Composition, Antifungal, Phytotoxic, and Insecticidal Activities of Thymus kotschyanus Essential Oil.

Essential oils (EOs) are some of the outstanding compounds found in Thymus that can exert antifungal, phytotoxic, and insecticidal activities, which encourage their exploration and potential use for agricultural and food purposes. The essential oils (EO) obtained from Thymus kotschyanus collected in the East Azerbaijan Province (Iran) were characterized using a gas chromatography-mass spectrometry (GC-MS) analysis. Thymol was the most important compound (60.48%), although 35 other active compounds were identified in the EO. Significant amounts of carvacrol (3.08%), p-cymene (5.56%), and γ-terpinene (6.67%) were found in the EO. The T. kotschyanus EO was tested against important phytopathogenic fungi (Botrytis cinerea, Aspergillus niger, and Penicillium expansum). The antifungal assay showed that the use of ≥500 ppm of EO resulted in a fungicidal effect against all funguses tested. In a similar way, the use of ≥500 ppm of EO inhibited the germination of all crop weed seeds (Amaranthus retroflexus L. and Panicum miliaceum L.) and their subsequent growth, which demonstrated its herbicidal effect. Finally, the insecticidal capacity of T. kotschyanus EO was also observed against selected insects (Oryzaephilus surinamensis and Sitophilus oryzae). O. surinamensis was more susceptible to the effect of EO (LC50 = 4.78 µL/L air) than S. oryzae (LC50 = 13.20 µL/L air). The obtained results of the present study can provide new safe resources to the development of new products for the food, agriculture, and pharmaceutical industries.


Introduction
The Genus Thymus L., family Labiatae, consists of more than 215 herbaceous perennial species mainly distributed in the Mediterranean region [1,2]. In Iran, fourteen species of Thymus L. are known [3]. Thymus kotschyanus represents one of the most diffused Thymus species. It is exploited mainly, but not only, in folk medicine to improve the digestive process and to treat respiratory disorders. Furthermore, its use as an aromatic ingredient, spice, or to prepare an herbal tea is
The yield of EO and thymol content found in this study were significantly higher compared to other previous studies evaluating T. kotschyanus. Therefore, T. kotschyanus collected from the Shabestar region could be identified as a new thymol chemotype. For instance, other authors found variable amounts in T. kotschyanus that ranges from 1.1% in plants collected in Yarz (Iran) [52] to 54.66% in plants collected in the highlands of Bojnurd (Iran) at an altitude of 1700 m [8]. In addition, Tohidi et al. [53] analyzed the EOs of ten Thymus species from different areas of Iran and reported that thymol contents ranged from 12.4% (EO of T. fedtschenkoi) to 79.74% (EO of T. migricus).

Antifungal Activity
The antifungal activity of EOs against mycelial growth is shown in Figure 2. Among the fungal strains tested, B. cinerea ( Figure 3A) was the most susceptible fungus, and it was completely inhibited by all EO treatments. In the cases of A. niger ( Figure 3B) and P. expansum ( Figure 3C), mycelial growth was reduced at 250 ppm, being completely inhibited at concentrations of ≥500 ppm (fungicidal effect). In addition, the effects of EOs at 250 ppm were considered fungistatic for P. expansum and A. niger that were partially inhibited.

