Effects of Origanum vulgare essential oil and its two main components, carvacrol and thymol, on the plant pathogen Botrytis cinerea

Inhibitory activity of different plant EOs on mycelium growth of B. cinerea

Seventeen plant EOs were involved in the current study, namely O. vulgare, C. cassia, Perilla frutescens, Saussurea costus, Mentha spicata, L. cubeba, Asarum sieboldii, Illicium verum, Foeniculum vulgare, Angelica dahurica, Curcuma zedoaria, Mentha haplocalyx, Artemisia argyi, Eucalyptus globulus, Syzygium aromaticum, Acorus tatarinowii and turpentine (mixture) EOs. All the EOs were purchased from Cedar pharmaceutical Co., LTD., Jiangxi, China. Isolates of B. cinerea were prepared from tomato fruits (Luoyang, China) and deposited at the Key Laboratory of Creation and Application of Novel Pesticides, Henan. The preliminary antifungal activities of these EOs against B. cinerea were determined according to the method described by Farzaneh et al. (2015). Emulsion of the EOs were prepared by sterile water with 0.5% (v/v) acetone and Tween 80, blended with Potato Dextrose Agar (PDA, 200 g extract of boiled potatoes, 20 g dextrose and 20 g agar powder in 1,000 mL distilled water) at 40–45 °C to obtain final concentrations of 0 (control), 0.5 and 2 mg/mL. A five mm mycelial disc from young cultures of target fungi (3 d) was placed in treated sterile plates (D = 7 cm) after medium solidification. The petri dishes were sealed with sealing film and cultured upside-down in dark at 26.5 °C.

The mycelial growth was measured by a nonius using decussation way at 2, 4 and 6 d, respectively. Three replicates per treatment were used in each experiment and the experiments were performed three times. Growth inhibition of each EO was computed by the formula (1): (1) $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\mathrm{\%}\right)=\left(D_{\mathrm{c}}-D_{\mathrm{t}}\right)/\left(D_{\mathrm{c}}-0.5\right)\times 100$$

Where D_c is the colony diameter of the control group; and D_t is the colony diameter of the treatment groups with EO.

GC–MS analysis

The chemical compositions of OVEO was analyzed by GC–MS (Agilent-7890B, Agilent Technology Co., LTD., USA) according to the method reported by Scalas et al. (2018). Chemicals in OVEO were separated with HP-5 MS capillary column (50 m × 0.25 mm × 0.25 μm), high-purity Helium (99.999%) as the carrier gas. The helium carrier gas rate was 1.0 mL/min at a split ratio of 20.4:1, and the injection volume was 1.0 μL. The initial temperature was set at 60 °C for 25 min, then gradually increased to 150 °C at a rate of 1.2 °C·min⁻¹. Electron ionization was used as the ion source, and the ionization energy was set at 70 eV. The quadrupole temperature was 150 °C, and the ion source temperature was 230 °C. The sector mass analyzer was set to a range from 33 to 900 amu. Diluted samples (1/100, in acetone) at 1 μL each were injected and mass spectra were compared with the standard mass spectra from the NIST 2.2 database provided by the software of GC–MS system.

In vitro antifungal activities of OVEO, carvacrol and thymol against B. cinerea

OVEO, carvacrol (purity 99%) and thymol (purity 98.5%, Sigma Aldrich, St. Louis, MO, USA) were dissolved in sterile water containing 0.1% (V/V) acetone and Tween 80 to prepare the stock solution at the concentration of 1,000 μg/mL (the stock solution shall be used for dilution below unless otherwise specified). The fungi were grown on a PDA plate for 5 d, and then harvested using a five mm sterilized puncher along the edges of the colonies, and the fungal blocks were placed in the center of PDA plates containing 0, 2.5, 5, 10, 15, 20, 40, 50 μg/mL carvacrol and 0, 2.5, 5, 10, 20, 40, 60, 80 μg/mL thymol. Cultures with 0.005% (0.008%) acetone and Tween 80 were used as the control, respectively. Incubation upside-down at 26.5 °C. The fungistatic effect was observed, and the colony diameter was measured and recorded when the diameter of the control was exceeded 7.5 cm. Then, calculations were made for B. cinerea mycelium inhibition ability of OVEO, carvacrol and thymol and regression equation and EC₅₀ values by probability value analysis. Three replicates per treatment were carried out in each experiment and the experiments were performed three times.

