Pestic. Sci. 1997, 49, 119È124

Effectiveness of Carvacrol Derived from Thujopsisdolabrata var. hondai Sawdust against

japonensisThecodiplosis (Diptera: Cecidomyiidae)Sang G. Lee,a Soon I. Kim,b Young J. Ahn,b* Joon B. Kima & Boum Y. Leea

a Forestry Research Institute, Seoul 130-114, Republic of Koreab Department of Agricultural Biology and Research Center for New Biomaterials in Agriculture, College ofAgriculture and Life Sciences, Seoul National University, Suwon 441-744, Republic of Korea

(Received 7 January 1996 ; accepted 27 September 1996)

Abstract : The larvicidal component from sawdust of T hujopsis dolabrata var.hondai (Family Cupressaceae) against the pine needle gall midge (T hecodiplosisjaponensis) was isolated by chromatographic techniques and characterized byspectral analysis as carvacrol. In a laboratory study using the impregnated Ðlterpaper method, carvacrol was more toxic to T . japonensis larvae than b-thujaplicine, cedrol, a-terpinol, thujone or thymol. In Ðeld studies with soil injec-tions of carvacrol, this compound exhibited potent larvicidal activity, suggestingthat this activity might be attributable to fumigant action. In a test with trunkimplantation, a mixture of carvacrol and phosphamidon (0É15 ] 0É15 ml cm~1diameter at breast height) revealed much more potent larvicidal activity thanphosphamidon alone (0É3 ml cm~1 DBH) in spite of little or no larvicidal activ-ity of carvacrol alone (0É3 ml cm~1 DBH), indicating a possible synergistic e†ect.As a naturally occurring insecticide, carvacrol could be useful as a new preven-tive agent against damage caused by T . japonensis.

Key words : T hecodiplosis japonensis, T hujopsis dolabrata var. hondai, carvacrol,larvicidal activity, trunk implantation

1 INTRODUCTION

The pine gall midge (T hecodiplosis japonensis Uchida &Inouye) is one of the most serious insect pests of pinesin Korea.1 In the spring (May and June), just as the leafbuds swell, emerging adults deposit eggs on developingneedles. After hatching, young larvae crawl down to theleaf sheath and feed by sucking sap, resulting in the for-mation of galls. Large numbers of galls on pine treescause premature defoliation which results in simulta-neous retardation in both terminal and cambial growthof the tree.2h4 In 1995, approximately 212 000 ha of redpines (Pinus densiÑora Sieb. et Zucc.) and black pines (P.thunbergii Parl.) were infested by this species.5

Control is primarily dependent upon repeated appli-cations of soil insecticides against T . japonensis larvaein March to April and/or October to November and

* To whom correspondence should be addressed.

trunk implantation of systemic insecticides such asphosphamidon in mid-June.5 Although they have e†ec-tively controlled this gall midge species, their extensiveuse for the past decades has led to the development ofresistance to insecticides, adverse e†ects on non-targetorganisms, environmental problems and human healthhazards. Besides these problems, factors such as labourand insecticide costs should be considered. This eco-nomic consideration and increasing concern over pos-sible adverse e†ects of the earlier types of insecticidehave brought about the need for the development ofnew types of selective alternatives or biorational man-agement methods without, or with reduced, use of con-ventional insecticides.

Plants may provide alternatives to currently usedinsect control agents, because these are often activeagainst a limited number of species, including speciÐctarget insects, are often biodegradable to non-toxic pro-ducts, and potentially suitable for use in IPM. There-fore, much e†ort has been focused on plant-derived

119Pestic. Sci. 0031-613X/97/$09.00 1997 SCI. Printed in Great Britain(

120 Sang G. L ee et al.

materials for potentially useful products as commercialinsecticides or as lead compounds.6h8 We reported in aprevious paper9 that, among 190 plant species, crude oilof T hujopsis dolabrata (L.) Sieb. & Zucc. var. hondaisawdust had potent larvicidal activity against T . japon-ensis.

