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Short Communication

Composition and acaricidal activity of Lippia sidoides essential oil againsttwo-spotted spider mite (Tetranychus urticae Koch)

S.C.H. Cavalcanti a, E. dos S. Niculau b, A.F. Blank c, C.A.G. Cmara d, I.N. Arajo d, P.B. Alves b,*

a Departamento de Fisiologia, Universidade Federal de Sergipe, Av. Marechal Rondon S/N, CEP 49100-000, So Cristvo-SE, Brazilb Departamento de Qumica, Universidade Federal de Sergipe, Av. Marechal Rondon S/N, CEP 49100-000, So Cristvo-SE, Brazilc Departamento de Engenharia Agronmica, Universidade Federal de Sergipe, Av. Marechal Rondon S/N, CEP 49100-000, So Cristvo-SE, Brazild Departamento de Qumica, Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros S/N, CEP 52171-900, Recife-PE, Brazil

a r t i c l e i n f o

Article history:

Received 12 March 2008

Received in revised form 6 August 2009

Accepted 7 August 2009

Available online 15 September 2009

Keywords:

Lippia sidoides

Essential oil

Acaricidal

Tetranychidae

Spider mite

a b s t r a c t

The essential oils from accessions of Lippia sidoides Cham. (Verbenaceae) were characterized by GC and

GC/MS and investigated for their acaricidal activity against the two-spotted spider mite (Tetranychus urti-

cae Koch). Twenty-nine compounds were identified with potential acaricidal activity. Glass receptacles

were used as test chambers. For each dose and exposure time combination, three replicates were used.

Each replicate consisted of 30 adult females ofT. urticae, 10 mites in each leaf disk ofCanavalia ensiformis

placed in a Petri dish. Increasing amounts of oil or terpene were applied on a blotting paper strip, fixed on

the inner surface of the glass recipient cover, corresponding to 2, 4, 6, 8, and 10 lL/L of air, respectively.

Exposure periods were 24, 48, and 72 h. Data obtained in these experiments were submitted to probit

analysis. The essential oil of L. sidoides, thymol and carvacrol exhibited potent acaricidal activity against

T. urticae.

2009 Elsevier Ltd. All rights reserved.

1. Introduction

Tetranychus urticae Koch (Acari: Tetranychidae) is one of the

most important pests of greenhouse plants in the world and is

associated with 900 plant species (Jeppson et al., 1975). These

pests are commonly controlled by applications of synthetic acari-

cides (Pontes et al., 2007b). However, resistance to pesticides has

guided research to find new methods intended to control T. urticae.

Additionally, the indiscriminate use of different synthetic acari-

cides to avoid deterioration of stored products may result in seri-

ous health hazards for mammalians. The search for new

vegetable species with insecticide properties has been increasing

in the past few years due to the indiscriminate use of synthetic

pesticides for crop protection. Among bioactive natural com-

pounds, several plant essential oils (Calmasur et al., 2006;

ElGengaihi et al., 1996; Pontes et al., 2007a,b), plant extracts (Shi

et al., 2006), and microbial secondary metabolites (Villanueva

and Walgenbach, 2006) were evaluated as acaricidal. Natural prod-

ucts have been used as templates for semisynthetic acaricidal

agents (Tsukamoto et al., 1997a,b).

The family Verbenaceae comprises approximately 175 genera

and 2300 species, distributed in tropics and subtropics, mainly in

the temperate zones of the Southern Hemisphere. Species of this

genus are rich in aromatic essential oils. Lippia sidoides Cham.

(Verbenaceae) is a perennial bushy plant native of the Caatinga

known in Brazil as alecrim-pimenta. Its antimicrobial and larvicidal

properties are the results of the presence of thymol and carvacrol

in the essential oil (Botelho et al., 2007; Carvalho et al., 2003).

Plant-based pesticides appear to have no ill effect on non-target

populations and are biodegradable, in addition to being locally

available in many parts of the world. Many natural pesticides act

by interfering with the growth and reproduction of the pest and

are effective against different stages of their growth (Pope et al.,

2005; Ullrich et al., 2002).

The search for efficient natural larvicide or pesticide sub-

stances with low environmental toxicity has increased (Chant-

raine et al., 1998; Kabir et al., 2003; Silva et al., 2004). Plants

essential oils are optimal candidates, since they are, in some

cases, highly active, and economically viable. Since plant essential

oils have been found to be active against T. urticae (Calmasur

et al., 2006; Pontes et al., 2007a), the acaricidal activity of the

essential oil of L. sidoides was analyzed by measurement of their

lethal concentration that kills 50% of subjects (LC50). Additionally,

the essential oil composition was obtained and its major com-

pounds LC50 were evaluated.

