# Essential Oil Constituents ofTanacetum cilicicum: Antimicrobial and Phytotoxic Activities

Authors: Zeynep Ulukanli; Salih Demirci; Murat Yilmaztekin
Journal: Journal of Food Quality
 (2017)

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## Abstract

Aerial parts ofTanacetum cilicicum were hydrodistillated for 3 h using Clevenger. Essential oil (EO) yield was 0.4% (v/w). According to the GC/MS analyses, EO ofT. cilicicum consisted of monoterpenes [α-pinene (2.95 ± 0.19%), sabinene (2.32 ± 0.11%), and limonene (3.17 ± 0.25)], oxygenated monoterpenes [eucalyptol (5.08 ± 0.32%), camphor (3.53 ± 0.27%), linalool (7.01 ± 0.32%), α-terpineol (3.13 ± 0.23%), and borneol (4.21 ± 0.17%)], and sesquiterpenes [sesquisabinene hydrate (6.88 ± 0.41%), nerolidol (4.90 ± 0.33%), α-muurolol (4.57%  ± 0.35), spathulanol (2.98 ± 0.12%), juniper camphor (2.68 ± 0.19%), (-)-caryophyllene oxide (2.64 ± 0.19%), 8-hydroxylinalool (2.62 ± 0.15%), and Δ-cadinene (2.48 ± 0.16%)]. In the antimicrobial assay, MIC/MBC values of the EO were the most significant onB. subtilis(0.39/0.78 µL/mL) andB. cereus (0.78/1.56 µL/mL).The most prominent phytotoxic activities of the EO were observed onL. sativa,L. sativum, andP. oleracea. The results of the present study indicated that EO ofT. cilicicum includes various medicinally and industrially crucial phytoconstituents that could be in use for industrial applications. The finding of this study is the first report on this species from the East Mediterranean region.


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## Body

## 1. Introduction

Essential oils are the secondary metabolites of the aromatic and medicinal plants. In the literature, there are reports on the characterization of the chemical composition of the essential oil as well as some bioactivities of the genusTanacetum (Asteraceae). Those species wereT. annuum (Granada Alfacar region, Spain) [1],T. santolinoides(DC.) Feinbr. and Fertig. (Wadi Elarbaeen, St. Catherine, Sinai Peninsula, Egypt) [2],T. gracile (Himalaya-Ganglas, Ladakh region, India) [3],T. vulgare L. (Canada, Finland, Slovakia, and Brazil) [4–7],T. tabrisianum (Iran) [8],T. annuum(Larache region, Northwest of Morrocco) [9],T. pinnatum(Lorestan region, Iran) [10],T. parthenium(L.) (Kamenica region, Kosova) [11], andT. parthenium (Hamedan region, Iran) [12].In Turkey, various species of the genusTanacetum have been also reported both for the chemical composition and/or antimicrobial, insecticidal, herbicidal, and antioxidant activities as well. Those species wereT. sorbifolium [13],T. balsamita subsp.balsamita andT. chiliophyllum var.chiliophyllum[14],T. argenteumsubsp.flabellifolium[15],T. aucheranum andT. chiliophyllum[16],T. argenteumsubsp.argenteum[17],T. zahlbruckneri[18],T. chiliophyllumvar.chiliophyllum[19],T. chiliophyllumvar.monocephalum[20],T. heterotomum, T. zahlbrucknei, T. densumsubsp.amani, T. cadmeumsubsp.orientale[21],T. argenteumsubsp.argenteum, and T. argenteumsubsp.canum[22] andT. alyssifolium[23], andT. cilicicum [24] (Table 1).Table 1
Main components in the essential oil of someTanacetum sp.
Tanacetum sp. EO yield and main components References T. sorbifolium EO yield: 0.85%Main components: camphor (54.3), pinocarvone (5.1), chrysanthenone (4.7), bornyl acetate (3.9), camphene (3.4), β-pinene (2.8) [13]  T. argenteumsubsp. flabellifolium EO yield: 0.36%Main components: α-pinene (29.1), (E)-sesquilavandulol (15.9), camphor (14.0), (E)-lavandulyl acetate (4.9), terpinen-4-ol (3.1), β-caryophyllene (3.1) [15]  T. aucheranum EO yield: 0.15%Main components: 1,8-cineole (23.8), camphor (11.6), terpinen-4-ol (7.2), α-terpineol (6.5), borneol (3.8), (E)-thujone (3.2), epi-α-cadinol (3.1), artemisia ketone (3.0) [16]  T. chiliophyllum EO yield: 0.22%Main components: camphor (17.9), 1,8-cineole (16.6), borneol (15.4), dihydro-α-cyclogeranyl pentanoate (3.0), dihydro-α-cyclogeranyl hexanoate (10.1) [16]  T. alyssifolium Main components: borneol (35.2), α-thujone (24.6), camphor (12.4), β-eudesmol (6.1), 1,8-cineole (4.8), thymol (4.1), β-thujone (3.3) [23]  T. balsamita subsp. balsamita Main components: trans-chrysanthenol (22.3), chrysanthenyl acetate (19.7), linalool oxide (11.5), 2H-pyran-3(4H)-one (9.7), camphor (7.5), 1,8-cineole (2.7) [14]  T. chiliophyllum var. chiliophyllum Main components: camphor (28.5), 1,8-cineole (17.1), camphene (7.1), isobornyl propionate (5.4), carveol (4.5), benzene, 1-methyl-2 (2.9) [14]  T. nitens Main components: 1,8-cineole (27.57), α-pinene (4.62), trans-verbenol (4.34), spathulenol (4.14), trans-pinocarveol (4.13), 3-cyclohexzan-1-ol (3.81), α-terpineol (3.68), caryophyllene oxide (3.23) and oplophenon (3.01) benzene, 1-methyl-2 (2.75), δ-cubebene (1.55) [17]  T. argenteumsubsp.argenteum Main components: α-pinene (27.86), santolinatriene (8.82), 1,8-cineole (6.82), chrysanthenone (3.83), cadina-1,4-dien (3.52) β-pinene (3.16), spathulenol (2.83), borneol (2.68), cis-sabinenehydrate (2.66), trans-verbenol (2.52), trans-pinocarveol (2.40), α-terpineol (2.41) [17]  T. tabrisianum EO yield of the flower part: 0.16%Main components: 1,8-cineole (17.6), hexadecanoic acid (10.3), borneol (6.9), decanoic acid (5.8), trans-linalooloxide acetate (5.3) [18]  T. zahlbruckneri EO yield of the stem part: 0.10% Main components: 1,8-cineole (22.5), hexadecanoic acid (8.0), trans-linalooloxide acetate (4.0), borneol (3.0) [18]  T. chiliophyllumvar. monocephalum EO yield of the flower part: 0.06%Main components: camphor (17.3), 1,8-cineole (8.3), unknown I (6.6), (E)-β-ionone cubenol unknown III (5.2), camphene (3.4), marsupellol unknown IV (3.0), borneol (2.9) [20]  T. chiliophyllumvar. monocephalum EO yield of the stem part: 0.05% Main components: camphor (10.4), (E)-β-ionone unknown I (10.4), cubenol unknown II (9.2), marsupellol unknown IV (7.4), hexadecanoic acid (3.5), (E)-nerolidol (3.2), phytol (2.8) [20]  T. chiliophyllumvar. monocephalum EO yield of the root part: <%0.01 Main components: hexadecanoic acid (37.5), cubenol unknown III (8.7), alismol (6.3), geranyl isovalerate (5.3), (E)-nerolidol (3.3), marsupellol unknown IV (3.1), (E)-β-ionone unknown I (2.6) [20]  T. chiliophyllumvar.chiliophyllum (A) EO yield of the flower and stem part: 0.1 and 0.2%Main components: camphor (32.5, 36.2), 1,8-cineole (1.6, 16.1), chamazulene (9.2, 2.9), pinocarvone (3.2, 2.4), hotrienol (2.7, 0.3), borneol (2.7, 2.8), chrysanthenyl isovalerate I (2.0, 3.0), chrysanthenyl isovalerate II (2.1, 2.8), β-eudesmol (4.7, 1.1) [19]  T. chiliophyllumvar.chiliophyllum (B) EO yield of the flower and stem part: 0.6 and 0.1%Main components: 1,8-cineole (12, 18.4), terpinen-4-ol (10.3, 9.0), hexadecanoic acid (4.2, 7.6), (E)-sesquilavandulol (5.8, 1.6), α-thujone (3.0, 1.2), trans-chrysanthenyl acetate (3.5, 2.8), α-eudesmol (3.4, 1.4) [19]  T. chiliophyllumvar.chiliophyllum (C) Main components: 1,8-cineole (22.1, 28.9), α-pinene (5.3, 1.5), terpinen-4-ol (6.5, 5.6), p-cymene (4.2, 4.3), trans-chrysanthenyl acetate (3.7, 2.0), pinocarvone (1.8, 2.8), (E)-sesquilavandulol (3.6, 0.0) [19]  T. heterotomum Main components: α-pinene (3.4), camphene (1.2), β-pinene (4.5), 1,8-cineole (17.9), α-terpinolene (1.2), cis-sabinenehydrate (1.7), camphor (22.4), pinocarvone (1.2), borneol (18.8), α-terpineol (1.3), α-copaene (1.3), β-selinene (1.4), caryophyllene oxide (1.1), hexadecanoic acid (1.6) [21]  T. zahlbrucknei α-Thujene (1.2), α-pinene (1.0), camphene (2.3), 1,8-cineole (1.3), bicyclo (3.1) hexan-3-one (2.1), trans-chrysanthemol (1.7), camphor (3.8), borneol (21.3), 3-cyclohexen-1-ol (2.4), α-terpineol (1.9), bicyclo (2.2) heptan-2-ol (1.1), thymol (2.7), germacrene D (21.4), spathulenol (16.2), aromadendrene (1.2) [21]  T. densumsubsp. amani α-Pinene (6.7), camphene (1.5), β-pinene (8.9), 1,8-cineole (16.7), cyclohexene (1.2), cis-sabinenehydrate (1.3), 4-acetyl-1-methylcyclohexan (1.2), camphor (26.8), pinocarvone (2.4), cis-chrysanthemol (1.3), borneol (3.8), α-copaene (2.6), β-bourbenene (1.4), trans-β-farnesene (1.2), germacrene D (3.9), spathulenol (1.4), β-bisabolene (1.1) [21]  T. cadmeumsubsp.orientale Main components: α-pinene (7.6), camphene (5.4), β-pinene (1.8), α-phellandrene (1.1), limonene (1.2), 1,8-cineole (19.6), trans-chrysanthemol (3.1), camphor (17.2), pinocarvone (1.8), 2-cyclohexen-1-ol (1.7), borneol (5.3), α-copaene (2.8), β-elemene (1.9), β-cubebene (1.3), isoledene (1.5), muurolene (1.6), epi-bicyclosesquiphellandrene (1.7), α-cadinol (1.3), aromadendrene (1.3) [21]  T. argenteumsubsp. argenteum EO yield: 0.32%Main components: α-pinene (67.9), β-pinene (4.8), α-phellandrene (2.8), limonene (2.0), 1,8-cineole (2.2), p-cymene (2.1), β-caryophyllene (1.5), germacrene D (2.2), δ-cadinene (1.6), caryophyllene oxide (1.1), T-cadinol (1.4), α-cadinol (1.6) [22]  T. argenteumsubsp. canum EO yield: 0.35%Main components: α-pinene (53.6), α-thujene (1.1), camphene (1.1), β-pinene (1.0), α-phellandrene (2.5), 1,8-cineole (14.8), p-cymene (2.1), trans-sabinene hydrate (2.9), camphor (4.7), terpinen-4-ol (2.1), trans-pinocarveol (1.2), trans-verbenol (1.0), α-terpineol (1.2), spathulenol (1.2) [22]  T. cilicicum Main components: bicyclo (31.1), hept-2-en-4-ol (21.92), camphor (15.56), 1,8-cineole (13.45) [24]Essential oils of aromatic and medicinal plants have been receiving increasing attention in many industries such as medicine, agriculture, food, and cosmetics [25]. In nature, many plants have been still awaiting for the exploration of their bio-benefits. To date, there has been no previous study indicating the chemical composition as well as antimicrobial and phytotoxic activities ofT. cilicicum from the East Mediterranean region. Hence, the essential oil composition as well as bioactivities of the aerial parts ofT. cilicicum could be the first report.