In the laboratory and Ðeld studies described herein,we assessed the e†ectiveness of the T hujopsis sawdust-derived materials against T . japonensis.

2 MATERIALS AND METHODS

2.1 Laboratory studies

2.1.1 InsectsTwigs of red pine trees severely infested with T . japon-ensis at Mikum-Si (Kyungi Province) were collected on13 September 1994, and brought to the laboratory. Thegall-formed needles only were selected and cut by arazor, and then larvae in the galls were carefully col-lected with a camelhair brush in Petri dishes(11 cm dia.) containing Ðlter paper (Toyo No. 2 ;6 cm dia.) moistened with 3 ml distilled water.

2.1.2 ChemicalsChemicals used in this study were as follows : b-Thujaplicine and a-terpineol (ExtraSynthese, France),cedrol (Karlsruhe Roth, Germany), thujone and carva-crol (Tokyo Kasei, Japan) and thymol (Wako, Japan).Phosphamidon 500 g litre~1 SL was supplied by HanKook Sam Gong (Seoul, Korea). All other chemicalswere of reagent grade.

2.1.3 Isolation and identiÐcationThe sawdust of T . dolabrata var. hondai was obtainedfrom Taiyo Kagaku Central Laboratories, Yokkaich,Mie Prefecture, Japan. PuriÐcation and isolation ofinsecticidal component(s) from the steam distillate ofT hujopsis sawdust active against T . japonensis larvaewas performed as previously described.10 The fractionsand isolates dissolved in methanol were bioassayed at arate of 10 mg per paper by the impregnated Ðlter papermethod (Section 2.1.4).

Structural determination of the active isolate wasmade by spectroscopic analysis. [1H] and [13C]NMRspectra were recorded in deuterochloroform with aJEOL GSX-400 FT-NMR (400 MHz) spectrometer.UV spectra were obtained with a Unikon 942 spectro-photometer, IR spectra on a Biorad FT-80 spectrometerand EI-MS spectra on a JEOL JMS-AX 505 spectro-meter.

2.1.4 BioassayLarvicidal activities of the T hujopsis sawdust-derivedmaterials against T . japonensis were tested by the

impregnated Ðlter paper method. Appropriate doses ofthe materials dissolved in methanol were applied toÐlter papers (Toyo No. 2 ; 6 cm dia.) by syringe. Afterevaporation, larvae were placed on to the papers inindividual Petri dishes (6 cm dia.). Controls receivedmethanol. All treatments were conducted in triplicate,and 20 larvae were used in each assay. Treated larvaewere held in a room at 25(^ 1)¡C, 50È60% RH under a16 : 8 h light : dark cycle. Mortalities were determined48 h after treatment. Data from all bioassays were cor-rected for control mortality using AbbottÏs formula.11

The larvicidal activity of the most active isolate wasexamined and compared with that of the T hujopsis-derived terpenoids (b-thujaplicine, cedrol and a-terpineol) and two commercial terpenoids (thujone andthymol) as previously described.10

2.2 Field studies

2.2.1 Soil applicationCarvacrol efficacy was evaluated by soil injection atdamaged sites of Mikum-Si (Kyunggi Province) andKangnung-Si (Kwangwon Province) on 20 May and 25November 1994, respectively. Three replicate plots ofeach of the treatments plus three checks, each 5] 5 m,were laid out in a completely randomized block. Thenumber of larvae per plot in the soil was determinedbefore treatment. Injections of carvacrol were made10 cm deep with an injector at 30-cm intervals in rows30 cm apart at rates of 1, 2É5 and 5 ml. After injection,the soil was tamped by stamping. Polyethylene traps(20 cm dia.) for emerging midge adults were placed onthe treated and untreated soils (Fig. 1). These traps wereplaced 1É5 m apart in the Ðelds at Mikum-Si andKangnung-Si on 31 May 1994 and on 1 June 1995,respectively. Traps were checked twice weekly beforecapture of the Ðrst midge adults and every day there-after. Midge adults were removed from the traps andcounted.