Antifungal activity of OVEO, carvacrol and thymol on spore germination of B. cinerea

B. cinerea isolates were cultured on PDA plates at 20 °C for 14 d. Then, the plates were injected with five ml sterile water and gently scraped with a sterile swab and filtered through four layers of sterile cheesecloth to obtain spore suspension (Jing et al., 2018). The spore suspension concentration was adjusted to 10⁵~10⁶ spores mL⁻¹ by using a hemocytometer and continuously adding sterile water. OVEO, carvacrol and thymol were blended with acetone and Tween 80 and sterile water, respectively, and the concentration was adjusted to 100, 200, 300, 400, 500, 600 μg/mL at the same time, a 0.5 mL aliquot of conidium suspension of B. cinerea plus 0.5 mL liquid were added to a tube. Then, 60 μL was transferred onto a double concave glass plate with pipette gun and placed in a wet sterile culture dish. Cultures with 0.03% acetone and Tween 80 were used as the control. The cultures were incubated at 28 °C. After incubating for 8 h, spores of B. cinerea were observed with a microscope (Nikon Eclipse Ti-S, Tokyo, Japan) at a 200× magnification. The spore germination percentage was calculated by counting germinated spores among 200 spores. Three replicates per treatment were conducted in each experiment and the experiments were performed three times.

Effects of carvacrol and thymol on fresh and dry mycelial weight of B. cinerea

The fresh and dry mycelial weight of the tested pathogen treated with carvacrol and thymol were measured (Chen et al., 2014). According to the EC₅₀ and EC₉₀ of carvacrol and thymol, two components were dissolved and diluted in sterile water with 0.1% acetone and Tween 80, and transferred into Erlenmeyer flasks with 25 mL of Potato Dextrose Broth (PDB, 200 g extract of boiled potatoes, 20 g dextrose in 1,000 mL distilled water) medium to obtain carvacrol concentrations of 0 (control), EC₅₀ (9.38 μg/mL) and EC₉₀ (43.49 μg/mL) and thymol concentrations of 0 (control), EC₅₀ (21.32 μg/mL) and EC₉₀ (65.13 μg/mL), respectively. The pathogen was harvested along the edge of the colony (3 d) with a 5 mm sterilized punch, then inoculated into each flaskand the flasks were incubated at 120 r/min shaking and 26.5 °C. The mycelium groups were harvested on 3 d and strained off excess culture. The agar blocks were removed with tweezers, and each mycelium groups were washed with sterile water at least three times, and then filtered for 30 min to remove excess water on the surface. The fresh mycelia were weighed and the dry mycelia as well after drying at 60 °C for 12 h. Each treatment consisted of three replicates and the experiments were performed three times.

