Dichloromethane and anhydrous sodium sulfate were purchased from VWR (Darmstadt, Germany) whereas sulfuric acid and sodium carbonate were from Roth (Karlsruhe, Germany). The reference substances were obtained from the following suppliers: acetic acid, 2-phenylethanol, 3-methyl-1-butanol (isoamyl alcohol), hexanoic acid, phenylacetic acid, ethyl 2-methylbutanoate, γ-decalactone, γ-undecalactone, 2-methylbutanoic acid and 3-(methylthio)-propanal (methional) were from Sigma-Aldrich (Steinheim, Germany), butanoic acid and ethyl butanoate from Fluka (Steinheim, Germany), 2-methylpropanoic acid (isobutanoic acid) from SAFC (Steinheim, Germany), and 4-hydroxy-3-methoxybenzaldehyde (vanillin) from ABCR (Karlsruhe, Germany). The isotopically labeled standards were purchased from the following suppliers given in parentheses: 3,4-²H₂-3-methyl-1-butanol (²H₂₋isoamyl alcohol), 1,3,4,5,6-²H₅-2-phenylethanol, 3-(methyl-²H₃-thio)-propanal (²H₃-methional), 5,5,6,6-²H₄-y-decalactone, 6,7-²H₂-y-undecalactone (Aromalab, Freising, Germany), 6,6,6-²H₃-hexanoic acid, 3,3,3-²H₃-2-methylpropanoic acid (²H₃-isobutanoic acid), and ²H₆-ethanol (CDN Isotopes, Pointe-Claire, Quebec, Canada), 1,2-¹³C₂-butanoic acid, α,β-¹³C₂–phenylacetic acid, ¹³C₁-acetic acid, and 4-hydroxy-3-methoxy-¹³C₆₋benzaldehyde (¹³C₆-vanillin) (Sigma-Aldrich, Steinheim, Germany).

²H₅-Ethyl butanoate and ²H₅-ethyl 2-methylbutanoate were synthesized in the following way: [²H₆]-ethanol (25 mmol, 1.30 g) and concentrated sulfuric acid (1 mmol, 0.098 g) were mixed with either butanoic acid (5 mmol, 0.44 g) to give ²H₅-ethyl butanoate or 2-methylbutanoic acid (5 mmol, 0.51 g) to give ²H₅-ethyl 2-methylbutanoate; the mixtures were stirred at 90°C for 2 h. To obtain the final products, 50 mL of sodium carbonate water solution (0.5 mol/L) were added, and the resulting mixtures were extracted thrice with 50 mL of dichloromethane. After vigorous shaking, the organic phase was separated from the aqueous phase using a separatory funnel. The combined organic phases were dried over anhydrous sodium sulfate. The products were concentrated using a R-114 rotary evaporator (Buechi, Essen, Germany) without application of vacuum at 50°C to a final volume of approximately 50 mL. The identity of the synthesized compounds was confirmed by mass spectrometry. For ²H₅-ethyl butanoate the following data was obtained: MS-EI: 43 (100), 71 (91), 93 (68), 41 (49), 39 (48), 42 (41), 74 (31), 40 (27), 91 (23), 106 (15), 92 (14), 67 (12), 56 (11), 69 (11); MS-CI: 122 (100, M⁺⁺¹), 121 (18), 71 (10). For ²H₅-ethyl 2-methylbutanoate the following data was obtained: MS-EI: 107 (100), 57 (87), 41 (48), 75 (37), 39 (26), 88 (19), 56 (18), 120 (17), 76 (12); MS-CI: 136 (100, M⁺⁺¹), 135 (21).

The effects of bunch rot were studied in three grape varieties: White Riesling, Red Riesling and Gewürztraminer. In the case of powdery mildew, the hybrid Gm 8622-3 was analyzed. Sample selection and grape harvest procedures have already been described in our previous study (Lopez Pinar et al., 2016). In brief, samples affected by the fungus to a very high degree were compared with their respective healthy control. In each case, the pairs of samples were collected separately, but both were harvested and processed identically. The degree of fungal infection of the grape berries was visually assessed as described in our previous study (Lopez Pinar et al. (2016), providing photographs with representative examples of grapes affected with bunch rot and powdery mildew). Grapes were crushed, pressed in a pneumatic press and left to settle with 50 mg/L SO₂ for approximately 24 h at 10°C, followed by filtering through filter paper. The grape musts were subsequently fermented at 17°C for 2–3 weeks in 20 L glass balloons by inoculation of the reactivated pure yeast culture (20 g/hL) Lalvin EC 1118 (Lallemand, Vienna, Austria). After fermentation, wines were racked and the content of SO₂ was adjusted to achieve a final concentration of approximately 50 mg/L free SO₂. Finally, samples were filled in brown glass bottles which were closed with screw caps. The wines were stored for approximately 1 year at room temperature. Residual sample material from opened bottles was kept at −80°C if required.