Antifungal Activity
The antifungal activity of EOs against mycelial growth is shown in Figure 2. Among the fungal strains tested, B. cinerea ( Figure 3A) was the most susceptible fungus, and it was completely inhibited by all EO treatments. In the cases of A. niger ( Figure 3B) and P. expansum ( Figure 3C), mycelial growth was reduced at 250 ppm, being completely inhibited at concentrations of ≥500 ppm (fungicidal effect). In addition, the effects of EOs at 250 ppm were considered fungistatic for P. expansum and A. niger that were partially inhibited.
Some components of the EO, such as thymol, γ-terpinene, and carvacrol, display strong antifungal activity [53][54][55][56][57]. Particularly for thymol, a monoterpene phenolic compound, previous studies indicated an important effect against the growth of important fruit and food-spoiling fungi, such as A. niger, Alternaria alternata, B. cinerea, Fusarium oxysporum, and Rhizopus oryzae, that are [58][59][60][61][62]. In a similar way as the results obtained by us, other studies observed that thymol and carvacrol exhibit an important antifungal activity against postharvest pathogens Botrytis cinerea [63] and also against spoilage yeasts in wine [64]. Additionally, other research found that carvacrol inhibits the growth of Penicillium expansum spores, which agree with our results [65]. The protective effect of T. kotschyanus EO can be explained by irreversible damage to fungi membrane and the consequent leakage of the cytoplasmic contents [64], although the effects of other minor components should not be overlooked. Some components of the EO, such as thymol, γ-terpinene, and carvacrol, display strong antifungal activity [53][54][55][56][57]. Particularly for thymol, a monoterpene phenolic compound, previous studies indicated an important effect against the growth of important fruit and food-spoiling fungi, such as A. niger, Alternaria alternata, B. cinerea, Fusarium oxysporum, and Rhizopus oryzae, that are [58][59][60][61][62]. In a similar way as the results obtained by us, other studies observed that thymol and carvacrol exhibit an important antifungal activity against postharvest pathogens Botrytis cinerea [63] and also against spoilage yeasts in wine [64]. Additionally, other research found that carvacrol inhibits the growth of Penicillium expansum spores, which agree with our results [65]. The protective effect of T. kotschyanus EO can be explained by irreversible damage to fungi membrane and the consequent leakage of the cytoplasmic contents [64], although the effects of other minor components should not be overlooked.

