toxicity of two essential oils from eucalyptus globulus origanum

Int. J. Med. Arom. Plants, ISSN 2249 – 4340 RESEARCH ARTICLE

Vol. 2, No. 2, pp. 240-245, June 2012

*Corresponding author: (E-mail) ascientific <A.T.> aec.org.sy http://www.openaccessscience.com ©2012 Open Access Science Research Publisher [email protected]

Toxicity of two essential oils from Eucalyptus globulus Labail and

Origanum syriacum L. on Larvae of Khapra beetle

Ghaleb TAYOUB*, Amer ABU ALNASER, Iyad GHANEM

Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria, Damascus, PO

Box 6091, Syria

*Corresponding Author, Phone: 00963-11-2132580. Fax: 00963-11-6112289

Article History: Received 4th April 2012, Revised 22nd April 2012, Accepted 23rd April 2012.

Abstract: The fumigants activity of essential oil vapours distilled from Eucalyptus globulus Labail (Myrtaceae) and Origanum syriacum L. (Lamiaceae) were tested against larvae which is the infective stage of stored product insect Trogoderma granarium Everts. The larvae were exposed to essential oil vapours of eucalyptus and oregano resulted in more than 95% mortality at concentrations 312 µl/l air and 187.5 µl/l air, respectively. The LC50 values of Trogoderma

granarium Everts larvae were 28.75 and 176.3 µl/l air for oregano and eucalyptus oils, respectively. This study proved that the essential oils from these two plants are effective against Trogoderma granarium Everts larvae.

Keywords: Trogoderma granarium; Essential oil; Eucalyptus; Oreganum; Toxicity.

Introduction

Khapra beetle (Trogoderma granarium Everts) is an important and economic storage pest. It attacks grains in stores causing severe damage and loss storage. Larvae, a voracious feeder and cause of damage, are able to avoid adverse conditions by entering into diapause. Also, the insect has become tolerant to conven-tional pesticides including fumigants such as phosphine and methyl bromide (Banks 1977; Bell et al. 1983). The pest is common in geo-graphical areas characterized by high tempera-ture and low humidity, an average temperature of 20 °C for no less than four months and rela-tive humidity (RH) <50% during this period Howe and Lindgren (1957). It is present in Syria [personal communication] and a serious out-break is always a possibility because of the suit-ability of the prevailing climatic conditions in the area.

Insect infestation of food commodities is an important quality control problem of concern for food industries. In industrialized countries like Canada and Australia, there is zero tolerance for insects in food grains (White 1995; Pheloung

and Macbeth 2002; Rajendran and Sriranjini 2008).

Fumigation is an essential tool for control of insect pests in stored products. Currently, phos-phine and methyl bromide are the two common fumigants used for stored product protection. Insect resistance to phosphine is a global issue now, and control failures have been reported in some countries. Methyl bromide has been de-clared an ozone depleting substance and is being phased out completely (Tayoub et al. 2012).

More recent research showed that essential oils and their constituents may have potential as alternative compounds to currently used fumi-gants (Lee et al., 2001). The aromatic plant Origanum syriacum L. also known as Mayodan

syria L. Rafin belongs to this family (Mouterde 1983; Post 1932). Oregano essential oil has been tested on several stored product insect. For ex-ample the LC50 for oregano essential oil was 4.3 µl/l air, 8.4 µl/ l air, 15.4 µl/ l air and > 15 µl/ l air for Oryzaephilus surinamensis, Rizhopus

dominica, Sitophilus oryzae, and Tribolium

castanum, respectively. The genus Eucalyptus (Myrtaceae) is native to Australia and is mainly cultured for paper, pharmaceutical and cosmet-

241 Int. J. Med. Arom. Plants Toxicity of two essential oils on Larvae of Khapra beetle

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ics industry (Hasegawa et al. 2008).Eucalyptus

globulus essential oil was tested as a fumigant to find out its toxicity on eggs of Acanthoscelide

ohtectus (Say) (Papachristos and Stamopoulos 2004). Also, the ovicidal activity of the essential oils of Eucalyptus camoldulensis and Origanum

syriacum were tested on eggs of the confused flour beetle (Tribolium confusum) and the Medi-terranean flour moth (Ephestia kuehniella) (Tunç et al. 2000). Lee et al. (2004) reported toxicity of 42 essential oils extracted from Myrtaceae was tested against S. oryzae, T.

castaneum. Kambouzia et al. (2009) also report-ed toxicities of essential oils as fumigants from Iranian Myrtaceae species (Eucalyptus

leucoxylon) against three major stored insects: C. maculates, S. oryzae and T. castaneum. A recent study by Tayoub et al. (2012) reported the fumigant toxicity effect of essential oil ex-tracted from leaves M. communis on the khapra beetle. Neither of these two essential oils was tested on larvae of the khapra beetle. Therefore, the purpose of this study is to assess the larvicidal effect of these two essential oils on the destructive instars of T. granarium, i.e. the larvae.

