3.1. Chemical analysis of essential oils
The major constituents of the essential oils and their percentages are given in Table 2. Some components were standard in most of the tested plants, such as carvacrol, thymol and p-cymene, but others were specific for the plant species.
Table 2
Main chemical compounds contained in the essential oils of Thymus capitatus (TC), Coridothymus capitatus (CC), T. vulgaris (TV), and T. vulgaris Demeter (TVD).
EO TC | EO CC | EO TV | EO TVD |
Component | % | Component | % | Component | % | Component | % |
carvacrol | 68.6 | carvacrol | 76.9 | thymol | 47.9 | p-cymene | 26.7 |
p-cymene | 7.7 | α-bisabolene | 3.7 | p-cymene | 15.8 | thymol | 20.7 |
γ-terpinene | 6.8 | caryophyllene oxide | 3.3 | γ-terpinene | 10.0 | limonene | 5.6 |
β-caryophyllene | 2.6 | β-bisabolene | 3.2 | carvacrol | 4.4 | α-terpinolene | 5.0 |
β-myrcene | 1.8 | β-caryophyllene | 2.8 | linalool | 4.1 | carvacrol | 3.8 |
linalool | 1.5 | carvacrol acetate | 1.4 | β-caryophyllene | 2.1 | β-caryophyllene | 2.9 |
α-thujene | 1.2 | L-terpinen-4 ol | 0.6 | β-myrcene | 2.0 | camphene | 2.3 |
α-terpinene | 1.1 | eugenol | 0.3 | borneol | 1.3 | α-pinene | 2.1 |
α-pinene | 0.9 | borneol | 0.3 | α-terpinene | 1.3 | borneol | 2.1 |
terpinene 4-ol | 0.7 | δ-cadinene | 0.2 | α-thujene | 1.2 | linalool | 2.0 |
thymol | 0.6 | cedrenol | 0.7 | camphene | 1.1 | β-pinene | 1.9 |
In total, essential oils from TC contained 28 chemical compounds of which most belong to the terpenoids class, and 24 are present in percentages less than 3%. Therefore, TC consists mainly of carvacrol, p-cymene and γ-terpinene. On the other hand, essential oils from CC contained only 11 different compounds with a dominant content of carvacrol (Table 2). Essential oils from TC and CC contained carvacrol as the main component but they differed in their other constituents, mostly sesquiterpenes (α- and β-bisabolene, β-caryophyllene and caryophyllene oxide) and monoterpenes (p-cymene, γ-terpinene, β-myrcene, and linalool) in CC and TC essential oils, respectively. TV and TVD essential oils consisted of around 30 and 20 compounds, respectively, with only five compounds occurring in a percentage higher than 3%. T. vulgaris essential oils cultivated under different conditions (TV-EO vs. TVD-EO) presented a very similar composition regarding their main compounds p-cymene, thymol and carvacrol, which are present in differing percentages.
Table 2
Figure 1, color, single-column
PCA analysis highlighted the differentiation between essential oils based on their chemical characterisation. The first component PC1, which described 95.7% of the total variance, was positively correlated with phenolic monoterpenes (0.99), the major compounds found in essential oils of T. capitatus (TC = 69.1% and CC = 76.9%), and with sesquiterpenes which were mainly present in CC essential oils (13.0%). The PC2, explaining 4.1% of the total variance, was positively correlated with phenolic monoterpenes (0.12), hydrocarbons (0.18) and sesquiterpenes (0.11). The PCA analysis allowed us to confirm two different chemotypes for each thyme species.
3.3. FTIR spectra of fungi under control and stress conditions
IR bands were assigned to specific compounds in the fungal cell wall, i.e. lipids (blue column), polyphosphates (red), chitin/carbohydrates (green), based on the respective functional groups (Table 4).
Table 4
Main IR absorption bands and signal assignments of chemical structures deriving from the respective biopolymers (Bhat, 2013; Dzurendova et al., 2020; Gupta et al., 2022).
