ijmap_2_1_29_myrtuscommunis syria plants

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

Vol. 2, No. 1, pp. 207-213, March 2012

*Corresponding author: (E-mail) ascientific <A.T.> aec.org.sy http://www.openaccessscience.com

©2012 Open Access Science Research Publisher [email protected]

Fumigant activity of leaf essential oil from Myrtus communis L. against

the 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, Fax: 00963-11-6112289, Tel: 00963-11-2132580

Article History: Received 29

th February 2012, Revised 19

th March 2012, Accepted 20

th March 2012.

Abstract: The fumigant toxicity of leaf essential oil from Myrtus communis (L.) was assessed against khapra beetle Tro-

goderma granarium Everts. The essential oil was active against different life stages of khapra beetle T. granarium. Adult

stage was the most sensitive of all developmental stages to essential oil vapours. Whereas, larvae were the most tolerant

with 94% and 100% mortality obtained after exposure of larvae to 562.5 µl/l air for 24 and 48h respectively. At 48h ex-

posure period, LC50 and LC90 were 221 µl/l air and 487 µl/l air respectively. The results suggest a possibility for using

leaf essential oil of the myrtle (M. communis) as an insecticide against Trogoderma granarium Everts in grain stores.

Keywords: Myrtus communis; Essential oil vapours; Trogoderma granarium; Fumigant toxicity.

Introduction

Khapra beetle Trogoderma granarium

Everts. (Coleoptera: Dermestidae), is the most

serious pest in stored products throughout the

world. It is a major threat to stored wheat, and

has been considered as one of the 100 most in-

vasive pests in the world (Lowe et al. 2000).

Development rates and survival of various

stages of the khapra beetle vary considerably

depending upon temperature, light, moisture,

season, and host species. Khapra beetle may

have one to nine or more generations per year as

a result of high humidity which has a depressing

effect on population buildup (Ramzan and

Chahal 1986). At favorable temperatures, eggs,

pupae, and adults each take about a week to de-

velop, while the larval stage may last for a

month and it may survive for several years un-

der diapause condition (Burges 1962). The in-

sect is present in Syria and the prevailing cli-

matic conditions in the area are conducive to

serious outbreaks (Ghanem and Shamma 2007).

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

(Rajendran and Sriranjini 2008). Insect resis-

tance to phosphine is a global issue now, and

control failures have been reported in some

countries (Tayler 1989; Collins et al. 2002; Ra-

jendran and Sriranjini 2008). Methyl bromide

has been declared an ozone depleting substance

and is being phased out completely (Rajendran

and Sriranjini 2008).

Therefore, there is a growing interest in al-

ternative strategies including development of

chemical substitutes, the use of ionizing radia-

tion and exploitation of controlled atmosphere

(MBTOC 2002; Aoki et al. 1977; Libby and

Black 1978). Interest has been shown in the po-

tential of essential oils of plants as fumigants for

the control of stored product pests since it is

believed that natural compounds from plants

sources may be less toxic to mammals (though

this is not always true, degrade rapidly and

available in abundance (Rajendran and Sriran-

jini 2008).

Myrtus communis L. (Myrtaceae), a peren-

nial shrub is widely distributed in the Mediter-

ranean area, and has been traditionally used as

an antiseptic and disinfectant drug. The leaves

contain tannins, flavonoids such as quercetin,

catechin and myricetin derivatives and volatile

oils (Baytop 1999; Romani et al. 1999). The es-

sential oil is obtained from leaves and mainly

used in the treatment of lung disorders (Gauthier

208

Int. J. Med. Arom. Plants Leaf essential oil from Myrtus communis against Khapra Beetle

Tayoub et al. http://www.openaccessscience.com

[email protected]

et al. 1989) and has been found to possess anti-

bacterial (Chevolleau et al. 1993; Hayder et al.

2003), anti candida, anti-inflammatory, antihe-

morrhagic (Ghannadi and Dezfuly 2011), anti-

louse (Lauk et al. 1996), antimutagenic and an-

tioxidant activities (Mimica-Dukia et al. 2010;

Romani et al. 2004). The myrtle essential oil

showed toxicity after 24h of fumigation of

adults Mediterranean flour moth Ephestia kueh-

niella Zeller, the Indian meal moth Plodia inter-

punctella Hübner, and the bean weevil Acan-

thoscelides obtectus Say (Ayvaz et al. 2010). A

recent study by, Zayzafoon et al. (2011) has elu-

cidated the chemical composition of the essen-

tial oil extracted from M. communis leaves in

Syria.

In the present study, we report the fumigant

toxicity effect of essential oil extracted from

leaves M. communis on the khapra beetle (T.

granarium Everts).

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

Leaves of M. communis, were harvested at

the flowering stage in May-June 2009 from

Wat-Alkhan (Lattakia - Syria). Collection was

made from three individual plants growing wild.

