Chemical Composition, Phytotoxic, Antimicrobial and Insecticidal Activity of the Essential Oils of Dracocephalum integrifolium

The present investigation studied the chemical composition of the essential oils extracted from Dracocephalum integrifolium Bunge growing in three different localities in northwest China and evaluated the phytotoxic, antimicrobial and insecticidal activities of the essential oils as well as their major constituents, i.e., sabinene and eucalyptol. GC/MS analysis revealed the presence of 21–24 compounds in the essential oils, representing 94.17–97.71% of the entire oils. Monoterpenes were the most abundant substances, accounting for 85.30–93.61% of the oils; among them, sabinene (7.35–14.0%) and eucalyptol (53.56–76.11%) were dominant in all three oils, which occupied 67.56–83.46% of the total oils. In general, phytotoxic bioassays indicated that the IC50 values of the oils and their major constituents were below 2 μL/mL (1.739–1.886 mg/mL) against Amaranthus retroflexus and Poa annua. Disc diffusion method demonstrated that the oils and their major constituents possessed antimicrobial activity against Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Saccharomyces cerevisiae, and Candida albicans, with MIC values ranging from 5–40 μL/mL (4.347–37.712 mg/mL). The oils, sabinene and eucalyptol also exhibited significant pesticidal activity, with the mortality rates of Aphis pomi reaching 100% after exposing to 10 μL oil/petri dish (8.694–9.428 mg/petri dish) for 24 h. To the best of our knowledge, this is the first report on the chemical composition, phytotoxic, antimicrobial and insecticidal activity of the essential oils extracted from D. integrifolium; it is noteworthy to mention that this is also the first report on the phytotoxicity of one of the major constituents, sabinene. Our results imply that D. integrifolium oils and sabinene have the potential value of being further exploited as natural pesticides.


Introduction
Yield losses caused by agricultural pests including arthropods, diseases, and weeds are estimated to account for about 35% in major crops worldwide [1]. Synthetic chemical pesticides are widely accepted in prevention of diseases and insect pests in crop growth; however, extensive application of synthetic chemicals has led to the development of resistance in insects and weeds [2,3]. Besides, wide use of synthetic pesticides has also caused many problems including the acute and chronic toxicity to human and mammals, as well as adverse and residual effects on the environment [4].
Traditionally, numerous plant products have a record of safe application in various industries [5]. Plant derived substances, including essential oils, have been an increasing interest in search of alternatives to chemical pesticides because of their innate biodegrade ability, minimal effects on non-target organisms and the environment [6,7]. In fact, essential oils as well as oil constituents/derivatives have successfully been commercialized as environment friendly pesticides such as clove oil, eucalyptol and so on [8,9].
Among various Dracocephalum species, D. integrifolium Bunge is an aromatic plant that has been used traditionally as an important medicinal herb known as "Marzan Juxi" in Uighur in treating cough and asthmadistributes in Central Asia [30]. Phytochemical study indicated that this plant mainly contains flavonoids, volatile oils, lactones and other compounds [11]. So far, there are no reports on the chemical composition and biological activities of the essential oil of D. integrifolium. In this study, we aim to: (i) analyze the phytochemical profile of the essential oils extracted from D. integrifolium growing in 3 different localities in northwest China; (ii) evaluate the phytotoxic, antimicrobial and pesticidal activities of these essential oils as well as their major constituents. The possibility of utilizing the essential oils and their major constituents as environment friendly pesticides is also discussed.