Phytotoxic Activity
The effect of EOs on weeds germination indices is shown in Table 2. In both weed species, germination and growth indices were affected by T. kotschyanus EO treatments. The final germination percentage (GP) varied significantly (p < 0.01) among the different EO concentrations used. For example, seed germination of A. retroflexus ( Figure 4A) and P. miliaceum ( Figure 4B) were completely inhibited when the concentrations exceeded 500 ppm. Likewise, the percentage germination of A. retroflexus was 81.33% and 0% for control and essential oils more than 500 ppm, respectively. For P. miliaceum, such index was 92.00% and 0% for control and EO more than 500 ppm, respectively. Moreover, mean germination time was significantly (p < 0.01) influenced by T. kotschyanus EO concentration.  Control ≥500 ppm *** *** *** *** *** *** *** A-C mean values not followed by a common letter differ significantly (*** significant at p < 0.001).  Moreover, it was also found that several concentrations of EOs had a significant effect (p < 0.01) on the fresh weight (FW) of seedlings. FW of samples treated with EOs were significantly lower than control for both weeds. Likewise, the vigor index (VI) was affected by EO concentration in both weeds. While the highest VI index was obtained in control (248.33 and 803.67 units for A. retroflexus and P. miliaceum, respectively), the lowest means values were observed in treatments with more than 500 ppm (0.00 units) of EO. The time to achieve 50% germination (T50 index) varied between the treatments for both studied species. The T50 index increased from 2.57 (control) to 4.17 (250 ppm) days for A. retroflexus seeds and from 1.86 (control) to 3.06 (250 ppm) days. Seeds treated with more than 500 ppm of T. kotschyanus EO did not germinate. The results obtained for T. kotschyanus EO phytotoxic activity are in agreement with other studies in scientific literature. An experiment with four Thymus daenensis ecotypes collected in Iran indicated that applying between 400 and 600 µL/L of EO was associated with complete inhibition of A. retroflexus seed GP, which consequently reduced the shoot and root fresh weight [66]. In the same line, the EO extracted from Thymus vulgaris displayed one of the lowest ED50 (concentration that causes 50% inhibition of seed germination; 0.16 g/L) values to inhibit the germination of A. retroflexus seeds among selected essential oils (lemon balm, sage, and tansy, for instance) [67]. The GRI was significantly (p < 0.01) influenced by EO concentration, being the highest value found for control (6.50 and 9.65 units for A. retroflexus and P. miliaceum, respectively), and the lowest was obtained from EO treatments with 500, 750, 1000, and 1500 ppm (0.00 units) for both A. retroflexus and P. miliaceum. A similar outcome was obtained for root length (RL) and shoot length (ShL). The seeds treated with T. kotschyanus EO displayed lower mean values than obtained in the control. The complete inhibition of root and shoot lengths were observed for seeds treated with ≥500 ppm of T. kotschyanus EO. Statistically significant differences (p < 0.01) among treatments were also observed in the seedling length (SLL) in both weeds. The highest length was observed in control (3.05 and 8.87 cm), while the lowest values were obtained after the exposition of seeds to more than 500 ppm of EO (0.00 cm) for A. retroflexus and P. miliaceum, respectively. The EOs decreased (p < 0.01) the SLL in a concentration-dependent manner, according to the weeds.
Moreover, it was also found that several concentrations of EOs had a significant effect (p < 0.01) on the fresh weight (FW) of seedlings. FW of samples treated with EOs were significantly lower than control for both weeds. Likewise, the vigor index (VI) was affected by EO concentration in both weeds. While the highest VI index was obtained in control (248.33 and 803.67 units for A. retroflexus and P. miliaceum, respectively), the lowest means values were observed in treatments with more than 500 ppm (0.00 units) of EO. The time to achieve 50% germination (T50 index) varied between the treatments for both studied species. The T50 index increased from 2.57 (control) to 4.17 (250 ppm) days for A. retroflexus seeds and from 1.86 (control) to 3.06 (250 ppm) days. Seeds treated with more than 500 ppm of T. kotschyanus EO did not germinate. The results obtained for T. kotschyanus EO phytotoxic activity are in agreement with other studies in scientific literature. An experiment with four Thymus daenensis ecotypes collected in Iran indicated that applying between 400 and 600 µL/L of EO was associated with complete inhibition of A. retroflexus seed GP, which consequently reduced the shoot and root fresh weight [66]. In the same line, the EO extracted from Thymus vulgaris displayed one of the lowest ED50 (concentration that causes 50% inhibition of seed germination; 0.16 g/L) values to inhibit the germination of A. retroflexus seeds among selected essential oils (lemon balm, sage, and tansy, for instance) [67].
The allelopathic effect of T. kotschyanus EO on A. retroflexus and P. miliaceum seeds could be explained by the individual activity of monoterpenes. A study about the phytotoxic effect of pure thymol (10 mg/Petri dish) reported GP of 0.00% and root growth of 0.00% in [68]. Likewise, the treatment with γ-terpinene inhibited the GP (76.5 vs. 32.0 and 21.3% for control and 10 and 20 µL, respectively) and seedling root growth (28.9 vs. 22.0 and 14.5 mm for control and 10 and 20 µL, respectively) of A. retroflexus seeds [69].
Moreover, seems reasonable to consider that these two monoterpenes are the main compounds associated with T. kotschyanus EO allelopathic activity due to low phytotoxic activity reported for p-cymene on A. retroflexus, Chenopodium album, and Rumex crispus seeds [68]. Although the inhibitory mechanism exerted by terpenes on weed seeds remains unclear, previous studies reported relevant effects on cellular proliferation, induction of oxidative stress, and inhibition of DNA synthesis on weed seeds after terpene treatment [70,71].