Effect of carvacrol and thymol on the relative leakage of B. cinerea

The permeability of the membrane was expressed in terms of relative extracellular conductivity and tested according to the method of Zhou et al. (2018) by using a Seven Excellence Multiparameter Tester (METTLER TOLEDO Instrument Co., Ltd., Shanghai, China) with some modifications. Carvacrol and thymol stock solution were prepared with sterile water and 10% dimethyl sulfoxide, and the final concentrations were 10, 200, 500 μg/mL and 70, 200, 500 μg/mL, respectively. Vinclozolin (500 μg/mL) was prepared with sterile water and 10% dimethyl sulfoxide as solvent. Fungal samples were collected 3 d after inoculation on PDA medium. Then the collected mycelium was inoculated in PDB and shaken at 120 rpm and 26.5 °C for 108 h. Remove the agar blocks with tweezers, wash each mycelium groups with sterile water at least three times, and filter for 30 min to remove excess water on the surface. The mycelia (0.1 g) were separately put into the solution with the above series concentrations into glass tubes of 10 mL, and 10% dimethyl sulfoxide (DMSO) sterile distilled water was the solvent control. Vinclozolin containing 10% DMSO was used as a positive control. Extracellular conductivity was measured at 0, 10, 40, 140, 220, 360 and 400 min. Finally, the electrical conductivity was measured again after dead treatment (temperature ≥ 95 °C, 25 min). Three replicates per treatment were carried out in each experiment and the experiments were performed three times. The relative leakage was determined according to the formula (2): (2) $$\mathrm{R}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{k}\mathrm{a}\mathrm{g}\mathrm{e}\left(\mathrm{\%}\right)=\left(C_{\mathrm{t}}-C_0\right)/C_{\mathrm{d}}\times 100$$

Where C_t is the conductivity of current time samples, C₀ is the conductivity of initial time (0 min) samples, and C_d is the conductivity of dead treatment conductivity samples.

Scanning electron microscopy and fluorescent microscopy

Carvacrol and thymol were dissolved and diluted in sterile water with acetone and Tween 80. One mL of mixture and nine mL PDA medium were mixed and poured into a 9 mm diameter Petri dishes. The final concentrations of 0 (control), 25 and 40 μg/mL of carvacrol and thymol were obtained, respectively. Medium containing 0.004% acetone and tween-80 was used as solvent control. The blocks of B. cinerea were inoculated on the medicated medium and cultured upside down at 26.5 °C for 5 d. The effects of carvacrol and thymol on mycelial morphology were analyzed using the modified method of Li et al. (2017). For SEM observation, PDA blocks about 5 × 7 mm were cut from the edge of mycelium. Mycelium exposed to different treatments was fixed with 25% glutaraldehyde for 48 h at low temperature. Samples were washed for 3 h (10 min/time) in 0.1 molL⁻¹ phosphate buffer (pH 7.2). After being fixed with 1% osmium for 2 h, the samples were washed with distilled water for three times (3 min/time) and dehydrated in a graded series of ethanol concentrations (30%, 50%, 70%, 80%, 90% and twice at 100%) for 15 min at each stage. Samples were dried by CO₂ critical point drier (CPD 030, Leica, Wetzlar, Germany), then gold coated using a sputter coating machine (MC1000, Hitachi, Tokyo, Japan). All samples were viewed in a SEM (Model SU8010-3400N, Hitachi) operating at 10 kV at 2.5 k× magnification.

The fluorescence intensity of H2DCFDA dye under specific excitation wave is related to the content of ROS, and the accumulation of ROS can be judged by the fluorescence intensity (Li et al., 2017). SYTOX Green is a fluorescent dye commonly used to study membrane integrity. It can pass through damaged membrane cells but cannot penetrate the complete cell membrane and emit Green fluorescence. Rhodamine-123 is a cationic dye that can penetrate the cell membrane and is an indicator of mitochondrial transmembrane potential. Mitochondrial matrix fluorescence intensity weakened or disappeared in normal cells, and strong yellow-green fluorescence was released when mitochondrial membrane was destroyed (Tian et al., 2012).

Mycelium was observed by Nikon Eclipse Ti-S inverted fluorescence microscope (Eclipse Ti-S, Tokyo, Japan) and slightly modified (Jing et al., 2018). The fungal blocks were inoculated into 50 mL erlenmeyer flasks with PDB culture medium and the flasks were incubated at 120 r/min and 26.5 °C for 2 d. Then the samples were incubated in PDB with the carvacrol and thymol (100 and 200 μg/mL) for 12 h, 0.1% acetone and Tween 80, where sterile water was used as the blank control. The collected mycelia were stained with 1 μg/mL H2DCFDA, SYTOX Green or Rhodamine 123 for 30 min at 4 °C in darkness. After the dying, the mycelia were washed with phosphate buffered saline to remove the residual dye and then observed by fluorescence microscope. The views were randomly selected from each group, and all experiments were repeated six times and the experiments were performed three times.