Phytotoxic Activity of the Essential Oils and their Major Constituents
Phytotoxic activity of the essential oils (concentrations tested ranged from 0.125 to 5 µL/mL, 0.109-4.714 mg/mL) as well as their major constituents was determined by comparing their plant regulatory effect on shoot and root length of two receiver plants, i.e., A. retroflexus and P. annua. At the lowest concentration tested (0.125 µL/mL, 0.91-0.938 mg/mL), all 3 oils significantly promoted root elongation of A. retroflexus; however, sabinene, eucalyptol and their mixture did not exert any promotive activity. With the increase of concentration, inhibitory effects were observed when oils were applied starting from 0.5-1 µL/mL (0.455-0.938 mg/mL), whereas the effective dose for sabinene, eucalyptol and their mixture was 2 µL/mL (1.739 mg/mL), 0.25 µL/mL (0.236 mg/mL), and 0.5 µL/mL (0.457 mg/mL), respectively. In fact, 2 µL/mL (1.739-1.886 mg/mL) oils or the major constituents resulted in over 50% reduction on root length: DI1, DI2, DI3 oils inhibited root elongation of A. retroflexus by 86.5%, 100%, 100%, meanwhile sabinene, eucalyptol and their mixture exerted 66.7%, 63.0%, and 76.0% reduction on root length of A. retroflexus, respectively. At the highest concentration tested (5 µL/mL, 4.347-4.714 mg/mL), seed germination of A. retroflexus was markedly inhibited by the oils and the major constituents. Shoot growth responded similarly to the oils and the major constituents but to a lesser extent (Figures 4 and 5). mixture, significant plant growth suppressive effect can be detected starting from 0.25-0.5 μL/mL (0.217-0.471 mg/mL). Like A. retroflexus, over 50% reduction on root length was triggered by 2 μL/mL (1.739-1.886 mg/mL) oils or the major constituents: the inhibition rates of 3 oils on root length of P. annua were 65.8% (DI1), 100% (DI2) and 80.5% (DI3), respectively, whilst at the same concentration, seedling height were reduced to 61%, 100% and 65.3% of the control, respectively. When the concentration reached 5 μL/mL (4.347-4.714 mg/mL), seed germination of P. annua was basically completely prohibited (Figures 6 and 7).
Strength of the phytotoxic activity was compared by calculating the IC50 values for the oils and the major constituents ( Figure 8). Among the three oils, DI3 showed the most potent phytotoxic activity on root growth of both receiver species, whereas DI1 exhibited the weakest activity. Slight promotive activity of the mixture of sabinene and eucalyptol was observed on root elongation of A. retroflesus and shoot growth of both test species.    0μL/mL 0.125μL/mL 0.25μL/mL 0.5μL/mL 1μL/mL 2μL/mL 5μL/mL Similarly, DI1 and DI2 oils significantly stimulated root growth of the other tested species, the monocot plant P. annua; DI3 oil, on the contrary, exhibited obvious phytotoxic activity starting from the lowest concentration tested, 0.125 µL/mL (0.117 mg/mL). For sabinene, eucalyptol and their mixture, significant plant growth suppressive effect can be detected starting from 0.25-0.5 µL/mL (0.217-0.471 mg/mL). Like A. retroflexus, over 50% reduction on root length was triggered by 2 µL/mL (1.739-1.886 mg/mL) oils or the major constituents: the inhibition rates of 3 oils on root length of P. annua were 65.8% (DI1), 100% (DI2) and 80.5% (DI3), respectively, whilst at the same concentration, seedling height were reduced to 61%, 100% and 65.3% of the control, respectively. When the concentration reached 5 µL/mL (4.347-4.714 mg/mL), seed germination of P. annua was basically completely prohibited ( Figures 6 and 7).

Figure 5.
Phytotoxic effects of the essential oils of D. integrifolium and their major constituents, sabinene, eucalyptol, and their mixture on shoot growth of A. retroflexus examined by ANOVA (p < 0.05) and analyzed using Fisher's LSD test at p < 0.05 level. Each value is the mean of five replicates ± SE (n = 50). Means with different letters indicate significant differences at p < 0.05 level according to Fisher's LSD test. 0μL/mL 0.125μL/mL 0.25μL/mL 0.5μL/mL 1μL/mL 2μL/mL 5μL/mL Root length (cm) of Poa annua Figure 6. Phytotoxic effects of the essential oils of D. integrifolium and their major constituents, sabinene, eucalyptol, and their mixture on root growth of P. annua examined by ANOVA (p < 0.05) and analyzed using Fisher's LSD test at p < 0.05 level. Each value is the mean of five replicates ± SE (n = 50). Means with different letters indicate significant differences at p < 0.05 level according to Fisher's LSD test.
Strength of the phytotoxic activity was compared by calculating the IC 50 values for the oils and the major constituents ( Figure 8). Among the three oils, DI3 showed the most potent phytotoxic activity on root growth of both receiver species, whereas DI1 exhibited the weakest activity. Slight promotive activity of the mixture of sabinene and eucalyptol was observed on root elongation of A. retroflesus and shoot growth of both test species. and analyzed using Fisher's LSD test at p < 0.05 level. Each value is the mean of five replicates ± SE (n = 50). Means with different letters indicate significant differences at p < 0.05 level according to Fisher's LSD test.