For each individual leaf collected. Voucher spe-

cimens have been deposited in the laboratory of

the plant biotechnology department at the Atom-

ic 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 hydro-

distillation for 3h using a Clevenger-type appa-

ratus (Clevenger 1928). Oil yields (1.68 % w/w)

were then estimated on the basis of the dry

weight of the plant material. Hydro-distillated

mass was about 100 g DW (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

Petri 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.

Fumigation of larvae

T. granarium larvae were divided into 11

groups, each group consisted of 10 larvae, ten

group treated with : 10, 20, 30, 40, 50, 60, 70,

80, 90 and 100 µl / 160 cm3, and one group as

control. All treated and control larvae were in-

cubated at 37 ±1 ºC for 5 days before the num-

ber of dead larvae was counted. Each treat-

ment was replicated ten times.

Fumigation of pupae

Newly formed pupae were collected and di-

vided into 6 different groups. Each group con-

sisted of 10 pupae, five groups were fumigated

with: 5, 10, 15, 20, and 30 µl / 160 cm3 each,

and one group was control. All treated and con-

trol pupae were incubated at 37 ±1 ºC. Mortality

was determined by adult emergence after 48 h.

each treatment was replicated ten times.

Fumigation of eggs

Newly laid eggs were collected and divided

into 7 different groups. Each group consisted of

10 eggs, six groups were fumigated with: 1, 2.5,

5, 7.5, 10, and 12.5 µl / 160 cm3 each, and one

group was control. All treated and control eggs

were incubated at 37±1 ºC, until mortality could

be determined by the presence or absence of

hatching. Each treatment was replicated ten

times.

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Fumigation of adults

1 to 5 days old adults collected and divided

into 8 groups. Each group consisted of 10

adults, 7 groups fumigated with: 1.5, 2.5, 5, 7.5,

10, 20 and 30 µl / 160 cm3 each, and one group

was control. The treated and control adults were

incubated at 37±1 ºC; mortality was recorded

after 48 h of exposure to essential oil vapour.

Each treatment run ten times in duplicate. Mor-

tality data for all treatments were corrected for

natural mortality in controls and were subjected

to probit analysis to estimate LC50 and LC90 and

slopes were generated (Finney 1971).

Results

Vapour of essential oil of M. communis

showed various levels of toxicity at the same

exposure period depending on the treated instar

of the insect.

Exposure of larvae to the highest dose of 90

µl/160 cm3 air (562.5 µl /l air) resulted in 94%

and 100% mortality at 24 h and 48 h-exposure

period, respectively. Whereas, the lowest con-

centration used, i.e. 10 µl/160cm3 air (65.5µl/l

air) had no effect after 24 h-exposure period and

resulted in only 4% mortality at 48 h-exposure

period (Figure 1). There was an evident positive

dose effect relationship for the range of doses

between the lowest and the highest dose used

(Fig1). At 48 h-exposure period, LC50 for M.

communis essential oil on larvae was 221 µl/l

air, whereas the LC90 was 487 µl/l air. However,

at 24 h, LC50 was 307.4 µl/l air and LC90 was

645.5 µl/l air (Table 1).

Table 1: Fumigant toxicity of essential oil of M. communis against T. granarium Everts larvae,

adults, eggs and pupae.

T. grana-

rium

exposure

time

LC50

µl/l air

LC90

µl/l air Slope±SE Fcal F 05 d.f χ

2

Larvae 48h 221 487.8 3.8±0.4 105.1 5.6 8 14.0

24h 307.4 645.5 4.0±0.4 94.7 5.6 8 11.0

Adults 48h 25.6 48.2 4.6±0.6 53.8 7.7 5 9.49

24h 68.4 319.9 1.9±0.2 66.3 6.6 6 11.0

Eggs 48h 38 67.1 5.2±0.6 46.1 7.7 5 9.5 24h 54.7 122.6 3.7±0.5 126.1 7.7 5 9.5

Pupae 48h 83.5 162.1 4.5±0.6 55.98 10.13 4 7.8

24h 328.6 1090.6 2.5±0.6 19.7 10.13 4 7.8

Units LC50 and LC90 =µl/l air, applied for 24 and 48h at 37 ºC. d.f = Degrees of freedom

Adults were more sensitive to the vapour of

M .communis essential oil where up to 80% and

100% of exposed adults died following expo-

sure to 30 µl/160 cm3 air for 24 h and 48 h-

exposure period, respectively. The lowest con-

centration of oil vapour (1.5 µl/160cm3 air) to

which adult khapra beetle was exposed resulted

in only 2% death at 24 and 48 h-exposure pe-

riod. Adults remained relatively insensitive to

the vapour at concentrations less than 5 µl/160

cm3 air, then after the toxic effect of the essen-

tial oil became more pronounced reaching 30%

and 74% at 24 h and 48 h-exposure period, re-

spectively. At 48 h-exposure period, LC50 for M.

communis essential oil on adults was 25.6 µl/l

air, whereas the LC90 was 48.2 µl/l air. Howev-

er, at 24 h, the LC50 was 68.4 µl/l air and the

LC90 was 319.9 µl/l air (Table 1).