## 2. Experimental

### 2.1. Plant Material and Essential Oil Distillation

Aerial parts ofT. cilicicum were collected from Bağlıca Plateau (Amanos Mountains, Hatay-İskenderun, 36°34′48,52′′N, 36°16′05,87′′E). Essential oil (EO) from air-dried aerial parts ofT. cilicicum was obtained with Clevenger for 3 h distillation period, which was followed by calculating the oil yield (v/w). A total of 100 g plant material was used during each distillation cycle. The blue coloured EO was dried with anhydrous sodium sulphate to remove the water from the distillate and then preserved in amber vials at +4°C for further analyses.


### 2.2. Analyses of the EO ofT. cilicicum Using Gas Chromatography and Mass Spectrometry (GC/MS)

The characterization of the compounds in the EO was obtained with Gas Chromatography and Mass Spectrometry. The conditions used during the analyses were described in Table2 [26]. n-Alkane mixture was used to determine and calculate the linear retention index (Kovats Indices) of each compound in the EO under the same temperature programme used for the analyses [27]. Mass spectra of the EO constituents were compared with those of the references documented in NIST (National Institute of Standards and Technology, 2013, Gaithersburg, MD, USA) and Wiley database. Tentative identification as well as the retention indices of the compounds in this assay was compared with those of NIST. In addition, quantitative analyses of the EO constituents (% area) as average of 3 repeated analytical assays were carried out with the measurement of the peak space normalization.Table 2
GC/MS conditions.
System Shimadzu QP 2010 Plus (Shimadzu, Kyoto, Japan) GC Colon TRB-Wax (Teknokroma, Barcelona, Spain) fused silica capillary column (60 m × 0.25 mm i.d. and film thickness, 0.25μm) Temperature programme 40°C/5 min, 3°C/1 min, 240°C/15 min (total running: 86 min) Injector AOC-20i/20s autosampler Injection volume 1µl (1 part of EO/100 part of n-hexane, v/v) Carrier gas Helium (flow rate, 1 mL/min) Detector MS-QP 2010 series mass-selective detector Split ratio 1/50 Electron energy 70 eV Mass spectra 35–450 m/z Scanning rate 1 scan/s


### 2.3. Antimicrobial Assays and Test Microorganisms

Six Gram-positive bacteria (Bacillus subtilis ATCC 6633,Bacillus cereus EU,Enterococcus faecalis ATCC 29212,Enterococcus casseliflavus ATCC 700327,Staphylococcus aureus ATCC 29213, andStaphylococcus aureus ATCC BAA 977), four Gram-negative bacteria (Enterobacter hormaechei ATCC 700323,Klebsiella pneumoniae ATCC 700603,Pseudomonas aeruginosa ATCC 27853, andEscherichia coli ATCC 25922), and two yeast species (Candida parapsilosis ATCC 22019 andCandida albicans ATCC 14053) were used in the assays.


### 2.4. Disc Diffusion Assay

The turbidity of the freshly bacterial culture grown on Nutrient Agar medium (37°C/ 24 h) and Sabouraud Dextrose Agar was adjusted to 0.5 McFarland Unit. 100µL of the suspension of each microorganism in sterile saline water was distributed on Mueller Hinton Agar for bacterial cultures and Sabouraud Dextrose Agar for fungal cultures. After spreading homogenously by drigalski spatula, a sterile antibiotic assay disc was placed on each medium. EO ofT. cilicicum (15 µL) was pipetted onto each disc. The petri dish was then sealed with parafilm in order to retain the EO in the glass petri atmosphere. Throughout the assay, controls were (I) only test medium in a petri plate, (II) essential oil impregnated assay disc on an uninoculated medium, and (III) test culture plated on appropriate medium. All treatments were maintained at 35.5°C for 24 h [28]. The diameters of the zones around the discs were measured as mm. Trimethoprim/Sulfamethoxazole (SXT5), Vancomycin (VA30), Teicoplanin (TEC30), and Nystatin (100 IU) were used as the standard antibiotics during the disc diffusion assay.


### 2.5. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal/Fungicidal Concentrations (MBC/MFC) of the EO fromT. cilicicum

Two-fold concentrations of the EO ranging from 0.0485 to 200.0µL/mL in Mueller Hinton Broth + 0.5% Tween 80 for bacteria and Sabouraud Dextrose Broth + 0.5% Tween 80 for yeast were prepared before assay. Each tube received 100 µL of microbial suspension. The following were used to check the test results for comparison: (I) medium including different essential oil concentrations dissolved in 0.5% Tween 80: MHB or SDB + Tween 80 + different essential oil concentrations, (II) MHB or SDB + Tween 80 + test strain, and MHB or SDB + Tween 80, and (III) only MHB or SDB. All treatments were incubated at 37°C for 24 h. Tube without any visible growth was treated as Minimum Inhibitory Concentration of the test EO in concern. The broth in test tubes was plated onto appropriate media to determine MBC/MFC [29]. In the broth dilution assay, commercially available antibiotics such as Ampicillin (A6140, Sigma) were used in Mueller Hinton Broth for bacteria, and Clotrimazole (CG019, Sigma) was tested in Sabouraud Dextrose Broth for yeast species.


### 2.6. Phytotoxicity Assay

Seeds ofLactuca sativa (lettuce),Lepidium sativum (cress), andP. oleracea (common purslane) were used in the direct-contact assay of the essential oil [30, 31]. Two round filter papers were placed into each glass petri dish (90 mm in diameter) and then sterilized at 121°C for 15 min. Afterwards, sterilized seeds with aqueous sodium hypochlorite solution (1.5%, v/v) for 10 min were aseptically placed onto double layered membranes [32]. EO ofT. cilicicum was dissolved in 0.5% Tween 80 at the concentrations from 0.062 to 2.0 mg/mL. A total of 10 mL from each concentration was aseptically placed to lower layer of the filter paper by using a sterile glass pipette. Throughout the assay, control did include only distilled water. All petri plates were sealed with parafilm. Treated petri dishes as well as the control groups were maintained for 7 days at 24°C, 12/12 h, 1.500 lux light/dark period, and about 80% relative humidity. At the final day of maintenance, germinated seeds were counted and then recorded. The length of seedling growths was measured as mm. Glyphosate N-(phosphonomethyl) glycine was tested as the commercial herbicide.


### 2.7. Statistical Analysis

Assessment of the test results was carried out using SPSS17 programme. Variance analyses (ANOVA) of the results of the test parameters were performed. Tukey Multiple Comparative test was employed to determine the differences in test parameters. The mean and standard deviations of the results were calculated. The statistical differences were indicated as a superscript in the mean values. All assays in this study were carried out in triplicate.


## 2.1. Plant Material and Essential Oil Distillation

Aerial parts ofT. cilicicum were collected from Bağlıca Plateau (Amanos Mountains, Hatay-İskenderun, 36°34′48,52′′N, 36°16′05,87′′E). Essential oil (EO) from air-dried aerial parts ofT. cilicicum was obtained with Clevenger for 3 h distillation period, which was followed by calculating the oil yield (v/w). A total of 100 g plant material was used during each distillation cycle. The blue coloured EO was dried with anhydrous sodium sulphate to remove the water from the distillate and then preserved in amber vials at +4°C for further analyses.


