Study of Allelopathic Interaction of Essential Oils from Medicinal and Aromatic Plants on Seed Germination and Seedling Growth of Lettuce

Medicinal and aromatic plants have the ability to transmit volatile allelochemicals and affect their surrounding organisms. In this regard, their interaction should also be considered. The inhibitory effects of 112 essential oils on lettuce seed and seedling were investigated by cotton swab method. Germination (G%), Mean germination time (MGT), Lethal of embryo (L%), dormancy (D%), radicle growth (R%), and hypocotyl growth (H%) were measured. Two methods were used for evaluating allelopathic interaction effects: the simplified modified dilution check-board technique (SMCT) and the isobologram. Thymus daenensis had the highest inhibitory effect on G% (IC50 = 2.9 ppm) and the most lethal effect on the embryo (LC50 = 7.2 ppm). Thymus transcaspicus, Dracocephalum moldavica, Artemisia sieberi and Amomum subulatum had the greatest effect on MGT. Ziziphora tenuior, Trachyspermum ammi and Pelargonium graveolens had the highest effect on D%. Origanum vulgare was the strongest growth inhibitor. The highest synergistic effect on G% was in A. subulatum + Mentha suaveolens, on H% was related to Perovskia abrotanoides + T. daenensis, and on R% was observed in Artemisia vulgaris + M. suaveolens. The results of this study can lead to identification of new phytotoxic compounds in EOs and control weeds more effectively.


Introduction
Growth interactions between weeds and crops lead to high costs for agricultural systems [1]. Herbicides have always played a key role in weed management. This event is one of the major causes of environmental damage and public health, leading to environmental pollution, product insecurity and human health hazards [2,3]. Furthermore, every year, a new list of pesticide-resistant weeds is released. According to the 2017 report by the International Herbicide-Resistant Weed site, a list of 36 new cases of weed resistance to herbicides was released [4]. This causes us to always look for new herbicides to control resistant weed populations.
Today, the global effort in modern agriculture is to reduce the use of harmful pesticides by introducing new biological and ecological methods. One of these methods is the use of chemical interactions among plants [5]. Interactions between plants in a common ecosystem are side effects that each plant exerts on its neighboring plants, and these include competition and allelopathy. Competition involves the active absorption of limited resources by one organism, which leads to a decrease in supply and thus to the growth inhibition of other organisms, but when one species stops growing due for 1 to 3 days). EO extraction was done by hydro distillation method for 4 h using Clevenger-type apparatus at a distillation rate of 3 mL/min according to the European Pharmacopoeia method [21]. The EOs were collected in dark sealed air-tight glass vials, dried with anhydrous sodium sulfate and stored in the refrigerator at 4 • C.

Cotton Swab Method
To evaluate the phytotoxicity of volatile constituents on germination, 112 EOs were evaluated in two different amounts of 1 and 3 µL in vial compared to the control. The experiment was conducted as a factorial experiment in completely randomized design with four replications. Allelopathic effects of volatile compounds were evaluated by cotton swab method [22] (Figure 1). For this purpose, 20 mL glass vials were disinfected in the oven after washing. The 0.75% (w/v) agar solution was prepared using agar powder (Merck Inc., Kenilworth, NJ, USA) and twice distilled water and then sterilized in autoclave. Due to the high sensitivity of agar volume precision in the vial, the vial filling step was carried out after sterilization and when the agar temperature reached about 40 • C. Using a graduated pipette, 10 mL of agar (equal to half the volume of vial) was poured into each vial. After the agar is cooled and solidified, seven lettuce seeds (Lactuca sativa, cultivar Great Lakes No. 366) were placed on the agar so that one third of the seed tip was immersed in the agar. Then a double-tipped cotton swab was cut in half and placed in the center of the agar so that its cotton tip was in the middle of the void above the agar surface. The EO was injected into a cotton swab in a specified amount (1 or 3 µL) using a Hamilton capillary syringe. Immediately, the rubber cap and aluminum seal were placed on the vial lid and sealed with Crimper ( Figure 1). The vials were placed in a germinator at 21 ± 2 • C under dark conditions. The control vials were filled with agar, seeds were sown in, the swabs were laid, then were sealed and incubated just like other vials. The only difference with the treated vials was that the swabs were not injected with essential oils.
Agronomy 2019, 9, x FOR PEER REVIEW 3 of 26 the volatile compounds, the plants were dried according to the type of tissue (in oven at 30-60 °C for 1 to 3 days). EO extraction was done by hydro distillation method for 4 hours using Clevenger-type apparatus at a distillation rate of 3 ml/min according to the European Pharmacopoeia method [21]. The EOs were collected in dark sealed air-tight glass vials, dried with anhydrous sodium sulfate and stored in the refrigerator at 4 °C.

