mg/mL at 25 °C (est). Vapor pressure is 3 hPa at 20 °C. The LD50 doses are 56, 340 and 3300 mg/Kg in mouse, rat and rabbit, respectively [55 ].
3-Octanone, also known as ethyl amyl ketone appears as a clear colorless liquid with a pungent odor. Partially soluble in alcohol and insoluble in water, solubility of 2.6 mg/mL. Vapors are denser than air and may have a narcotic effect in high concentrations. It is used in perfumes and as a solvent for nitrocellulose and vinyl resins. It has the same molecular weight and formula of 1-Octen-3-ol (C8H16O). Vapor pressure is 2.7 hPa at 20 °C. The LD50 is 0.406 and 5 g/Kg in mouse and rat, respectively [56 ].

Analytical-grade chemicals and reagents for preparing standard solutions for GC-MS analysis were used without further purification. Standard 1-octen-3-one (96%), 1-octen-3-ol (≥98%), 3-octanone (≥98%), benzaldehyde (≥99.5%), trans-2-hexen-1-ol (96%), benzyl alcohol (99%+), 1,5-dimethyl naphthalene (98%), 3-methyl-1-butanol (99%+), 1,5-dimethyltetralin (≥90%), phenylethyl alcohol (≥99%), 4-methyl-2-pentanol (98%), glucose (≥99.5%), fructose (≥99%), tartaric acid (99%), naphthalene-d8 (99 atom% D, ≥98% (CP)), 3-octanol (99%), 2-octanol (≥99.5%), zinc nitrate hexahydrate (98%), 2-methylimidazole (98%), and glass wool were purchased from Sigma-Aldrich (Sydney, NSW, Australia). Hexanal (98%), 1-hexanol (99%), menthol (HPLC), and N,N-dimethylformamide (DMF) (99%) were purchased from Alfa Aesar (Scoresby, VIC, Australia). Hexanal-d12 (98.5 atom % D) and 2,4,6-tribromoanisol-d5 (99 atom % D) were purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). Milli-Q™ water (18 MΩ·cm) was collected through a Millipore™ water purification system (Merk, Bayswater, VIC, Australia). Synthetical grape juice was made using 100 g glucose, 100 g fructose, and 4 g tartaric acid. All solutions were prepared in Milli-Q™ water, with the pH adjusted to 7.0.

Similarly, initial assays were performed to determine the optimal concentration of Metarhizium-derived VOCs for plant growth promotion of M. brunneum. Two key Metarhizium-derived VOCs [30 (link)]—3-octanone (Sigma-Aldrich, Gillingham, UK, 106-68-3, ≥98%) and 1-octen-3-ol (Sigma-Aldrich, Gillingham, UK 3391-86-4, ≥98%)—were prepared for assay using air-dried compost containing John Innes (Westfield, UK). Dried compost was mixed with either of the VOCs at a rate of 0.01, 0.1, 1 or 10 µL per 100 g compost. Compost was mixed thoroughly with the VOCs and wetted to 60 ± 5 % soil RH. Oilseed rape seeds were placed in Petri dishes to germinate as described in Section 2.1. Resultant germinated seedlings were selected, and into each prepared 0.75 L square pots (80 × 80 × 120 mm) pot a divot was made 10 mm deep before a seedling was transplanted and re-covered with compost. Plants were grown in a glasshouse as previously described, and fresh weights were recorded. Control experiments were grown in manufacturer compost without additional treatment. Five plants were used per replicate, with three technical replicates per experiment. The whole experiment was repeated twice (n = 30).

2-Methyl-1-propanol, 3-methyl-1-butanol, 3-methyl-butanal, 3-octanone, p-xylene, styrene, benzeneacetaldehyde, phenylethyl alcohol, 4-ethyl-phenol, 2,5-dimethyl-pyrazine, trimethyl-pyrazine, and 3-ethyl-2,5-dimethyl-pyrazine were purchased from Sigma-Aldrich China Inc. (Beijing, China). The 18 amino acid standards, including glycine (Gly), asparagine (Asn), alanine (Ala), glutamic acid (Glu), lysine (Lys), arginine (Arg), proline (Pro), valine (Val), tyrosine (Tyr), isoleucine (Ile), leucine (Leu), tryptophan (Try), serine (Ser), histidine (His), threonine (Thr), methionine (Met), cysteine (Cys) and phenylalanine (Phe), were all purchased from Biosharp.
High-performance liquid chromatography (HPLC)-grade acetonitrile was supplied by Tedia Company (Fairfield, OH, USA). Phenylisothiocyanate (PITC, lot code: 10113227) was purchased from Alfa Aesar Chemical Co. Ltd (Tianjin, China), with a purity higher than 97%. Triethanolamine (TEA, analytical grade) and n-hexane (analytical grade) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Pure water was obtained using a Milli-Q purification system (Millipore, Bedford, MA, USA).

The volatile collections were analyzed with gas chromatography combined with mass spectrometry (HP Agilent 5890 GC and 5975 MS, Agilent Technologies) equipped with HP-5 UI capillary column (30 m × 0.25 mm × 0.25 μm, J&W). The injector temperature was set to 250 °C and operated in splitless mode for 30 s for solvent injection (1 μl was injected with 3 min solvent delay) and for 1 min for SPME injection. The oven temperature was maintained at 50 °C for 1 min, then increased at 10 °C min^-1 to 280 °C and held for 4 min. The flow rate of the helium was 1.0 ml min^-1. Positive electron ionisation (EI+) was used, with an electron energy level of 70 eV, 2 scans s^-1 were recorded in the range of 29–300 m/z.
Compounds were tentatively identified by matching their mass spectra with those in the MS Libraries (NIST 11 and Wiley) using ChemStation (D.01.02.16, Agilent USA). The samples were also verified by injection of synthetic standards and compared to published and calculated Kováts index (KI) values using C8-C40 alkanes calibration standards. The identification of electrophysiologically active compounds was subsequently verified by testing the synthetic standards with GC-EAD/FID. 1-octen-3-ol (98%, CAS 3391-86-4), 3-octanone (≥98%, CAS 106–68-3) and 1-hepten-3-ol (≥98%, CAS 4938-52-7) were purchased from Sigma-Aldrich and were diluted in n-hexane (HPLC grade, Merck).