## 2.2. Analyses of the EO ofT. cilicicum Using Gas Chromatography and Mass Spectrometry (GC/MS)

The characterization of the compounds in the EO was obtained with Gas Chromatography and Mass Spectrometry. The conditions used during the analyses were described in Table2 [26]. n-Alkane mixture was used to determine and calculate the linear retention index (Kovats Indices) of each compound in the EO under the same temperature programme used for the analyses [27]. Mass spectra of the EO constituents were compared with those of the references documented in NIST (National Institute of Standards and Technology, 2013, Gaithersburg, MD, USA) and Wiley database. Tentative identification as well as the retention indices of the compounds in this assay was compared with those of NIST. In addition, quantitative analyses of the EO constituents (% area) as average of 3 repeated analytical assays were carried out with the measurement of the peak space normalization.Table 2
GC/MS conditions.
System Shimadzu QP 2010 Plus (Shimadzu, Kyoto, Japan) GC Colon TRB-Wax (Teknokroma, Barcelona, Spain) fused silica capillary column (60 m × 0.25 mm i.d. and film thickness, 0.25μm) Temperature programme 40°C/5 min, 3°C/1 min, 240°C/15 min (total running: 86 min) Injector AOC-20i/20s autosampler Injection volume 1µl (1 part of EO/100 part of n-hexane, v/v) Carrier gas Helium (flow rate, 1 mL/min) Detector MS-QP 2010 series mass-selective detector Split ratio 1/50 Electron energy 70 eV Mass spectra 35–450 m/z Scanning rate 1 scan/s


## 2.3. Antimicrobial Assays and Test Microorganisms

Six Gram-positive bacteria (Bacillus subtilis ATCC 6633,Bacillus cereus EU,Enterococcus faecalis ATCC 29212,Enterococcus casseliflavus ATCC 700327,Staphylococcus aureus ATCC 29213, andStaphylococcus aureus ATCC BAA 977), four Gram-negative bacteria (Enterobacter hormaechei ATCC 700323,Klebsiella pneumoniae ATCC 700603,Pseudomonas aeruginosa ATCC 27853, andEscherichia coli ATCC 25922), and two yeast species (Candida parapsilosis ATCC 22019 andCandida albicans ATCC 14053) were used in the assays.


## 2.4. Disc Diffusion Assay

The turbidity of the freshly bacterial culture grown on Nutrient Agar medium (37°C/ 24 h) and Sabouraud Dextrose Agar was adjusted to 0.5 McFarland Unit. 100µL of the suspension of each microorganism in sterile saline water was distributed on Mueller Hinton Agar for bacterial cultures and Sabouraud Dextrose Agar for fungal cultures. After spreading homogenously by drigalski spatula, a sterile antibiotic assay disc was placed on each medium. EO ofT. cilicicum (15 µL) was pipetted onto each disc. The petri dish was then sealed with parafilm in order to retain the EO in the glass petri atmosphere. Throughout the assay, controls were (I) only test medium in a petri plate, (II) essential oil impregnated assay disc on an uninoculated medium, and (III) test culture plated on appropriate medium. All treatments were maintained at 35.5°C for 24 h [28]. The diameters of the zones around the discs were measured as mm. Trimethoprim/Sulfamethoxazole (SXT5), Vancomycin (VA30), Teicoplanin (TEC30), and Nystatin (100 IU) were used as the standard antibiotics during the disc diffusion assay.


## 2.5. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal/Fungicidal Concentrations (MBC/MFC) of the EO fromT. cilicicum

Two-fold concentrations of the EO ranging from 0.0485 to 200.0µL/mL in Mueller Hinton Broth + 0.5% Tween 80 for bacteria and Sabouraud Dextrose Broth + 0.5% Tween 80 for yeast were prepared before assay. Each tube received 100 µL of microbial suspension. The following were used to check the test results for comparison: (I) medium including different essential oil concentrations dissolved in 0.5% Tween 80: MHB or SDB + Tween 80 + different essential oil concentrations, (II) MHB or SDB + Tween 80 + test strain, and MHB or SDB + Tween 80, and (III) only MHB or SDB. All treatments were incubated at 37°C for 24 h. Tube without any visible growth was treated as Minimum Inhibitory Concentration of the test EO in concern. The broth in test tubes was plated onto appropriate media to determine MBC/MFC [29]. In the broth dilution assay, commercially available antibiotics such as Ampicillin (A6140, Sigma) were used in Mueller Hinton Broth for bacteria, and Clotrimazole (CG019, Sigma) was tested in Sabouraud Dextrose Broth for yeast species.


## 2.6. Phytotoxicity Assay

Seeds ofLactuca sativa (lettuce),Lepidium sativum (cress), andP. oleracea (common purslane) were used in the direct-contact assay of the essential oil [30, 31]. Two round filter papers were placed into each glass petri dish (90 mm in diameter) and then sterilized at 121°C for 15 min. Afterwards, sterilized seeds with aqueous sodium hypochlorite solution (1.5%, v/v) for 10 min were aseptically placed onto double layered membranes [32]. EO ofT. cilicicum was dissolved in 0.5% Tween 80 at the concentrations from 0.062 to 2.0 mg/mL. A total of 10 mL from each concentration was aseptically placed to lower layer of the filter paper by using a sterile glass pipette. Throughout the assay, control did include only distilled water. All petri plates were sealed with parafilm. Treated petri dishes as well as the control groups were maintained for 7 days at 24°C, 12/12 h, 1.500 lux light/dark period, and about 80% relative humidity. At the final day of maintenance, germinated seeds were counted and then recorded. The length of seedling growths was measured as mm. Glyphosate N-(phosphonomethyl) glycine was tested as the commercial herbicide.


## 2.7. Statistical Analysis

Assessment of the test results was carried out using SPSS17 programme. Variance analyses (ANOVA) of the results of the test parameters were performed. Tukey Multiple Comparative test was employed to determine the differences in test parameters. The mean and standard deviations of the results were calculated. The statistical differences were indicated as a superscript in the mean values. All assays in this study were carried out in triplicate.


## 3. Results and Discussion

### 3.1. Essential Oil Composition ofT. cilicicum

As shown in Table3 and Figure 1, EO of the aerial parts fromT. cilicicum consisted of mainly (%) linalool (7.01 ± 0.32), sesquisabinene hydrate (6.88 ± 0.41), eucalyptol (5.08 ± 0.32), nerolidol (4.90 ± 0.33), α-muurolol (4.57 ± 0.35), borneol (4.21 ± 0.17), camphor (3.53 ± 0.27), limonene (3.17 ± 0.25), α-terpineol (3.13 ± 0.23), spathulanol (2.98 ± 0.12), α-pinene (2.95 ± 0.19), juniper camphor (2.68 ± 0.19), (-)-caryophyllene oxide (2.64 ± 0.19), 8-hydroxylinalool (2.62 ± 0.15), Δ-cadinene (2.48 ± 0.16), sabinene (2.32 ± 0.11), trans-verbenol (2.06 ± 0.11), and alloaromadendrene (2.0 ± 0.16). β-Pinene (1.89 ± 0.10), cis-caryophyllene (1.58 ± 0.09), epiglobulol (1.58 ± 0.11), α-cadinol (1.51 ± 0.13), 4-terpineol (1.45 ± 0.09), germacrene D (1.26 ± 0.09), α-copaene (1.24 ± 0.09), α-cedrene (1.21 ± 0.08), and spathulenol (1.21 ± 0.07) were other significant compounds in the EO ofT. cilicicum. EO yield of the aerial parts ofT. cilicicumwas 0.4% (v/w). In previous reports, essential oil yield and chemical constituents ofTanacetum sp. were reported in different species belonging to genusTanacetum (Table 1). It seemed that yield and composition differed from previous reports owing to the species differences belonging to this genus.Table 3
Essential oil composition ofT. cilicicum.
Number Retention index Compound % 1 1010 α-Pinene 2.95 ± 0.19 2 1015 α-Thujene 0.08 ± 0.01 3 1052 Camphene 0.73 ± 0.03 4 1095 β-Pinene 1.89 ± 0.10 5 1109 Sabinene 2,32 ± 0,11 6 1113 Verbenene 0,08 ± 0,01 7 1154 β-Myrcene 0,07 ± 0,01 8 1168 α-Terpinene 0,19 ± 0,02 9 1188 Limonene 3,17 ± 0,25 10 1198 Eucalyptol 5,08 ± 0,32 11 1235 γ-Terpinene 0,36 ± 0,02 12 1260 para-Cymene 0.54 ± 0.03 13 1286 Amyl isovalerate 0.06 ± 0.01 14 1345 3-Methyl-3-butenyl isovalerate 0.11 ± 0.01 15 1383 Nonanal 0.23 ± 0.01 16 1429 trans-Linalool oxide (fur.) 0.12 ± 0.01 17 1450 trans-Sabinene hydrate 0.80 ± 0.02 18 1455 Menthone 0.16 ± 0.03 19 1487 α-Copaene 1.24 ± 0.09 20 1509 Camphor 3.53 ± 0.27 21 1513 Benzaldehyde 0.13 ± 0.01 22 1533 Linalool 7.01 ± 0.32 23 1545 4-Acetyl-1-methyl-1-cyclohexene 0.29 ± 0.01 24 1550 trans-p-Mentha-2-en-1-ol 0.13 ± 0.01 25 1560 Pinocarvone 0.40 ± 0.02 26 1574 Bornyl acetate 0.52 ± 0.07 27 1590 4-Terpineol 1.45 ± 0.09 28 1593 Terpendiol I 0.64 ± 0.04 29 1614 trans-p-Mentha-2.8-dien-1-ol 0.52 ± 0.03 30 1621 Myrtenal 0.25 ± 0.01 31 1639 Pulegone 0.33 ± 0.07 32 1643 Alloaromadendrene 2.00 ± 0.16 33 1655 Isoledene 0.48 ± 0.03 34 1666 trans-Verbenol 2.06 ± 0.11 35 1671 cis-Citral 0.18 ± 0.02 36 1684 α-Terpineol 3.13 ± 0.23 37 1690 Borneol 4.21 ± 0.17 38 1706 Germacrene D 1.26 ± 0.09 39 1718 α-Muurolene 0.94 ± 0.06 40 1726 Carvone 0.28 ± 0.01 41 1729 Bicyclogermacrene 0.20 ± 0.02 42 1737 Verbenol 0.21 ± 0.04 43 1746 Neryl acetate 0.30 ± 0.03 44 1754 Δ-Cadinene 2.48 ± 0.16 45 1756 γ-Cadinene 0.81 ± 0.07 46 1764 1.7.7-Trimethylbicyclo[2.2.1]hept-5-en-2-ol 0.27 ± 0.06 47 1767 Ar-Curcumene 0.61 ± 0.05 48 1780 Myrtenol 0.76 ± 0.04 49 1785 Isocarveol 0.42 ± 0.06 50 1821 trans-Carveol 0.32 ± 0.02 51 1828 cis-Calamenene 0.13 ± 0.01 52 1831 Geraniol 0.84 ± 0.07 53 1859 Benzyl isovalerate 0.57 ± 0.03 54 1874 trans-p-Mentha-1(7).8-dien-2-ol 0.36 ± 0.03 55 1879 Spathulanol 2.98 ± 0.12 56 1883 benzyl-Isovalerate 0.52 ± 0.04 57 1912 α-Calacorene 0.22 ± 0.06 58 1923 Palustrol 0.13 ± 0.02 59 1932 Sesquisabinene hydrate 6.88 ± 0.41 60 1981 (-)-Caryophyllene oxide 2.64 ± 0.19 61 1989 Benzyl tiglate 0.51 ± 0.04 62 2020 Tridecanal 0.38 ± 0.03 63 2026 Nerolidol 4.90 ± 0.33 64 2041 cis-Caryophyllene 1.58 ± 0.09 65 2053 (-)-Cedreanol 0.70 ± 0.04 66 2059 α-Cedrene 1.21 ± 0.08 67 2067 Elemol 1.00 ± 0.09 68 2120 8-Hydroxylinalool 2.62 ± 0.15 69 2141 Spathulenol 1.21 ± 0.07 70 2153 Eugenol 0.54 ± 0.04 71 2163 α-Muurolol 4.57 ± 0.35 72 2171 Cubenol 0.35 ± 0.02 73 2177 α-Cadinol 1.51 ± 0.13 74 2193 ar-Turmerol 0.47 ± 0.03 75 2221 Epiglobulol 1.58 ± 0.11 76 2245 Juniper camphor 2.68 ± 0.19 77 2289 Isoaromadendrene epoxide 0.69 ± 0.04 78 2294 Tricosane 0.13 ± 0.01 79 2327 α-Santalol 0.34 ± 0.02 80 2407 Nerolidol 0.56 ± 0.04 81 2494 Pentacosane 0.21 ± 0.02 82 2570 1-Octadecanol 0.47 ± 0.03 83 2597 Phytol 0.19 ± 0.02 Total   100.00Figure 1
GC/MS chromatogram of the EO fromT. cilicium.The peaks in the chromatogram belong to the main compounds which are more than 1% of the EO chemical composition: (1) α-pinene, (2) β-pinene, (3) sabinene, (4) limonene, (5) eucalyptol, (6) α-copaene, (7) camphor, (8) linalool, (9) 4-terpineol, (10) alloaromadendrene, (11) trans-verbenol, (12) α-terpineol, (13) borneol, (14) germacrene D, (15) Δ-cadinene, (16) spathulanol, (17) sesquisabinene hydrate, (18) (-)-caryophyllene oxide, (19) nerolidol, (20) cis-caryophyllene, (21) α-cedrene, (22) 8-hydroxylinalool, (23) spathulenol, (24) α-muurolol, (25) α-cadinol, (26) epiglobulol, and (27) juniper camphor (the peak numbering given here is not in accordance with peak numbers in Table 3).


