2 methyl 1 butanol

Each commercial volatile compound, namely, acetic acid, 2-methyl-1-butanol (Sigma Aldrich, Saint Louis, MO, USA), 2-pentylfuran (Sigma Aldrich, Saint Louis, MO, USA), and 6-pentyl-2H-pyran-2-one (6-PP) (Sigma Aldrich, Saint Louis, MO, USA), was used for the antifungal bioassay, enzyme assay, and phenotypic measurements. Each volatile compound was diluted as 10⁻³ (v/v or w/v) and 10 µL was applied in a sterile cotton pad [4 (link),35 (link)] exposed to A. thaliana instead of culturing T. asperelloides PSU-P1, similar to the method in Section 2.4. The phenotypic characteristics were measured according to the method in Section 2.4. An enzyme assay was conducted to verify the enzyme activity between the control and treatment, as shown in Section 2.7.

Isolated and identified yeast strains growing over-night (Sabouraud Dextrose Broth;BIOCORP, Warsaw, Poland) were centrifuged (735 g), resuspended in Ringer’s solution and 6 log CFU mL⁻¹ were inoculated into YNB solution (Yeast Nitrogen Base; Sigma-Aldrich, Saint Louis, MI, USA) with 0.55% of sucrose, 0.25% of glucose and 0.2% of fructose as a carbon source (average ratio of fermenting sugars in plum mashes). After 10 days of incubation (25 °C), the samples were centrifuged (735 g), and the supernatants were analyzed by SPME-GC-MSTOF. Determination of the volatiles was carried out according to the method described by Zdaniewicz et al. [15 (link)]. Compounds were identified using mass spectral libraries and Linear Retention Indices, calculated from a series of n-alkanes from C6 to C30.
The qualitative and quantitative identification of volatile substances (ethyl acetate, isobutyl acetate, isopentyl acetate, ethyl hexanoate, ethyl octanoate, 2-phenylethyl acetate, ethyl decanoate, ethyl dodecanoate, isobutanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 1-hexanol, 1,6-heptadien-4-ol, acetic acid, hexanoic acid, octanoic acid, decanoic acid, diethyl acetal; Sigma-Aldrich, Saint Louis, MI, USA) was based on the comparison of retention times and peak surface area read from sample and standard chromatograms. All tests were carried out in triplicate.

To test the effect of dominant volatile compound participated in antifungal activity against F. incarnatum, the sealed plate method was conducted as shown in Section 2.3. The compound phenylethyl alcohol (PEA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The effect of commercial PEA versus the other volatile antifungal compounds 2-ethylhexanol, 1-nonanol, 6-PP, and 2-methyl-1-butanol (Sigma-Aldrich, St. Louis, MO, USA) [6 (link)] was tested through the sealed plate method. PEA was dissolved in 95% ethanol and we adjusted the dilution to 10⁻¹, 10⁻², and 10⁻³. Each volatile compound was applied on a sterile cotton pad (20 µL) and subjected to the method of Wonglom et al. [6 (link)] Application of 95% ethanol served as a negative control. The tested plates were then incubated at 28 ± 2 °C for seven days. Each treatment was composed of five replicates and the experiment was repeated twice. Colony diameters of F. incarnatum were measured and the percentage inhibition was calculated as described in Section 2.2.

A mixture of fungal VOCs containing fusel alcohols and acetates: 3-methyl-1-butanol (Sigma- Aldrich, 95%), 2-methyl-1-butanol (Sigma-Aldrich, 9%), 3-methyl-1-butyl acetate (Sigma-Aldrich, 95%), 2-phenylethanol Sigma-Aldrich, 99%), 2-methyl-1-butyl acetate (Sigma-Aldrich, 99%), and 2-phenylethyl acetate (Sigma-Aldrich, 98%) was prepared at a ratio of 1:1:1:1:1:1 (v/v). The actual release rates of the dispensers were determined in the laboratory under ambient conditions (25°C, 0.5 m/s airflow) using the gravimetric method (Table 1). Three replicates of each dispenser type were weighed for 10 days at regular intervals, and weight loss was recorded to the nearest 0.0001 g using a fine balance. The average release rate for each dispenser type was calculated based on the average weight loss over 24 h. Additionally, the average release rate of dispensers, under natural field conditions, was determined using the gravimetric method by periodically weighing the dispensers used in the field.

