Nematicidal and Propagation Activities of Thyme Red and White Oil Compounds toward Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae)

Abstract

The toxic and propagation effects on Bursaphelenchus xylophilus of 28 Thymus vulgaris red oil and white oil compounds were examined using direct contact and cotton ball bioassays. Results were compared with those of the trunk-injection nematicides emmamectin benzoate, levamisol hydrochloride and morantel tartrate. In direct contact bioassays, geraniol (LC₅₀, 0.47 mg/ml) was the most toxic compound, followed by thymol (1.08 mg/ml), carvacrol (1.23 mg/ml) and terpinen-4-ol (2.61 mg/ml). In cotton ball tests with 20 inactive compounds at 2 mg/cotton ball, p-cymene significantly inhibited propagation (propagation ratio [PR] 8), compared with the castor oil-ethanol-treated control (PR 56). Propagation stimulation was observed with (–)-caryophyllene oxide, (+)-ledene, (+)- and (–)-limonene, linalool oxide, β-myrcene, (–)-α-phellandrene, (+)-α-pinene and γ-terpinene (PR 63–100). The other 10 compounds exhibited low to moderate levels of propagation inhibition (PR 36–56). At 0.1 μg/cotton ball, emmamectin benzoate and morantel tartrate exhibited complete suppression of propagation, whereas a very low level of propagation inhibition was obtained from levamisol hydrochloride (PR 6). In conclusion, propagation-stimulating compounds can exist in plants in addition to nematicidal compounds, and careful use of plant preparations containing high quantities of these compounds is mandatory.

Pinewood nematode, Bursaphelenchus xylophilus (Sterner and Buhrer, 1934) Nickle, 1970, is associated with dead and dying conifers, particularly pines. The nematode is the causal agent of pine wilt disease (PWD) (Mamiya and Kiyohara, 1972; Linit, 1988; Dwinell, 1997). In Korea, PWD was first recorded from Pinus densiflora Siebold & Zuccarini collections made at Mt. Geumjeong in Busan in 1988 (Yi et al., 1989). There were more than 25,000, 115,000, 150,000 and 1.37 million P. densiflora trees killed by PWD in 1999, 2002, 2004 and 2006, respectively (KFRI, 2007). Among the cerambycid beetle species of the genus Monochamus, M. alternatus Hope and M. carolinensis Oliver are primary vectors of B. xylophilus in East Asia (Kobayashi et al., 1984) and the United States (Linit, 1988), respectively. Control of PWD in Korea has been principally provided by killing the vector beetles by aerial and ground applications of conventional insecticides, by fumigation of pine trees killed by the disease, or by eradicating B. xylophilus by trunk injection of nematicides such as morantel tartrate and emmamectin benzoate (Lee et al., 2003a, 2003b). Certain trunk-injection nematicides often caused phytotoxicity (Kishi, 1995). Additionally, factors such as increased costs of trunk-injection nematicide and labor and growing public concern for the environmental effects of pesticides have made PWD control difficult. These problems highlight the need for the development of biorational control alternatives for B. xylophilus.

Plant essential oils may be alternative sources for nematode control products because they are widely available and because some are selective, degrade to nontoxic products and have few harmful effects on nontarget organisms and the environment (Isman, 2000; Chitwood, 2002). These potential, new nematicidal products can be applied to pine trees in the same manner as the currently used trunk-injection nematicides. In part, because certain plant essential oils meet the criteria of minimum risk pesticides (US EPA, 2004), much effort has been focused on them and their constituents as potential sources of commercial nematode control products. Additionally, many plant preparations manifest propagation-stimulating and -inhibiting activities toward B. xylophilus (Kawazu et al., 1980a; Chitwood, 2002). Previously, the present authors reported that red oil of thyme, Thymus vulgaris L. (Lamiaceae, formerly Labiatae), had good nematicidal activity toward adult B. xylophilus (Kong et al., 2006). Very little work has been done on the effects of nonnematicidal constituents of thyme essential oils on stimulating or inhibiting B. xylophilus propagation.

This study was aimed at isolating nematicidal constituents from thyme red oil and white oil active against adult B. xylophilus. Also, the propagation-inhibiting or -stimulating activities of thyme oil compounds were compared with those of the most commonly used trunk-injection nematicides: emmamectin benzoate, levamisol hydrochloride and morantel tartrate salt.