Cotton Swab Method
To evaluate the phytotoxicity of volatile constituents on germination, 112 EOs were evaluated in two different amounts of 1 and 3 μL in vial compared to the control. The experiment was conducted as a factorial experiment in completely randomized design with four replications. Allelopathic effects of volatile compounds were evaluated by cotton swab method [22] (Figure 1). For this purpose, 20 mL glass vials were disinfected in the oven after washing. The 0.75% (w/v) agar solution was prepared using agar powder (Merck Inc., Kenilworth, NJ, USA) and twice distilled water and then sterilized in autoclave. Due to the high sensitivity of agar volume precision in the vial, the vial filling step was carried out after sterilization and when the agar temperature reached about 40 °C. Using a graduated pipette, 10 mL of agar (equal to half the volume of vial) was poured into each vial. After the agar is cooled and solidified, seven lettuce seeds (Lactuca sativa, cultivar Great Lakes No. 366) were placed on the agar so that one third of the seed tip was immersed in the agar. Then a doubletipped cotton swab was cut in half and placed in the center of the agar so that its cotton tip was in the middle of the void above the agar surface. The EO was injected into a cotton swab in a specified amount (1 or 3 µL) using a Hamilton capillary syringe. Immediately, the rubber cap and aluminum seal were placed on the vial lid and sealed with Crimper ( Figure 1). The vials were placed in a germinator at 21 ± 2 °C under dark conditions. The control vials were filled with agar, seeds were sown in, the swabs were laid, then were sealed and incubated just like other vials. The only difference with the treated vials was that the swabs were not injected with essential oils. Each experiment unit consisted of a vial containing seven seeds in which germination status was monitored daily for five days and germination-related traits were measured.

Germination and Seed Traits
Germination percentage (G%) [23]: G%: germination percentage; N: total germinated seeds by the end of experiment; S: total seeds Each experiment unit consisted of a vial containing seven seeds in which germination status was monitored daily for five days and germination-related traits were measured.

