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Insecticidal toxicities of carvacrol and thymol derived from Thymus vulgaris Lin. against Pochazia shantungensis Chou & Lu., newly recorded pest

Scientific Reports volume 7, Article number: 40902 (2017) | Download Citation

Abstract

The insecticidal toxicities of five essential oils against Pochazia shantungensis adults and nymphs, newly recorded pests, were evaluated. The LC50 values of Thymus vulgaris, Ruta graveolens, Citrus aurantium, Leptospermum petersonii and Achillea millefolium oils were recorded as 57.48, 84.44, 92.58, 113.26 and 125.78 mg/L, respectively, against P. shantungensis nymphs using the leaf dipping bioassay, and 75.80, 109.86, 113.26, 145.06 and 153.74 mg/L, respectively, against P. shantungensis adults using the spray bioassay method. Regarding volatile components identified in T. vulgaris oil, the LC50 values of carvacrol and thymol using the leaf dipping bioassay against P. shantungensis nymphs were 56.74 and 28.52 mg/L, respectively. The insecticidal action of T. vulgaris oil against P. shantungensis could be attributed to carvacrol and thymol. Based on the structure-toxicity relationship between thymol analogs and insecticidal toxicities against P. shantungensis nymphs similar to the LC50 values against P. shantungensis adults, the LC50 values of thymol, carvacrol, citral, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol were 28.52, 56.74 and 89.12, 71.41, 82.49, and 111.28 mg/L, respectively. These results indicate that the insecticidal mode of action of thymol analogs may be largely attributed to the methyl functional group. Thymol analogues have promising potential as first-choice insecticides against P. shantungensis adults and nymphs.

Introduction

Pochazia shantungensis Chou & Lu. is a hemipterous insect that belongs to the Ricaniidae insect family, which includes about 400 described species in over 40 genera1. P. shantungensis is a newly recorded pest that is economically devastating for various trees including apple, blueberry, peach and persimmon, mainly in Wanjugun, Korea2. P. shantungensis was first recorded in Chungcheongnamdo in 20103. In later outbreaks of exotic invasive insects, P. shantungensis has subsequently occurred sporadically in several other provinces across Korea2,3. In spite of this, the species identification has not yet been confirmed, mostly due to the impossibility of identifying the genital characteristics of externally similar species2. As such, an insecticide has yet to be developed for long-term control of P. shantungensis adults and nymphs, despite the urgent demand4. Discovering selective and natural insecticides that are safe for the environment and other organisms is essential for the management of P. shantungensis.

Plant oil is a very complex mixture that can contain approximately 30–65 constituents at various concentrations3,5,6. Two or three major constituents are featured at 25–60% of the concentration compared to other components present in trace quantities3. Acetovanillone (47.98%) and 2′-hydroxy-5′-methoxyacetophenone (49.23%) are the major components of Cynanchum paniculatum oil7, menthol (59%) and menthone (19%) are the major components of Mentha piperita oil8, and carvone (59.79%) and limonene (25.40%) are the major components of Mentha spicata oil9. The essential oils and major components derived from plants are significant insecticidal activity against diverse insect species and have been developed as ecologically potential pesticides3,7,8,9,10.

So for no report has been received about the insecticidal toxicities of Thymus vulgaris oil-derived constituents against P. shantungensis. Therefore, the aims of the present study were first to investigate the insecticidal properties of T. vulgaris oil-derived components against P. shantungensis adults and nymphs, and then to determine the structure-activity relationship between thymol analogs and insecticidal toxicities.