### 3.2. Antimicrobial Activity Results of the EO ofT. cilicicum

Antibiotics raise serious concerns in relation to antimicrobial resistance problems for all group of organisms. Therefore, natural sources of the environments have been intensively studied by many scientists to combat their undesirable effects to the health of living organisms and their environments as well [33–35]. In this study, EO ofT. cilicicum was tested on twelve microorganisms, with two methods: disc diffusion and macrobroth dilution. In addition, standard antibiotics were used for comparison during the assays. As shown in Table 4, the results of the disc diffusion assay indicated that the most sensitive microorganisms wereB. subtilis andS. aureus 29213. This was followed byB. cereus=S. aureus BAA >E. faecalis >E. casseliflavus. The susceptibilities of the Gram-negative bacteria towards the essential oil were less than Gram-positive bacteria. The most susceptible Gram-negative bacterium wasE. hormaechei >E. coli >K. pneumonia =P. aeruginosa. In addition,C. parapsilosiswas also more susceptible than that ofC. albicans.Table 4
Antimicrobial activities of the EO fromT. cilicicum using disc diffusion assay.
Microorganisms Source EO(15 µL) SXT5 VA30 TEC30 NS 100 IU Inhibition zones (mm) B. subtilis ATCC 6633 19.33 ± 0.57 a B 34.66 ± 0.57 a A 19.33 ± 0.57 c B 15.33 ± 0.57 e C — B. cereus EU (food isolate) 16.00 ± 0.00 b C 23.33 ± 1.15 e A 20.00 ± 0.00 b c B 22.33 ± 0.57 a A — E. faecalis ATCC 29212 15.67 ± 0.57 b c C 25.00 ± 1.00 d e A 21.00 ± 1.00 b c B 17.66 ± 0.57 d C — E. casseliflavus ATCC 700327 14.33 ± 0.57 c D 26.66 ± 0.57 d A 24.66 ± 0.57 a B 21.00 ± 1.00 a b C — S. aureus ATCC 29213 19.66 ± 0.57 a B 32.00 ± 0.00 b A 21.66 ± 1.52 b B 20.00 ± 1.00 b c B — S. aureus ATCC BAA977 16.33 ± 0.57 b D 33.00 ± 0.00 a b A 20.00 ± 0.00 b c B 18.66 ± 0.57 c d C — K. pneumoniae ATCC 700603 10.33 ± 0.57 e B 18.00 ± 1.00 f A 0.00 ± 0.00 d C 0.00 ± 0.00 f C — E. hormaechei ATCC 700323 12.00 ± 0.00 d B 26.00 ± 0.00 d A 0.00 ± 0.00 d C 0.00 ± 0.00 f C — P. aeruginosa ATCC 27853 10.00 ± 0.00 e A 0.00 ± 0.00 g B 0.00 ± 0.00 d B 0.00 ± 0.00 f B — E. coli ATCC 25922 10.66 ± 0.57 d e B 28.66 ± 0.57 c A 0.00 ± 0.00 d C 0.00 ± 0.00 f C — C. parapsilosis ATCC 22019 29.00 ± 0.00 a A — — — 16.00  ±  1.00 b B C. albicans ATCC 14053 16.33 ± 0.5 7 b B — — — 19.66 ± 1.15 a A EO: essential oil; SXT5: Trimethoprim/Sulfamethoxazole; VA30: Vancomycin; TEC30: Teicoplanin; NS 100 IU: Nystatin. The small case letters as the superscript in the columns and capital letters as the superscript in the columns showed the statistical differences in each column and row, respectively (p<0.05). — indicates not tested.As shown in Table5, according to MIC/MBC (µL/mL) assays, it was found that the most potent activity was observed onB. subtilis (0.39/0.78) andB. cereus (0.78/1.56). Higher inhibitory and cidal values of the EO fromT. cilicicum were observed for other tested microorganisms. This was statistically the same forS. aureus BAA (5.20/6.25) =E. casseliflavus (6.25/12.5) =S. aureus 29213 (6.25/6.25) =E. hormaechei (6.25/25.00) =E. coli (6.25/6.25) =E. faecalis (6.25/12.5). Less activities were determined for two Gram-positive bacteria and the activity was statistically the same forK. pneumoniae (10.41/50.00) andP. aeruginosa (12.5/50.00). MIC and MFC values (µL/mL) ofT. cilicicum were 1.56/6.25 forC. albicans and 3.12/12.5 forC. parapsilosis.Table 5
Minimum Inhibitor Concentration and Minimum Bactericidal/Fungicidal Concentration values ofT. cilicicum essential oil.
Microorganisms  EO (µL/mL) AMP (µg/mL) CLO (µg/mL) MIC MBC/MFC MIC MBC MIC MFC B. subtilis ATCC 6633 0.39 ± 0.00 a 0.78 ± 0.00 a 15.62 ± 0.00 d 31.25 ± 0.00 a — — B. cereus EU (food isolate) 0.78 ± 0.00 a 1.56 ± 0.00 a 1000 ± 0.00 g >1000 ± 0.00d — — E. faecalis ATCC 29212 6.25 ± 0.00 b 12.50 ± 0.00 c 0.48 ± 0.00 a 125.0 ± 0.00 b — — E. casseliflavus ATCC 700327 6.25 ± 0.00 b 12.50 ± 0.00 c 0.48 ± 0.00 a 31.25 ± 0.00 a — — S. aureus ATCC 29213 6.25 ± 0.00 b 6.25 ± 0.00 b 31.25 ± 0.00 e 250.0 ± 0.00 c — — S. aureus ATCC BAA977 5.20 ± 1.80 b 6.25 ± 0.00 b 125.0 ± 0.00 f 250.0 ± 0.00 c — — K. pneumoniae ATCC 700603 10.41 ± 3.60 c 50.00 ± 0.00 e >1000 ± 0.00g >1000 ± 0.00d — — E. hormaechei ATCC 700323 6.25 ± 0.00 b 25.00 ± 0.00 d 7.81 ± 0.00c 41.66 ± 8.04 a — — P. aeruginosa ATCC 27853 12.5 ± 0.00 c 50.00 ± 0.00 e >1000 ± 0.00g >1000 ± 0.00d — — E. coli ATCC 25922 6.25 ± 0.00 b 6.25 ± 0.00 b 3.90 ± 0.00 b 125.0 ± 0.00 b — — C. parapsilosis ATCC 22019 3.12 ± 0.00 a b 12.5 ± 0.00 c — — 300 ± 0.00 a >300 ± 0.00a C. albicans ATCC 14053 1.56 ± 0.00 a 6.25 ± 0.00 b — — >300 ± 0.00a >300 ± 0.00a In each column, small case letters as the superscript in the columns showed the statistical differences (p<0.05). — indicates not tested. MIC: Minimum Inhibitory Concentration; MFC: Minimum Fungicidal Concentration; AMP: Ampicillin; CLO: Clotrimazole.In previous studies, EO ofTanacetum sp. was assayed on various microorganisms. The EO fromT. balsamita subsp.balsamita andT. chiliophyllum var.chiliophyllumrevealed inhibitory zones on the growth ofB. subtilis ATCC 6633 (13-17 mm),S. aureus ATCC 6538 P (17–15),E. coli ATCC 25922 (15-16 mm),C. glabrata ATCC 66032 (15–18 mm), andC. tropicalis ATCC 13803 (14–17 mm) [14]. In another study, EO from the flowers and stems ofT. chiliophyllumvar.chiliophyllum grown in three different localities showed MIC values (µg/mL) as follows:S. aureus ATCC 6538 (250/500, 150/500, and 250/>500), meticillin resistantS. aureus(clinical isolate) (500/125, 750/500, and 250/250),S. epidermidis ATCC 12228 (250/250, 150/500, and 500/31.2),B. cereus NRRL B-3711 (250/125, 150/500, and >500/125),B. subtilis NRRL B-4378 (500/125, 375/500, and 500/62.5),E. coli NRRL B-3008 (>500/62.5, >150/500, and >500/62.5),P. aeruginosa ATCC 27853 (500/250, 150/500, and 500/31.25), andE. aerogenes NRRL 3567 (500/500, >150/>500, and >500/62.5) [19]. EO from the flowers and stems ofT. chiliophyllumvar.monocephalum had MIC values (µg/mL):S. aureus ATCC 6538 (>500, 1000), meticillin resistantS. aureus (125, >500),B. cereus NRRL B-3711 (62.5, 1000),B. subtilis NRRL B-4378 (250, >1000),E. coli NRRL B-3008 (>500, >1000),P. aeruginosa ATCC 27853 (500, 1000), andE. aerogenes NRRL 3567 (>500, >1000) [20]. MIC values (µg/mL) of the EO fromT. argenteumsubsp.flabellifoliumwere as follows:E. coliATCC 25922 (250),S. aureusATCC 6538 (125),P. aeruginosaATCC 27853 (125),E. aerogenesNRRL 3567 (125), andC. albicansO.G.U (125) [15].In another study, the EO ofT. aucheranum andT. chiliophyllum revealed various level of antimicrobial activities on test microorganisms exceptP. cichorii andB. subtilis ATCC 6633 using the disc diffusion and broth dilution assay. Inhibitory zones (as mm) and MIC values (µL/mL) of the essential oils fromT. aucheranum andT. chiliophyllum were as follows, respectively:P. chlororaphis (13.5 mm, 166.7) (13.3 mm, 500.0),P. syringae pv.syringae (7.8 mm, 500.0), (7.5 mm, 500.0),B. coagulans (11.8 mm, 166.7), (10.2 mm, 166.7),E. faecalis ATCC 29122 (9.0 mm, 1000.0), (0.0, 0.0),S. aureus ATCC 29213 (9.5 mm, 1000,0), (11.5 mm, 1000.0),E. intermedius (10.7 mm, 166.7), (10.2 mm, 54.4),E. coli (9.8 mm, 166.7), (8.8 mm, 500.0),P. aeruginosa ATCC 27859 (19.8 mm, 166.7), (16.5 mm, 500.0),P. aeruginosa ATCC 9027 (11.0 mm, 1000.0), (12.0 mm, 1000.0), andK. trevisanii (9.3 mm, 500.0), (8.2 mm, 166.7) [16]. The differences of the results of the present study when compared to the previous reports could be attributed to the chemical differences in the species assayed.