Insecticidal Activity
The T. kotschyanus EO was lethal for both insect species (O. surinamensis and S. oryzae) used in the experiments. O. surinamensis was more susceptible to the effect of EOs than S. oryzae, wherein the values of LC 50 were 4.78 and 13.20 µL/L air, respectively (Table 3).
Moreover, LT 50 values highlighted that the EOs killed O. surinamensis faster than S. oryzae. Cumulative mortality of O. surinamensis ( Figure 5A) and S. oryzae ( Figure 5B) increased daily. Half the population of O. surinamensis adults died within 1.57 days, and 95% of insects were killed within 9.17 days after exposure to EO vapors. However, 50 percent and 95 percent of adult rice weevils were killed after 2.36 and 14.60 days of treatment, respectively (Table 4 and Figure 6).
These results are in agreement with data reported by other authors. For instance, the fumigant toxicity effects of commercial thyme EO and selected terpenes, particularly p-cymene and thymol, were evaluated against S. oryzae [72]. The authors obtained LC 50 and LC 95 concentrations of 63.9 and 89.5 µL/L air for thyme EO, respectively. Regarding the individual terpenes, p-cymene displayed lower LC 50 and LC 95 concentrations (25 and 39 µL/L air, respectively) than those obtained for thymol (69 and 174 µL/L air, respectively). A similar fumigant toxicity effect of thyme EO was reported against Callosobruchus maculatus and Sitophilus granaries (relevant pests in the storage of legumes and wheat). In this case, the EO extracted from T. daenensis Celak displayed LC 50 concentrations of 4.22 and 6.55 µL/L air for C. maculatus and S. granaries, respectively. The authors also obtained the LC 90 concentrations for C. maculatus and S. granaries (8.21 and 8.73 µL/L air, respectively).  These results are in agreement with data reported by other authors. For instance, the fumigant toxicity effects of commercial thyme EO and selected terpenes, particularly p-cymene and thymol, were evaluated against S. oryzae [72]. The authors obtained LC50 and LC95 concentrations of 63.9 and 89.5 µL/L air for thyme EO, respectively. Regarding the individual terpenes, p-cymene displayed lower LC50 and LC95 concentrations (25 and 39 µL/L air, respectively) than those obtained for thymol (69 and 174 µL/L air, respectively). A similar fumigant toxicity effect of thyme EO was reported against Callosobruchus maculatus and Sitophilus granaries (relevant pests in the storage of legumes and wheat). In this case, the EO extracted from T. daenensis Celak displayed LC50 concentrations of 4.22 and 6.55 µL/L air for C. maculatus and S. granaries, respectively. The authors also obtained the LC90 concentrations for C. maculatus and S. granaries (8.21 and 8.73 µL/L air, respectively).
The key role of T. kotschyanus EO compounds in the mortality of crop pests is also associated with impairment of important molecular pathways. Particularly for thymol, it was reported that this terpene can influence the GABA-gated chloride channel, which causes hyperexcitation of the central nervous system and can lead to convulsions and death. Another related effect of thymol exposure is modulation of a tyramine receptors cascade that eventually blocks the octopamine receptors and undermines neurological insect functions [73]. Finally, our results illustrated that T. kotschyanus has an insecticidal activity against insect pests.  Figure 6. Evolution of pest mortality (%) exposed to 3 µL/L air of T. kotschyanus essential oil.

Plant Materials
Aerial parts of Thymus kotschyanus plants (voucher number: UHDH-101) in the flowering stage were taken from the Shabestar Region in the East Azerbaijan Province, Iran in 2016. Having a classically semi-arid climate and at the altitude of 1352 m above the sea level (Latitude: 38°19′ N; Longitude: 45°18′ E), this region has a high precipitation rate that often takes place throughout the autumn and winter, whereas there is little rainfall in the summer. Figure 6. Evolution of pest mortality (%) exposed to 3 µL/L air of T. kotschyanus essential oil.

Extraction of Essential Oil
The key role of T. kotschyanus EO compounds in the mortality of crop pests is also associated with impairment of important molecular pathways. Particularly for thymol, it was reported that this terpene can influence the GABA-gated chloride channel, which causes hyperexcitation of the central nervous system and can lead to convulsions and death. Another related effect of thymol exposure is modulation of a tyramine receptors cascade that eventually blocks the octopamine receptors and undermines neurological insect functions [73]. Finally, our results illustrated that T. kotschyanus has an insecticidal activity against insect pests.

Plant Materials
Aerial parts of Thymus kotschyanus plants (voucher number: UHDH-101) in the flowering stage were taken from the Shabestar Region in the East Azerbaijan Province, Iran in 2016. Having a classically semi-arid climate and at the altitude of 1352 m above the sea level (Latitude: 38 • 19 N; Longitude: 45 • 18 E), this region has a high precipitation rate that often takes place throughout the autumn and winter, whereas there is little rainfall in the summer.