Antimicrobial Activity of the Essential Oils and Their Major Constituents
Disc diffusion method demonstrated that the essential oils and their major constituents had suppressive effect on tested microorganisms. In general, DI2 oil showed the strongest activity against and analyzed using Fisher's LSD test at p < 0.05 level. Each value is the mean of five replicates ± SE (n = 50). Means with different letters indicate significant differences at p < 0.05 level according to Fisher's LSD test.

Antimicrobial Activity of the Essential Oils and their Major Constituents
Disc diffusion method demonstrated that the essential oils and their major constituents had suppressive effect on tested microorganisms. In general, DI2 oil showed the strongest activity against

Antimicrobial Activity of the Essential Oils and Their Major Constituents
Disc diffusion method demonstrated that the essential oils and their major constituents had suppressive effect on tested microorganisms. In general, DI2 oil showed the strongest activity against the tested strains compared with other oils; meanwhile, the major constituents were also very effective; for example, sabinene resulted in the largest inhibition zone (6.63 cm) in the assay ( Table 2). The minimal inhibitory concentration (MIC) of D. integrifolium oils and their major constituents was also evaluated in order to assess the strength of their antimicrobial activity (Table 3). B. subtillis turned out to be the most sensitive microorganism with the lowest MIC values (5 µL/mL, 4.347-4.714 mg/mL) for the oils and the major constituents; in comparison, S. cerevisiae was the most tolerant strain, with MIC values of 40 µL/ mL for sabinene (34.776 mg/mL) and the mixture of sabinene and eucalyptol (36.576 mg/mL).

Pesticidal Activity of Essential Oils and their Major Constituents
The insecticidal effect of D. integrifolium oils against A. pomi was summarized in Figures 9-14. Results showed that all three essential oils as well as the major constituents posed toxic effect on A. pomi, and higher mortality was observed as the doses of essential oils and exposure period increased. After 48 h exposure to the oils at 2 µL/petri dish (1.820-1.876 mg/petri dish), the mortality rates of A. pomi were 56.0%, 72.6% and 78.3%, respectively; and when the concentration reached 10 µL (9.100-9.380 mg), the mortality rates were 100% for all 3 oils after 24 h exposure. In order to compare the strength of the oils, LC 50     Percentages of mortality of DI2     Percentages of mortality of DI2   Percentages of mortality of eucalyptol   Percentages of mortality of eucalyptol