Residual sugar, titratable acidity and alcoholic content (v/v) were determined using a Winescan FT-2 Fourier Transform Infrared Spectrometer (FTIR) (Foss, Hillerød, Denmark) as described previously (Patz et al., 1999, 2004).

Mass spectra were recorded using a 5975C MSD quadrupole system combined with a 7890A GC system (Agilent Technologies, Waldbronn, Germany). The instrument was equipped with a Gerstel CIS 4 injection system and a Gerstel MPS 2 auto sampler (Gerstel, Duisburg, Germany), and a DB-FFAP (30 m, 0.25 mm, film thickness 0.25 μm; J&W Scientific, Fisons Instruments, Mainz-Kastel, Germany) as the analytical capillary column. An uncoated, deactivated fused silica capillary column was used as precolumn (2–3 m, 0.53 mm). The carrier gas was helium and the flow rate was 1 mL/min. The temperature program used was as follows: the initial 40°C were held for 2 min, then the temperature was raised at 8°C/min until 240°C, and held for 5 min. Injection volume was 1 μL. EI-mass spectra were generated in full scan mode (m/z range 40–400) at 70 eV ionization energy. The scan rate was 3.94 scan/s.

Two-dimensional gas chromatographic analyses were carried out for quantification of trace constituents and specifically for those compounds that could not be fully resolved due to interferences from coeluting substances during one dimensional GC-MS analysis (Table 1). The system consisted of two Varian 450-GCs (Varian, Darmstadt, Germany) combined with a Saturn 2200 MS (Varian). The first GC was equipped with a Gerstel multi-column switching system MCS 2 and connected to the second GC by a Gerstel cryo-trap system CTS 1 (Gerstel). The system was further equipped with a Gerstel CIS 3 injection system and Gerstel MPS 2 auto sampler (Gerstel). The analytical capillaries used were a DB-FFAP capillary (30 m, 0.32 mm, film thickness 0.25 μm; J&W Scientific) in the first oven and a Rxi®-5HT (30 m, 0.25 mm, film thickness 0.25 μm; Restek, Homburg, Germany) in the second oven. At the end of the capillary, the effluent was split between a sniffing port and a FID, or a MS, in the first and second oven, respectively, using two deactivated but uncoated fused silica capillaries (100 cm, 0.20 mm). Application of the samples to the GC system was performed at 40°C using the cold-on-column technique. The GC temperature programs were as follows: the initial 40°C were held for 2 min, then the temperature was raised at 8°C/min until 240°C (for DB-FFAP measurements) or 250°C (for Rxi®-5HT measurements) were reached; the final temperature was then held for 5 min. The flow rate of the helium carrier gas was 2.0 mL/min. The FID and the sniffing port were held at 240 and 260°C, respectively. Mass spectra were generated at 70 eV ionization energy in electron ionization (EI) mode (m/z range 30–300) or in the positive chemical ionization (CI) mode with methanol as the reactant gas (m/z range 35–300).

In the first step, the isotopically labeled compounds dissolved in dichloromethane were mixed to the wine samples containing the odorant at comparable concentrations, as determined in preliminary experiments. Volumes of wines employed in the analyses were 1, 5, and 60 mL, respectively, depending on the concentrations of the compounds in the wine samples (cf. Table 1). For subsequent extraction of the volatile compounds, wine samples were mixed with dichloromethane in the ratio 1:1 (v/v), or 1:5 (v/v) in case that the volume of extracted wine was only 1 mL. These mixtures were stirred for 1 h at room temperature, and then submitted to solvent assisted flavor evaporation (SAFE) (Engel et al., 1999). The obtained dichloromethane phases were separated in each case, and dried over anhydrous sodium sulfate. Finally, each extract was concentrated to a final volume of 100 μL at 51°C by means of Vigreux distillation followed by micro-distillation.

The selected ions of the labeled and unlabeled compounds were monitored by one- or two-dimensional GC-MS (cf. Table 1), and their intensities recorded in relation to each other. The absolute concentrations were then calculated based on these relative intensities and the amount of labeled standard that had been added in each case, and were corrected by using calibration factors obtained from the mixtures of labeled and unlabeled compounds (cf. Table 1) as described in Spitzer et al. (2013). Analyses were performed per triplicate in each case.

The statistical significance of the effects of the two fungal diseases on the concentrations of the target aroma compounds was analyzed based on a comparison of the mean quantitative values from affected and unaffected samples using Student's t-test. Data were evaluated with regard to a significance level of p < 0.05 and p < 0.01.

Three basic oenological parameters were analyzed: residual sugar, titratable acidity and alcoholic percentage (Table 2).

