Biological Activity of Essential Oils of Four Juniper Species and Their Potential as Biopesticides

The objective of this study was to assess the biological activity of essential oils (EOs) of four Juniperus species obtained via two different distillation methods and their potential as biopesticides. The studied factors were juniper species (Juniperus communis L., J. oxycedrus L., J. pygmaea C. Koch., and J. sibirica Burgsd), plant sex (male (M) and female (F)), and distillation method (hydrodistillation via a standard Clevenger apparatus (ClevA) and semi-commercial (SCom) steam distillation). The hypothesis was that the EO will have differential antioxidant, antimicrobial, and insecticidal activities as a function of plant species, plant sex, and distillation method. The two distillation methods resulted in similar EO composition within a given species. However, there were differences in the EO content (yield) due to the sex of the plant, and also differences in the proportions of some EO components. The concentration of α-pinene, β-caryophyllene, δ-cadinene and δ-cadinol was dissimilar between the EO of M and F plants within all four species. Additionally, M and F plants of J. pygmaea, and J. sibirica had significantly different concentrations of sabinene within the respective species. The EOs obtained via ClevA extraction showed higher antioxidant capacity within a species compared with those from SCom extraction. All of the tested EOs had significant repellent and insecticidal activity against the two aphid species Rhopalosiphum padi (bird cherry-oat aphid) and Sitobion avenae (English grain aphid) at concentrations of the EO in the solution of 1%, 2.5%, and 5%. The tested EOs demonstrated moderate activity against selected pathogens Fusarium spp., Botrytis cinerea, Colletotrichum spp., Rhizoctonia solani and Cylindrocarpon pauciseptatum. The results demonstrate that the standard ClevA would provide comparable EO content and composition in comparison with SCom steam distillation; however, even slight differences in the EO composition may translate into differential bioactivity.

. Essential oil (EO) content of Juniperus oxycedrus, J. communis, J. pygmaea, and J. sibirica as a function of the sex of the tree and the extraction method.
As shown in Table 2, α-pinene was the predominant constituent of the monoterpenes in all four species (J. communis, J. pygmaea, J. sibirica, J. oxycedrus), with the highest concentration being in J. communis (F) EO. α-pinene varied from 14.9% (in J. sibirica F) to 34.9% (in J. communis F). The concentration of sabinene was generally above 9% of the EO, except in J. oxycedrus (M, F) ( Table 2). Overall, the concentration of limonene was higher in J. communis (M) and J. oxycedrus (M) and lower in J. sibirica (M, F) ( Table 3). α-terpinene, p-cymene, bornyl acetate, β-elemene were minor (below 2% of the EO) constituents in the EOs of all four species (Tables 2 and 4). β-myrcene and terpinen-4-ol were found at higher concentrations in the EO of J. communis (Tables 2 and 3). Overall, the monoterpenes in the EOs obtained via ClevA were slightly higher compared to those in the EO obtained through SCom. One sample of J. communis-M obtained through ClevA was an exception (Table S5). Table 2. Mean concentration (%) of α-pinene, camphene, sabinene, β-pinene, β-myrcene, α-terpinene, and p-cymene (extracted using the commercial steam extraction method) obtained from the eight combinations of species and sex.
The diterpenes class was the highest in J. oxycedrus (M) EO (~20%) (Table S5). Oxygenated diterpenes were found only in J. oxycedrus and J. communis EO, while monocyclic diterpenes were found in J. sibirica EO.
Generally, the EO composition of the respective samples obtained by the two extraction methods (ClevA and SCom) was very similar. We found differences in the proportions of EO constituents, with the exception of the J. communis EO. As mentioned in the Introduction section, J. communis is one of the most widely distributed species on the planet [20], and therefore its EO composition has been reported most often. Previous studies on J. communis utilized different EO extractions methods [29][30][31][32][33]. It has been demonstrated that supercritical fluid extraction and hydrodistillation methods result in dissimilar leaf EO composition [32].
Our results disprove the working hypothesis, because the EO profile of the samples extracted via the two different methods was very similar within a juniper species. In this study, the main EO constituents of the different extraction methods were α-pinene, sabinene, limonene, β-myrcene. Previous research identified the same composition of EOs obtained by micro-distillation and extraction, and supercritical carbon dioxide extraction from juniper needles [31]. Differences in the EO composition of J. communis have been reported following supercritical fluid extraction (SFE) and hydrodistilled EOs [32]. The cited authors found a total of 22 compounds in the EOs from SFE, and only 11 in the hydrodistilled EO [32]. The EO of J. communis berries (galbuli) had different qualitative compositions when extracted via hydrodistillation and hexane extraction methods [30]. The concentrations of α-pinene, sabinene, myrcene were higher in the hydrodistilled EO, while some less volatile compounds were present in the extracts, especially in the hexane extract [30].

