Doctor of Philosophy In ENTOMOLOGYprr.hec.gov.pk/jspui/bitstream/123456789/7721/1/Sohil Akhtar UAF fi… · Sohail Akhtar M.Sc. (Hons.) Agri. Entomology Regd. No. 2003-ag-1985 Thesis

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INSECTICIDAL, REPELLENT, ANTIFEEDANT AND GROWTH

REGULATORY INFLUENCES OF ESSENTIAL OIL OF INDIGENOUS

MEDICINAL PLANTS AGAINST STORED GRAIN INSECT PESTS

BY

Sohail Akhtar M.Sc. (Hons.) Agri. Entomology Regd. No. 2003-ag-1985

Thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

In

ENTOMOLOGY

DEPARTMENT OF ENTOMOLOGY

FACULTY OF AGRICULTURE

UNIVERSITY OF AGRICULTURE, FAISALABAD

PAKISTAN

2015

2

The Controller of Examinations, University of Agriculture, Faisalabad.

We, the Supervisory Committee, certify that the contents and form of thesis submitted by

Mr. Sohail Akhtar, Registration No. 2003-ag-1985 have been found satisfactory and recommend

that it be processed for evaluation, by the External Examiner(s) for the award of degree.

Supervisory Committee:

1. Chairman:

(Prof. Dr. Mansoor-ul-Hasan)

2. Member:

(Dr. Muhammad Sagheer)

3. Member:

(Prof. Dr. Nazir Javed)

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This thesis is dedicated with love and respect to my

mother, Father and my family

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ACKNOWLEDGEMENTS

First of all I would like to bow my head before “ALMIGHTY ALLAH” the Omnipotent, the Omnipresent, the Merciful, the Beneficial who presented me in a Muslim community and also bestowed and blessed me with such a lucid intelligence as I could endeavor my services toward this manuscript. Countless salutations are upon the HOLY PROPHET MUHAMMAD (May Peace Be Upon Him), the fountains of knowledge, who has guided his “Ummah” to seek knowledge for cradle to grave. The work presented in this manuscript was accomplished under the sympathetic attitude, animate directions, observant pursuit, scholarly criticism, cheering perspective and enlightened supervision of Dr. Mansoor-ul-Hasan, Professor, Department of Entomology, University of Agriculture, Faisalabad. His thoughtful guidance helped me in all the time of research, writing of dissertation/publications etc. and his rigorous critique improved my overall understanding of the subject. I am grateful to his ever inspiring guidance, keen interest, scholarly comments and constructive suggestions throughout my Ph.D. studies. I wish to acknowledge my deep sense of profound gratitude to the worthy member of my research supervisory panel Dr. Muhammad Sagheer, Lecturer, Department of Entomology, Dr. Nazir Javed, Professor, Department of Plant Pathology, U.A.F., moreover I would like to include Dr. Mazhar Rangha, Assistant Professor, Department of Entomology, U.A.F. for their constructive criticism, illuminating and inspiring guidance and continuous encouragement throughout course of my Research. I really have no words to express my sincere thankful feelings and emotions for all my seniors and friends especially Dr. Muhammad Rafay, Dr. Ch.M. Shahid Hanif, Dr. Shahazad Saleem, Hafiz Salman Saleem, Ali bin Suhail, Capt. Muhammad ibraheem, Irfan Ali Muhammad Aslam Farooqi, Qurban Ali, Kazim Ali, Imran Faraz, Rana Mubashir and Rauf Shah for their cooperation, well wishes and moral support from time to time during the course of study. Words are lacking to express my humble obligation to my affectionate Father, Mother, Brothers Javed Akhtar Malik, Dr. Imran Ali, Sikandar Zaman Khan and Malik Rashid and Sisters for their love, good wishes, inspirations and unceasing prayers for me, without which the present destination would have been mere a dream. I would like to express my deepest gratitude to the Higher Education Commission, Islamabad (Pakistan) for the scholarship under Ph.D. Indigenous Fellowship Program is greatly acknowledged.

Sohail Akhtar

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENT……………………………………………………….. TABLE OF

CONTENTS………………………………………………………… LIST OF

TABLES………………………………………………………………..

LIST OF FIGURES………………………………………………………………

Abstract……………………………………………………………..