### 3.3. Phytotoxicity Assay ofT. cilicicum

As shown in Table6, EO ofT. cilicicum as well as the herbicide (glyphosate (N-(phosphonomethyl) glycine) at 0.062, 0.125, 0.25, 0.50, 1.0, and 2.0 mg/mL were assayed on the seeds ofL. sativa,L. sativum, andP. oleracea. Effects of test substances were dependent on the dose activity. EO at 0.25 mg/mL and the lower concentrations did not reveal any inhibitory effects on the seeds ofL. sativa,L. sativum, andP. oleracea. Higher concentrations ranging from 0.50 to 2.0 mg/mL had inhibitory effects on the growth ofL. sativa,L. sativum, andP. oleracea. Compared to the control, 0.50, 1.0, and 2.0 mg/mL doses of the EO inhibited the seed germination by 87.97 and 100% inL. sativa, by 55, 86, and 92% inL. sativum, and by 19, 50, and 89% inP. oleracea, respectively.Table 6
Effects of the essential oil fromT. cilicicum on the seed germination (%), radicle, and plumule growth of L. sativa, L. sativum,andP. oleracea.
Lactuca sativa Lepidium sativum Portulaca oleracea SG (%) RL(mm) (mean ± SD) PL(mm) (mean ± SD) SG (%) RL(mm) (mean ± SD) PL(mm) (mean ± SD) SG (%) RL(mm) (mean ± SD) PL(mm) (mean ± SD) Control 100.0 ± 0.00 a ∗ 40.73 ± 1.04 a 27.33 ± 1.77 a 100.0 ± 0.00 a 50.86 ± 3.14 a 39.33 ± 1.54 a 100.0 ± 0.00 a 43.36 ± 1.20 a 20.13 ± 1.88 a EO (mg/mL) 0.062 100.0 ± 0.00 a 40.70 ± 1.16 a 20.33 ± 1.30 b 100.0 ± 0.00 a 49.73 ± 2.31 a 30.90 ± 2.80 b 100.0 ± 0.00 a 33.36 ± 1.64 b 16.20 ± 3.10 a b 0.125 100.0 ± 0.00 a 33.30 ± 1.63 b 17.83 ± 1.27 b 100.0 ± 0.00 a 39.73 ± 1.08 b 24.23 ± 1.90 c 100.0 ± 0.00 a 31.40 ± 1.58 b 15.63 ± 2.64 a b 0.25 100.0 ± 0.00 a 29.16 ± 1.86 c 14.16 ± 1.49 c 100.0 ± 0.00 a 34.16 ± 1.52 c 21.73 ± 2.36 c 100.0 ± 0.00 a 20.33 ± 2.59 c 11.76 ± 2.52 b 0.50 13.33 ± 2.00 b 10.75 ± 1.59 d 04.00 ± 0.81 d 45.53 ± 1.15 b 07.44 ± 1.82 d 09.61 ± 2.00 d 81.10 ± 1.65 b 02.33 ± 0.84 d 01.25 ± 0.44 c 1.00 03.00 ± 0.00 c 02.00 ± 0.00 e 00.00 ± 0.00 e 14.43 ± 2.88 c 02.00 ± 0.74 e 00.00 ± 0.00 e 50.00 ± 3.56 c 01.09 ± 0.29 d 00.00 ± 0.00 c 2.00 00.00 ± 0.00 d 00.00 ± 0.00 e 00.00 ± 0.00 e 07.76 ± 0.57 d 01.00 ± 0.00 e 00.00 ± 0.00 e 11.10 ± 1.73 d 01.00 ± 0.00 d 00.00 ± 0.00 c Herbicide (mg/mL) 0.062 100.0±0.00a 07.23 ± 1.40 b 26.13 ± 1.25 a 100.0 ± 5.01 a 33.33 ± 1.14 b 22.00 ± 1.57 b 100.0 ± 0.00 a 11.00 ± 2.82 b 14.63 ± 1.80 b 0.125 100.0 ± 0.00 a 05.56 ± 1.04 b c 24.36 ± 1.55 a b 100.0 ± 4.35 a 29.06 ± 1.06 b c 20.53 ± 1.52 b 100.0 ± 0.00 a 07.96 ± 1,71c 10.50 ± 1.35 c 0.25 100.0 ± 0.00 a 04.80 ± 0.84 c 20.10 ± 1.96 b 100.0 ± 1.52 a 26.20 ± 2.63 c 18.88 ± 1.99 b 100.0 ± 0.00 a 07.26 ± 1.30 c 08.11 ± 0.96 c d 0.50 90.00 ± 0.57 a 04.74 ± 0.81 c 10.59 ± 2.76 c 86.66 ± 1.52 b 06.85 ± 1.26 d 04.83 ± 1.62 c 100.0 ± 0.00 a 05.60 ± 1.29 c d 07.70 ± 1.27 c d 1.00 90.00 ± 1.52 a 04.20 ± 0.95 c 08.37 ± 1.84 c d 86.66 ± 1.52 b 05.86 ± 0.89 d 04.00 ± 0.90 c 100.0 ± 0.00 a 04.28 ± 0.71 d 05.05 ± 1.25 d e 2.00 80.00 ± 2.04 b 03.85 ± 0.74 c 05.35 ± 1.53 d 86.66 ± 0.57 b 05.00 ± 1.08 d 03.63 ± 1.21 c 100.0 ± 0.00 a 04.00 ± 0.69 d 03.73 ± 0.73 e SG: seed germination; RL radicle length; PL plumule length; SD: standard deviation.In∗ the same column, different letters showed significant statistical differences for the applied doses of the essential oil (p<0.05).None of the doses of the herbicide revealed any inhibitory effects on the seed germination ofP. oleracea. As the EO, lower concentrations of the herbicide ranging from 0.062 to 0.25 mg/mL had no inhibitory effect on the seed germination ofL. sativa andL. sativum. At 0.50 mg/mL and higher doses, herbicide inhibited the seed germination by 10, 10, and 20% inL. sativa and by 13, 13, and 13% inL. sativum, respectively. It appeared that the inhibitory effects of the essential oil on the germination of test seeds seem to be more noteworthy than those of the herbicide.