Extraction of Essential Oil
Aerial parts of T. kotschyanus were harvested and dried at room temperature, preventing them from taking light. Dried leaves (20 g) were subjected to hydrodistillation through a Clevenger instrument (Urmia University, Urmia, Iran) for 3 h. The resulting EO was poured into screw-capped vials and maintained in darkness at 4 • C for further analysis.

GC and GC-MS Analysis
The analysis of the oil was performed using an Agilent gas chromatograph (GC-FID) (Agilent Technologies, Santa Clara, CA, USA) with a DB-5-fused silica column (30 m × 0.25 mm; 0.25 µm film thickness). Nitrogen was used as the gas carrier at a constant flow of 1.1 mL/min. The oven temperature was programmed from 60 to 250 • C at 5 • C/min and then isothermaled for 10 min. The injector and FID temperatures were set at 250 • C and 280 • C, respectively. The injection volume was 0.1 mL. Samples were injected by splitting, and the split ratio was 1:100. GC-MS analysis was carried out on a Thermoquest Finnigan Trace GC-MS instrument equipped with a DB-5 column (30 m × 0.25 mm; 0.25 µm film thickness) programmed as above, with helium as the carrier gas with a flow rate of 1.1 mL/min and a split ratio of 1:50. The MS operating parameters were: ionization voltage, 70 eV and ion source temperature, 200 • C. Identification of the compounds was performed by comparison of the retention indexes (relative to a homologue C 6 -C 24 n-alkane series) obtained in the same column with those of reference compounds. Additionally, each mass spectra obtained was compared with those from the usual electronic libraries [74,75]. Relative area percentages obtained from GC were used for quantification of the components.

Fungal Isolates
Three post-harvest pathogens fungal isolates, Bot-245 g (Botrytis cinerea Pers.), Pen-653mb (Penicillium expansum Link), and As-88ma (Aspergillus niger Tiegh.), purchased from the fungal culture collection of the Plant Pathology Department, Urmia University (Urmia, Iran) were used in our experiments. The pathogenicity of fungal isolates was previously confirmed (data not shown). Fungal isolates were grown in potato dextrose agar (PDA) medium at 25 ± 2 • C. Only actively growing colonies were used in bioassays [76].

In vitro Antifungal Assays
The antifungal activity of the T. kotschyanus essential oil was evaluated through the poison food medium method. Different concentrations of T. kotschyanus essential oil (0, 250, 500, 750, 1000, and 1500 µL/L) were prepared in sterile water containing Tween 80 (0.5%, v/v) and aseptically added to sterile, cooled, molten potato dextrose agar (PDA Merck, Darmstadt, Germany) medium (45 • C). The resulting mixture (EO plus medium) were instantly dispensed onto sterilized glass petri plates (90 mm diameter, 20 mL each) and allowed to solidify under aseptic conditions. A mycelial disk (6-mm-diameter) of the tested fungi, taken from the margins of the actively growing cultures, was placed upside-down at the center of the petri plates.
Inoculated petri plates were incubated in darkness at 25 ± 2 • C. The control was composed of 0.5%Tween 80 in sterile water. Four replicates were used for each treatment, and all the experiments were repeated twice. Antifungal activity of essential oil was measured taking into account the percentage of the mycelial growth inhibition (MGI), calculated by the following the formula [77]: where dc was the colony growth diameter in the control and dt represented the diameter of colony growth in the treatment.
In the case of no visible growth detected after the incubation with the essential oil, and in order to determine fungistatic and/or fungicidal effects of the essential oil against the tested fungi, the inoculated discs were transferred to new PDA plates and incubated again at 25 ± 2 • C for more than 72 h. If mycelial growth was restarted in PDA medium, the effect was considered fungistatic; otherwise, it was considered fungicidal.