Insecticidal Action and Repellent Activities of the EOs from the Semi-Commercial Steam Extraction against Rhopalosiphum padi (Bird Cherry-Oat Aphid) and Sitobion avenae (English Grain Aphid)
Aphids are some of the main pests on cereal crops and can significantly reduce yields [34,35]. The interaction effect of species-sex and EO concentration was highly significant on % number (nb) for the repellent action of EO on leaf aphids (Table 5), which suggests that the ideal EO concentration that needs to be used in repellent varies with the combination of the species and the sex of the plant. There are no previous reports concerning repellent and insecticidal action of the EOs of Bulgarian conifers. The insect repellent activity of EOs is commonly used to deter insects. The results of the repellent activity for the EO and constituents against Rhopalosiphum padi and Sitobion avenae are presented in Table 6. The data of the tested EO of Rh. padi showed that at a 5% concentration, the EOs of J. oxycedrus (M), J. sibirica (M) and J. pygmaea (F, M) had a strong repellent activity (Table 6), where the main compound EO in all three species was α-pinene (Table 2). However, at concentrations of 1-2.5%, the EOs J. communis (M), J. sibirica (F) (1%), J. oxycedrus (M), and J. sibirica (F) (2.5%) demonstrated a lower repellent action (Table 6). Similar results were obtained by Carroll et al. [36]. The cited authors tested junipers EOs (J. chinensis, J. communis) for repellency for Aedes aegypti, Amblyomma americanum and Ixodes scapularis and concluded that the oils were repellent to both species of ticks, but EO of J. communis had a minimum effective dosage for repellency [36]. However, in this study, S. avenae was more sensitive to the tested EOs at lower concentrations. The three tested concentrations (1%, 2.5% and 5%) of the EOs of J. communis (F), J. sibirica (F), and J. oxycedrus (M) had a strong repellent activity, and α-pinene and sabinene were main EO constituents in the three species. The oils of J. communis (M) (2.5% and 5%), J. pygmaea (F) (1% and 5%), J. pygmaea (M) (2.5% and 5%), and J. sibirica (M) (2.5% and 5%) had a lower repellent activity (Table 6). Table 6. Mean Rhopalosiphum padi and Sitobion avenae for the repellent action obtained from the 28 combinations of species-sex and EO concentration (Conc).  (Table 2). Over 50% of the composition of the J. communis, J. pygmaea and J. sibirica oils consists of α-pinene, sabinene, germacrene D, and δ-cadinene. α-pinene was reported as a repellent in an insect activity study [37]. Obviously, the synergism between the individual compounds in the EOs of J. communis, J. pygmaea and J. sibirica is the reason for their activity. Current thinking is that although some specific compounds may have a repellent effect, in some instances, the interaction between them causes greater bioactivity [38]. The EOs of junipers can be used in lower doses to achieve a repellent effect in agricultural management practices. The insecticidal activity of the EOs obtained from the four juniper species (M, F) was established by testing oil efficacy at different concentrations. The effect was calculated using Abbot's formula [39]. The tested EOs demonstrated a very good insecticidal effect 24 h after the treatment of the aphids ( Table 7). The EOs most often act as neurotoxins on the insects, and they affect their physiological processes [40,41]. The EOs' efficacy was 100% on both aphids (S. avenae and Rh. padi). An exception was the EO of J. sibirica (M) at 2.5% and J. communis (F) at 1%, with efficiencies around 90%. After 72 h, the efficiency in all treatments and concentrations was 100% ( Table 7). The juniper EOs have mostly been tested for the control of mosquitoes and ticks, and less for the control of agricultural pests. Athanassiou et al. [42] suggested that the simultaneous use of silica gel and EO of J. oxycedrus ssp. oxycedrus significantly enhanced the activity against Sitophilus oryzae. Therefore, the results suggest that the tested EOs could be utilized at their lowest dose (1%) to achieve a very good insecticidal effect [42]. We observed the excellent insecticidal effect of juniper EOs on both species of aphids. The efficacy was 100% after 24 h of the EO applications. These results are not accidental, because α-pinene, sabinene, limonene, β-myrcene are the main constituents of monoterpenes in juniper species and they have been reported to have insecticidal activity [43][44][45]. Monoterpenes and sesquiterpenes (α-pinene, terpineol, 1,8-cineole, p-cymene, limonene, α-terpinene, thymol, carvacrol) were found to have a high fumigant activity against Musca domestica, Tribolium confusum and Sitophilus oryzae [43,45].