Protective and therapeutic effects of OVEO, carvacrol and thymol on tomato gray mold caused by B. cinerea

Protective effect experiment: tomato fruits with same size and weight were selected and put in sterile plastic boxes (9 tomatoes in each box) with gauze at the bottom wetted with sterile water to keep moisture. Tomatoes were sprayed with OVEO, carvacrol or thymol at 500 and 1,000 μg/mL, respectively. Pyrimethanil at 400 μg/mL was used as the standard control, and 0.1% acetone plus Tween 80 with sterile water was used as the solvent control. Sterile water was used as the blank control. Each box had one treatment, nine repetitions for each treatment, and three fungal blocks for each repetition. After the liquid was dried, fungal blocks were placed on each tomato by acupuncture method and placed in the artificial climate box with humidity over 85% and temperature at 26.5 °C. After 48 h, the diameter of the spot was measured and the control effect was calculated. Therapeutic effect experiment: Similar to protection, the above mentioned tomato fruits were inoculated with fungal blocks and cultured under the above conditions. After 24 h, the above mentioned concentration solution was taken out and sprayed on the tomato fruits for further culture. After 48 h, the size of diseased spots was measured. The protective and therapeutic effects were calculated by the following formula (3): (3) $$\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{c}\mathrm{y}\left(\mathrm{\%}\right)=\left(D_{\mathrm{c}}-D_{\mathrm{t}}\right)/D_{\mathrm{c}}\times 100$$

Where D_c is the diameter of disease spots in the control group; and D_t is the spot diameter in the treatment group.

Statistical analysis

The statistical software SPSS (V 20.0; Chicago, IL, USA) was used for all data analyses. Data were analyzed by Duncan’s multiple range test at the level P < 0.05.

Inhibitory activities of 17 plant EOs on mycelial growth of B. cinerea

The antifungal activities of 17 plant EOs against B. cinerea are shown in Fig. 1. C. cassia, L. cubeba and O. vulgare EOs completely inhibited the mycelial growth at the concentration of 0.5 mg/mL. In addition, the inhibition rate of M. spicata and S. aromaticum EOs reached 91.70 and 87.55%, respectively. At the concentration of 2 mg/mL, the inhibition rate of M. haplocalyx, I. verum and A. sieboldii EOs was 100%, 99.56% and 82.53%, respectively.

GC–MS analysis of OVEO

In total, 21 different chemical components are identified from the OVEO (Table 2; Fig. 2). Carvacrol (89.98%) was found to be the major component, followed by β-caryophyllene (3.34%), thymol (2.39%), α-humulene (1.38%) and 1-methyl-2-propan-2-ylbenzene isopropyl benzene (1.36%). In addition, the isomers carvacrol and thymol had adjacent peaks.

Antifungal activities of the OVEO, carvacrol and thymol against mycelial growth of B. cinerea in vitro

The effects of the OVEO and its main components on B. cinerea are shown in Table 3. Among the four main constituents, OVEO, carvacrol and thymol could significantly inhibit the mycelial growth of B. cinerea, where the effect increased with increasing the concentration, whereas β-caryophyllene showed no inhibitory effects on B. cinerea. The EC₅₀ of carvacrol and thymol were 9.09 and 21.32 μg/mL, respectively, lower than that of OVEO (140.04 μg/mL). Carvacrol and thymol as main antifungal components have been demonstrated, and were chosen for further study.

Antifungal activities of OVEO, carvacrol and thymol on spore germination of B. cinerea

Carvacrol and thymol at all tested concentrations (50–300 μg/mL) result a significant low germination of B. cinerea spores compared with the untreated ones (Fig. 3). Thymol at 250 μg/mL and carvacrol at 300 μg/mL can completely inhibit spore germination. However, the low concentration of OVEO had no significant effect on the spore germination rate of B. cinerea. The spore germination was 80.03% at the concentration of 300 μg/mL.