Materials and Methods

Materials: The 28 compounds used in this study were as follows: (+)-aromadendrene (≥97% purity), (+)-camphor (≥97% purity), (–)-camphor (≥99% purity), carvacrol (≥97% purity), (+)-ledene (≥95% purity), (+)-menthone (≥98.5% purity), (–)-menthone (≥99% purity), (–)-α-phellandrene (≥95% purity), (+)-α-pinene (≥99.5% purity), (–)-α-pinene (≥99% purity), (+)-β-pinene (≥98.5% purity) and (–)-β-pinene (≥99% purity) purchased from Fluka (Bucks, Switzerland); (+)-borneol (98% purity), (–)-borneol (99% purity), camphene (95% purity), (–)-caryophyllene oxide (99% purity), p-cymene (99% purity), (–)-linalool (≥95% purity), β-myrcene (90% purity), γ-terpinene (97% purity), (+)-α-terpineol (≥97% purity) and thymol (≥99.5% purity) purchased from Sigma-Aldrich (St. Louis, MO); β-caryophyllene (≥80% purity), geraniol (≥96% purity), linalool oxide (≥97% purity) and terpinen-4-ol (≥95% purity) purchased from Tokyo Chemical Industry (Tokyo, Japan); and (+)-limonene (≥95% purity) and (–)-limonene (≥99% purity) purchased from Wako (Osaka, Japan). Analytical emmamectin benzoate (99.6% purity), levamisol hydrochloride (≥99% purity) and morantel tartrate salt were obtained from Riedel-de Haén (Seelze, Germany), Tokyo Chemical Industry and Sigma-Aldrich, respectively. Polyoxyethylene hydrogenated castor oil (HCO–50), a solubilizer, was a gift from Nikko Chemical (Tokyo). Thyme red and white oils were purchased from Berjé (Bloomfield, NJ). Potato dextrose agar (PDA) was supplied by Becton, Dickinson and Company (Sparks, MD). All other chemicals were of reagent grade and available commercially.

Nematodes: A colony of B. xylophilus was maintained in the laboratory on PDA plates cultured with Botrytis cinerea Persoon at 25 ± 1°C in darkness (Kong et al., 2007). Briefly, the cultured nematodes were separated from the culture medium by the Baermann funnel technique (Viglierchio and Schmitt, 1983). The standard nematode suspension was prepared by appropriate dilution with distilled water, and the number of nematodes was counted under a binocular microscope (×20).

Gas chromatography (GC): Chemical constituents of thyme red and white oils were identified based on retention time and by co-injection with authentic compounds. A GC-2010 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with split injector was used. Analytes were separated with a 60 m × 0.32 mm i.d. (d _f = 0.25 μm) DB-1 MS bonded-phase fused-silica capillary column (J&W Scientific, Ringoes, NJ). The oven temperature program used for the analysis was as follows: initial temperature at 80°C, held for 5 min and ramped at 5°C/min to 210°C, held for 5 min and ramped at 30°C/min to 300°C and held at 10 min. The linear velocity of the helium carrier gas was 26.1 cm/sec (30°C) at a split ratio of 1:10. The injector temperature was 210°C.

Gas chromatography-mass spectroscopy (GC-MS): Constituents of thyme red and white oils were identified by comparison of mass spectra of each peak with those of authentic samples in a mass spectra library (Anonymous, 2000). GC-MS analysis was performed using a GC-2010 GC-GCMS QP-2010 mass spectrometer (Shimadzu, Kyoto). The capillary column and temperature conditions for the GC-MS analysis were the same as described above for GC analysis. Helium carrier gas was used at a column head pressure of 42.6 kPa. The ion source temperature was 200°C. The interface was kept at 280°C, and mass spectra were obtained at 70 eV. The effluent of the capillary column was introduced directly into the ion source of the mass spectrometer. The sector mass analyzer was set to scan from 40 to 800 amu every 0.5 sec.

Contact toxicity bioassay: A direct-contact bioassay (Kong et al., 2007) was used to evaluate the toxicity of thyme red and white oils and their constituents to adult B. xylophilus. An aqueous nematode suspension (approx. 3,000 nematodes/ml) was prepared by diluting the standard nematode suspension. The nematode suspension (99 μl) was added separately to each well prior to toxicity tests. Five to seven concentrations of each test material in 1 μl of polyoxyethylene hydrogenated castor oil-ethanol solution (1 mg/ml) (Takai et al., 2000) were added to the wells. The well plate was then covered with a solid lid. Controls received 1 μl of the castor oil-ethanol solution.

Treated and control (castor oil-ethanol only) nematodes were held at the same conditions used for colony maintenance. Mortalities were determined 24 hr post-treatment under a binocular microscope. Nematodes were considered to be dead if their bodies showed straight shapes without movement when they were prodded with fine wooden dowels (Kong et al., 2007). All treatments were replicated three to five times.