Antifungal Activity of Juniper EOs on Plant Pathogenic Fungi
Screening of the antifungal activity of the four juniper species EOs (1 µL mL −1 ) on the studied plant pathogens showed a varying positive effect ( Table 8). The most substantial inhibitory effect on the radial mycelial growth was found against C. pauciseptatum and Fusarium sp. Significantly different inhibitory effects (p < 0.05) on the mycelial growth of Fusarium sp. (36.6% and 34.5%) were observed after the application of the EOs from J. sibirica (F) and J. pygmaea (M). Regarding the other tested EOs, the closest values to that already mentioned were obtained from the EOs of J. oxycedrus (M) and J. communis (M), and the differences in their antifungal activity were small. The largest difference in the antifungal activity of EOs obtained from M and F juniper species was between EOs from J. pygmaea (M) and J. pygmaea (F) (16.9%). The weakest inhibitory effect was found for the J. communis (F) EO against Fusarium spp. (Table 8).
The highest antifungal efficacy against C. pauciseptatum was observed in J. oxycedrus  Juniperus pygmaea (M) EO showed a strong inhibitory efficacy against four of the studied pathogens (Fusarium sp., R. solani, C. pauciseptatum and Colletotrichum sp.), while J. sibirica (F) showed a similar effect against Fusarium sp. and C. pauciseptatum ( Table 8). As shown in Table 2, the concentrations of sabinene (25.06), terpinen-4-ol (6.01) and α-pinene (19.64) were higher in J. pygmaea (M), and these compounds probably interact with each other. Botrytis cinerea was the most resistant species against the used juniper EOs. None of the tested EOs showed a significant inhibitory effect on mycelial growth of B. cinerea.
The available information on the biological activity of the tested juniper EOs against phytopathogenic fungi is scarce. Zabka et al. [46] reported relatively low inhibitory activity of EO from J. communis L. on Alternaria alternata, Cladosporium cladosporioides and Aspergilus niger, 11.8%, 31.0% and 1.8%, respectively, at 1 µL/mL. According to these authors, the high antifungal effect of Origanum vulgare, Thymus vulgaris and Pimenta racemosa EO of the 20 studied types of oils was due to the high content of the phenolic monoterpenes thymol, carvacrol or eugenol. Gleń-Karolczyk and Boligłowa [47] reported a strong inhibitory effect of J. communis EO, ranging from 87.1% at 0.1 mm 3 cm −3 to 93.1% at 1 mm 3 cm −3 on R. solani and from 42% at 0.1 mm 3 cm −3 to 85.4% at 1 mm 3 cm −3 on C. destructans isolated from horseradish seedlings. Analyzing EOs from green and ripe berries of J. communis, an antifungal effect was found, manifested against Sclerotium rolfsii [48]. Based on their own results and on those obtained by Jing et al. [49], the latter authors suggested that sesquiterpenes were the phytochemical components that show a toxic effect on fungal pathogens. It can be seen that EOs mainly from J. communis were tested to establish the antifungal effect against phytopathogens.
In the present study, the most significant inhibition of the radial mycelial growth of the studied phytopathogens was demonstrated by the EOs of J. sibirica (F) (36.6%) and J. pygmaea (M) (34.5%) on Fusarium spp., by the EO of J. oxycedrus (M, 29.8%) and J. pygmaea (M, 29.0%) on C. pauciseptatum, as well as by the EO of J. pygmaea (M, 31.97%) on R. solani.
In most cases, there were statistically significant differences between the means of the inhibition coefficient of the juniper EO from M and F plants against the tested pathogens (Table 8). This was probably due to the different chemical composition of the EOs obtained from M and F plants. An exception was found in EO from J. communis against C. pauciseptatum and J. sibirica against R. solani and C. pauciseptatum, in which, under the conditions of the present experiment, the differences between the inhibitory effect of EO obtained from M and F juniper plants were not significant.
Overall, we cannot find a clear pattern between the chemical composition of EOs of Juniperus species and their inhibitory effect. J. pygmaea and J. sibirica are characterized with a high content of sabinene (Table 2). In the studied species, the predominant component of EOs was the class monoterpenes, especially α-pinene. In order to determine exactly which compounds exhibit antifungal efficacy, these compounds should be tested individually. As indicated by Tripathietii et al. [50] and Sharma and Tripathi [51], the fungicidal activity of EO is often the result of the synergistic activity of their compounds.
The results from this study on the inhibitory effect of EOs from four species of juniper, M and F plants (1 µL/mL) on phytopathogens and polyphages, causing diseases both during vegetation and the storage of plant products, are encouraging and call for further research.