Band number | Wavenumber [cm− 1] | Signal assignment | Biopolymer contribution |
- | 3500–3200 | O-H stretching | carbohydrates |
- | 3275 | N-H stretching | chitin/chitosan |
- | 3105 | N-H stretching | chitin/chitosan |
- | 2955 | =C-H stretching | lipids |
- | 2925 | -C-H (CH3) stretching | lipids |
- | 2855 | -C-H (CH2) stretching | lipids |
1 | 1745 | -C = O stretching in esters | lipids |
2 | 1680–1630 | -C = O stretching, Amide I | proteins, chitin |
3 | 1560–1530 | C-N-H deformation, Amide II | proteins, chitin |
4 | 1465 | -C-H (CH2, CH3) bending | lipids |
5 | 1402 | C = O pf COO− groups | lipids |
6 | 1377 | -C-H (CH3) bending | chitin |
7 | 1320 | Amide III | proteins, chitin |
8 | 1265 | P = O stretching | polyphosphates, phospholipids |
9 | 1180 | C-O-C stretching in esters | lipids |
10 | 1150 | C-O and C-O-C stretching | carbohydrates |
11 | 1075 | PO2 symmetric stretching | polyphosphates, phospholipids |
12 | 1043 | C-O stretching | carbohydrates |
13 | 930 | glycosidic linkages | carbohydrates |
14 | 894 | P-O-P stretching | polyphosphates |
Table 4
FTIR spectra of the four different fungal strains grown on pure MEA showed significant differences in the lipids region (3000–2800 cm− 1) and especially in the fingerprint region from 1800 cm− 1 to 800 cm− 1 (Fig. 3A). When comparing the respective FTIR spectra of colonised MEA with the one of pure MEA, it was evident from the overlapping bands that a major part of the IR absorbance of the four different fungi samples derived from the culture medium (Fig. 3B). Thus, the spectra of the colonised media were corrected by subtracting the respective spectra of pure media in order to illustrate only the growth response of the fungi under the different treatments. Further baseline correction resulted in the FTIR spectra in Fig. 3C showing the respective growth response of the four fungi on pure MEA.
Figure 3, color, 2-column fitting
As indicated by the differing absorbance of the specific IR bands in Fig. 3C, there were biosynthetic differences between the four fungi growing on MEA. Poria monticola apparently synthesised more lipids (band 1 at 1740 cm− 1) in the cell wall, while chitin biosynthesis seemed reduced, based on the absorbance at 1650 cm− 1, compared to the other investigated strains (Fig. 3C, band 2). Moreover, high absorbance around 1250–1220 cm− 1 (Fig. 3C, band 8) reflected a cell wall composition in favour of polyphosphates and phospholipids, respectively. These observations could indicate a significantly different cell wall architecture in P. monticola with a more pronounced plasma membrane layer (phospholipids) and a thinner chitin layer. Similarly, P. monticola and G. trabeum showed increased absorbance at 1080 cm− 1, another band assigned to polyphosphates in the fungal cell wall. Gloeophyllum trabeum further showed higher absorbance at 1540 cm− 1 (chitin), 980–920 cm− 1 (carbohydrates) and 900–880 cm− 1 (polyphosphates). Concerning P. ostreatus, lower band intensities for the bands 3, 8, 11 and 12 were observed compared to the other three fungal strains while the bands 13 and 14 were rather intense (Fig. 3C).
Figure 4, color, 2-column image
The FTIR spectra of T. versicolor grown under different treatment conditions are shown in Fig. 4. Growth of T. versicolor significantly changed under stress, depending also on the different thyme varieties used for the production of the respective essential oils. Essential oil from TC apparently had the biggest influence on T. versicolor biosynthesis, showing elevated absorbance in spectral regions assigned to cell wall and plasma membrane lipids (band 1, 4, 5, 9 and 10) but also in regions assigned to chitin and carbohydrates, respectively (band 6 and 12). This observation confirmed data from MIC values (Table 3), which were lowest for TC-EO and TV-EO, respectively, as well as the determined mycelial growth inhibition which was highest for TC-EO against T. versicolor (Fig. 4).