Germination and Seed Traits
Germination percentage (G%) [23]: G%: germination percentage; N: total germinated seeds by the end of experiment; S: total seeds. Mean germination time (MGT) [23]: MGT: Mean germination time; N i : number of germinated seeds on day T i ; T i : day during germination period; N: Total germinated seeds by end of experiment. These traits were reported relative to control: V%: relative value of trait; V T : average trait of treatment; V C : average trait of control. Lethal percentage of seed embryo (L%) and seed dormancy induction (D%); for this purpose, the seeds that did not germinate after five days were cultured in vial without EO for another three days. Seeds that did not germinate after this time were uncoated and immersed in 1% tetrazolium solution (pH = 7) and incubated for 6 h at 30 • C. The endosperm and embryo status were examined using a microscope and the lethality was determined (Formula no. 4). Only seeds that were completely and uniformly colored were considered as live seeds according to the ISTA instructions [24] (Figure 2). With respect to the number of germinated seeds and L%, some seeds were neither germinated nor showed sign of embryo death indicating that they were dormant-induced seeds (Formula no. 5): L%: Lethal percentage; n: non-colored seeds; S: total seeds. Mean germination time (MGT) [23]: MGT: Mean germination time; Ni: number of germinated seeds on day Ti; Ti: day during germination period; N: Total germinated seeds by end of experiment These traits were reported relative to control: V%: relative value of trait; VT: average trait of treatment; VC: average trait of control Lethal percentage of seed embryo (L%) and seed dormancy induction (D%); for this purpose, the seeds that did not germinate after five days were cultured in vial without EO for another three days. Seeds that did not germinate after this time were uncoated and immersed in 1% tetrazolium solution (pH = 7) and incubated for 6 h at 30 °C. The endosperm and embryo status were examined using a microscope and the lethality was determined (Formula no. 4). Only seeds that were completely and uniformly colored were considered as live seeds according to the ISTA instructions [24] (Figure 2). With respect to the number of germinated seeds and L%, some seeds were neither germinated nor showed sign of embryo death indicating that they were dormant-induced seeds (Formula no. 5): D%: dormancy percentage; S: total seeds; N: total germinated seeds by the end of experiment; A: germinated seeds after removal of EO treatment; L: Seeds with dead embryos

Evaluation of Allelopathic effect of EOs on Lettuce Seedling Growth
The experiment was conducted as same as the previous one except that in this experiment germinated seeds were used to investigate the effect of EOs on lettuce seedling growth. Lettuce seeds were germinated 24 h prior to the experiment and those with 2 mm radicle were used. Each test unit was a vial containing five germinated seeds that the EO treatment was applied on and sealed. The vials were incubated at 21 °C under dark conditions for three days.
After three days, seedlings were photographed and radicle and hypocotyl length were measured using Image J software using formula no 6 and 7 respectively.

Evaluation of Allelopathic Effect of EOs on Lettuce Seedling Growth
The experiment was conducted as same as the previous one except that in this experiment germinated seeds were used to investigate the effect of EOs on lettuce seedling growth. Lettuce seeds were germinated 24 h prior to the experiment and those with 2 mm radicle were used. Each test unit was a vial containing five germinated seeds that the EO treatment was applied on and sealed. The vials were incubated at 21 • C under dark conditions for three days.
After three days, seedlings were photographed and radicle and hypocotyl length were measured using Image J software using formula no 6 and 7 respectively.
R%: radicle length percentage; R T : treatment radicle length; R C : control radicle length.

Statistical Analysis
The effect of treatments on different traits was compared and classified according to mean and standard deviation (sd) of each trait. The inhibitory effect of treatment on germination and growth increased some traits (MGT%, D%, L%) and decreased some others (G%, H%, R%, VI%). Intensity of inhibitory effect was considered based on the increase and decrease of these traits compare to the mean, respectively. The severity of inhibition was defined at five levels: from the highest to the least effect respectively, level five (*****) Mean ± 2 sd, level four (****) Mean ± 1.5 sd, level three (***) Mean ± 1 sd, Level two (**) Mean ± 0.5 sd, and level one (*) Mean ± 0.25 sd, respectively. It should be noted that MGT was not defined in some test units due to zero germination percent; for this reason, only EOs with at least three replications of germination above zero percent were compared in this trait. SPSS, Graphpad Prism and Excel were used for statistical analysis and graphing.

Headspace Gas Chromatography-Mass Spectrometry (HS-GC-MS)
Headspace GC-MS was performed to analyze the chemical composition of the strongest samples from the screening stage (experiment 1 and 2). For chemical analysis, 1 microlitre (equivalent to 1 mg) of each EO was added to the tip of a cotton swab, placed in a 20 mL vial then sealed and incubated at 21 • C for 1 h. After 1000 µL of head space from each vial was injected into GC-MS (Shimadzu QP 2010, Tokyo, Japan) using a 5 mL SGE 5MDR-HSV syringe. The injection condition was as follow: equity five capillary columns (30 m × 250 µm × 0.25 µm) and use of helium as carrier gas. GC-MS Operating Conditions: The temperature of the GC cooker was adjusted from 50 to 220 • C with a rise of 3 • C/min, held for 10 min and then increased to 250 • C with a rise of 10 • C/min. The compounds were identified from the mass spectra of NIST/NBS. Mass spectra were recorded at 70 eV with a mass range of 50 to 400 m/z, compared to an internal spectral library (NIST and Wiley). Then they are validated by comparing retention times with authentic standards.