Results and Discussion

This study was undertaken within the framework of a more general study involving the natural products for insecticidal toxicities against P. shantungensis adults and nymphs. Essential oils of Achillea millefolium flowers, Citrus aurantium fruits, Leptospermum petersonii leaves, Ruta graveolens leaves and T. vulgaris leaves were analyzed (Table 1). The yields of A. millefolium, C. aurantium, L. petersonii, R. graveolens and T. vulgaris oils were 0.658, 1.451, 0.984, 0.924, and 1.122%, respectively. The insecticidal toxicities of the five oils against P. shantungensis adults and nymphs were evaluated after 48 and 72 h exposure (Table 2). From the leaf dipping and spray bioassays against P. shantungensis adults and nymphs, the insecticidal responses and the LC50 values increased from 48 to 72 h exposure. The LC50 values of T. vulgaris, R. graveolens, C. aurantium, L. petersonii and A. millefolium oils at 72 h exposure were 75.80, 109.86, 113.26, 145.06 and 153.74 mg/L, respectively, in the spray bioassay against P. shantungensis adults, and 57.48, 84.44, 92.58, 113.26 and 125.78 mg/L, respectively, in the leaf dipping bioassay against P. shantungensis nymphs. Based on the LC50 values against P. shantungensis adults and nymphs, T. vulgaris oil had the highest insecticidal toxicity followed by R. graveolens, C. aurantium, L. petersonii and A. millefolium oils. The insecticidal toxicity of T. vulgaris oil against P. shantungensis nymphs was about 1.3-fold more than that against P. shantungensis adults. There was no insect mortality in the distilled water treatment (negative control) of P. shantungensis adults and nymphs. Differences in the insecticidal toxicities of plant-derived oils may be explained on the basis of species-specific responses to plant species, phytochemicals, and the weight and size of P. shantungensis adults and nymphs11.

Table 1: List of five plants tested and yields of essential oils (Yield (%) = (Dried weight of essential oil/dried weight of sample) × 100).
Table 2: Insecticidal toxicities of five essential oils against P. shantungensis adults and nymphs at 48 and 72 h exposure times (LC50 is the average of 3 determinations, with 30 nymphs and 20 adults per replication).

To further explore the insecticidal toxicities of the five essential oils against P. shantungensis adults and nymphs, the components of A. millefolium, C. aurantium, L. petersonii, R. graveolens, and T. vulgaris oils were investigated by GC-MS analysis. The identified components, together with the percentages present in the essential oils are displayed in Table 3. The major components were α-pinene (15.49%), β-caryophyllene (13.35%), sabinene (11.12%), camphor (9.65%), 1,8-cieole (9.37%), bornyl acetate (5.88%), and 2,2-dicyclohexylmalononitrile (5.58%) in A. millefolium oil; limonene (87.75%), citral (3.21%), limonene oxide (2.29%), and (−)-carveol (1.75%) in C. aurantium oil; citral (48.12%), β-citronellal (19.50%), isopulegol (8.14%), geraniol (4.64%), and linalool (3.33%) in L. petersonii oil, and thymol (23.34%), undecyl trichloroacetate (18.52%), methyltridecyl pentanoate (16.18%), and 2,2-dimethylpropanoic acid (7.03%), 2-acetoxytetradecane (6.98%), palmitic acid (5.07%), and methyl linolenate (4.78%) in R. graveolens oil. The major components in T. vulgaris oil were thymol (40.04%), ρ-cymene (29.97%), γ-terpinene (8.17%), linalool (4.99%), terpinolene (3.26%), α-pinene (2.84%), β-caryophyllene (2.50%), carvacrol (2.45%), limonene (1.25%), α-phellandrene (1.20%), myrcene (0.91%), camphene (0.89%), and caryophyllene oxide (0.21%). Together, thymol, ρ-cymene and γ-terpinene made up 78.18% of T. vulgaris oil. The volatile components consisted of 8 monoterpene hydrocarbons (camphene, α-pinene, limonene, myrcene, terpinolene, γ-terpinene, α–phellandrene, and ρ-cymene), 1 monoterpene alcohol (linalool), 2 monoterpene phenols (carvacrol and thymol) and 2 sesquiterpene hydrocarbons (β-caryophyllene and caryophyllene oxide). Venskutonis9 reported that the main chemicals identified in the T. vulgaris oil were borneol (0.98%), camphene (0.60%), carvacrol (2.81%), β-caryophyllene (2.39%), 1,8-cineol (0.96%), p-cymene (25.2%), myrcene (1.93%), linalool (2.86%), α-pinene (1.16%), 1-octen-3-ol (1.19%), α-terpinene (1.02%), γ-terpinene (6.37%), α-thujene (1.50%) and thymol (42.27%). The fact that the essential oil of T. vulgaris leaves dried more at 45 °C than at 30 °C can be explained mainly by the increase of thymol by 8% and carvacrol by 12%, while the quantity of γ-terpinene was decreased by 4.9%9. In previous and present studies, the quantities of volatile chemicals derived from T. vulgaris were affected by the environmental conditions, including harvest time, genotype, storage period, handling method and intraspecific variability, and the experimental conditions, which included the extraction method, extracted plant parts, and plant tissue drying temperature12,13.