Discussion
The chemical composition of essential oils produced by a number of Dracocephalum species have been reported, especially D. moldavica and D. kotschyi, mostly because of their important value as traditional medical herbs [19][20][21]27,28,31,32]. Suleimen et al. [26] investigated the chemical composition of D. peregrinum and detected 1, 8-cineol (synonym for eucalyptol, 18.5%), α-pinene (8.4%), limonene (5.8%), β-caryophyllene oxide (5.5%), and spathulenol (3.3%) as the major constituents; however, the major constituents of some other Dracocephalum species differ greatly from D. integrifolium. Mahmood et al. [12] found citronellol (74.9%), Citronellal (6.7%) and Citronellyl formate (6.7%) were abundant in D. heterophyllum oil; Nezhadali et al. [18] reported that the major constituents of the essential oil obtained from D. subcapitatum were geranial (63.4%), limonene (23.4%), p-Menth-1-en-9-ol (4.4%) and caryophyllene E (4.3%). Various factors such as geographic locality and surrounding climate, species variety or ecotype, seasonal variations, stress, post-harvest processing, etc. might influence the chemical profile of essential oils [33] In terms of D. integrifolium, VOCs released by powdered dry plant materials were studied by Liu et al. [30] using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) method, and eucalyptol and cymene were discovered as the most abundant ingredients. To the best of our knowledge, our study is the first to report the chemical profile of the essential oil of D. integrifolium, with eucalyptol and sabinene being the dominant compounds in the oils; both compounds are common constituents of plant essential oils and have been demonstrated to attribute to certain biological activities of the oils. Essential oils are produced by plants from the family Compositae, Umbelliferae, etc., that function as pollinator attractants, determinants of vegetation patterning or regulatory factor of community structure via allelopathy [34]. Further study revealed that essential oils possess various biological activities such as antioxidant, cytotoxic, anti-inflammatory, anti-microbial activity, etc. [35,36]. In terms of the phytotoxic activity of essential oils produced by Dracocephalum species, there was one single report on the significant allelopathic activity of the essential oil produced by a new chemotype of D. kotschyi that was characterized with abundant limonene-10-al and limonene [27]. D. integrifolium has the ability to emit phytotoxic volatile compounds into the environment, indicating the possible involvement of allelopathy in its competition against neighboring plants. Allelopathy refers to any direct and indirect harmful or beneficial effect by one plant on another through the production of chemical compounds that are released into environment, which is often found to contribute to successful establishment of dominance in a certain habitat [37,38]. Meanwhile, it is noteworthy to mention that the mechanism of allelopathy is rather complicated and may be very difficult to be confirmed, mainly because of the uncertainty of the fate of the potential volatile allelochemicals after they are released into the environment. On the one hand, these compounds might act directly as phytotoxins to affect other plants' growth if they can accumulate to effective doses in the soil; on the other hand, they might indirectly impact on neighbor's growth by altering the soil's physico-chemical properties as well as the community structure and function of soil biota [39,40].
In most cases biological activities of a particular essential oil is determined by one or two of its major components, however synergistic effect may occur in other circumstances [33,41]. Essential oil constituents such as eucalyptol, camphor, β-myrcene, limonene, thymol, geraniol, αand β-pinene, bornyl acetate, citronellal, menthol, borneol and a-terpineol, etc. have been found to exhibit plant growth inhibitory activity [42,43]. Eucalyptol and sabinene are common components present in a number of essential oils [44][45][46][47][48][49]. So far, there is no report on the phytotoxicity of sabinene, therefore we are the first to study this biological activity of this compound; however, there are some essential oils with phytotoxic activity have sabinene as the major constituents, implying that sabinene might play an important role as the active compound in the oils. For instance, the essential oil produced by Ravensara aromatic that was rich in sabinene (the sabinene chemotype) caused 52.5% mortality of rice seedlings at a dose of 100 µL/L, and completely killed the garden cress seedlings at a dose of only 5 µl/L [46]. The essential oil of Ferulago angulate also exhibited considerable phytotoxic activity, with sabinene (6.89%) being one of its major constituents [50]. Zingiber montanum oil reduced germination and inhibited seedling growth of lettuce significantly, which is abundant with sabinene 13.5-38.0% [51]. Eucalyptol is also known as 1, 8-cineole, which has been previouslyt demonstrated to be phytotoxic [52][53][54][55]; this compound was considered an active constituent of cyanobacterial VOCs that negatively affect other algae's growth by reducing photosynthetic abilities and inducing degradation of photosynthetic pigments [35,56]; Barton et al. [53] found that eucalyptol posed post-emergence herbicidal activity against annual ryegrass and radish in a dose-dependent manner, and suppression of seedling growth was significant at and above 0.1 mol/L on radish, meanwhile application of eucapyptol prohibited seedling growth of ryegrass when the concentration was above 0.1 mol/L, with root suppression first occurring at 0.0316 mol/L and shoot suppression at 0.1 mol/L. Martino et al. [43] compared the antigerminative activity of 27 monoterpenes and found that eucalyptol suppressed radicle elongation of both radish and garden cress in a significant way at the lowest concentration tested (10 −6 M).