### 3.4. Effects on the Seedling Growth

In the agriculture, herbicides have been utilised in the fields to overcome undesirable attack of various organisms to crops. Detrimental effects of these chemicals are not questionable anymore because of long term negative effects on the soil structure and community [36, 37]. Natural compounds are very attractive constituents to overcome the negative effects of the synthetic chemicals. In this study, inhibitory effects at the lowest dose of the EO (0.0625 mg/mL) were not observed on the radicle growth ofL. sativa seeds. Two-fold increase onwards showed decreasing effects on the radicle. The most significant decreasing effects were 95% at 1.0 mg/mL and 100% at 2.0 mg/mL, respectively. Plumules ofL. sativa were also inhibited significantly by 85% at 0.50 mg/mL; however, a complete inhibition was observed at 1.0 and 2.0 mg/mL. Herbicide in this assay was not as effective as the EO at the same doses (Table 6). A complete inhibition was not observed on neither the length of radicle nor the plumule ofL. sativa. Herbicide seemed to have more inhibitory effects on the radicle growth ofL. sativa rather than the plumule growth at 0.0625–2.0 mg/mL. It appeared that herbicide was not found to be as effective as the essential oil.EO revealed significant inhibitory effects on radicle growth ofL. sativum by 93% at 0.50 mg/mL, 98% at 1.0 mg/mL, and 99% at 2.0 mg/mL. It was also found to be very active on the plumule growth ofL. sativum by 91% at 0.50 mg/mL onwards. A complete inhibition of the plumule growth ofL. sativum was observed at 1.0 and 2.0 mg/mL. It seemed that EO seemed to be more effective on the plumule growth ofL. sativum. Compared to the EO, herbicide revealed similar inhibitory effects on the radicle growth, whereas inhibition on both the radicle and the plumule was not effective as the EO.EO ofT. cilicicum inhibited the growth ofP. oleracea at all doses; however, the most significant inhibitory effects on the radicle growth ofP. oleracea were observed at 0.5 mg/mL onwards. At 0.50, 1.0, and 2.0 mg/mL, inhibitions of the radicle were 95%, 97%, and 99%, respectively. The plumule growth ofP. oleracea was also inhibited by 94% at 0.50 mg/mL; however, a total inhibition was observed at 1.0 and 2.0 mg/mL of the EO doses. The present findings indicated that effects of the EO were more prominent on the plumule growth. Unlike EO, different doses of the herbicide had no inhibitory effect on neither the radicle nor the plumule growth ofP. oleracea; however, inhibitory effects of the herbicide were more prominent on radicle growth rather than plumule growth ofP. oleracea.In a previous study, the bioherbicidal potential of two species of the genusTanacetum has been reported by Salamci et al. (2007), who found that EO ofT. aucheranum andT. chiliophyllum at the dose of 30 µL per petri receiving 50 test seeds completely inhibited the seed germination, radicle, and plumule growth ofA. retroflexus,C. album, andR. crispus. The findings of the previous and the present results indicated thatTanacetum species has active bioherbical constituents and deserves to be studied in more detail in advance.


## 3.1. Essential Oil Composition ofT. cilicicum

As shown in Table3 and Figure 1, EO of the aerial parts fromT. cilicicum consisted of mainly (%) linalool (7.01 ± 0.32), sesquisabinene hydrate (6.88 ± 0.41), eucalyptol (5.08 ± 0.32), nerolidol (4.90 ± 0.33), α-muurolol (4.57 ± 0.35), borneol (4.21 ± 0.17), camphor (3.53 ± 0.27), limonene (3.17 ± 0.25), α-terpineol (3.13 ± 0.23), spathulanol (2.98 ± 0.12), α-pinene (2.95 ± 0.19), juniper camphor (2.68 ± 0.19), (-)-caryophyllene oxide (2.64 ± 0.19), 8-hydroxylinalool (2.62 ± 0.15), Δ-cadinene (2.48 ± 0.16), sabinene (2.32 ± 0.11), trans-verbenol (2.06 ± 0.11), and alloaromadendrene (2.0 ± 0.16). β-Pinene (1.89 ± 0.10), cis-caryophyllene (1.58 ± 0.09), epiglobulol (1.58 ± 0.11), α-cadinol (1.51 ± 0.13), 4-terpineol (1.45 ± 0.09), germacrene D (1.26 ± 0.09), α-copaene (1.24 ± 0.09), α-cedrene (1.21 ± 0.08), and spathulenol (1.21 ± 0.07) were other significant compounds in the EO ofT. cilicicum. EO yield of the aerial parts ofT. cilicicumwas 0.4% (v/w). In previous reports, essential oil yield and chemical constituents ofTanacetum sp. were reported in different species belonging to genusTanacetum (Table 1). It seemed that yield and composition differed from previous reports owing to the species differences belonging to this genus.Table 3
Essential oil composition ofT. cilicicum.
Number Retention index Compound % 1 1010 α-Pinene 2.95 ± 0.19 2 1015 α-Thujene 0.08 ± 0.01 3 1052 Camphene 0.73 ± 0.03 4 1095 β-Pinene 1.89 ± 0.10 5 1109 Sabinene 2,32 ± 0,11 6 1113 Verbenene 0,08 ± 0,01 7 1154 β-Myrcene 0,07 ± 0,01 8 1168 α-Terpinene 0,19 ± 0,02 9 1188 Limonene 3,17 ± 0,25 10 1198 Eucalyptol 5,08 ± 0,32 11 1235 γ-Terpinene 0,36 ± 0,02 12 1260 para-Cymene 0.54 ± 0.03 13 1286 Amyl isovalerate 0.06 ± 0.01 14 1345 3-Methyl-3-butenyl isovalerate 0.11 ± 0.01 15 1383 Nonanal 0.23 ± 0.01 16 1429 trans-Linalool oxide (fur.) 0.12 ± 0.01 17 1450 trans-Sabinene hydrate 0.80 ± 0.02 18 1455 Menthone 0.16 ± 0.03 19 1487 α-Copaene 1.24 ± 0.09 20 1509 Camphor 3.53 ± 0.27 21 1513 Benzaldehyde 0.13 ± 0.01 22 1533 Linalool 7.01 ± 0.32 23 1545 4-Acetyl-1-methyl-1-cyclohexene 0.29 ± 0.01 24 1550 trans-p-Mentha-2-en-1-ol 0.13 ± 0.01 25 1560 Pinocarvone 0.40 ± 0.02 26 1574 Bornyl acetate 0.52 ± 0.07 27 1590 4-Terpineol 1.45 ± 0.09 28 1593 Terpendiol I 0.64 ± 0.04 29 1614 trans-p-Mentha-2.8-dien-1-ol 0.52 ± 0.03 30 1621 Myrtenal 0.25 ± 0.01 31 1639 Pulegone 0.33 ± 0.07 32 1643 Alloaromadendrene 2.00 ± 0.16 33 1655 Isoledene 0.48 ± 0.03 34 1666 trans-Verbenol 2.06 ± 0.11 35 1671 cis-Citral 0.18 ± 0.02 36 1684 α-Terpineol 3.13 ± 0.23 37 1690 Borneol 4.21 ± 0.17 38 1706 Germacrene D 1.26 ± 0.09 39 1718 α-Muurolene 0.94 ± 0.06 40 1726 Carvone 0.28 ± 0.01 41 1729 Bicyclogermacrene 0.20 ± 0.02 42 1737 Verbenol 0.21 ± 0.04 43 1746 Neryl acetate 0.30 ± 0.03 44 1754 Δ-Cadinene 2.48 ± 0.16 45 1756 γ-Cadinene 0.81 ± 0.07 46 1764 1.7.7-Trimethylbicyclo[2.2.1]hept-5-en-2-ol 0.27 ± 0.06 47 1767 Ar-Curcumene 0.61 ± 0.05 48 1780 Myrtenol 0.76 ± 0.04 49 1785 Isocarveol 0.42 ± 0.06 50 1821 trans-Carveol 0.32 ± 0.02 51 1828 cis-Calamenene 0.13 ± 0.01 52 1831 Geraniol 0.84 ± 0.07 53 1859 Benzyl isovalerate 0.57 ± 0.03 54 1874 trans-p-Mentha-1(7).8-dien-2-ol 0.36 ± 0.03 55 1879 Spathulanol 2.98 ± 0.12 56 1883 benzyl-Isovalerate 0.52 ± 0.04 57 1912 α-Calacorene 0.22 ± 0.06 58 1923 Palustrol 0.13 ± 0.02 59 1932 Sesquisabinene hydrate 6.88 ± 0.41 60 1981 (-)-Caryophyllene oxide 2.64 ± 0.19 61 1989 Benzyl tiglate 0.51 ± 0.04 62 2020 Tridecanal 0.38 ± 0.03 63 2026 Nerolidol 4.90 ± 0.33 64 2041 cis-Caryophyllene 1.58 ± 0.09 65 2053 (-)-Cedreanol 0.70 ± 0.04 66 2059 α-Cedrene 1.21 ± 0.08 67 2067 Elemol 1.00 ± 0.09 68 2120 8-Hydroxylinalool 2.62 ± 0.15 69 2141 Spathulenol 1.21 ± 0.07 70 2153 Eugenol 0.54 ± 0.04 71 2163 α-Muurolol 4.57 ± 0.35 72 2171 Cubenol 0.35 ± 0.02 73 2177 α-Cadinol 1.51 ± 0.13 74 2193 ar-Turmerol 0.47 ± 0.03 75 2221 Epiglobulol 1.58 ± 0.11 76 2245 Juniper camphor 2.68 ± 0.19 77 2289 Isoaromadendrene epoxide 0.69 ± 0.04 78 2294 Tricosane 0.13 ± 0.01 79 2327 α-Santalol 0.34 ± 0.02 80 2407 Nerolidol 0.56 ± 0.04 81 2494 Pentacosane 0.21 ± 0.02 82 2570 1-Octadecanol 0.47 ± 0.03 83 2597 Phytol 0.19 ± 0.02 Total   100.00Figure 1
GC/MS chromatogram of the EO fromT. cilicium.The peaks in the chromatogram belong to the main compounds which are more than 1% of the EO chemical composition: (1) α-pinene, (2) β-pinene, (3) sabinene, (4) limonene, (5) eucalyptol, (6) α-copaene, (7) camphor, (8) linalool, (9) 4-terpineol, (10) alloaromadendrene, (11) trans-verbenol, (12) α-terpineol, (13) borneol, (14) germacrene D, (15) Δ-cadinene, (16) spathulanol, (17) sesquisabinene hydrate, (18) (-)-caryophyllene oxide, (19) nerolidol, (20) cis-caryophyllene, (21) α-cedrene, (22) 8-hydroxylinalool, (23) spathulenol, (24) α-muurolol, (25) α-cadinol, (26) epiglobulol, and (27) juniper camphor (the peak numbering given here is not in accordance with peak numbers in Table 3).