Table 3: Volatile components of T. vulgaris oil identified by GC-MS (RI, retention indices in elution order from the DB-5 column; RT, comparison with pure standard retention time; MS, mass spectrometry).

The insecticidal toxicities of 34 major commercial components (bornyl acetate, camphene, camphor, carvacrol, (−)-carveol, (+)-carvone, β-caryophyllene, caryophyllene oxide, β-citronellal, citral, 1,8-cineole, p-cymene, decyl chloroformate, dodecanoic acid, β-farnesene, geranyl acetate, geraniol, isopulegol, linalool, limonene, limonene oxide, methyl linolenate, myrcene, myristic acid, palmitic acid, α-phellandrene, α-pinene, pivalic acid, sabinene, α-terpineol, γ-terpinene, 4-terpineol, terpinolene, and thymol) derived from the five essential oils were evaluated using spray and leaf dipping bioassays against P. shantungensis adults and nymphs (Table 4). Based on the LD50 values against P. shantungensis nymphs, the LC50 values of thymol, carvacrol and citral identified in C. aurantium, L. petersonii, R. graveolens, and T. vulgaris oils using the leaf dipping bioassay were 28.52, 56.74 and 89.12 mg/L respectively. Using the spray bioassay against P. shantungensis adults, the LC50 values of thymol, carvacrol and citral were 42.12, 75.62, and 102.74 mg/L, respectively. The insecticidal toxicity of thymol against P. shantungensis nymphs and adults was approximately 1.8–3.3 times greater than that of carvacrol and citral. In contrast, the other components (β-caryophyllene, camphene, caryophyllene oxide, ρ-cymene, linalool, limonene, myrcene, α-phellandrene, α-pinene, terpinolene, γ-terpinene, sabinene, β-pinene, camphor, (−)-carveol, geraniol, and bornyl acetate) did not exhibit any insecticidal toxicity against P. shantungensis adults and nymphs (data not shown). The insecticidal toxicities of the essential oils appear to be connected to their chemical composition. The insecticidal toxicities of T. vulgaris and R. graveolens oils could be due to the existence of thymol and carvacrol, which exhibited the greatest insecticidal toxicities. The essential oils of C. aurantium and L. petersonii contain citral, which showed insecticidal toxicities against P. shantungensis adults and nymphs, however its toxicity was weaker than thymol and carvacrol. Furthermore, P. shantungensis nymphs were more susceptible to T. vulgaris oil, carvacrol and thymol, when compared to P. shantungensis adults (Fig. 1). In a previous study, the differential susceptibility shown by P. shantungensis adults and nymphs to thymol and carvacrol was attributed to differences in the weights and sizes of P. shantungensis adults and nymphs, as well as the potential to detoxify glutathione S-transferase and hydrolase7,8,9. The synergetic effect of thymol combined with carvacrol has previously been reported for other insects, such as beetles14 and lepidopterans6. Medeiros et al.15 suggested that thymol and carvacrol to different species of insects are connected with the insecticidal effect of these monoterpenes on the cells of target insects, since they cause disorganization in the cell membrane, leading it to lose permeability15. In contrast, although the A. millefolium oil did not contain thymol, carvacrol and citral, the insecticidal properties of A. millefolium against P. shantungensis adults and nymphs could be due to internal synergy or blend effect of their constituents. Previous study reported internal synergy or a blend effect of the main constituents of plant oil for Ocimum kenyenst16, Zanthoxylum armatum17 and Plectranthus marruboides18 oils against the mosquito species, Aedes aegypti and Anopheles gambiae. Our results indicate that some terpenes containing the other tested components may correlate with the detoxification mechanisms of P. shantungensis adults and nymphs by several terpenes. Treatment with terpenes of Melia azedarach against Spodoptera littoralis can significantly increase the activities of α-esterase and β-esterase, which are important detoxifying enzymes19 and significant decreased the acid phosphatases, alkaline phosphatases, adenosine triphosphatases and the lactate dehydrogenase of Cnaphalocrocis medinalis20.