Antimicrobial Activity
The antimicrobial activity of the F and M Juniperus EOs (J. communis, J. oxycedrus, J. pygmaea, J. sibirica) obtained through the commercial steam extraction method was evaluated in this study. Among the combinations of the levels of the two factors (sex, species), the highest antimicrobial activity was observed in J. communis (M) and J. sibirica  [14,52], while Glišič et al. [53] found higher antimicrobial activity for Juniperus EOs compared with that of commercial antibiotics. Overall, the results of this study confirm our working hypothesis.

Antioxidant Activity
Essential oils, including those from some of the juniper species, have been utilized in the pharmaceutical, cosmetic and food industries [5]. The antioxidant capacity (ORAC) of J. communis, J. oxycedrus, J. pygmaea, and J. sibirica EOs obtained by different extraction methods were evaluated in this study. The ORAC values of Clevenger method EOs were higher than that of semi-commercial method EOs (Figure 1). The results on the antioxidant capacity confirm our working hypothesis that the EOs extracted via different methods would have dissimilar bioactivity. Overall, the values of monoterpenes in the tested EOs obtained by the Clevenger method were higher than the concentration of monoterpenes in the EOs obtained using the semi-commercial method, and α-pinene was the predominant constituent. Reports on the antioxidant capacity of junipers EOs can be found in the literature [7][8][9][10]14,23,24]. It is well known that the qualitative and quantitative profiles of junipers can be influenced by a number of factors, including genetic, environmental, and post-harvest processing such as drying and extraction. The higher antioxidant capacity of the galbuli EO from J. sibirica and J. communis were reported by Zheljazkov et al. [14], where α-pinene was the dominant compound in the EOs. Due to the high level of γ-terpinene, Emami et al. [54] reported the strong antioxidant capacity of the leaves J. communis subsp. hemisphaerica [54]. In this study, the highest ORAC value was obtained from EOs of J. oxycedrus (M) (Figure 1), where α-pinene and limonene were the dominant compounds ( Table 2). Current thinking is that the antioxidant capacity in EOs may depend strongly on the interaction between several compounds. The demonstrated high antioxidant capacity of some of the juniper oils opens the door for their wider utilization in various products. the pharmaceutical, cosmetic and food industries [5]. The antioxidant capacity (ORAC) of J. communis, J. oxyсedrus, J. pygmaea, and J. sibirica EOs obtained by different extraction methods were evaluated in this study. The ORAC values of Clevenger method EOs were higher than that of semi-commercial method EOs (Figure 1). The results on the antioxidant capacity confirm our working hypothesis that the EOs extracted via different methods would have dissimilar bioactivity. Overall, the values of monoterpenes in the tested EOs obtained by the Clevenger method were higher than the concentration of monoterpenes in the EOs obtained using the semi-commercial method, and α-pinene was the predominant constituent. Reports on the antioxidant capacity of junipers EOs can be found in the literature [7][8][9][10]14,23,24]. It is well known that the qualitative and quantitative profiles of junipers can be influenced by a number of factors, including genetic, environmental, and post-harvest