0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2009.08.053

* Corresponding author. Tel.: +55 79 2105 6825; fax: +55 79 2105 6641.

E-mail address: pericles@ufs.br (P.B. Alves).

URL: http://www.ufs.br (P.B. Alves).

Bioresource Technology 101 (2010) 829832

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2. Methodology

2.1. Plant and biological materials

Four accessions of L. sidoides leaves, collected at different sites,

(LISID1, LISID2, LISID3, and LISID4) were cultivated in the Research

Farm of the Federal University of Sergipe, Department of Agronom-

ical Engineering, So Cristvo, Brazil and harvested for essentialoil extraction at the flowering stage. Plants were transplanted into

the experimental field in rows 100 cm apart with inter-row spac-

ing of 100 cm. Voucher specimens of each L. sidoides genotype were

deposited at the Federal University of Sergipe Herbarium, Biology

Department. Voucher numbers and collection data are listed in Ta-

ble 1. Prior to hydrodistillation, leaves were dried at 40 C in a

forced air oven (Marconi MA 037) for 48 h and pulverized using

a mill (Grindomix GM 200, Haan, Germany). Specimens ofT. urticae

used for the bioassays were grown in plants ofCanavalia ensiformes

L. at 27 0.5 C, relative humidity of 75 5% and 12:12 h light/dark

cycle.

2.2. Terpenes

Thymol was purchased from Merck. Carvacrol,b-caryophyllene,

and p-cymene were purchased from SigmaAldrich. All com-

pounds were directly added to blotting paper strips attached to

the inner surface of the lids.

2.3. Essential oil extraction

The dried plant powder was submitted to hydrodistillation in a

Clevenger-type apparatus consisting of a 500 mL distillation bottle,

a 5 mL graduated receiver, and a jacketed-coil condenser. A total of

100 g of dried plant material and 250 mL of H2O were used, and the

distillation was carried out for 4 h after the mixture had reached

boiling. Condensation of the steam followed by accumulation of

the essential oil/water system in the graduated receiver resultedon separation of the essential oil from the water, allowing further

manual collection of the organic phase. Traces of water were re-

moved by freezing the sample below 0 C followed by transferring

unfrozen essential oil to a new vial to yield yellowish volatile oils.

Samples were kept in freezer until further analysis. Yields were cal-

culated from weight of dried material.

2.4. Analytical conditions

The essential oils obtained by hydrodistillation were analyzed

by GC/MS on a Shimadzu QP5050A (Shimadzu Corporation, Kyoto,

Japan) system equipped with a AOC-20i autosampler under the fol-

lowing conditions: J&W Scientific DB-5MS fused silica capillary

column (30 cm 0.25 mm i.d., composed of 5%-phenyl95%-meth-ylpolysiloxane) operating in electron impact mode at 70 eV. He-

lium (99.999%) as the carrier gas at a constant flow of 1.2 mL/

min. The injection volume was 0.5lL (split ratio of 1:100) and

the injector temperature was 250 C and the ion-source tempera-

ture was 280 C. The oven temperature was programmed from

50 C (isothermal for 2 min), with an increase of 4 C/min to

200 C, then 10 C/min to 300 C, ending with a 10 min isothermal

at 300 C. Mass spectra were taken at 70 eV with a scan interval of

0.5 s and fragments from 40 to 550 Da. Quantitative analysis of the

chemical constituents was performed by flame ionization gas chro-matography (FID), using a Shimadzu GC-17A (Shimadzu Corpora-

tion, Kyoto, Japan) instrument, under the following operational

conditions: capillary ZB-5MS column (5%-phenyl-arylene95%-

dimethylpolysiloxane) fused silica capillary column

(30 m 0.25 mm i.d. 0.25 lm film thickness) from Phenomenex

(Torrance, CA, USA), under same conditions as reported for the GC

MS. Quantification of each constituent was estimated by area nor-

malization (%). Compound concentrations were calculated from the

GC peak areas and they were arranged in order of GC elution.

2.5. Identification of essential oil constituents

Identification of individual components of the essential oil was

performed by computerized matching of the acquired mass spectrawith those stored in NIST21 and NIST107 mass spectral library of

the GC/MS data system. Retention indices (RI) for all compounds

were determined according to Vandendool and Kratz (1963) for

each constituent as previously described (Adams, 2007).