Fig. 1. Polyethylene trap for emerging T hecodiplosis japon-ensis adults.

E†ectiveness of carvacrol against T. japonensis 121

Emergence rate was calculated as (total number ofmidge adults captured at each trap for 30 days aftertreatment/number of gall midge larvae in soil beforetreatment) ] 100. However, efficacy was expressed asprotective value (PV) using the formula

PV\ [1 [ (Un[ Ue/Un)/1 [ (Tn [ Te/Tn)]] 100,

in which of gall midge larvae in untreatedUn \ numberplot before treatment ; number of midgeUe \ totaladults captured at each trap in untreated plot aftertreatment ; of gall midge larvae in treatedTn\ numberplot before treatment and number of midgeTe\ totaladults captured at each trap in treated plot after treat-ment.

2.2.2 T runk implantationAll treatments were conducted on uniform 20- to 25-year-old red pine stands (c.12È15 cm dia. and 5È6 mhigh) at Pyungchang-Gun and Kangnung-Si (KangwonProvince) on 15 June 1994 and on 20 June 1995, respec-tively. Phosphamidon alone and in mixture with carva-crol was applied as trunk implantations to matureÐve-tree plots in a completely randomized block withthree replications. Carvacrol was introduced directlyinto the trees, while phosphamidon was introduced as acommercial 500 g litre SL. Implantations were madewith a motor-driven injector (Kawasaki TD33D, Japan)after drilling holes (1 cm dia.) in the trunk at 60 cmabove soil level. The holes were drilled 8 cm deep andat 45¡ to the main axis of the tree. Control trees weredrilled without the introduction of chemical. Carvacrol,phosphamidon and carvacrol ] phosphamidon (1 ] 1by volume) were applied at 0É3 ml per cm of trunkdiameter at breast height (DBH). In a preliminary test,little or no larval mortality occurred in pine treestreated with 0É15 ml cm~1 DBH.

The treatments at Pyungchang-Gun and Kangnung-Si were sampled at random from each treatment on 20September 1994 and 22 September 1995, respectively.The gall-formed needles from twigs of the red pine treeswere cut with a razor, and larvae in the galls whichfailed to move when prodded with a camelhair brushwere considered dead.

To determine phytotoxic e†ect, the presence orabsence of leaf burn and damage in treated trees wasassessed after each chemical application and comparedwith that of the untreated trees.

2.3 Statistical analysis

The percentage mortality and emergence rate weredetermined and transformed to arcsine values foranalysis of variance. Means(^SEM) of untransformeddata were reported. Treatment means were compared

and separated by TukeyÏs studentized range test atP\ 0É05.12

3 RESULTS

3.1 Laboratory studies

The crude oil of the T hujopsis sawdust revealed potentlarvicidal activity against T . japonensis and was separat-ed into four fractions, using a centrifugal thin-Ðlmevaporator. The fractions and isolates dissolved inmethanol were bioassayed by the impregnated Ðlterpaper method (Table 1). At 10 mg per paper, fractions Iand II caused 100 and 58% mortalities against theselarvae, respectively. PuriÐcation of the biologicallyactive component(s) from the most potent fraction I wasdone by silica gel column chromatography and HPLC.

Bioassay-guided fractionation and HPLC of the T hu-jopsis crude oil a†orded an active compound which wascharacterized as the terpenoid carvacrol by spectro-scopic methods including MS and NMR, and compari-son of the spectral data with literature.10 The larvicidalactivity of carvacrol was compared with that of otherT hujopsis-derived and commercial terpenoids (Fig. 2) aspreviously described10 and the data are presented in

TABLE 1Larvicidal Activity of the Crude Fractions from T hujopsis dol-abrata var. hondai Sawdust against T hecodiplosis japonensis

Larvae by Impregnated Filter Paper Bioassay

Fractiona Mortality (%) (^SEM)b

I 100aII 58É3 (^4É4)b

III 26É7 (^3É3)cIV 10É0 (^0É0)d

a Rate\ 10 mg per paper (6 cm dia.).b Means followed by the same letter in column are not signiÐ-cantly di†erent (P\ 0É05 ; TukeyÏs studentized range test) (20larvae per replicate ; three replicates per treatment : n \ 60).