processing such as drying and extraction. The higher antioxidant capacity of the galbuli EO from J. sibirica and J. communis were reported by Zheljazkov et al. [14], where α-pinene was the dominant compound in the EOs. Due to the high level of γ-terpinene, Emami et al. [54] reported the strong antioxidant capacity of the leaves J. communis subsp. hemisphaerica [54]. In this study, the highest ORAC value was obtained from EOs of J. oxycedrus (M) (Figure 1), where α-pinene and limonene were the dominant compounds ( Table 2). Current thinking is that the antioxidant capacity in EOs may depend strongly on the interaction between several compounds. The demonstrated high antioxidant capacity of some of the juniper oils opens the door for their wider utilization in various products.

Preparation of Juniperus Samples for the Extraction of the Essential Oil (EO)
The samples of the four Juniper species were collected in June and were placed in a shady area at a temperature of below 35 • C for air-drying.

Hydrodistillation Extraction of EO
The Clevenger-type (ClevA) hydrodistillation extraction of EO was performed according to the British Pharmacopoeia [56] by using 800 mL of water in 2 L Clevenger-type hydrodistillation units. The plant tissue samples consisted of 100 g of air-dried leaves in 2 L flasks for the ClevA distillation. All samples were distilled for 2 h in two replicates.

Steam Distillation Semi-Commercial Extraction (SCom) of EO
The SCom steam distillation was conducted in a semi-commercial 100 L steam distillation units using 20 kg of leaves and small twigs, steam distilled for 3 h, the usual steam distillation time used by industry for junipers. The resulting EO from the above extractions was collected, separated from the remaining water, and kept in a freezer until the gas chromatography (GC)-mass spectroscopy (MS) analyses were performed. The EO was measured both by volume and by weight.

Gas Chromatography (GC)-Mass Spectroscopy (MS) Analyses of the Essential Oils (EO)
The chemical profile of the four Juniperus EOs in three replications was determined by GC-FID and GC/MS techniques, according to the methods described previously [14]. Identification of the components present in the EO samples was performed by comparing the mass spectra of components in the EOs with those from the National Institute of Standards and Technology (NIST 08) and Adams mass spectra libraries [57], by AMDIS (Automated Mass spectral Deconvolution and Identification System) and by comparing the literature and estimated Kovat s (retention) indices that were determined using mixtures of homologous series of normal alkanes from C8 to C40 in hexane, under the same conditions mentioned above. The percentage ratio of EO components was computed using the normalization method of the GC/FID peak areas (Supplementary Materials). Rh. padi and S. avenae. The repellence of the EO was assessed by using the Petri dish assay [58]. The seven EOs were diluted with an aqueous solution with an emulsifier, 0.1% polysorbate 80. Two microliters of each EO of the target species with 0%, 1%, 2.5%, and 5% concentrations in three replicates were used, according to the methods described previously [10]. One treated leaf and one non-treated leaf plus 10 leafless aphids were introduced into each Petri dish. After that, the Petri dishes were covered with cheesecloth (44 g/m 2 ). The repellent effect was observed and recorded after 24 h.