Effects of carvacrol and thymol on the mycelial morphology of B. cinerea

The mycelial morphology of B. cinerea treated with carvacrol and thymol at the concentration of 40 μg/mL is observed by SEM (Fig. 4). There were regular, uniform and complete mycelia with smooth surfaces in the control group, while the mycelia treated with carvacrol and thymol showed great morphological changes, including irregular growth of mycelium, formation of verrucous surface, shrinkage, collapse and hollowing of hyphae.

Effects of carvacrol and thymol on fresh and dry mycelium weight of B. cinereal

The mycelial growth of B. cinerea in PDB medium containing carvacrol and thymol for 3 d is significantly inhibited (Fig. 5). The fresh and dry weights of the mycelia treated with carvacrol were 21.93 and 5.83 mg, 8.30 and 4.13 mg, respectively and had EC₅₀ of 9.09 μg/mL and EC₉₀ of 40.62 μg/mL. The inhibition rates of fresh and dry weights were 86.88%, 96.51% and 78.53%, 89.31%, respectively. After being treated with thymol, the mycelial inhibitions of fresh and dry weights were 78.06% and 98.23%, 69.94% and 95.97%, respectively where the EC₅₀ reached 21.32 μg/mL and EC₉₀ reached 65.13 μg/mL.

Effects of carvacrol and thymol on cell leakage of B. cinerea

A linear relative conductivity response with the increasing concentration of three reagents are found in all treatments (Fig. 6). Compared with the blank control, the two treatments with carvacrol and thymol maintained a higher level of relative conductivity. When carvacrol and thymol were at the lowest concentrations of 10 μg/mL and 70 μg/mL, at the treatment time points of 10, 40, 140, 220, 360 and 400 min, the relative conductivity values were 2.98%, 10.26%, 32.48%, 37.97%, 47.68%, 51.51% and 10.87%, 15.41%, 23.58%, 28.81%, 37.87%, 39.83%, respectively. The relative conductivity values were comparable to that of the positive control (vinclozolin) group at the same time. There was a higher relative conductivity values when carvacrol and thymol increased to 200 μg/mL and 500 μg/mL, compared with that of the vinclozolin. This indicates that there was more leakage of cell contents.

Effects of carvacrol and thymol on reactive oxygen species (ROS) accumulation, fungal membrane integrity and mitochondrial injury

The results of ROS accumulation, fungal membrane integrity and mitochondria damage experiments are presented in Figs. 7–9. Compared with the untreated hyphae, the treated hyphae gave off strong green fluorescence, indicating there was a large amount of ROS accumulation in the mycelium (Fig. 7). SYTOX Green penetrated the mycelium treated with carvacrol and thymol, indicating that the integrity of the mycelial membrane was damaged (Fig. 8). Compared with normal mitochondria, rhodamine 123, which entered the mitochondria from the outside, was re-released after the membrane was damaged, emitting yellow-green fluorescence (Fig. 9).

Application of fungicides and disease assessment in vivo

The results of in vivo conditions showed that OVEO, carvacrol and thymol have different protective and therapeutic effects on tomato gray mold caused by B. cinerea (Table 4). At the concentration of 500 μg/mL, the protective and therapeutic effects of OVEO were significantly lower than pyrimethanil at 400 μg/mL. Although the protective effect of carvacrol was comparable to pyrimethanil, its therapeutic effect was lower. The therapeutic and protective effects of thymol were lower than that of the chemical control. At the concentration of 1,000 μg/mL, OVEO and thymol had the same protective and therapeutic effects as 400 μg/mL pyrimethanil. The protective effect of carvacrol was significantly higher than pyrimethanil while the therapeutic effect was similar to that of the chemical control. Among all the treatment groups, the protective effect of carvacrol (77.98%) was the best.