## 3.2. Antimicrobial Activity Results of the EO ofT. cilicicum

Antibiotics raise serious concerns in relation to antimicrobial resistance problems for all group of organisms. Therefore, natural sources of the environments have been intensively studied by many scientists to combat their undesirable effects to the health of living organisms and their environments as well [33–35]. In this study, EO ofT. cilicicum was tested on twelve microorganisms, with two methods: disc diffusion and macrobroth dilution. In addition, standard antibiotics were used for comparison during the assays. As shown in Table 4, the results of the disc diffusion assay indicated that the most sensitive microorganisms wereB. subtilis andS. aureus 29213. This was followed byB. cereus=S. aureus BAA >E. faecalis >E. casseliflavus. The susceptibilities of the Gram-negative bacteria towards the essential oil were less than Gram-positive bacteria. The most susceptible Gram-negative bacterium wasE. hormaechei >E. coli >K. pneumonia =P. aeruginosa. In addition,C. parapsilosiswas also more susceptible than that ofC. albicans.Table 4
Antimicrobial activities of the EO fromT. cilicicum using disc diffusion assay.
Microorganisms Source EO(15 µL) SXT5 VA30 TEC30 NS 100 IU Inhibition zones (mm) B. subtilis ATCC 6633 19.33 ± 0.57 a B 34.66 ± 0.57 a A 19.33 ± 0.57 c B 15.33 ± 0.57 e C — B. cereus EU (food isolate) 16.00 ± 0.00 b C 23.33 ± 1.15 e A 20.00 ± 0.00 b c B 22.33 ± 0.57 a A — E. faecalis ATCC 29212 15.67 ± 0.57 b c C 25.00 ± 1.00 d e A 21.00 ± 1.00 b c B 17.66 ± 0.57 d C — E. casseliflavus ATCC 700327 14.33 ± 0.57 c D 26.66 ± 0.57 d A 24.66 ± 0.57 a B 21.00 ± 1.00 a b C — S. aureus ATCC 29213 19.66 ± 0.57 a B 32.00 ± 0.00 b A 21.66 ± 1.52 b B 20.00 ± 1.00 b c B — S. aureus ATCC BAA977 16.33 ± 0.57 b D 33.00 ± 0.00 a b A 20.00 ± 0.00 b c B 18.66 ± 0.57 c d C — K. pneumoniae ATCC 700603 10.33 ± 0.57 e B 18.00 ± 1.00 f A 0.00 ± 0.00 d C 0.00 ± 0.00 f C — E. hormaechei ATCC 700323 12.00 ± 0.00 d B 26.00 ± 0.00 d A 0.00 ± 0.00 d C 0.00 ± 0.00 f C — P. aeruginosa ATCC 27853 10.00 ± 0.00 e A 0.00 ± 0.00 g B 0.00 ± 0.00 d B 0.00 ± 0.00 f B — E. coli ATCC 25922 10.66 ± 0.57 d e B 28.66 ± 0.57 c A 0.00 ± 0.00 d C 0.00 ± 0.00 f C — C. parapsilosis ATCC 22019 29.00 ± 0.00 a A — — — 16.00  ±  1.00 b B C. albicans ATCC 14053 16.33 ± 0.5 7 b B — — — 19.66 ± 1.15 a A EO: essential oil; SXT5: Trimethoprim/Sulfamethoxazole; VA30: Vancomycin; TEC30: Teicoplanin; NS 100 IU: Nystatin. The small case letters as the superscript in the columns and capital letters as the superscript in the columns showed the statistical differences in each column and row, respectively (p<0.05). — indicates not tested.As shown in Table5, according to MIC/MBC (µL/mL) assays, it was found that the most potent activity was observed onB. subtilis (0.39/0.78) andB. cereus (0.78/1.56). Higher inhibitory and cidal values of the EO fromT. cilicicum were observed for other tested microorganisms. This was statistically the same forS. aureus BAA (5.20/6.25) =E. casseliflavus (6.25/12.5) =S. aureus 29213 (6.25/6.25) =E. hormaechei (6.25/25.00) =E. coli (6.25/6.25) =E. faecalis (6.25/12.5). Less activities were determined for two Gram-positive bacteria and the activity was statistically the same forK. pneumoniae (10.41/50.00) andP. aeruginosa (12.5/50.00). MIC and MFC values (µL/mL) ofT. cilicicum were 1.56/6.25 forC. albicans and 3.12/12.5 forC. parapsilosis.Table 5
Minimum Inhibitor Concentration and Minimum Bactericidal/Fungicidal Concentration values ofT. cilicicum essential oil.
Microorganisms  EO (µL/mL) AMP (µg/mL) CLO (µg/mL) MIC MBC/MFC MIC MBC MIC MFC B. subtilis ATCC 6633 0.39 ± 0.00 a 0.78 ± 0.00 a 15.62 ± 0.00 d 31.25 ± 0.00 a — — B. cereus EU (food isolate) 0.78 ± 0.00 a 1.56 ± 0.00 a 1000 ± 0.00 g >1000 ± 0.00d — — E. faecalis ATCC 29212 6.25 ± 0.00 b 12.50 ± 0.00 c 0.48 ± 0.00 a 125.0 ± 0.00 b — — E. casseliflavus ATCC 700327 6.25 ± 0.00 b 12.50 ± 0.00 c 0.48 ± 0.00 a 31.25 ± 0.00 a — — S. aureus ATCC 29213 6.25 ± 0.00 b 6.25 ± 0.00 b 31.25 ± 0.00 e 250.0 ± 0.00 c — — S. aureus ATCC BAA977 5.20 ± 1.80 b 6.25 ± 0.00 b 125.0 ± 0.00 f 250.0 ± 0.00 c — — K. pneumoniae ATCC 700603 10.41 ± 3.60 c 50.00 ± 0.00 e >1000 ± 0.00g >1000 ± 0.00d — — E. hormaechei ATCC 700323 6.25 ± 0.00 b 25.00 ± 0.00 d 7.81 ± 0.00c 41.66 ± 8.04 a — — P. aeruginosa ATCC 27853 12.5 ± 0.00 c 50.00 ± 0.00 e >1000 ± 0.00g >1000 ± 0.00d — — E. coli ATCC 25922 6.25 ± 0.00 b 6.25 ± 0.00 b 3.90 ± 0.00 b 125.0 ± 0.00 b — — C. parapsilosis ATCC 22019 3.12 ± 0.00 a b 12.5 ± 0.00 c — — 300 ± 0.00 a >300 ± 0.00a C. albicans ATCC 14053 1.56 ± 0.00 a 6.25 ± 0.00 b — — >300 ± 0.00a >300 ± 0.00a In each column, small case letters as the superscript in the columns showed the statistical differences (p<0.05). — indicates not tested. MIC: Minimum Inhibitory Concentration; MFC: Minimum Fungicidal Concentration; AMP: Ampicillin; CLO: Clotrimazole.In previous studies, EO ofTanacetum sp. was assayed on various microorganisms. The EO fromT. balsamita subsp.balsamita andT. chiliophyllum var.chiliophyllumrevealed inhibitory zones on the growth ofB. subtilis ATCC 6633 (13-17 mm),S. aureus ATCC 6538 P (17–15),E. coli ATCC 25922 (15-16 mm),C. glabrata ATCC 66032 (15–18 mm), andC. tropicalis ATCC 13803 (14–17 mm) [14]. In another study, EO from the flowers and stems ofT. chiliophyllumvar.chiliophyllum grown in three different localities showed MIC values (µg/mL) as follows:S. aureus ATCC 6538 (250/500, 150/500, and 250/>500), meticillin resistantS. aureus(clinical isolate) (500/125, 750/500, and 250/250),S. epidermidis ATCC 12228 (250/250, 150/500, and 500/31.2),B. cereus NRRL B-3711 (250/125, 150/500, and >500/125),B. subtilis NRRL B-4378 (500/125, 375/500, and 500/62.5),E. coli NRRL B-3008 (>500/62.5, >150/500, and >500/62.5),P. aeruginosa ATCC 27853 (500/250, 150/500, and 500/31.25), andE. aerogenes NRRL 3567 (500/500, >150/>500, and >500/62.5) [19]. EO from the flowers and stems ofT. chiliophyllumvar.monocephalum had MIC values (µg/mL):S. aureus ATCC 6538 (>500, 1000), meticillin resistantS. aureus (125, >500),B. cereus NRRL B-3711 (62.5, 1000),B. subtilis NRRL B-4378 (250, >1000),E. coli NRRL B-3008 (>500, >1000),P. aeruginosa ATCC 27853 (500, 1000), andE. aerogenes NRRL 3567 (>500, >1000) [20]. MIC values (µg/mL) of the EO fromT. argenteumsubsp.flabellifoliumwere as follows:E. coliATCC 25922 (250),S. aureusATCC 6538 (125),P. aeruginosaATCC 27853 (125),E. aerogenesNRRL 3567 (125), andC. albicansO.G.U (125) [15].In another study, the EO ofT. aucheranum andT. chiliophyllum revealed various level of antimicrobial activities on test microorganisms exceptP. cichorii andB. subtilis ATCC 6633 using the disc diffusion and broth dilution assay. Inhibitory zones (as mm) and MIC values (µL/mL) of the essential oils fromT. aucheranum andT. chiliophyllum were as follows, respectively:P. chlororaphis (13.5 mm, 166.7) (13.3 mm, 500.0),P. syringae pv.syringae (7.8 mm, 500.0), (7.5 mm, 500.0),B. coagulans (11.8 mm, 166.7), (10.2 mm, 166.7),E. faecalis ATCC 29122 (9.0 mm, 1000.0), (0.0, 0.0),S. aureus ATCC 29213 (9.5 mm, 1000,0), (11.5 mm, 1000.0),E. intermedius (10.7 mm, 166.7), (10.2 mm, 54.4),E. coli (9.8 mm, 166.7), (8.8 mm, 500.0),P. aeruginosa ATCC 27859 (19.8 mm, 166.7), (16.5 mm, 500.0),P. aeruginosa ATCC 9027 (11.0 mm, 1000.0), (12.0 mm, 1000.0), andK. trevisanii (9.3 mm, 500.0), (8.2 mm, 166.7) [16]. The differences of the results of the present study when compared to the previous reports could be attributed to the chemical differences in the species assayed.