Allelopathic Interaction Effects of EOs
After screening the EOs the strongest inhibitors on G%, L%, R%, H% and S%, which caused the highest level of inhibition at 1 µL, were identified. In order to investigate the allelopathic interaction of these EOs, effective inhibitory and lethal concentration were first determined.
2.6.1. Determination of IC 50 and LC 50 IC 50 or inhibitory concentration (the concentration of the EO that results in 50% inhibition) and LC 50 or effective lethal concentration (the concentration of EO that causes the death of embryo in 50% of the seeds) were determined through applying different amounts of each EO (0.01 µL to 1 µL per vial) so that at least five effective concentrations were in the range of 20% to 80% inhibitory. To increase the accuracy it was necessary to dilute the EOs, which was done with dimethyl sulfoxide (DMSO). The results were presented in ppm and only the void space of each vial was considered to determine the concentration. The results were analyzed using GraphPad Prism 8 software to determine IC 50 and LC 50 . In addition, the values of IC 25 and IC 90 , and LC 25 and LC 90 were calculated with Quick Calcs online software. The most effective EOs were identified after determination of IC 50 and LC 50 .

Essential oils Combinations
Some of the most effective EOs were selected and their combination effects were evaluated. The experiment was conducted as factorial in a completely randomized design with four replications. Simplified modified dilution check-board technique (SMCT) in cotton swab method was used and two different EOs (A and B) were evaluated at their IC 25 concentration. Experiments were performed separately on seed germination, radicle growth, and hypocotyl growth. For germination evaluation the method was similar to the first experiment, and for the radicle and hypocotyl growth it was done like second experiment. The allelopathic interaction effect of the EOs was determined using two methods: determination of the combination effect (CE), and drawing the isobologram curves.
• Combination effect (CE) was calculated using the following equation: In this equation if CE ≥ 1.1 then the interaction was considered as synergistic effect, if 0.9 < CE < 1.1 was then considered as additive effect, and if CE ≤ 0.9 then the interaction was considered as antagonistic effect.

• Isobologram curves
To draw these curves, the inhibitory effect of two EOs (A and B) was considered as the basis, then the concentration of A and B independently leading to similar inhibition was calculated using Quick Calcs software. Thus, there were three concentrations (A alone, B alone, and A or B in combination state) for a fixed amount of inhibition (inhibition caused by the combination of two EOs), using in isobologram curve. This curve can have three different states: (1) no curvature means additive effect, (2) upward curvature means antagonistic effect, and (3) downward curvature means synergistic effect ( Figure 3). with Quick Calcs online software. The most effective EOs were identified after determination of IC50 and LC50.

Essential oils Combinations
Some of the most effective EOs were selected and their combination effects were evaluated. The experiment was conducted as factorial in a completely randomized design with four replications. Simplified modified dilution check-board technique (SMCT) in cotton swab method was used and two different EOs (A and B) were evaluated at their IC25 concentration. Experiments were performed separately on seed germination, radicle growth, and hypocotyl growth. For germination evaluation the method was similar to the first experiment, and for the radicle and hypocotyl growth it was done like second experiment. The allelopathic interaction effect of the EOs was determined using two methods: determination of the combination effect (CE), and drawing the isobologram curves.