Phytotoxicity of the essential oils and their major ingredients may lead to the discovery of environment-friendly natural herbicides. For example, the high effectiveness of eucalyptol and its analogs led to the successful commerciliazation of a herbicide, cinmethylin, which was designed based on the chemical structure of eucalyptol, as the active ingredient 1, 4-cineole is a 2-benzyl ether-substituted analog of eucalyptol [9]. Another herbicide, Burnout II (Bonide Products Inc., New York) used clove oil (common volatile oil constituent) as the major component in the ingredients [8].
Essential oils have been reported to possess antimicrobial activity, including a number of Dracocephalum species [15,19,20,57]. Essential oils produced by two Dracocephalum species (D. polychaetum Bornm. and D. surmandinum Rech.f.) exhibited significant antibacterial activity against both gram-positive and gram-negative bacteria [17]. Essential oil obtained from another Dracocephalum species, D. foetidumby, also showed inhibitory activity against most of the tested pathogenic bacteria and yeast strains with MIC values ranging from 26 to 2592 µg/mL; furthermore, the oil was found to exhibit significant antimicrobial activity against methicilin-resistant Staphylococcus aureus (MRSA) strains, indicating this oil has potential medical value [20]. Farimani et al. [14] examined the antimicrobial activity of D. kotschyi essential oils against gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria and found S. aureus was the most sensitive strain with an IC 50 value of 2 mg/mL. Zhang et al. [19] tested the antimicrobial activity of the essential oil of D. heterophyllum against nine bacterial strains, one yeast, three fungi, and found that MIC values for bacteria, yeast, and fungi strains were 0.039-0.156, 0.156, and 0.313-2.500 mg/mL, respectively. Besides frequently reported antibacterial effect, the essential oil of D. heterophyllum also showed promising antifungal activity against Colletotrichum species [15].
Antimicrobial activity of essential oils could be attributed to the high content of certain components; for instance, the high antimicrobial activity of Thymus pulegioides oil was caused by one the its abundant constituents, thymol [58,59]. Essential oils rich in eucalyptol are reported to possess antimicrobial activity; eucalyptol was found to exhibit inhibitory activity against various microorganisms such as E. coli, S. aureus, B. cereus, P. aeruginosa, etc. [60]. Trinh et al. [61] found that eucalyptol significantly decreased the amount of viable Gardnerella vaginalis and Candida albicans in the vaginal cavity and myeloperoxidase activity in mouse vaginal tissues compared with controls. Vimal et al. [62] discovered that eucalyptol and sabinene could be potent inhibitors of salmonella target protein L-asparaginase, and sabinene was even better than the standard drug, Ciprofloxacin and the natural substrate L-asparagine. Some essential oils that are rich in sabinene were found to possess antimicrobial activity: Artemisia kulbadica oil (sabinene 25.1%) showed inhibitory activity against six bacterial strains and one fungal [63]; Alpinia nutans oil (sabinene 27.8%) was effective against Pasteurella multocida, Escherichia coli, Salmonella enterica, Shigella fluxneri and Staphylococcus aureus [64]; Laserpitium latifolium oil (sabinene 26.8%) inhibited growth of Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus [65]. The effectiveness of D. integrifolium oil and its major constituents implies their potential value to be further explored as natural antimicrobial agents.
Essential oils of Dracocephalum plants have been demonstrated to have insecticidal activity. D. Kotschyi essential oil was effective against green peach aphids, and D. ruyschiana, D. foetidum, D. moldavica, D. fruticztlosum and D. peregrinum essential oils had good insecticidal effect on mosquito larvae [24,27]; Chu et al. [25] found D. moldavica essential oil exhibited strong fumigant toxicity against Sitophilus zeamais and Tribolium castaneum adults with LC 50 values of 2.65 and 0.88 mg/L, respectively. The two major components of the essential oils, sabinene and eucalyptol, also have insecticidal activity. Sukontason et al. [66] studied the effects of eucalyptol on house fly (Musca domestica) and blow fly (Chrysomya megacephala) and found for M. domestica, males were more susceptible than females, with the LD 50 being 118 and 177 µg/fly, respectively; as of C. megacephala, the LD 50 values were 197 µg/fly for males and 221 µg/fly for females. Another research showed that eucalyptol possessed strong fumigant toxicity against two insects, Tribolium castaneum (LC 50 = 5.47 mg/L air) and Lasioderma serricorne (LC 50 = 5.18 mg/L air; [67]). Another major constituent, sabinene, was also reported to be insecticidal. Wang et al. [68] found sabinene was strongly repellent against Tribolium castaneum with the LC 50 value of 18.2 mg/L of air, although its effect was weaker compared with the positive control, DEET (N, N-diethyl-3-methylbenzamide). Sabinene also exhibited strong fumigant toxicity against the maize weevils with LC 50 value of 9.12 mg/L [69]. In another study, sabinene possessed noticeable repellent activity against adults of the granary weevil (Sitophilus granaries) at low dosages [70]. Therefore, the dominant components of D. integrifolium, eucalyptol and sabinene, are quite likely the main responsible active ingredients contributing to the oils' insecticidal activity.