Propagation effect of test compounds: A cotton ball bioassay (Kawazu et al., 1980a) was used to determine whether inactive thyme oil compounds exhibited propagation inhibition or stimulation toward B. xylophilus. Briefly, an aqueous nematode suspension (approx. 6,000 nematodes/ml) was prepared by diluting the standard nematode suspension. A 2 mg quantity of each test compound was dissolved in 10 μl of polyoxyethylene hydrogenated castor oil-ethanol solution. The test compound solution (10 μl) was injected into an 8-mm-diam. cotton ball that was placed at the center of the fungal mat in the 8.7-cm-diam. petri dish, and the aqueous nematode suspension (90 μl) was then injected into the ball. Controls received castor oil-ethanol solution and aqueous nematode suspension. Because of their anti-nematodal activity (Takai et al., 2000), emmamectin benzoate, levamisol hydrochloride and morantel tartrate served as positive controls for comparison in propagation inhibitory tests. Treated and control nematodes were kept at 25 ± 1°C for 5 d in darkness. The living nematodes were separated from the culture by the Baermann funnel technique and were counted. All treatments were replicated four times.

Data analysis: Data were corrected for control mortality using Abbott's formula (Abbott, 1925). LC₅₀ values were calculated by probit analysis (OnlineDoc, v8.01, SAS Institute, Cary, NC). Nematicidal activity was considered to be significantly different when 95% confidence limits of the LC₅₀ values failed to overlap. The propagation ratio (PR) was determined using the formula: PR = (T_a – T_b)/T_b × 100, where T_a is the number of nematodes after chemical treatment and T_b is the number of nematodes before chemical treatment. The propagation percentages were transformed to arcsine square root values for analysis of variance (ANOVA). The Bonferroni multiple-comparison method was used to test for significant differences among the test compounds.

Results

Chemical constituents of thyme red and white oils: Thyme red and white oils were composed of three and four major constituents, respectively (Table 1). Thymol, p-cymene and γ-terpinene comprised 58.02, 17.47 and 8.83% of thyme red oil. Thymol, p-cymene, linalool and γ-terpinene comprised 47.52, 17.46, 10.56 and 6.84% of white oil. They constituted approx. 83% of the total red and white oils.

Table 1.

Chemical constituents of thyme red and white oils identified by GC-MS^a and GC.

Nematicidal activity of thyme oils and their constituents: The nematicidal activity of thyme red and white oils and their 28 constituents was evaluated by comparing the LC₅₀ values estimated from the contact bioassay toward adult B. xylophilus (Table 2). As judged by 24 hr LC₅₀ values, thyme red oil (1.39 mg/ml) and white oil (1.64 mg/ml) exhibited good nematicidal activity. Of the oil compounds tested, geraniol (LC₅₀ 0.47 mg/ml) was the most toxic compound, followed by thymol (1.08 mg/ml) and carvacrol (1.23 mg/ml). Moderate activity was produced from terpinen-4-ol and (+)-α-terpineol, whereas weak nematicidal activity was obtained from linalool. The body of a nematode treated with these compounds usually showed a straight shape without movement when prodded with wooden dowels. Little or no nematicidal activity was produced from the other 22 test compounds. Mortality in the castor oil-ethanol treated controls was less than 2%.

Table 2.

Toxicity of thyme essential oils and their constituents to adult Bursaphelenchus xylophilus using the direct contact toxicity bio-assay during a 24 hr exposure.

Propagation effect of test compounds: Because of little or no nematicidal activity of 22 test compounds as stated above, propagation responses of B. xylophilus to these inactive compounds were compared with those of three trunk-injection nematicides, emmamectin benzoate, levamisol hydrochloride and morantel tartrate (Fig. 1). Propagation responses varied according to the compound used. At 2 mg/cotton ball, p-cymene significantly inhibited propagation (PR 8) compared with the castor oil-ethanol-treated control (PR 56). Low to moderate levels of propagation inhibition were produced from (+)-aromadendrene, (+)- and (–)-borneol, camphene, (+)- and (–)-camphor, β-caryophyllene, (+)- and (–)-menthone and (+)-β-pinene (PR 36–56). Moderate to high propagation stimulation was observed in (–) caryophyllene oxide, (+)-ledene, (+)-limonene, (–)-limonene, linalool oxide, β-myrcene, (–)-α-phellandrene, (+)-α-pinene and γ-terpinene (PR 63–100). At 0.1 μg/cotton ball, emmamectin benzoate and morantel tartrate exhibited complete suppression of propagation, whereas a very low level of propagation inhibition was obtained from levamisol hydrochloride (PR 6).

Fig. 1.

Propagation responses of thyme oil compounds and three trunk-injection nematicides to Bursaphelenchus xylophilus using the cotton ball bioassay. A 2 mg quantity of each thyme oil compound and 0.1 μg of each nematicide were injected into a cotton ball that was placed at the center of the fungal mat. n = 4. Horizontal bar is the standard error. Different letters indicate significant differences (P = 0.05; Bonferroni method).