Combination effect (CE) was calculated using the following equation: In this equation if CE ≥ 1.1 then the interaction was considered as synergistic effect, if 0.9 < CE < 1.1 was then considered as additive effect, and if CE ≤ 0.9 then the interaction was considered as antagonistic effect.

Isobologram curves
To draw these curves, the inhibitory effect of two EOs (A and B) was considered as the basis, then the concentration of A and B independently leading to similar inhibition was calculated using Quick Calcs software. Thus, there were three concentrations (A alone, B alone, and A or B in combination state) for a fixed amount of inhibition (inhibition caused by the combination of two EOs), using in isobologram curve. This curve can have three different states: (1) no curvature means additive effect, (2) upward curvature means antagonistic effect, and (3) downward curvature means synergistic effect ( Figure 3).

Allelopathic Effects of Essential Oils on Lettuce Seed and Germination Characteristics
All germination traits were significantly affected by plant EO treatment at both amount of 1 and 3 µ L (Table 1).

Germination Percentage (G%)
Most EOs reduced G% and some had no effect on it. In 1 µL, 36 EOs were ineffective on this trait and 76 EOs decreased the index compared to the control. At this amount, the highest degree of reduction (less than 22% compared to control) was observed in 12 EOs of Lamiaceae (Melissa officinalis, Thymus daenensis, Thymus transcaspicus, Origanum vulgare leaf, Ziziphora tenuior, Mentha suaveolens), Geraniaceae (Pelargonium graveolens leaf and flower), Apiaceae (Pimpinella anisum and Trachyspermum ammi), Liliaceae (Allium hirtifolium) and Rutaceae (Ruta graveolens). The inhibitory effects were greater in 3 µL, 89 EOs reduced this trait and 23 EOs were ineffective compared to control. At this amount, the highest degree of reduction (less than 5% compared to control) was observed in 21 EOs of Lamiaceae (Dracocephalum moldavica and Zataria multiflora in addition to six EOs that were effective at 1 µL), Geraniaceae (as same as the EOs that were effective at 1 µL), Apiaceae (Cuminum cyminum, Bunium persicum, and Ferula foetida in addition to two EOs that were effective at 1 µL), Liliaceae (A. hirtifolium) and Rutaceae (R. graveolens and Citrus aurantifolia leaf), Asteraceae (Artemisia sieberi and Tanacetum balsamita), and Zingiberaceae (Amomum subulatum).

Mean Germination Time (MGT)
The EOs also had a significant effect on MGT. The values of this trait were 1.7 to 5 days in 1 µL, 2 to 5 days in 3 µL, and 2.1 days in control. In 1 µL, 95 EOs increased and four EOs decreased this trait compared to control and the rest had no effect. At this amount, the highest increase (more than 224% compared to control) belonged to four EOs of Lamiaceae (T. transcaspicus and D. moldavica), Asteraceae (A. sieberi), and Zingiberaceae (A. subulatum). At 3 µL, 70 EOs increased and 40 EOs reduced MGT compared to control. At this amount, the highest degree of increase (more than 204% compared to control) belonged to nine EOs of Lamiaceae (Rosmarinus officinalis leaf and flower, and Perovskia abrotanoides), Asteraceae (A. wilhelmsii, Artemisia aucheri, and Artemisia scoparia), Cupressaceae (Chamaecyparis lawsoniana and Thuja occidentalis), and Apiaceae (Apium graveolens).

Allelopathic Effects of Essential Oils on Lettuce Seedling Growth
Seedlings growth were also significantly affected by treatments (Table 1).