2.6. Fumigant assay

The method to evaluate the activity of the oils or terpenes was

adapted from Aslan et al. (2004). Tests were performed at 25 2 C,

relative humidity of 70 8%, and 12:12 h light/dark cycle. Glass

receptacles with a capacity of 2.5 L were used as test chambers.

For each dose and exposure time combination, three replicates

were used. Each replicate consisted of 30 adult females ofT. urticae,

Table 1

Collection data of four accessions of L. sidoides.

Accession Collection site Geographical

coordinates

Date Voucher

LISID1 Mossor county/Rio

Grande do Norte

5 070 26,700 S; 37

240 14,600 W

10/22/

2006

8219

LISID2 Mossor county/Rio

Grande do Norte

5 080 28,300 S; 37

230 58,000 W

10/22/

2006

8220

LISID3 Limoeiro do Norte

county/Cear

5 090 47,800 S; 38

060 31,000 W

10/22/

2006

8222

LISID4 Poo Redondo county/

Sergipe

9 580 09,200 S; 37

510 50,300 W

12/08/

2006

8226

Table 2

Essential oil composition from accessions of L. sidoides characterized by GC/MS.

RI Compound LISID1 (%) LISID2 (%) LISID3 (%) LISID4 (%)

931 Tricyclene 0.67 0.65 0.65 1.59

938 a-Pinene 0.38 0.43 0.40 0.42984 b-Pinene 0.10 0.00 0.13 0.00

986 3-Octanone 0.12 0.00 0.14 0.00

996 Myrcene 1.57 1.63 1.40 3.68

1013 a-Phelandrene 0.00 0.00 0.00 0.171015 d-(3)-Carene 0.00 0.00 0.07 0.10

1023 a-Terpinene 1.01 1.15 1.05 2.791031 p-Cymene 10.02 10.56 8.36 15.06

1036 Limonene 0.44 0.43 0.45 0.19

1039 1,8-Cineole 0.54 0.55 0.54 0.00

1043 b-(Z)-Ocimene 0.08 0.00 0.10 0.28

1053 b-(E)-Ocimene 0.00 0.00 0.07 0.08

1064 c-Terpinene 2.34 2.61 2.69 15.491092 Terpinolene 0.00 0.00 0.00 0.06

1149 Ipsdienol 0.82 0.26 0.63 0.00

1188 Terpin-4-ol 0.99 1.07 0.83 0.81

1237 Methyl thymol 0.66 0.58 0.59 4.40

1246 Methyl carvacrol 0.00 0.00 0.00 0.16

1290 Thymol 68.81 68.40 70.36 7.25

1307 Carvacrol 0.34 0.40 0.30 46.09

1429 b-Caryophyllene 8.13 8.81 8.01 0.19

1448 b-(Z)-Farnesene 0.50 0.50 0.61 0.00

1501 a-Selinene 0.40 0.43 0.52 0.00Monoterpenes 88.89 88.72 88.76 98.62

Sesquiterpenes 9.03 9.74 9.14 0.19

Total 97.92 98.46 97.90 98.81

RI: Relative retention index calculated against n-alkanes applying the Vandendool

equation.%: Compound percentage.

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10 mites in each leaf disk (2.5 cm diameter) ofC. ensiformis, within

a Petri dish (9 cm). To maintain the leaf turgor and avoid the exit of

mites, filter paper disks saturated with water were used under the

leaves. Each Petri dish was brought into the glass recipient. The

amounts of essential oil or terpene applied on a blotting paper strip

(5 2 cm), fixed on the inner surface of the glass recipient cover,

by an automatic pipette were 5, 10, 15, 20, and 25 lL in each test

chamber, corresponding to 2, 4, 6, 8, and 10 lL/L of air, respec-tively. Since thymol is solid, the used volumes were converted to

mass and weighted in an analytical balance. No material was ap-

plied to the control glass recipient. Exposure periods were 24, 48,

and 72 h. Evaluation to determine mortalities, in each exposure

time, was made with a slight touch on the mite with a fine haired

brush. If they were black and did not move, they were considered

as dead. Data obtained in these experiments were submitted to

probit analysis (Finney and Stevens, 1948).