Fig. 2. Structures of compounds tested. 1 Carvacrol ; 2 b-Thujaplicine ; 3 Cedrol ; 4 a-Terpineol ; 5 Thujone ; 6 Thymol.

122 Sang G. L ee et al.

TABLE 2Larvicidal Activity of T hujopsis dolabrata var. hondai-derived Materialsand Commercial Terpenoids against T hecodiplosis japonensis Larvae by

Impregnated Filter Paper Bioassay

Mortality (%) (^SEM)a

Compound 1b 5b 10b

Carvacrol 75É5 (^2É3)a 79É0 (^3É8)a 93É3 (^1É7)ab-Thujaplicine 67É3 (^3É7)ab 70É0 (^5É8)a 73É3 (^4É4)aCedrol 59É3 (^5É8)abc 70É0 (^5É8)a 70É0 (^7É6)aa-Terpineol 52É7 (^6É4)abc 55É0 (^5É0)a 70É0 (^7É6)aThujone 47É7 (^3É9)bc 65É0 (^5É0)a 67É7 (^4É3)aThymol 42É3 (^6É2)c 60É0 (^5É8)a 76É3 (^4É5)a

a Means followed by the same letter in column are not signiÐcantly di†er-ent (P\ 0É05 ; TukeyÏs studentized range test) (20 larvae per replicate ;three replicates per treatment : n\60).b Rate\ mg per paper (6 cm dia.).

TABLE 3E†ect of Carvacrol on Emergence Rate of T hecodiplosis japonensis Larvae by

Soil Injection

Experiment Ib Experiment IIc

ERd (%) (^SEM)e PV f (%) ER (%) (^SEM) PV (%)Ratea(ml)

0 84É8 (^4É1)a 84É0 (^3É2)a1 Èg 33É7 (^3É6)b 59É02É5 È 6É5 (^1É0)c 92É35 15É7 (^3É1)b 81É5 3É2 (^1É3)c 96É2

a Injections of carvacrol were made 10 cm deep with an injector at 30-cmintervals in rows 30 cm apart.b Treated on 20 May 1994.c Treated on 25 November 1994.d Emergence rate ; see Section 2.2.1.e Means followed by the same letter in column are not signiÐcantly di†erent(P\ 0É05 ; TukeyÏs studentized range test).f Protective value ; see Section 2.2.1.g Not determined.

TABLE 4Susceptibility of T hecodiplosis japonensis Larvae to Phosphamidon Alone and in

Mixture with Carvacrol by Plant Implantation Application

Mortality (%) ( ^ SEM)a

Experiment Ib Experiment IIcRate, ml cm~1

Chemical DBH

Carvacrol 0É3 10É8 ( ^ 1É5)c ÈdCarvacrol ] phosphamidon 0É15 ] 0É15 95É0 ( ^ 1É2)a 92É3 ( ^ 2É5)bPhosphamidon 0É3 81É8 ( ^ 6É1)b 83É4 ( ^ 1É6)cUntreated È 1É8 ( ^ 0É1)c 2É0 ( ^ 0É6)a

a Means followed by the same letter in column are not signiÐcantly di†erent(P \ 0É05 ; TukeyÏs studentized range test).b Treated on 15 June and determined on 20 September 1994.c Treated on 20 June and determined on 25 September 1995.d Not determined.

E†ectiveness of carvacrol against T. japonensis 123

Table 2. Of the terpenoids tested, carvacrol revealed themost potent larvicidal activity.

3.2 Field studies

The efficacy of carvacrol against T . japonensis larvaewas investigated by soil injection (Table 3). In a Ðeldtest at Pyungchang-Gun, carvacrol was highly e†ectiveagainst T . japonensis larvae at a rate of 5 ml as com-pared with control. Therefore, titration studies wereperformed at heavily damaged sites in Kangnung-Si.SigniÐcant di†erences in emergence rates were observedfor each treated plot. Over 90% control was achieved inthe plots treated with 2É5 and 5 ml of carvacrol,whereas treatment with 1 ml produced 59% control.