Table 4: Insecticidal toxicities of major commercial components of five essential oils and thymol analogs against P. shantungensis adults and nymphs at 72 h exposure time (LC50 is the average of 3 determinations after 72 h exposure, with 30 nymphs and 20 adults per replication).
Figure 1: Structures of thymol structural related analogs.
Figure 1

(a) 2-isopropyl-5-methylphenol (thymol); (b) 5-isopropyl-2-methylphenol (carvacrol); (c) 2-isopropylphenol; (d) 3-isopropylphenol; (e) 4-isopropylphenol.

In order to establish the structure-toxicity relationship between thymol analogs and insecticidal toxicities against P. shantungensis adults and nymphs, thymol, carvacrol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol were selected as thymol analogs for testing (Fig. 1). The insecticidal toxicities of thymol structurally related analogs and how activity varies with structure were investigated using leaf dipping and spray bioassays against P. shantungensis adults and nymphs (Table 4). Based on the LC50 values of thymol analogs against P. shantungensis nymphs, the LC50 values of thymol, carvacrol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol using the leaf dipping bioassay were 28.52, 56.74, 71.41, 82.49, and 111.28 mg/L, respectively. Using the spray bioassay against P. shantungensis adults, the LC50 values of thymol, carvacrol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol were 42.12, 75.62, 85.77, 104.65, and 122.36 mg/L, respectively. The insecticidal toxicity of thymol against P. shantungensis nymphs and adults was approximately 1.80–3.90 times greater than that of carvacrol, citral, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol. While the functional group in thymol was necessary for insecticidal toxicity, the removal of the methyl functional group reduced in insecticidal toxicity. Furthermore, the position of the methyl and isopropyl functional group in the phenol ring altered insecticidal toxicity. These results indicate that the insecticidal mode of action of thymol analogs may be largely attributable to the methyl functional group. This observation contrasts to an earlier finding that the isopropyl functional group in thymol analogs is key in imparting insecticidal toxicity against stored-food pests21.

The present results implicate T. vulgaris oil, thymol and thymol structurally related analogs as promising natural products of insecticides against exotic insects. Others have found visual evidence of leaf phytotoxicity caused by T. vulgaris oil to the host plant of P. shantungensis, grape leaf22. The LD50 values of carvacrol, thymol, and T. vulgaris oil against rat are 810, 980 and 2,840 mg/kg, respectively, by oral administration and the dermal LD50 value of thymol and T. vulgaris oil exceeds 2,000 mg/kg against rat and 5,000 mg/kg against rabbit, respectively23. These results suggest that T. vulgaris oil, carvacrol, thymol and thymol analogs have a relatively low acute toxicity in mammals.

Our study is the first to investigate the insecticidal toxicities of T. vulgaris oil, thymol and thymol analogs against P. shantungensis adults and nymphs. Considering the fact that T. vulgaris is a very inexpensive plant to acquire and is easily cultivated, and are not barriers for the commercial development of carvacrol, thymol, and T. vulgaris oil isolated from T. vulgaris. Further study is required to decrease the human toxicity of the T. vulgaris oil, thymol and thymol analogs and establish the insecticidal mode of action of thymol analogs against P. shantungensis adults and nymphs.

Materials and Methods

Chemicals and material preparation

Bornyl acetate (99%), camphene (95%), camphor (96%), carvarol (98%), (−)-carveol (95%), (+)-carvone (96%), β-caryophyllene (98.5%), caryophyllene oxide (95%), β-citronellal (95%), citral (95%), 1,8-cineole (99%), p-cymene (99%), decyl chloroformate (97%), dodecanoic acid (98%), β-farnesene (90%), geranyl acetate (97%), geraniol (98%), isopulegol (98%), linalool (97%), limonene (97%), limonene oxide (97%), methyl linolenate (99%), myrcene (90%), myristic acid (99%), palmitic acid (99%), α-phellandrene (85%), α-pinene (98%), pivalic acid (99%), sabinene (75%), α-terpineol (90%), γ-terpinene (97%), 4-terpineol (95%), terpinolene (90%), and tymol (99%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Achillea millefolium L. flowers, Citrus aurantium L. fruits, Leptospermum petersonii F. M. Bailey leaves, Ruta graveolens L. leaves, and Thymus vulgaris L. leaves were collected from a local store in Chonju, Korea. Sample specimens were authenticated by Jeongmoon Kim at Chonbuk National University, Korea. Essential oils of the five plants were obtained by steam distillation extraction, and finally dried over Na2SO4 to extract the pure essential oils (Table 1).