Materials and methods

Insects

A culture of T. granarium insects was reared in the lab in 3 liter glass jars covered with a piece of muslin and placed in an incubator in continuous darkness at 37±1 0C. Larvae were isolated using a sieve that allowed their separa-tion from wheat grains.

Plant materials

The aerial parts of Origanum syriacum L

and leaves of Eucalyptus globulus Labail were harvested at the flowering stage from Lattakia (Syria) in May-June 2009. Collection was made from three individual plants growing wild. On each individual leaves were collected. Voucher specimens have been deposited in the laboratory of the plant biotechnology department at the Atomic Energy Commission of Syria (AECS).

Essential oil extraction

Samples were initially air-dried for 6 days at room temperature until they were crisp, and then powdered. Oil samples were obtained by hydrodistillation for 3h, using a Clevenger-type apparatus (Clevenger 1928). Oil yields (1% for O. syriacum, 1.5% for E. globulus w/ w ) were then estimated on the basis of the dry weight of the plant material. Hydro-distillated mass was about 100 g dry weight (Tayoub et al. 2006).

Fumigation bioassay

Fumigation bioassays were conducted by placing insect instars inside glass Petri dishes (9 cm diameter) with wheat provided as source of feed when needed. Petri dishes containing the insects were placed inside other larger glass Pe-tri dishes (11 cm diameter). The essential oil droplets were deposited on the inner surface of the larger Petri dish. The whole system was sealed by parafilm. The volume of the large Pe-tri dish was 160 cm3 air (Tayoub et al. 2012).

Fumigation of larvae

In case of E. globulus, Khapra larvae were divided into 6 groups; each group consisted of 10 larvae, five groups treated with 10, 20, 30, 40, and 50 µl of essential oils/160 cm3 air, and one group as control. While O. syriacum, the Khapra larvae were divided into 8 groups; each group consisted of 10 larvae, seven groups treated with 0.5, 1, 2.5, 5, 10, 20 and 30 µl/160 cm3 air, and one group as control. All treated and control larvae were incubated at 37 ±1 ºC for 48h before the number of dead larvae was counted. Each treatment was replicated ten times.

Treatment of Khapra larvae with O. syriacum

essential oil in the presence of wheat grains

A chromatography glass tube, 56 x 2.5 cm, was filled with clean wheat grains. Wheat grains were infested with second and third instar larvae (30). Larvae were left for 3h to infiltrate within the grains. The whole preparation was covered with aluminum foil to prevent light. O. syriacum essential oil was pumped into the grain filled



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glass tube using a vacuum pump, then the tube was sealed. Three concentrations of essential oil, 100, 200 and 300 µ/250 cm3 (column vol-ume) were tested, and three replicates were used for each concentration. Exposure period was 58h, after which number of dead larvae was scored by emptying wheat grains from the tube and the total number of larvae was accounted. Mortality data were corrected for natural mortal-ity in controls and were subjected to probit analysis to estimate LC50, LC90 and slopes were generated (Finney 1971).

Results

Vapors of essential oils of O. syriacum and E. globulus showed variable toxicity to larvae of T. granarium (Figure 1). For example: The va-pour dose needed to achieve near 100% death following 48h exposure time was 30 and 50 µl/160 cm3 air for O. syriacum and E. globulus, respectively (Figure 1) which equals to 187.5 and 375 µl/l air, respectively. In the present ex-periment a 24h-exposure period was used as a preliminary indication of toxicity, it is evident from Figure1 that oregano (O. syriacum) showed a higher toxicity to khapra larvae than E. globulus since at 30 µl/160 cm3 air, the % mortality by oregano vapour was around 60%, compared to 19 % mortality caused by the same concentration of E. globulus vapour. The above results were reflected in the LC50 values repre-senting toxicity levels of the two essential oils. Table 1 shows the LC50 values for O. syriacum and E. globulus as calculated by probit analysis. LC50 for O. syriacum was 28.75 µl/l air com-pared to LC50 of 176.3 µl/l air for E. globulus at 48h exposure period (Table 1). So LC50 for O.

syriacum was 6 times lower, i.e. more toxic,

than that of E. globulus at 48h exposure period. However, at 24h xposure period LC50 for O.

syriacum was only 2.3 times lower than that of E. globulus (Table 1). Indicating that full toxici-ty effect of O. syriacum vapour is time depend-ent, and that to achieve a full toxicity effect time of exposure is an important factor.

30 20

10 5

2.5

1

0.5

Con

tro

0123456789

10

Mort

alit

y (

%)

24h

48h

O.syriacum

Dose (µl/160 cm3 air )

50

40

30

20

10C

onte

rol

0123456789

10

Mort

alit

y (

%)

24h

48h

E. globulus

Dose (µl/160 cm3 air)

Figure 1: Percent mortality in the larvae of T.

granarium exposed to four essential oils at dif-ferent concentrations and exposure times.

Table 1: Fumigant Toxicity of two essential oils against T. granarium Everts larvae.