Testing the Insecticidal Action of the Essential Oils (EO) from the SCom Steam Distillation against Rhopalosiphum padi and Sitobion avenae
The insecticidal activity of EOs was determined following a method described by Konstantopoulou et al. [59]. Each of the seven EOs was tested on the adult wingless forms of two aphid species at a concentration of 0%, 1%, 2.5%, and 5% in three replicates. The insecticidal actions of the EO on pests were evaluated on leaves of H. vulgare subsp. distichum var. erectum, cv. Obzor. The EOs were diluted in aqueous solution with an emulsifier of 0.1% polysorbate 80. The control (0%) was treated with a 0.1% aqueous solution of polysorbate 80. Two microliters of the solution (0%, 1%, 2.5%, and 5%) were applied directly on barley leaves with the aphid colonies. After the treatments, the leaves were dried on filter paper and transferred to Petri dishes [59]. The Petri dishes were covered with cheesecloth (44 g/m 2 ). The effect of the application (knock-down or mortality) was observed after 24 and 72 h. The results (knock-down or mortality) were compared with the controls. The effect of the application of EOs at different concentrations was calculated using Abbott's formula [38]: where E is aphid mortality; x is the percentage of live aphids in the control; and y is % of live aphids in the treatment with the EOs.

Fungal Plant Pathogenic Strains
Five plant pathogenic fungal strains were obtained from a culture collection maintained in the Department of Phytopathology, Agricultural University, Plovdiv, Bulgaria. Fusarium sp. (fusarium dry rot) and Rhizoctonia solani (stem canker and black scurf) were isolated from stored potato tubers (Solanum tuberosum L.), Botrytis cinerea (grey mould) strain was isolated from infected stored tomato fruits (Lycopersicon esculentum Mill). The strain of a Colletotrichum sp. was isolated from anthracnose of banana fruit (Musa sp.), whereas Cylindrocarpon pauciseptatum strain was obtained from black foot disease of grapevine (Vitis vinifera L.). All strains were identified using cultural and morphological characteristics, as well as pathogenicity test. The Cylindrocarpon pauciseptatum was confirmed by DNA sequences [60]. Strains were preserved on potato dextrose agar (PDA) at 4 • C.

Agar Dilution Method (Antifungal Activity of Juniper EOs)
Preliminary study of inhibitory effect of seven juniper essential oils (J. oxycedrus L. M, J. communis L. M and F, J. pygmaea C. Koch., M and F, and J. sibirica Burgsd. M and F) on the mycelial radial growth of plant pathogenic fungi were tested by the agar dilution method [46]. The essential oils were diluted in PDA at 1 µL mL −1 concentration. Discs (5 mm/d) were cut out from the periphery of a 10 day-old culture of tested fungi and aseptically placed in the prepared Petri dishes (9 cm/d) with PDA and EO. Pure PDA medium (without essential oil) was used as the control. The inoculated Petri dishes were placed for incubation at 22 • C for 10 days. The percent inhibition of the radial mycelial growth of the tested fungal pathogens was calculated using the formula: where DC is the diameter of the colony of the control, and DT is the diameter of the colony of the treatment. The experiment was conducted in four replications.

Antioxidant Activity
The antioxidant activity of EOs of J. communis, J. oxycedrus, J. pygmaea and J. sibirica (Clevenger; Commercial) were analyzed at the University of Nebraska-Lincoln, Small Molecule Analysis Laboratory. The oxygen radical absorbance capacity (ORAC oil) was detected according to Huang et al. [61,62]. Briefly, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid), a polar derivative of vitamin E, was used as a standard, and the results were reported as µmole Trolox g −1 . All samples of EOs were prepared by mixing 10 ± 1 mg oil with 1 mL of water and acetone (1:1) with 7% methyl-β-cyclodextrins (w:v). The activity was started in a 96-well plate by first transferring 25 µL of 74 mM phosphate buffer, with pH 7.4, to each well. Thereafter the EO samples (25 µL) or Trolox (25 µL) were added at concentrations of 0.2, 0.4, 3.3, 6.5, 10, 13, 25, and 50 µg/mL, followed by 150 µL of fluorescein (8.16 × 10 −5 mM). Each sample was incubated at 37 • C for 10 min, with 3 min of alternating shaking. The 153 mM 2,2 -azobis (2-amidinopropane) hydrochloride (25 µL) was used for reaction activation. The standards and tested EO were monitored with a BMG Labtech FLUOstar Optima microplate reader (Durham, NC, USA). We measured fluorescence every 1.5 min at an excitation and emission wavelength of 485 and 520 nm, respectively, until the decreasing fluorescence values plateaued. The area under the curve was calculated. The EOs of the four junipers were analyzed in triplicate. The averages of the tested EOs in triplicate were used for the statistical analysis.