Radicle Growth (R%)
Radicle length compare to control (R%) was also affected by different amounts of the EOs. The radicle length was 26.5 mm in control, 0.1-28.3 mm in 1 µL, and 0-24.9 mm in 3 µL. The highest radicle inhibitory degree (less than 3.4% compared to control) in 1 µL was observed in two EOs of Lamiaceae (O. vulgare flower and T. daenensis). The only EO that increased radicle growth at 1 µL was coffee EO, which increased 6.9% this trait compared to control. At 3 µL, the highest radicle growth inhibition (less than 0.5% compared to control) belonged to eight EOs of Lamiaceae (L. angustifolia flower, O. vulgare flower and leaf, Salvia nemorosa, and T. daenensis) and Asteraceae (T. balsamita, A. aucheri, and A. deserti). In this amount of EOs T. balsamita, A. aucheri, and A. deserti and T. daenensis were the main radicle growth inhibitors of 100%.

Vigor Index (VI)
Vigor index (VI) was also significantly affected by essential oil treatment. The index was 4300 in control, 0-0.4399 in 1 µL, and 0-3487 in 3 µL. At 1 µL the highest degree of reduction (less than 12%

Allelopathic Interaction Effect of EOs
Based on the results of screening experiments, the interaction effect on seed germination was evaluated in eight EOs. Also, this effect was studied on root and hypocotyl growth separately in six EOs.

Simplified Modified Dilution Check-Board Technique (SMCT)
Results of SMCT and combination effect (CE) showed different states of the synergistic and antagonistic effect. In the combination of essential oils regarding germination inhibition, most of the combinations resulted in antagonistic interaction ( Table 5). The highest antagonistic effect was observed in the combination of R. graveolens + D. moldavica (CE = 0.56) followed by the R. graveolens + T. transcaspicus, and C. aurantifolia + T. daenensis (CE = 0.59 and CE = 0.66, respectively). The synergistic effect was observed only in five combinations, the strongest of which was the combination of A. subulatum + M. suaveolens (CE = 1.25). There were also different combination effects of essential oil on hypocotyl growth inhibition. Of the 15 compounds, five had additive interaction, 6 had synergistic effects, and 4 had antagonistic effects ( Table 5). The most synergistic interaction was related to P. abrotanoides + T. daenensis (CE = 1.25), and the highest antagonistic effect was observed in A. subulatum

Isobologram Curves
Isobologram curves showed different type of allelopathic interaction of EOs and they appropriately confirm the SMCT result ( Figures S1-S3). In the isobologram curves of germination inhibition ( Figure S1), eight EOs showed synergistic effects, 16 EOs had antagonistic effects and four others showed additive effects. Among the EOs, M. suaveolens had the most synergistic effect with other EOs (four synergistic effects), while A. subulatum, R. graveolens and T. transcaspicus EOs had the most antagonistic effects with the other EOs (five antagonistic effects). In the hypocotyl inhibitory isobologram ( Figure S2), four EOs indicating antagonistic status, seven curves indicating synergistic status between EOs, and four other curves indicating additive effects. A. subulatum showed the highest number of antagonistic effects with the other EOs (three antagonistic effects), so that black cardamom did not have synergistic effect with any other EOs. P. abrotanoides and T. daenensis had the highest number of synergistic effects in combination with other EOs (four and three synergistic effects, respectively), so that they showed no antagonistic effect with any other EOs. And finally, in the isobologram curves of radicle growth inhibition ( Figure S3) four curves showed antagonistic effects between EOs, five curves indicating synergistic effects and six other curves indicating additive effects. The highest number of antagonistic effects was related to P. abrotanoides EO (three antagonistic effects) and M. suaveolens EO had the highest number of synergistic effects with other EOs (three synergistic effects). In this regard, black cardamom EO had no synergistic effect, and T. daenensis EO showed no antagonistic effect.