Residual sugar was higher in the sample infected by bunch rot than in the corresponding healthy sample of the White Riesling and Red Riesling varieties, but in Gewürztraminer, residual sugar was moderately lower in the affected sample. In contrast to this, a much lower level of residual sugar was observed in the powdery mildew-affected sample.

Titratable acidity decreased moderately in the samples affected by bunch rot for White Riesling and Red Riesling. However, the Gewürztraminer sample again showed a different tendency with titratable acidity being higher in the affected sample. On the other hand, acidity in the powdery mildew-affected sample was lower than in the healthy sample.

Finally, the alcohol level was higher in all four fungi-affected samples in comparison to their respective healthy samples.

In order to characterize the effects of both fungi on the concentration of the selected aroma compounds, wine samples affected to a high degree by bunch rot (BW) or powdery mildew (PW) were compared with healthy ones (HW) of the same variety. Each pair of samples was collected and processed under identical conditions. In case of White Riesling, an additional intermediate state of bunch rot-infection (IW) was included (Table 3). Target odorants were selected based on our previous study where the main changes in odorant profile with fungi infection were elaborated by means of aroma extract dilution analyses and odorant identification studies (Lopez Pinar et al., 2017). Thereby, marker substances representative for acids, esters, alcohols, lactones and aldehydes were selected.

First, quantification experiments revealed that the most abundant odorants in the wine samples were isoamyl alcohol and acetic acid: both compounds were found in all samples at concentrations between 100 and 900 μg/g.

In the following, fungi effects on the respective odorant concentrations are presented. First and foremost, concentrations of phenylacetic acid and γ-decalactone were significantly increased (p < 0.01) by both fungi. Thereby, their concentrations in the fungi-infected samples increased by a factor of ~2–5 in comparison to the corresponding healthy samples. White Riesling was the sole exception where no significant change in γ-decalactone concentration was observed. In addition, acetic acid roughly doubled in concentration, in case of bunch rot infection (p < 0.05) whereas powdery mildew effects were only moderate (< factor 1.5).

Apart from that, vanillin concentration doubled in the Gewürztraminer bunch rot affected sample (p < 0.01). In contrast, its concentration remained unaffected in White Riesling and Red Riesling varieties and in the sample affected by powdery mildew.

In addition, the concentrations of methional were differently affected by both fungal infections. It was decreased (p < 0.05) by bunch rot in Red Riesling and Gewürztraminer, being lower by a factor of about 1.5–3. In contrast to this, its concentration was not affected in White Riesling variety. On the other hand, the concentration of methional doubled due to powdery mildew infection (p < 0.01).

The concentrations of several other compounds were also affected by both fungal diseases, but only moderately. Thereby, the changes described below were smaller than a factor of 2.

In the group of the cheesy smelling acids, inconsistent effects were recorded. Isobutanoic acid concentrations were higher (p < 0.05) in all four fungi-affected samples than in their corresponding healthy samples. In contrast to this, hexanoic acid concentrations decreased (p < 0.05) due to bunch rot infection, however, only in Red Riesling variety. Powdery mildew did not cause any significant effects on the concentration of this acid. Likewise, bunch rot induced a decline (p < 0.01) in butanoic acid in Gewürztraminer. Meanwhile, butanoic acid concentration was neither affected in the remaining bunch rot-affected samples nor in the sample affected by powdery mildew.

In the group of alcohols, isoamyl alcohol concentrations were lower (p < 0.05) in the Red Riesling and Gewürztraminer samples affected by bunch rot than in the corresponding healthy samples. In the White Riesling bunch rot-affected sample, however, the concentration did not vary from that in the healthy sample or in the sample infected to an intermediate state. Lastly, powdery mildew infection did not lead to any relevant differences in the concentration of this compound. Bunch rot effects on the concentration of 2-phenylethanol differed among the three varieties studied: it was increased (p < 0.05) in the White Riesling bunch rot-affected sample, unchanged in Red Riesling, and decreased (p < 0.01) in Gewürztraminer. In addition, a significant rise (p < 0.01) in concentration of this substance occurred in case of powdery mildew infection.

With regard to the ester substance group, variable results were obtained. Bunch rot led to an increase (p < 0.01) of ethyl butanoate in Red Riesling, a decrease (p < 0.01) in Gewürztraminer, whereas its concentration in White Riesling was not affected. Powdery mildew, on the other hand, caused no significant changes in the concentration of this compound. In addition, bunch rot caused a significant decrease (p < 0.05) of ethyl 2-methylbutanoate in White Riesling. However, significant effects on the concentration level of this substance were neither observed in the remaining varieties nor in the sample affected by powdery mildew.

Finally, γ-undecalactone was neither affected by bunch rot nor by powdery mildew.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YL and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.