Water (HPLC-grade), 1-propanol, 2-propanol and 2-pentanol were purchased from VWR (Darmstadt, Germany) and was used to prepare calibration and internal standard solutions. Ethanol, Ethanol-D6, mEthanol-D4, 2-methyl-1-butanol, and sodium sulfate (anhydrous, ≥ 90% purity) were purchased from Sigma Aldrich (St. Louis, USA), isobutanol, 2-butanone, 2-methyl- and 3-methyl-1-butanol from AppliChem (Darmstadt, Germany), mEthanol, 1-butanol, 2-butanol, acetone and tert.-butanol from Merck (Darmstadt, Germany). All chemicals were of analytical grade.
Serum (human/pig) was evaporated to dryness (Eppendorf® Concentrator Plus, Eppendorf, Hamburg, Germany) and redissolved with water (HPLC-grade) before analysis to guarantee congener free serum.

Apples (Malus domestica ‘Jonathan’, Csány 1) were collected beside the city of Székesfehérvár, located in central Hungary, in September 2019, and were transported to the laboratory for subsequent analysis. The Jonathan apple is a classic American variety, medium-sized apple, and widely regarded as one of the best flavored, with a good sweet/sharp balance. The Viniflora Concerto^TM (L. thermotolerans) and Melody^TM (mixed culture of T. delbrueckii, L. thermotolerans, and S. cerevisiae) starter cultures were obtained from Chr. Hansen A/S (Hoersholm, Denmark), while the Biodiva^TM Level 2 (Torulaspora delbrueckii TD291) and Uvaferm 228 (S. cerevisiae) of Lallemand Inc. (Montréal, QC, Canada) dry yeasts were purchased from Kokoferm Ltd. (Gyöngyös, Hungary). Standards (glucose, fructose, saccharose, acetic acid, lactic acid, succinic acid, acetaldehyde, methanol, isoamyl alcohol, 1-propanol, 1-butanol, 2-butanol, 2-phenylethanol, 1-hexanol, 2-methyl-1-butanol, trans-3-hexen-1-ol, cis-2-hexen-1-ol, benzyl alcohol, ethyl acetate, propyl acetate, ethyl hexanoate, ethyl butyrate, isoamyl acetate, phenylethyl acetate, diethyl succinate, ethyl octanoate, ethyl benzoate, ethyl formate, linalool), and all chemicals of analytical grade were obtained from Sigma-Aldrich (Steinheim, Germany).

The chemical standards dimethyl sulfide, carbon disulfide, 2‐methyl‐1‐propanal, 2,3‐butanedione, 2‐butanone, chloroform, 2‐methyl‐1‐propanol, 3‐methylbutanal, 3‐methyl‐2‐butanone, 2‐methylbutanal, 2‐pentanone, 1‐penten‐3‐ol, pentanal, 3‐pentanone, acetoin, 3‐methyl‐1‐butanol, methylcyclohexane, dimethyl disulfide, 2‐methyl‐1‐butanol, 2‐penten‐1‐ol, 3‐hexanone, hexanal, ethyl butyrate, 1,2‐dimethylcyclohexane, 1‐hexanol, 2‐heptanone, dimethyl trisulfide, octanal, 2‐ethyl‐1‐hexanol, 2‐nonanone, nonanal, decanal, 2‐undecanone, and 4‐heptanone were purchased from Sigma‐Aldrich. The chemical standards 2,4‐octadiene and 1,1,3‐trimethylcyclohexane were purchased from BOC Sciences. Pentane, n‐heptane, toluene, sodium sulfate (Na₂SO₄), and anhydrous powder (ACS grade) were purchased from Fisher Scientific.