Figure 5, color, 2-column image
In Fig. 5, the corrected FTIR spectra of G. trabeum under control conditions and in presence of essential oils were compared. Similar to the case of T. versicolor (Fig. 4), cell wall composition of G. trabeum changed substantially in presence of essential oils. Especially, the absorbance of the bands 1 (lipids), 2 (chitin), 3 (chitin), 4–6 (lipids and chitin), 11 (polyphosphates) and 12 (carbohydrates) differed from the control sample. Based on the FTIR spectra, the strongest cell wall modifications of G. trabeum derived from the essential oils from CC, TC and TV. Accordingly, the lowest MIC values against G. trabeum were determined for essential oils from CC and TV.
Figure 6, color, 2-column image
The biggest differences between the FTIR spectra of P. monticola grown on pure MEA and on modified MEA, respectively, are seen at the bands 1 (lipids), 2, 3 (chitin) and 13 (carbohydrates), 8, 11 and 14 (polyphosphates) (Fig. 6). While the chitin- and carbohydrates-related IR bands (2, 3 and 13) increased in absorbance, the absorbance of the bands 8 and 11 (polyphosphates) were higher in the control sample, indicating disturbance in the phospholipids biosynthesis caused by the presence of essential oils in the substrate. The strongest influence on P. monticola growth apparently was caused by the essential oils from TC, CC and TV, which was partly confirmed by in vitro inhibition experiments where the lowest MIC against P. monticola was determined for TC-EO.
Figure 6, color, 2-column image
Similar to the three other fungal strains reported above, also P. ostreatus showed significantly altered FTIR spectra in the fingerprint region when comparing spectra of the fungi grown on control substrate and modified substrates (Fig. 7). The biggest differences were found at the bands 1 (lipids), where the control sample showed almost no absorbance, bands 2, 3, 10 and 13 (chitin, carbohydrates), and the bands 8 and 14 (polyphosphates). The FTIR spectra indicated that the strongest impact on P. ostreatus growth came from essential oils from TC, TV and CC, confirming data obtained from the in vitro tests.
Table 5
Table 5
FTIR band area ratios of the second derivatives of the respective FTIR spectra of the four fungi under control conditions and in presence of essential oils (EOs) from T. capitatus (TC), T. vulgaris (TV), T. vulgaris Demeter (TVD) and C. capitatus (CC).
Fungus | Control / EOs | Lipids / Amide I | Lipids / Amide II | Amide I / Total Amides | Amide II / Total Amides | Lipids / Carbohydrates |
T. versicolor | TC | 1.633 | 4.967 | 0.507 | 0.167 | 0.482 |
TV | 0.787 | 2.354 | 0.518 | 0.173 | 0.244 |
TVD | 0.745 | 2.478 | 0.522 | 0.157 | 0.256 |
CC | 0.751 | 2.621 | 0.528 | 0.151 | 0.242 |
Control | 0.456 | 1.728 | 0.517 | 0.137 | 0.161 |
G. trabeum | TC | 0.750 | 1.313 | 0.552 | 0.315 | 0.343 |
TV | 0.539 | 1.526 | 0.632 | 0.223 | 0.287 |
TVD | 0.801 | 1.706 | 0.562 | 0.264 | 0.344 |
CC | 1.034 | 1.103 | 0.434 | 0.407 | 0.337 |
Control | 0.329 | 0.572 | 0.498 | 0.286 | 0.087 |
P. monticola | TC | 0.321 | 0.585 | 0.534 | 0.294 | 0.098 |
TV | 0.180 | 0.818 | 0.659 | 0.145 | 0.123 |
TVD | 0.325 | 0.897 | 0.587 | 0.213 | 0.153 |
CC | 0.383 | 1.156 | 0.615 | 0.203 | 0.250 |
Control | 0.247 | 0.730 | 0.595 | 0.201 | 0.111 |
P. ostreatus | TC | 0.712 | 1.220 | 0.548 | 0.319 | 0.158 |
TV | 0.544 | 1.351 | 0.598 | 0.241 | 0.218 |
TVD | 0.750 | 1.686 | 0.567 | 0.252 | 0.221 |
CC | 0.874 | 1.239 | 0.497 | 0.351 | 0.243 |
Control | 0.304 | 0.624 | 0.521 | 0.254 | 0.134 |
The second derivatives of the FTIR spectra from fungi grown under different conditions were used for the integration of specific band areas and their ratios (Table 5). The ratio between the lipids and the amide I IR bands changed in favour of lipids when essential oils were employed in the cultivation substrates. This was observed to a bigger extent for T. versicolor (x3.6 in the case of TC-EO), G. trabeum (x3.1 in the case of CC-EO) and P. ostreatus (x2.9 in the case of CC-EO) than for P. monticola (x1.6 in the case of CC-EO). This observation was already reported based on the corrected FTIR spectra shown in Figs. 5–8 and the respective IR band area ratios reported in Table 5 provided confirmation. Similarly, the ratio between the lipids and the amide II band was growing in favour of lipids. In contrast, the two ratios ''Amide I / Total Amides'' and ''Amide II / Total Amides'' remained rather constant, indicating that the applied EOs did not affect the protein profile of the four fungi too much. In one case, G. trabeum vs. TV-EO, the increase of the ratio ''Amide I / Total Amides'' relative to the control sample was substantially higher than for the other three essential oils, indicating a certain effect of TV-EO on the protein profile of G. trabeum. Further, a trend towards higher lipids to carbohydrates was observed, in concordance with reports from Sompong et al (2013) regarding elevated lipid synthesis under stress conditions.