In a test with trunk implantation in 1994 (Table 4),phosphamidon (0É3 ml cm~1 DBH) gave outstandingcontrol of T . japonensis (81É8% mortality), whereas nosigniÐcant efficacy (10É8% mortality) was produced bycarvacrol alone (0É3 ml cm~1 DBH). However, amixture (0É15 ] 0É15 ml cm~1 DBH) of carvacrol andphosphamidon was highly e†ective (95É0% mortality),more so than phosphamidon alone (0É3 ml cm~1 DBH).Similar results were also obtained from the 1995 experi-ments. No leaf burn or other symptom of phytotoxicitywas observed for any of the chemical treatments.

4 DISCUSSION

Various compounds, including phenolics, terpenoidsand alkaloids, exist in plants and jointly or indepen-dently contribute to their insecticidal activities.13 ManyT hujopsis species are rich in terpenoids.14h16 In ourlaboratory study with Ðeld-collected T . japonensislarvae, carvacrol with potent larvicidal activity was iso-lated from the crude oil from T hujopsis sawdust. This isthe Ðrst report on insecticidal activity of carvacrolagainst this insect species, although investigations haveshown that this plant-derived material has insecticidalactivity against termites17h19 and stored-insect pests19and antimicrobial20 and rodent-repellent e†ects.10

Ahn et al.10 pointed out that carvacrol might be usedas a pest-control agent in limited spaces such as storagebins, greenhouses or buildings, because of its high vola-tility. In our study with soil and impregnated Ðlterpaper applications, carvacrol was highly e†ective. Theseresults indicate that carvacrol might have both contactand fumigant action. It has been reported that the modeof action of carvacrol against termites and stored-product insects is attributable to both contact and fumi-gant e†ects.19

Control of the gall midge in Korea is most commonlydependent upon trunk implantation of systemic insecti-cides such as phosphamidon.5 Although much success

has been achieved using systemic insecticides for thecontrol of insects on coniferous trees when implantedinto the trunk,21 these compounds have attendantproblems such as human health hazards. Therefore,much attention has been focused on alternative man-agement methods without, or with reduced, use of con-ventional insecticides. In the present study with trunkimplantation, a mixture of carvacrol and phosphamidonrevealed much more potent larvicidal activity thanphosphamidon alone, in spite of little or no insecticidalactivity of carvacrol alone. These results suggest thatcarvacrol may promote rapid translocation of phos-phamidon into pine trees or prevent rapid degradationof this insecticide in T . japonensis larvae and trees. It ispossible that a mixture of insecticides may increaseinsecticidal activity, delay the development of insecticideresistance, or control insect species resistant to singleinsecticides.

It has been well acknowledged that certain plant-derived extracts and phytochemicals are potential alter-natives to insecticides,6h8,22 based upon the facts thatthey have selectivity towards the natural enemies ofpests, act in many ways on various types of pestcomplex, and may be applied to the plant in the sameway as other agricultural chemicals. In addition, plant-derived materials are found to be highly e†ective oninsecticide-resistant insect pests.23h26 Carvacrol has lowtoxicity27 and caused no mutagenicity, when testedagainst four strains of Salmonella typhimurium Castell.& Chalm.19 Based upon our data and earlier Ðndings,good potential exists for the control of Ðeld populationsof T . japonensis with a reduced level of phosphamidonwhen applied with carvacrol to soil in late Novemberand when a mixture of phosphamidon and carvacrol isimplanted in mid-June. An average of 4000 tonnes ofsawdust per year is produced as a by-product of thetimber industry, because T . dolabrata is an importanttimber species in Japan.28 Thus there is an adequatesource of the crude oil available.

ACKNOWLEDGEMENT

This research was supported by grants from ResearchCenter for New Biomaterials in Agriculture and Fore-stry Research Institute.

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