In the case of pupae, exposure to 30 µl/160

cm3 air ( 187.5 µl/l air) of essential oil vapour

resulted in the death of 24% , 98% of pupae at

24 h and 48 h-exposure period, respectively.

The lowest concentration of 5 µl/160cm3 air

(31.25 µl/l air) to which the pupae were exposed

resulted in 5% and 24% mortality at 24 h and

48 h-exposure period, respectively (Fig.1). The

LC50 of essential oil on pupae was 83.54 µl/l air

and 328.6 µl/l air at 48 h and 24 h-exposure pe-

riod, respectively (Table 1).

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30 20 107.

5

5

2.5

1.5

Con

trol

0123456789

10M

ort

ality

(%

)

Adult

24h

28h

Dose ( µl/160 cm3 air )

90

80 70 60

50 40

30 20 10co

ntro

l

0123456789

10

Mort

alit

iy (

%)

Larvae

24h

48h

Dose (µl /160 cm3 air )

12.5

10

7.5

5

2.5

1

Control

0123456789

10

Mortalit

y (%

)

Eggs

24h

48h

Dose ( µl/ 160 cm3 air )

30

20

15

10

5

Control

0

1

2

3

4

5

6

7

8

9

10

Mortality ( %

)

Pupae

24h

48h

Dose (µl/160 cm3 air)

Figure 1: Percent mortality in larvae, adult,

eggs, pupae of T. granarium exposed to essen-

tial oil of M. communis at four different concen-

trations and two exposure times.

Eggs appeared highly sensitive to the toxic

effect of M. communis essential oil vapour, so at

12.5 µl/160 cm3 air. 68% and 100% of exposed

eggs died at 24 h and 48 h-exposure period re-

spectively (Fig.1), with LC50 at 48 h and 24 h

reaching 38 µl/l air and 54.7 µl/l air, respective-

ly (Table 1).

Discussion

Essential oil of the myrtle plant showed a

highly toxic effect to all developmental stages

of Khapra beetle (T. granarium). The toxic ef-

fect of the oil was most effective on adults, fol-

lowed by eggs compared with other insect stag-

es. The toxic effect is almost certainly due to

one or more of the components of the essential

oil distilled from leaves of M. communis, partic-

ularly monoterpenes that are found in the oil and

from 85.5% of the 95.4% of the total com-

pounds contained in the essential oil (Zayzafoon

et al. 2011). It is worth noting that our samples

were collected from the same region in Syria

It is feasible that the essential oil of M.

communis with its monoterpenoid constituents

act against insects as neurotoxins (Ayvaz et al.

2010; Grundy and Still 1985; Enan 2001; Pa-

pachristos and Stamopoulos 2004). It was sug-

gested that natural terpenes isolated from essen-

tial oils could act as activators of octopaminer-

gic receptors in larvae of T. granarium (Kostyu-

kovsky et al. 2002; Shaaya et al. 2007).

A study on essential oil constituents isolated

from aromatic plants showed that two natural

terpenes termed ZP-51 and SEM-76 isolated and

cultivated from unidentified cultivated aromatic

plants belonging to Labiatae family have an out-

standing fumigant toxicity effect on T. grana-

rium larvae, at 1.5 µl/l air they showed 87% and

99% mortality for SEM-76 and ZP-51, respec-

tively (Kostyukovsky et al. 2002). M. communis

essential oil tested in our present study is less

toxic than the above mentioned two compounds

but still showed potent toxicity (LC50 =25.6, 38,

83,5 and 221 µ/l air on adults, eggs, pupae and

larvae, respectively), considering that it is only a

crude oil with no purification of any of its active

ingredient was attempted.

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Other studies have shown that essential oils

and their constituents may potentially be utilized

as alternative compounds to synthetic fumigants

in current use (Shaaya et al. 1997; Regnault-

Roger et al. 1993; Shakarami et al. 2005). The

major constituents from aromatic plants, primar-

ily monoterpenes, are of particular interest to

industrial market due to their potent biological

activities, in addition to their toxicity to insects

(Isman 2000; Weinzierl 2000). Monoterpenoids

are typically volatile and rather lipophilic com-

pounds, which can rapidly penetrate into insects

and interfere with their physiological functions

(Lee et al. 2002).

Conclusion

The present study showed that the essential

oil of Myrtus communis L. may be used as a

fumigant against Khapra beetle. Also, consider-

ing that essential oils of myrtle are being used in

the pharmaceutical and cosmetic industry

(Bayotop 1999; Aidi et al. 2007; Boelens and

Jimenes 1992; Flamini et al. 2004; Tuberoso et

al. 2006), it is thus considered to be less toxic

and harmful to humans and environment than

conventional insecticides (Isman 2006). Conse-

quently, the possibility of employing these natu-

ral fumigants to control stored products insects

may warrant further investigation.

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 Ni-

zar Mir Ali for support and encouragement.

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