Discussion

Highly complex mixtures of terpenoids, particularly monoterpenoids, and related phenols exist in plant essential oils (Sellar, 2001; Kubeczka and Formáček, 2002; Lawless, 2002), and jointly or independently they contribute to a variety of biological activities including repelling or attracting nematodes, stimulating or inhibiting egg hatch and nematicidal activity (Chitwood, 2002). Little work has been done with respect to managing B. xylophilus with thyme oils and their constituents despite excellent pharmacological actions of the oils (Sellar, 2001; Kubeczka and Formáček, 2002; Lawless, 2002). Much effort has been focused on determining the distribution, nature and practical use of plant-derived substances that have nematode-antagonistic activity. Naturally occurring nematicidal compounds toward B. xylophilus include the acetylenes cis-dehydromatricaria ester, 9,10-epoxyheptadec-16-en-4,6-diyn-8-ol, 1-phenylhepta-1,3,5-triyne, 2-phenyl-5-(1-propynyl)-thiophene and tridec-1-en-3,5,7,9,11-pentayne (Kawazu et al., 1980b); the quinolizidine alkaloids, such as aloperine, cytisine, N-methylcytisine and matrine (Matsuda et al., 1989, 1991; Zhao, 1999); the lignans (–)-nortrachelogenin and (+)-pinoresinol, the phenylpropanoid methyl ferulate and the stilbene pinosylvin monomethyl ether (Suga et al., 1993), the phenolics 3-undecylphenol and 3-(8Z-tridecenyl)phenol (Alen et al., 2000) and the phenylpropanoids (E) cinnamaldehyde, cinnamyl acetate and eugenol and the monoterpenoid α-terpineol (Kong et al., 2007). In the current study, thyme red and white oils showed good nematicidal activity toward adult B. xylophilus. The nematicidal principles of thyme oils were identified as the monoterpenoids carvacrol, geraniol, linalool, terpinen-4-ol, α-terpineol and thymol. Geraniol was the most toxic and linalool was the least toxic.

Available information is limited because immobile nematodes intoxicated by some plant-derived materials and chemicals were described as dead. Jourand et al. (2004) reported that an aqueous extract of Crotalaria virgulata subsp. grantiana Harvey leaves caused paralysis of Meloidogyne incognita (Kofoid & White) Chitwood juveniles but the effect was reversible, i.e., nematostatic. They suggested that pyrrolizidine alkaloids such as grantianine and grantaline of C. grantiana (Hartmann and Witte, 1995) might be responsible for the nematostatic effect on the basis that alkaloids of Fabaceae plants possess antinematodal properties (Fassuliotis and Skucas, 1969). Additionally, diverse chemical classes of compounds have been well noted as propagation stimulants or inhibitors for nematodes. For example, Takai et al. (2000) studied anti-nematodal activity of 58 compounds with different modes of action toward B. xylophilus. They reported that the γ-aminobutyric acid receptor agonists, such as abamectin, ivermectin and emmamectin benzoate, had better propagation inhibitory activity than compounds influencing glutamate, β-adrenergic, dopamine and acetylcholine receptors, as well as those inhibiting acetylcholinesterase, monoamine oxidase and ion channels. It has also been reported that nematode bodies treated with the muscle activity blockers levamisol hydrochloride and morantel tartrate usually exhibit semicircular and coiling shapes, respectively (Kong et al., 2006), whereas nematode bodies treated with cinnamaldehyde and its derivatives usually showed straight shapes (Kong et al., 2007).

In the present study, active thyme oil compounds caused straight shapes and rapid nematicidal action. The straight-shaped nematodes never recovered. Additionally, propagation inhibition was produced from (+)-aromadendrene, (+)- and (–)-borneol, camphene, (+)- and (–)-camphor, β-caryophyllene, p-cymene, (+) and (–)-menthone and (+)-β-pinene (PR 36–56), whereas propagation stimulation was observed in (–) caryophyllene oxide, (+)-ledene, (+)-limonene, (–)-limonene, linalool oxide, β-myrcene, (–)-α-phellandrene, (+)-α-pinene and γ-terpinene. This is the first report on the propagation stimulatory activity of these terpenoids toward B. xylophilus. Treatment with emmamectin benzoate, morantel tartrate or levamisol hydrochloride caused strong suppression of propagation at a very low concentration. Our present and previous other studies suggest that the nematicidal mode of action of thyme oil terpenoids might be different from that of three trunk-injection nematicides used, emmamectin benzoate, morantel tartrate and levamisol hydrochloride, or of plant alkaloids.

In conclusion, thyme red and white oils contain nematicidal compounds as well as propagation-stimulating compounds toward B. xylophilus. These results indicate that careful use of plant preparations containing high quantities of propagation-stimulating compounds is mandatory. Further research is necessary on the nematicidal mode of action and propagation-stimulating mechanism.