2.7. Statistical analyses

These results (n = 3) were further analyzed using oneway ANO-

VA analysis of variance, followed by Duncan test. A significance le-

vel of 5% was set for all analyses. It is important to note that each nconsists of 30 T. urticae individuals, representative in a composite

homogeneous sample.

3. Results and discussion

The essential oils ofL. sidoides accessions LISID1, LISID2, LISID3,

and LISID4 were obtained in 5.35%, 4.86%, 5.29%, and 8.00% w/v

yield, respectively. Twenty-four compounds, representing 97.90

98.81% of the essential oils were identified; their retention indices

and percentage composition, listed in order of elution in the ZB-

5MS column, are given in Table 2. In addition, a representative

example of LISID2 GC chromatogram with peak assignments is pre-

sented in Fig. 1.

The major components in L. sidoides essential oil were identifiedas thymol (7.2570.36%), carvacrol (0.3046.09%), p-cymene

(8.3615.06%), and b-caryophyllene (0.198.81%), which ac-

counted for 88.7298.62% of monoterpenes and 0.199.74% of ses-

quiterpenes. The essential oil composition L. sidoides herein

evaluated was similar to the one characterized by Botelho et al.

(2007), with thymol and carvacrol as major compounds. In search-

ing for new forms to control T. urticae propagation, the essential

oils ofL. sidoides accessions were tested and exhibited acaricidal ef-

fects compared to other plant essential oils (Calmasur et al., 2006;

ElGengaihi et al., 1996). At higher essential oil concentrations, the

T. urticae showed restless movement for some time and died. The

rate of mortality was directly proportional to concentration.

Results on mortality of T. urticae are shown in Table 3 along

with their confidence limits. The volatile constituents of L. sido-ides leaves exhibited potent acaricidal activity. No significant dif-

ferences were observed in the acaricidal activity of L. sidoides

accessions (p > 0.05). Selected compounds previously mentioned

were evaluated for their acaricidal potential (Table 3). Major com-

pounds in the essential oil of L. sidoides, thymol and carvacrol,

exhibited LC50 of 0.001 and 0.036 lL/L of air, respectively. Addi-

tionally, no significant differences were observed in the acaricidalactivities of L. sidoides accessions, thymol, carvacrol, and b-caryo-

phyllene. However, p-cymene exhibited significantly weaker aca-

ricidal potency (LC50 3.020 ppm, p < 0.05) and may not be the

principle responsible for the observed larvicidal actions. Conse-

quently, the evaluated components are probably acting synergis-

tically to achieve the acaricidal action observed. However,

synergistical action of other minor constituents cannot be fully

disregarded. However, ElGengaihi et al. (1996) found that the

essential oil of thyme was toxic against T. urticae. The predomi-

nance of thymol in thyme essential oil was believed to be deter-

minant for its activity.

4. Conclusions

Tetranychus urticae is susceptible to the composition of the

essential oil herein evaluated. The use of natural products may

be considered an important alternative acaricide to control T. urti-

cae, since they constitute a rich source of bioactive compounds

that are biodegradable, non-toxic, and potentially suitable for

use as pesticide. However, the cost of the essential oil may also

be an important factor for its implementation, which depends

on the availability of the plant and its yield/ha. Such studies are

currently being conducted by our research group. In accordance

with the present conclusions, L. sidoides essential oil may be used

as an ecologically safe alternative pesticide against T. urticae. The

evaluated monoterpenes thymol, carvacrol, and b-caryophyllene,

major compounds in the essential oil of L. sidoides, were found

to be acting synergistically to achieve the observed acaricidalaction.

Fig. 1. GC chromatogram of LISID1 with peak assignments.

Table 3

Acaricidal activities (LC50) of the essential oil of L. sidoides accessions, and their main

constituents.

Accession/terpene LC50 (CL)

LISID1 0.011 (0.0050.017)

LISID2 0.010 (0.0050.016)

LISID3 0.011 (0.0050.019)

LISID4 0.014 (0.0020.044)

Thymola 0.001 (0.0010.002)

p-Cymenea,* 3.020 (2.5703.580)

b-Caryophyllenea 0.080 (0.0520.120)

Carvacrola 0.036 (0.0290.044)

LC50 = lethal concentration (lL/L) at which 50% of the T. urticae showed mortality;

CL = confidence limits at 95% probability.a Commercial product.

* p < 0.05 (ANOVA, followed by Duncan).

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Acknowledgements

The authors wish to acknowledge CNPq (ID: 620212/2006/3),

PIBIC/CNPq and FAPITEC for supporting funds.

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