Statistical Methods for Aphids
The effect of the combinations of juniper species and sex (7 levels: J. communis-F, J. communis-M, J. oxycedrus-M, J. pygmaea-F, J. pygmaea-M, J. sibirica-F, and J. sibirica-M) and the concentration of their EOs (4 levels: 0%, 1%, 2.5%, and 5%) on nb/leaf of Sitobion avenae and Rhopalosiphum padi for repellent action was determined using a 7 × 4 factorial design with 3 replications. However, for the insecticidal action, only three concentrations (1%, 2.5%, and 5%) were used for each of the seven species by sex combinations. Additionally, the antifungal activity effect of juniper species and sex (7 levels) on plant pathogens was determined using a completely randomized design (a single factor with 7 levels).
The analysis was completed using the GLM procedure of SAS [63], and the validity of model assumptions (normal distribution and constant variance of the error terms) was verified by examining the residuals as described in Montgomery [64]. Since the twoway interaction effect for all constituents, antimicrobial activities, and aphids and the three-way interaction effect for antioxidant capacity were highly significant, a multiple means comparison of the corresponding treatment combinations was completed and letter groupings generated using the least squares means at the 1% level of significance to protect the Type I experiment-wise error rate from overinflation.

Conclusions
Generally, the EOs of J. communis, J. pygmaea, J. sibirica, and J. oxycedrus had similar chemical profiles between the two extraction methods (ClevA, SCom) within a species, but they had a different antioxidant capacity. The EOs obtained via ClevA extraction showed greater antioxidant capacity within a species compared with those from SCom extraction. Overall, the EOs among the two factors (sex, species) had different antimicrobial activity. All of the tested EOs had significant repellent and insecticidal activity against the two aphid species Rhopalosiphum padi (bird cherry-oat aphid) and Sitobion avenae (English grain aphid) at concentrations of the EO in the solution at 1%, 2.5%, and 5%. Most of juniper EOs were effective against the tested pathogens, Fusarium spp., Cylindrocarpon pauciseptatum Colletotrichum spp. and Rhizoctonia solani, especially J. pygmaea (M) and J. sibirica (F). These EOs show promise as alternatives to conventional synthetic pesticides and could be used in the development of biopesticide products.
Supplementary Materials: The following are available online, Table S1: Constituents and concentrations of J. communis extracted using the Clevenger and Commercial extraction methods; Table S2: Constituents and concentrations of J. sibirica extracted using the Clevenger and commercial extraction methods; Table S3: Constituents and concentrations of J. pygmaea extracted using the Clevenger and commercial extraction methods; Table S4: Constituents and concentrations of J. oxycedrus extracted using the Clevenger and Commercial extraction methods; Table S5 Acknowledgments: This study was supported in part by the Agricultural University Plovdiv, Bulgaria [Project № 11-17]. This work was supported by the Bulgarian Ministry of Education and Science under the National Research Program "Healthy Foods for a Strong Bio-Economy and Quality of Life" approved by DCM # 577/17.08.2018" and in part by the Oregon State University funds awarded to Valtcho Jeliazkov (Zheljazkov). The authors thank Yulia Yonkova for helping with the essential oil extraction.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Data of the compounds are available from the authors.