Discussion
The results of this experiment confirm, in many cases, other researchers' findings on the allelopathic effects of EOs and their compounds on seed germination. Monoterpenes can affect the germination of seeds at very low concentrations [25]. The EOs of T. daenensis and P. anisum were among the strongest germination inhibitors in this experiment. The potent microbicidal effects of these species, as well as their antimicrobial, inhibitory or stimulatory effect on enzymatic activity, toxic effects of estragol have been reported in previous studies [26][27][28][29]. Phytotoxic effects of T. daenensis EO have been reported in other allelopathic studies. The germination of some weed species has been showed to be strongly inhibited by this species EO [30], so that complete germination inhibition of Avena fatua occurred at 600 µL/L and complete germination inhibition of Amaranthus retroflexus and Datura stramonium occurred at 800 µL/L. Antibacterial effects on E. coli using this EO, have also been reported [28], achieving complete bacterial death within five minutes. These results seem to be caused by thymol, carvacrol, and carvone which are the compounds of T. daenensis EO that showed highest inhibitory effect on seed germination [31].
The insecticidal effects of P. anisum and P. graveolens, especially on larvae killing and oviposition-deterrent, are already known [32,33], as well as their fumigant-pesticide effect [34,35]. In our experiment, these species' EOs showed strong inhibitory and phytotoxic effects. According to Dhima et al. [36], P. anisum had significant inhibitory effect on germination and seedling of Echinochloa crus-galli, although Azirak and Karaman [31] observed effectiveness against seed germination only at high concentration, and not at low ones. Trans-anethole, the dominant constituent of P. anisum EO, is a type of phenylpropanoids that is toxic for some insects [37]. Since this compound was very high in headspace analysis of P. anisum EO (90%), it is most likely to be responsible for inhibition of the seed germination. Despite this, in another study, trans-anethole had no significant effect on seed germination inhibition [38]. The differences in the results may be, in part, due to the use of different plant species in the tests: and it should be noted that lettuce seed is more sensitive to allelochemicals. The way EO was applied in each study, fumigation in ours vs soaking in the others, might have also explain the differences in the results. Concerning the allelopathic effects of O. vulgare, its EO has potent antifungal effects on a wide range of fungi that have been attributed to thymol and carvacrol, among others [39]. These compounds also had germination inhibitory effects on some plant species, whereas p-cymene showed no phytotoxic effect [40]. But Elshafie et al. [41] saw moderate phytotoxic effects of O. vulgare EO. According to other studies, Z. multiflora significantly reduced germination rate, seedling length, root and shoot weight [42]. Allelopathic effects of leaves of this species on sandwich method have also been reported [43]. The antifungal effects of Z. multiflora EO were also investigated and showed complete inhibition of fungi growth [44]. Allelopathic studies of other researchers have reported phytotoxic effects of P. abrotanoides [11,43]. Its flower was one of the most effective inhibitors of lettuce seedling growth in sandwich method [43], and in dish pack method, it had an inhibitory effect of more than 80% on root growth, which, according to the experimental method, is due to its volatile compounds [11]. For other plants, many herbaceous species of Artemisia contain active compounds that have allelopathic and antifungal effects, and their allelochemicals are mainly volatile ethers and alkaloids. The allelopathic effect of A. ludoviciana showed inhibitory effects on plant growth [45]. The EO of this plant also had antibacterial effects and at a concentration of 10 µg/mL inhibited the growth of some bacteria [46].
Among major volatile compounds resulting from HS-GC-MS, alpha and beta pinene, which were dominant compound in most EOs, are oxygenated bicyclic monoterpenes, which are insoluble in water and highly volatile, and their allelopathic effects on the environment and insect repellent effects have been reported [47,48]. Other compounds, like limonene, an aliphatic and cyclic monoterpene, also present phytotoxic effects [49]. Eucalyptol or 1,8-cineole is an ether cyclic monoterpene that has insecticidal and insect repellent effects [50,51], as well as strong growth inhibitory effects on plants, affecting the activity of the asparagine synthase enzyme, thus being one of the most important allelochemical constituents [52]. The toxicity and lethal effects of eucalyptol on rats have been tested and, although it has a relatively high lethal dose (LD 50 = 2.48 g/kg bw, rat), its long-term effects on living cells can be extremely damaging and should be carefully considered [53] before using it against weeds. Borneol is an alcoholic monoterpene with reports of its inhibitory effects on Schizachyrium scoparium germination (8%) and radicle growth (47%), whereas it did not affect Leptochloa dubia germination and seedling growth [54]. Except for O. vulgare and P. abrotanoides, which inhibited the growth of both radicle and hypocotyl, the other EOs did not have the same inhibitory effects on these two seedling parts. Allium hirtifolium, L. angustifolia and P. graveolens had the greatest effect on decreasing hypocotyl growth, while A. ludoviciana, S. officinalis, and P. anisum were more effective in inhibiting radicle growth. This difference is probably due to the different role of the EOs in disrupting hormonal balance, and the effect of their compounds on growth regulators biosynthetic pathways.
Another notable point in this experiment was the different effect of the EO of different parts of a plant. In 11 plants, the EOs were extracted from two different parts of them, which in some cases had different effects. About flowers and leaves EO, in P. abrotanoides, A. vulgaris, and L. angustifolia, the allopathic effect of flower EO was greater than that of leaf, in O. vulgare the effect of the leaf EO was more than the flower, and there was no difference in geranium and rosemary in this regard. On the effect of leaf and fruit EO on germination, in C. aurantifolia, F. vulgare and Cupressus arizonica the inhibitory effect of leaf oil was greater than that of fruit, and in Juniperus horizontalis there was no difference. Regarding their differences in inhibition of seedling growth, in Juniperus horizontalis and C. arizonica the inhibitory effect of leaf EO was greater than fruit, in F. vulgare the fruit EO effect was more than leaf and in C. aurantifolia there was no difference. Regarding the different effects of Pinus eldarica leaf EO with its oleogum EO, the former had more inhibitory effects on germination than the later.
In this study, synergistic, additive and even antagonistic effects between plant EOs were observed. Synergistic effects between the EOs of some species of Apiaceae family such as Coriander and Cumin have been reported for antibacterial and antioxidant activity [16]. In another study, antifungal synergistic effects were observed between the EO of marjoram and thyme, peppermint and tea tree, and thyme and cinnamon [55]. Antimicrobial synergistic effects between EO components such as eugenol and linalool, eugenol and menthol, and carvacrol and thymol have also been reported [18].