Conclusions
Plant derived essential oils have the potential to be further explored as agricultural chemicals to control pathogenic fungi, pests and weeds to decrease the negative impact of synthetic agents due to the fact that they are effective, selective, biodegradable, and less toxic to the environment. To the best of our knowledge, this is the first report on the chemical composition, phytotoxic, antimicrobial and pesticidal activities of the essential oil extracted from D. integrifolium; this is also the first report on the phytotoxic activity of one of the oil's major constituents, sabinene. Our results imply that D. integrifolium oil and its major constituents are valuable candidates to be further explored as biopesticides.

Essential oil Extraction
Three hundred fresh stems and leaves of D. integrifolium were cut into fragments to extract essential oil using a Clevenger-type apparatus. The hydrodistillation procedure lasted for 4 h, followed by collection of the oil; the oil was then dried over anhydrous sodium sulfate and kept in a sealed vial at 4 • C until required. Materials collected from 3 sites were extracted and stored separately. The following formula was used to calculate the yield of D. integrifolium oil: Oil yield (%, V/W) = volume of essential oils (mL)/fresh weight of plant material (g) × 100.

GC/MS Analysis of the Essential Oils
The phytochemical profile of the oil was analyzed using a 7890A/5975C GC/MS system (Agilent Technologies, Palo Alto, CA, USA) equipped with FID and a DB-5MS 5% Phenyl Methyl Silox column (30 m × 0.25 mm; 0.25 µm film thickness). The carrier gas was helium (flow rate: 1 mL/min). The oven temperature was held at 60 • C for 5 min and then programmed from 60 • C to 280 • C at 3 • C/min. Mass spectra were taken at 70 eV with mass range from m/z 40-800 amu. The temperature of both injector port and detector port was kept at 280 • C; sample injection volume, 0.1µL; split ratio was 50: 1. Relative amounts of individual compound were calculated based on GC peak areas without FID response factor correction. Identification of single component was determined by comparison of their mass spectra and retention indices (RI, calculated by linear interpolation relative to retention times of a standard mixture of C7-C40 n-alkanes) with the data given in NIST (National Institute of Standards and Technology), and published literature [52,71].

Phytotoxic Activity of the Essential Oils and Their Major Constituents
The major constituents of the essential oil, i.e., sabinene and eucalyptol, were purchased from Sigma-Aldrich Co. (St. Louis, USA). Phytotoxic effect of the essential oils, their major constituents (sabinene and eucalyptol), along with their mixture prepared at 10.5: 65.1 (sabinene: eucalyptol; the average ratio of sabinene and eucalyptol in the oils) was evaluated by conducting bioassays against a dicot plant, Amaranthus retroflexus L., and a monocot plant, Poa annua L. Seeds were surface sterilized with 0.5% HgCl 2 before use. The essential oils, sabinene, eucalyptol, and mixture of sabinene and eucalyptol were diluted with distilled H 2 O to obtain 0.125 (0.109-0.118 mg), 0.25 (0.217-0.236 mg), 0.5 (0.435-0.471 mg), 1 (0.869-0.943 mg), 2 (1.739-1.886 mg), 5 (4.347-4.714 mg) µL/mL solutions, with acetone (final volume < 0.5%) as the initial solvent; a preliminary experiment demonstrated that acetone at such concentration did not significantly influence seedling growth of receiver plants. Five mL of 0.5% acetone in distilled H 2 O (control) or diluted solutions (treatments) were added to each petri dish (9 cm in diameter, lined with a single layer of Whatman No. 2 filter paper), followed by sowing of 10 seeds of receiver plants. Petri dishes were sealed with parafilm to keep the moisture and avoid oil evaporation. An incubator was used to store all the petri dishes in the dark at 25 • C. Seedlings were observed and measured after 5 days of cultivation for the dicot plant A. retroflexus, and 7 days for the monocot plant P. annua. Five replicates were made for all phytotoxic bioassays (in total 50 seedlings were measured).