## 3.3. Phytotoxicity Assay ofT. cilicicum

As shown in Table6, EO ofT. cilicicum as well as the herbicide (glyphosate (N-(phosphonomethyl) glycine) at 0.062, 0.125, 0.25, 0.50, 1.0, and 2.0 mg/mL were assayed on the seeds ofL. sativa,L. sativum, andP. oleracea. Effects of test substances were dependent on the dose activity. EO at 0.25 mg/mL and the lower concentrations did not reveal any inhibitory effects on the seeds ofL. sativa,L. sativum, andP. oleracea. Higher concentrations ranging from 0.50 to 2.0 mg/mL had inhibitory effects on the growth ofL. sativa,L. sativum, andP. oleracea. Compared to the control, 0.50, 1.0, and 2.0 mg/mL doses of the EO inhibited the seed germination by 87.97 and 100% inL. sativa, by 55, 86, and 92% inL. sativum, and by 19, 50, and 89% inP. oleracea, respectively.Table 6
Effects of the essential oil fromT. cilicicum on the seed germination (%), radicle, and plumule growth of L. sativa, L. sativum,andP. oleracea.
Lactuca sativa Lepidium sativum Portulaca oleracea SG (%) RL(mm) (mean ± SD) PL(mm) (mean ± SD) SG (%) RL(mm) (mean ± SD) PL(mm) (mean ± SD) SG (%) RL(mm) (mean ± SD) PL(mm) (mean ± SD) Control 100.0 ± 0.00 a ∗ 40.73 ± 1.04 a 27.33 ± 1.77 a 100.0 ± 0.00 a 50.86 ± 3.14 a 39.33 ± 1.54 a 100.0 ± 0.00 a 43.36 ± 1.20 a 20.13 ± 1.88 a EO (mg/mL) 0.062 100.0 ± 0.00 a 40.70 ± 1.16 a 20.33 ± 1.30 b 100.0 ± 0.00 a 49.73 ± 2.31 a 30.90 ± 2.80 b 100.0 ± 0.00 a 33.36 ± 1.64 b 16.20 ± 3.10 a b 0.125 100.0 ± 0.00 a 33.30 ± 1.63 b 17.83 ± 1.27 b 100.0 ± 0.00 a 39.73 ± 1.08 b 24.23 ± 1.90 c 100.0 ± 0.00 a 31.40 ± 1.58 b 15.63 ± 2.64 a b 0.25 100.0 ± 0.00 a 29.16 ± 1.86 c 14.16 ± 1.49 c 100.0 ± 0.00 a 34.16 ± 1.52 c 21.73 ± 2.36 c 100.0 ± 0.00 a 20.33 ± 2.59 c 11.76 ± 2.52 b 0.50 13.33 ± 2.00 b 10.75 ± 1.59 d 04.00 ± 0.81 d 45.53 ± 1.15 b 07.44 ± 1.82 d 09.61 ± 2.00 d 81.10 ± 1.65 b 02.33 ± 0.84 d 01.25 ± 0.44 c 1.00 03.00 ± 0.00 c 02.00 ± 0.00 e 00.00 ± 0.00 e 14.43 ± 2.88 c 02.00 ± 0.74 e 00.00 ± 0.00 e 50.00 ± 3.56 c 01.09 ± 0.29 d 00.00 ± 0.00 c 2.00 00.00 ± 0.00 d 00.00 ± 0.00 e 00.00 ± 0.00 e 07.76 ± 0.57 d 01.00 ± 0.00 e 00.00 ± 0.00 e 11.10 ± 1.73 d 01.00 ± 0.00 d 00.00 ± 0.00 c Herbicide (mg/mL) 0.062 100.0±0.00a 07.23 ± 1.40 b 26.13 ± 1.25 a 100.0 ± 5.01 a 33.33 ± 1.14 b 22.00 ± 1.57 b 100.0 ± 0.00 a 11.00 ± 2.82 b 14.63 ± 1.80 b 0.125 100.0 ± 0.00 a 05.56 ± 1.04 b c 24.36 ± 1.55 a b 100.0 ± 4.35 a 29.06 ± 1.06 b c 20.53 ± 1.52 b 100.0 ± 0.00 a 07.96 ± 1,71c 10.50 ± 1.35 c 0.25 100.0 ± 0.00 a 04.80 ± 0.84 c 20.10 ± 1.96 b 100.0 ± 1.52 a 26.20 ± 2.63 c 18.88 ± 1.99 b 100.0 ± 0.00 a 07.26 ± 1.30 c 08.11 ± 0.96 c d 0.50 90.00 ± 0.57 a 04.74 ± 0.81 c 10.59 ± 2.76 c 86.66 ± 1.52 b 06.85 ± 1.26 d 04.83 ± 1.62 c 100.0 ± 0.00 a 05.60 ± 1.29 c d 07.70 ± 1.27 c d 1.00 90.00 ± 1.52 a 04.20 ± 0.95 c 08.37 ± 1.84 c d 86.66 ± 1.52 b 05.86 ± 0.89 d 04.00 ± 0.90 c 100.0 ± 0.00 a 04.28 ± 0.71 d 05.05 ± 1.25 d e 2.00 80.00 ± 2.04 b 03.85 ± 0.74 c 05.35 ± 1.53 d 86.66 ± 0.57 b 05.00 ± 1.08 d 03.63 ± 1.21 c 100.0 ± 0.00 a 04.00 ± 0.69 d 03.73 ± 0.73 e SG: seed germination; RL radicle length; PL plumule length; SD: standard deviation.In∗ the same column, different letters showed significant statistical differences for the applied doses of the essential oil (p<0.05).None of the doses of the herbicide revealed any inhibitory effects on the seed germination ofP. oleracea. As the EO, lower concentrations of the herbicide ranging from 0.062 to 0.25 mg/mL had no inhibitory effect on the seed germination ofL. sativa andL. sativum. At 0.50 mg/mL and higher doses, herbicide inhibited the seed germination by 10, 10, and 20% inL. sativa and by 13, 13, and 13% inL. sativum, respectively. It appeared that the inhibitory effects of the essential oil on the germination of test seeds seem to be more noteworthy than those of the herbicide.


## 3.4. Effects on the Seedling Growth

In the agriculture, herbicides have been utilised in the fields to overcome undesirable attack of various organisms to crops. Detrimental effects of these chemicals are not questionable anymore because of long term negative effects on the soil structure and community [36, 37]. Natural compounds are very attractive constituents to overcome the negative effects of the synthetic chemicals. In this study, inhibitory effects at the lowest dose of the EO (0.0625 mg/mL) were not observed on the radicle growth ofL. sativa seeds. Two-fold increase onwards showed decreasing effects on the radicle. The most significant decreasing effects were 95% at 1.0 mg/mL and 100% at 2.0 mg/mL, respectively. Plumules ofL. sativa were also inhibited significantly by 85% at 0.50 mg/mL; however, a complete inhibition was observed at 1.0 and 2.0 mg/mL. Herbicide in this assay was not as effective as the EO at the same doses (Table 6). A complete inhibition was not observed on neither the length of radicle nor the plumule ofL. sativa. Herbicide seemed to have more inhibitory effects on the radicle growth ofL. sativa rather than the plumule growth at 0.0625–2.0 mg/mL. It appeared that herbicide was not found to be as effective as the essential oil.EO revealed significant inhibitory effects on radicle growth ofL. sativum by 93% at 0.50 mg/mL, 98% at 1.0 mg/mL, and 99% at 2.0 mg/mL. It was also found to be very active on the plumule growth ofL. sativum by 91% at 0.50 mg/mL onwards. A complete inhibition of the plumule growth ofL. sativum was observed at 1.0 and 2.0 mg/mL. It seemed that EO seemed to be more effective on the plumule growth ofL. sativum. Compared to the EO, herbicide revealed similar inhibitory effects on the radicle growth, whereas inhibition on both the radicle and the plumule was not effective as the EO.EO ofT. cilicicum inhibited the growth ofP. oleracea at all doses; however, the most significant inhibitory effects on the radicle growth ofP. oleracea were observed at 0.5 mg/mL onwards. At 0.50, 1.0, and 2.0 mg/mL, inhibitions of the radicle were 95%, 97%, and 99%, respectively. The plumule growth ofP. oleracea was also inhibited by 94% at 0.50 mg/mL; however, a total inhibition was observed at 1.0 and 2.0 mg/mL of the EO doses. The present findings indicated that effects of the EO were more prominent on the plumule growth. Unlike EO, different doses of the herbicide had no inhibitory effect on neither the radicle nor the plumule growth ofP. oleracea; however, inhibitory effects of the herbicide were more prominent on radicle growth rather than plumule growth ofP. oleracea.In a previous study, the bioherbicidal potential of two species of the genusTanacetum has been reported by Salamci et al. (2007), who found that EO ofT. aucheranum andT. chiliophyllum at the dose of 30 µL per petri receiving 50 test seeds completely inhibited the seed germination, radicle, and plumule growth ofA. retroflexus,C. album, andR. crispus. The findings of the previous and the present results indicated thatTanacetum species has active bioherbical constituents and deserves to be studied in more detail in advance.


## 4. Conclusion

In the present study, EO ofT. cilicicum includes significant amount of antimicrobial compounds against a wide range of microorganisms and therefore, it could be used in food, agricultural, and pharmaceutical applications and so forth. Furthermore, significant phytoconstituents of the EO fromT. cilicicum could be suggested for use in the various formulation of biopesticides. However, further studies are required for isolating the bioactive constituents in the EO and testing alone and/or their synergistic, antagonistic relationships as well as determining their toxic effects on test organisms before potential industrial benefits.



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*Source: 6214896-2017-01-16.xml*