Phytotoxic Activity
Ripe seeds of Amaranthus retroflexus L. (redroot pigweed) and Panicum miliaceum L. (millet) collected from the North West of Iran (West Azerbaijan Province) on September, 2016 were used to investigate the phytotoxic effect exhibited by the EO. Seeds were stored in paper bags for a span of four weeks at room temperature. The viability of the seeds and their germinability were checked prior to the experiments. Surfaces of seeds were sterilized through a two-step procedure (rinse for 30 s with 70% ethyl-alcohol and a subsequent treatment with a 10% sodium hypochlorite solution for 20 min), then washed three times with sterile distilled water, and finally, air-dried in aseptic conditions under a laminar hood. Fifty seeds from each weed were placed in Petri dishes containing two layers of filter-paper (Whatman No. 2). To make exact concentrations of EO in water (0, 250, 500, 750, 1000, and 1500 µL/L), first a stock of EO in dimethyl sulfoxide (DMSO)/water (1%, v/v) was prepared. Ultimately, 10 mL of each concentration was poured into the petri dishes. In the controls, 1% DMSO in water was used. Each treatment had five replicates, and all the experiments were replicated twice. The petri dishes containing seeds were sealed by plastic paraffin film tape. Then, petri dishes were kept in a germinator set at 25 • C with a 16-h photoperiod of 28-36 mM/m 2 s.
In this experiment, germination percentage (GP); mean germination time (MGT); germination rate index (GRI); vigor index (VI); root, shoot, and seedling lengths (RL, ShL, and SLL, respectively); and T50 index were measured. The GP was expressed as the ratio of germinated seed to the total of the seeds. The MGT and GRI were calculated using the following formula [78]: where n is the number of seeds germinated on each day, d is the days from the beginning of the germination test, and N represents the final germinated seeds. The GRI: GRI = (number of germinated seeds since n − 1) n where n represents the days of incubation. At the end of the incubation, root, shoot, and seedling lengths were also measured, and the seed vigor index (VI) was obtained using the equation [79]: The T50 value was calculated in terms of days needed for germination of 50% of the seeds.

Insecticidal Activity
The fumigant toxicity of T. kotschyanus EO was assessed using two model insect species, which are mainly the infesting of food products during their storage, namely the saw-toothed grain beetle (Oryzaephilus surinamensis) and the so-called rice weevil (Sitophilus oryzae). LC 50 (median lethal concentration) and LT 50 (median lethal time) values were used as parameters to calculate the insecticidal strength of the EO. After preliminary bioassays, 6-cm-diameter disks of filter papers (Whatman No. 1) were impregnated with different concentrations of the essential oil (2-87 µL/L air for S. oryzae and 2-12.6 µL/L air for O. surinamensis) without any solvent. The disks were mounted on the underside of tightly screwed caps of 250 mL glass vials. Ten newly emerged adults of each insect were introduced into each vial. Combinations of different concentrations and exposure times (1-7 days) were replicated five times. In the controls, only filter papers were used. Vials were kept in darkness, 70% ± 5% RH and 28 ± 1 • C. Mortality percentage was recorded at 24-h intervals until 7 days. Insects with no reaction after physical stimulation (leg or antennal movements) were considered as dead.

Statistical Analysis
Statistical analyses of the data (antifungal and phytotoxic activity) were performed using MSTAT-C statistical software (Michigan State University, East Lansing, MI, USA), and means were separated by DMRT at 0.01 probability level. In the case of insecticidal activity, all the experiments were replicated five times. Data were analyzed using SPSS V22.0 software (IBM, Armonk, NY, USA).

Conclusions
The analysis of T. kotschyanus EO (yield around 3.5%) composition collected in the Shabestar Region (Iran) revealed that thymol is the main compound, followed by γ-terpinene and p-cymene. Moreover, our results also showed that T. kotschyanus EOs can be considered as an efficient natural compound to control post-harvest fungal diseases (A. niger and P. expansum; fungicidal effect at concentration ≥500 ppm); weeds (A. retroflexus and P. miliaceum; ≥500 ppm); and harmful insects (O. surinamensis and S. oryzae). Therefore, taking into account the several issues related to the harmful effects on the environment and on the health associated with synthetic pesticides, and the remarkable fungicidal properties of T. kotschyanus active components, the EO evaluated in the present study can be certainly considered as a good alternative in the post-harvest pest management.