Strains
Five microorganisms, including Bacillus subtilis (ATCC6633), Pseudomonas aeruginosa (ATCC27853), Escherichia coli (ATCC25922), Saccharomyces cerevisiae (ATCC9763), and Candida albicans (ATCC10231) were chosen to evaluate the antimicrobial effect of the essential oils as well as their major constituents; all strains were obtained from China General Microbiological Culture Collection Center (CGMCC). Among the test strains, B. subtilis is a gram positive bacterium; P. aeruginosa and E. coli are gram negative bacteria; S. cerevisiae and C. albicans are fungi. Antimicrobial activity of the essential oils and their major constituents were tested using disc diffusion method (for measurement of radius of the inhibition zone) and tube dilution method (for measurement of MIC) [72,73].

Determination of Radius of Inhibition Zone
Bacteria or fungi suspensions were adjusted to 1 × 10 7 CFU/mL and spread on LB medium (for bacteria) or PDA medium (for fungi) using sterile glasses spreader. Subsequently, filter paper discs (5 mm in diameter, Whatman No. 2) that were impregnated with 20 µL (17.388-18.856 mg) of essential oils or the major constituents were placed on LB or PDA medium. Negative controls were prepared with distilled water. Petri dishes were then incubated at 37 • C for 24 h for bacteria, and 48 h for fungi. All assays were performed in triplicates. Antimicrobial activity was evaluated by measuring the radius of the inhibition zones to the nearest millimeter.

Determination of MIC
The essential oils and their major constituents were diluted with LB medium (for bacteria) and PDB medium (for fungi) supplemented with 0.5% of Tween-20 to give oil concentrations of 5 (4.347-4.714 mg), 10 (8.694-9.428 mg), 20 (17.388-18.856 mg), 40 (34.776-37.712 mg) µL/mL. Samples were first sterilized with a 0.22 µm Millipore before application, and then inoculated with 50 µL of fresh culture of the test microorganisms at 1 × 10 7 CFU/mL and incubated at 37 • C for 24 h for bacteria, and 48 h for fungi. MIC was defined as the lowest concentration of oils that had no macroscopically visible growth. All assays were performed in triplicates.

Insecticidal Bioassay
Aphis pomi was used to determine the insecticidal activity of the oils and their major constituents. Whatman No. 2 filter paper discs (1 cm × 1 cm) were soaked in D. integrifolium oils, sabinene, eucalyptol, and the mixture of sabinene and eucalyptol at the following concentrations: 0, 0.25 (0.217-0.236 mg), 0.5 (0.435-0.471 mg), 1 (0.869-0.943 mg), 2 (1.739-1.886 mg), 5 (4.347-4.714 mg) and 10 (8.694-9.428 mg) µL/disc. Paper discs were then placed onto the inner side of the lid of each petri dish (9 cm in diameter) using adhesive tape. This procedure was adopted in order to prevent direct contact between the oils and the insects. Treatments consisted of 20 adult aphids, which were placed onto fresh apple tree leaves that were kept on a layer of moist filter paper. Petri dishes were then covered and transferred to an incubator and kept at 25 ± 2 • C with a photoperiod L/D = 16:8 for 2 days. Each treatment was replicated three times (n = 60).

Statistical Analysis
The significance of the phytotoxic/antimicrobial/pesticidal activity was first examined by ANOVA (p < 0.05) and then analyzed using Fisher's LSD test at p < 0.05 level. All of the statistical analyses were performed using SPSS 13.0 software package.