Chi

square

χ2

d.f. F 05 Fcal Slope±SE

LC90

µL/l air

LC50

µL/l air

Exposure

time

Essential oil

11.07 6 6.6 167.2 1.8 ± 0.12 147.5 28.75 48h O. syriacum

11.07 6 6.6 92.8 1.6 ± 0.13 662.7 103.6 24h 7.8 4 10.1 17.1 5..3± 1.3 307.7 176.3 48h E. globulus

7.8 4 10.1 27.8 5.3 ± 1.0 405 233 24h Units LC50 and LC90 =µl/l air, applied for 24 and 48h at 37 ºC. d.f = Degrees of freedom.



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Results obtained from treatment of Khapra larvae infesting wheat grain in a glass tube with O. syriacum essential oil show that the vapour of the oil killed larvae in a dose dependent man-ner. At concentration of 100, 200 and 300µ/ 250 cm3, the average number of killed larvae was 13.7, 12.7 and 29.7 larvae, respectively. This is equivalent to 45.56, 75.56 and 98.89% respec-tively.

The concentrations used to achieve this level of effect were 10 times the concentrations used when larvae were treated in the absence of wheat grain. This increase in concentrations can be explained by the loss of essential oil on wheat grain surfaces. And possibly by larvae hiding within the grains.

Discussion

The essential oil of plants used in cosmetics and pharmaceutical hence less harmful to hu-man and environment than conventional pesti-cides (Yeğen et al. 1998). In the search for al-ternatives to conventional fumigants, essential oils extracted from aromatic plants have been widely investigated.

Most of these studies assessed fumigants ac-tivity of these compounds on adults but lesser extent larvae, which is the infective stage in case of Khapra beetle.

In this study showed that O. syriacum is rel-atively toxic to larvae of khapra beetle. Its LC50 was 6 times higher than that of E. globulus at 48h exposure time. The oil of E. globulus, was shown to have contact toxicity and a seed pro-tection effect against Callosobruchus chinensis Linn. (pulse beetle) (Srivastava et al. 1988). Re-pellency growth inhibition and oviposition ef-fects against bean Acanthoscelides obtectus

(say) bruchids (Regnault-Roger et al. 1993). Another genus of eucalyptus (E. camaldulen) exhibited fumigant toxicity on eggs of confused flour beetle (T. confusum Duval) and Mediterra-nean flour moth (Ephestia Kuechniella Zeller) (Tunç et al. 2000).

Ovicidal fumigant toxicity of O. syriacum essential oil was tested on eggs of confused flour beetle (T. confusum) and on Mediterranean flour moth (E. Kuechniella), it was found that at

a concentration of 196.9 µl/l air and 96 h expo-sure period, percentage of mortality was 77% and 89% for T. confusum and E. Kuechniella, respectively (Tunç et al. 2000). By comparison, the present study showed that 125µl/l air (20 µl/160cm3, see graph 1) of O. syriacum essential oil resulted in 61% mortality to larvae of T.

granarium when fumigated for 24h. It is evident that the fumigant toxicity of O. syriacum essen-tial oil was stronger on T. granarium larvae.

It was suggested that for fumigants, the ac-tive stages (adults and non-diapausing larvae) of insects are more susceptible than the sedentary stages (eggs and pupae) due to difference in their respiratory rate (Rajendran and Sriranjini 2008).

A study on the essential oil constituents iso-lated from aromatic plants showed that two nat-ural terpenes termed ZP-51 and SEM-76 isolat-ed and cultivated from unidentified cultivated aromatic plants belonging to Lamiaceae family have an outstanding fumigant toxicity effect on T. granarium larvae, at 1.5 µl/l air they showed 87% and 99% mortality for SEM-76 and ZP-51, respectively (Kostyukovsky et al. 2002). O.

syriacum essential oil tested in the present study is less toxic than the above mentioned two com-pounds but still showed potent toxicity (LC50 =28 µl/l air), considering that it is a crude oil without any isolation of an active ingredient.

It is feasible that the essential oil of O.

syriacum with its monoterpenoid constituents act against insects as neurotoxins (Grundy and Still 1985; Enan 2001; Papachristos and Stamopoulos 2004). It was suggested that natu-ral terpenes isolated from essential oils could act as activators of octopaminergic receptors in lar-vae of T. granarium (Kostyukovsky et al. 2002; Shaaya and Rafaeli 2007).

The present work has demonstrated that O.

syriacum essential oil is promising as a fumi-gant against T. granarium larvae. Further stud-ies involving isolation and purification of con-stituents of that essential oil could prove useful in identifying a more potent constituent against the destructive stage of dangerous stored prod-uct pest.



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Acknowledgement: The authors would like to express their thanks and gratitude to Professor Ibrahim Othman, Director General of Atomic Energy Commission of Syria and Professor Nizar Mir Ali for support and encouragement.

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toxicity of two essential oils from eucalyptus globulus origanum

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