Insects and bioassays

P. shantungensis adults and nymphs were collected from persimmon trees in Wanjugun, Korea and classified the fourth instar stages of P. shantungensis nymphs and adults as detailed elsewhere2,4. The insecticidal toxicities of the five essential oils against P. shantungensis adults and nymphs were assessed (Table 2). Experimental protocols were approved by the Korea National Institute of Agricultural Sciences and the Korea Centers for Disease Control and Prevention. Using the leaf-dipping method, the insecticidal toxicities of A. millefolium, C. aurantium, L. petersonii, R. graveolens and T. vulgaris oils were evaluated against P. shantungensis nymphs. Insecticidal toxicities against P. shantungensis nymphs were evaluated according to a prior bioassay method8. Eight dilutions of insecticidal constituent (1000 to 50 mg/L) were prepared by dissolving in distilled water. Rose leaf disk of sharon (3 cm) were dipped into each test sample for 4 min and allowed to dry. The treated leaf was placed into a petri dish (60 × 15 mm) and 30 nymphs were released. The nymphs affected by this treatment were evaluated for 48 and 72 h after treatment. All treatments were repeated twice at 21 °C. The insecticidal toxicity of each sample was evaluated against P. shantungensis adults with the spray bioassay8. Eight concentrations (1000 to 50 mg/L) of the insecticidal constituents were diluted in distilled water. An insect square dish (15 × 15 × 20 cm) which contained 20 adults, was sprayed with 200 μL of treatment solutions to run off with a hand-held sprayer. Assessment was carried out 24 and 48 h after treatment by counting the normal insects. The tests were conducted with two replicates and incubated at 21 °C.

Gas chromatography-mass spectrometry

The components of the essential oil extracted from T. vulgaris leaves were quantified using the Hewlett-Packard HP 6890 and H5973IV series (Agilent, Santa Clara, CA, USA) and were separated with HP-Innowax capillary column and DB-5 column (0.25 mm i.d. × 0.25 μm thickness × 2,990 cm L.). The conditions of the column were as follows: Helium at 0.75 mL/min; column temperature (51 to 201 °C) at 2 °C/min; injector temperature (211 °C); split ration (48:1); ion source temperature (231 °C); ionization potential (70e V); and mass spectra range (50–800 amu). The components of T. vulgaris oils were evaluated according to retention times, retention indices, and mass spectra and were identified by comparison with a spectrum library (Table 3). The relative composition of each T. vulgaris oil constituent (%) was measured by comparison with internal standards.

Statistical analysis

Data obtained for each dose response bioassay were subjected to probit analysis. The median lethal concentration (LC50) value and the slope of the regression lines were calculated using the statistical package SPSS, version 12.0 for Windows.

Additional Information

How to cite this article: Park, J.-H. et al. Insecticidal toxicities of carvacrol and thymol derived from Thymus vulgaris Lin. against Pochazia shantungensis Chou & Lu., newly recorded pest. Sci. Rep. 7, 40902; doi: 10.1038/srep40902 (2017).

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Acknowledgements

This research was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project title: Study on the ecology and development of management practice for an exotic and epidemic pest, Ricania sp., Project No. PJ0116902017) Rural Development Administration, Republic of Korea”.

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  1. Department of Bioenvironmental Chemistry, Chonbuk National University, Jeonju 54896, Korea

    • Jun-Hwan Park
    • , Ye-Jin Jeon
    • , Chi-Hoon Lee
    •  & Hoi-Seon Lee
  2. Division of Research Planning, National Research Institute of Health, Centers for Disease Control & Prevention, Chungbuk 28159, Korea

    • Chi-Hoon Lee
  3. Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea

    • Namhyun Chung

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Contributions

J.-H.P. carried out the experiments, prepared most of the data, and wrote the paper; Y.-J.J. carried out the experiments; C.-H.L. carried out the experiments for the bioassay and assisted in paper-writing; N.C. designed, carried out the experiments and assisted in paper-writing; H.-S.L. proposed the key idea of this paper, designed and carried out the experiments, prepared most of the data, managed the research process, and wrote the paper.

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The authors declare no competing financial interests.

Corresponding authors

Correspondence to Namhyun Chung or Hoi-Seon Lee.

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