Conclusions
This study represents new results in the allelopathy database. The results showed significant inhibitory effects of essential oils even at 1 µL concentration on the studied traits in gaseous phase. P. graveolens, P. anisum and T. daenensis EOs had the greatest inhibitory effect on germination; A. subulatum, A. sieberi, D. moldavica and T. transcaspicus EOs had the greatest effect on germination delay; P. anisum and T. daenensis EOs had the greatest effect on seed embryo lethality. P. anisum, O. vulgare, P. abrotanoides and T. daenensis EOs caused the greatest inhibition of seedling growth. In addition, the results of allelopathic interaction of the EOs showed that there were different antagonistic, synergistic, and additive interaction in their combination. M. suaveolens in inhibition of germination and radicle growth, and P. abrotanoides in inhibition of hypocotyl growth, showed the highest synergistic ability in combination with other EOs. A. subulatum, R. graveolens, and T. transcaspicus EOs had the highest antagonistic ability in combination with others. The interaction effects between the EOs can be of great interest in plant-based herbicides production. Such effects have a particular role in determining the appropriate dose so that synergistic effects can more effectively prevent germination and weed growth and reduce herbicide consumption.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/10/2/163/s1, Figure S1: Isobologram curves of allelopathic interaction effects of EOs on lettuce seed germination inhibition by cotton swab method, Figure S2: Isobologram curves of allelopathic interaction effects of EOs on lettuce hypocotyl growth inhibition by cotton swab method, Figure S3: Isobologram curves of allelopathic interaction effects of EOs on lettuce radicle growth inhibition by cotton swab method, Table S1: Analysis of variance of variables studied in the experiment to investigate the effects of 112 essential oils concentrations on seed germination and seedling growth of lettuce.