Figure 8, color, 2-column image
The PCA was conducted using the second derivatives of the FTIR spectra after correction for the substrate background and further baseline correction. The total variance was described to > 93% by the first three principal components. The PCA scatter plots in Fig. 8 showed clustering of the samples according to the essential oils added to the MEA substrate. Essential oil from TC apparently had the biggest effect on grouping fungal strains on the base of their fungal cell wall composition, while the essential oils from TV, TVD and CC seemed to group up in one single cluster. However, excluding the FTIR spectrum of T. versicolor cultivated on MEA amended with CC-EO (red square), which did not follow the trend of the other three fungi, the FTIR spectra of G. trabeum, P. monticola and P. ostreatus could be considered another single cluster (Fig. 8, left). Apparently, T. versicolor responded in a very similar way to the presence of the essential oils of TV, TVD and CC (Fig. 8, orange square, green square and red square). On the other side, the other three fungi showed distinct, but still neighbouring clusters for the respective three essential oils. When comparing principal components 1 and 3, the clusters got smaller but the overall pattern remained with the biggest difference deriving from TC-EO and the spectra of each fungal strain cultivated on MEA containing EO from TV, TVD and TC, respectively, showing grouped up clusters (Fig. 8, right). Considering the main components of the respective EOs, carvacrol (main component in EO from TC and CC) apparently had a bigger effect on fungal growth than thymol or p-cymene that presented the main constituents in EO from TV and TVD, respectively. Further estimating the effect of the EOs, the fungi P. monticola and P. ostreatus apparently were affected to a higher extent than T. versicolor and G. trabeum.
Figure 9, color, 2-column image
Pellets from cultivated control and EO-modified MEA were used for FTIR microscopy and mapping of specific IR band ratios. In the case of Fig. 9, the ratio of the IR band areas assigned to lipids (3045–2760 cm− 1) and amide I (1725–1580 cm− 1) respectively, was used for the mapping of the investigated sample area.
The control sample (Fig. 9, left) presented a uniform distribution of a generally low ratio of lipids to amide I, similar to the values that were obtained for the IR band ratios using the second derivatives of the respective FTIR spectra (Table 5). On the other hand, when P. ostreatus was cultivated on TC-EO modified MEA, the determined IR band ratios were much higher and the FTIR mapping showed a much less uniform distribution, indicating areas where fungal hyphae synthesised more lipids in the cell wall as a stress response (Fig. 9, right).
Beside cultivated MEA pellets, mycelium patches from liquid cultures of P. ostreatus in control medium and medium containing EO TC, respectively, were used for FTIR microscopy. Figure 10 shows an FTIR image mapped based on the IR band area at the wavenumber 1460 cm− 1, which was assigned to C-H bending vibrations in lipids (Dzurendova et al., 2020). The absorbance intensity scale was set to the same levels for both samples in Fig. 11 to illustrate the higher lipid presence in the P. ostreatus liquid culture sample (yellow-reddish tones) as a response to the presence of EO from TC in the cultivation medium (Fig. 10, right).
Figure 10, color, 2-column image