Chapter 1. Introduction……….………………………………………….. 1

1.1 Post harvest losses due to infestation of stored grain insect pests

during storage………………………………………………… ... 1

1.2 Major stored grain insect pests………………………….............. 1

1.2.1 Tribolium castaneum (Herbst) (Coleoptera:

Tenebrionidae)………………………………...…. 1

1.2.2 Rhyzopertha dominica (Fabr.) (Coleoptera:

Bostrichidae)…………………………....... 2

1.2.3 Trogoderma granarium (Everts) (Coleoptera:

Dermestidae)………………………………… 3

1.3 Conventional chemicals used to control stored grain insect pests…. 4

1.4 Plant derived insecticides: An alternative of conventional chemicals..4

Chapter 2. Review of literature….………………………………………….. 7

6

1.1 Repellency of essential oils of indigenous medicinal plants ….. 7

1.2 Contact toxicity of essential oils of indigenous medicinal plants 13

1.3 Fumigant toxicity of essential oils of indigenous medicinal plants 19

1.4 Anti-feedant effect of essential oils of indigenous medicinal plants 28

1.5 Growth regulatory influences of essential oil of indigenous

Medicinal plants …………………………………………………. 31

Chapter 3. Materials and methods

3.2.1 Plant Materials………………………………………………….. 35

3.2.2 Extraction………………………………………………………. 35

3.2.3 Test Insects……………………………………………………... 35

Chapter 4. Repellency of essential oil of five indigenous medicinal plants

against three stored grain insect pests.

Abstract…………………………………………………………………. 37

4.1 Introduction……………………………………………………………… 37

4.2 Materials and Methods………………………………………………….. 39

4.2.1 Bioassay………………………………………………………... 39

4.2.2 Statistical Analysis…………………………………………….. 40

4.3 Results…………………………………………………………………. 41

4.4 Discussion……………………………………………………………... 43

Chapter 5. Contact toxicity of essential oil of five indigenous medicinal

plants against three stored grain insect pests.

7

Abstract………………………………………………………………………. 50 5.1

Introduction……………………………………………………………… 51


5.2.1 Bioassay………………………………………………………... 52

5.2.2 Post treatment buildup of population………………………….. 52


5.3 Results…………………………………………………………………. 54

5.4 Discussion……………………………………………………………... 56

Chapter 6. Fumigant toxicity of essential oil of five indigenous medicinal

plants against three stored grain insect pests.

Abstract……………………………………………………………………… 68 6.1

Introduction……………………………………………………………… 69


6.2.1 Bioassay………………………………………………………... 70


6.3 Results…………………………………………………………………. 71

6.4 Discussion……………………………………………………………... 72

Chapter 7. Antifeedant effact of essential oil of five indigenous medicinal

plants against three stored grain insect pests

Abstract…………………………………………………………………….. 79 7.1

Introduction……………………………………………………………… 81


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7.2.1 Bioassay………………………………………………………... 81


7.3 Results…………………………………………………………………. 83

7.4 Discussion……………………………………………………………... 86

Chapter 8. Insect growth regulatory activities of essential oil of five indigenous

medicinal plants against three stored grain insect pests.

Abstract…………………………………………………………………. 98

8.1 Introduction……………………………………………………………… 99


8.2.1 Bioassay………………………………………………………... 101


8.3 Results…………………………………………………………………. 102

8.3.1 Larval emergence …………….……………..………………… 102

8.3.2 Pupae formation …………..…………………………….……. 103

8.3.3 Adult emergence ……………………..………………………. 103

8.4 Discussion…………………………………………………………….. 104 List of

Tables

Sr. No. Title Page

No.

Table

4.1

Percentage repellency (Mean ± SE) of T. castaneum (F(8,90) = 84.4, p

= 0.00003), T. granarium (F(8,90) = 54.3, p = 0.010059) and R.

dominica (F(8,90) = 6.21, p = 0.00002) against different exposure

times of essential oils.

47

9

Table

4.2


= 0.000983) and R. dominica (F(8,90) = 41.3, p = 0.039443) against

different concentrations of essential oils.

48

Table

4.3


= 0.015339) and R. dominica (F(4, 90) = 77.90, p = 0.004799) against

different concentrations and exposure times of essential oils.

49

Table.

5.1

Percentage contact mortality (Mean ± SE) of T. castaneum (F(8,90) =

15.98, p = 0.000), T. granarium (F(90, 8) = 3.36, p = 0.00562) and R.

dominica (F(8,90) = 8.70, p = 0.000) against of essential oils

indigenous medicinal plants.

64

Table

5.2

Percentage contact mortality (Mean ± SE) of T. castaneum (F(8, 90) =

4.35, p = 0.0182) and T. granarium (F(8,90) = 3.36, p = 0.00208)

against different concentrations and exposure times of essential oils.

65

Table

5.3

Percentage contact toxicity (Mean ± SE) of T. granarium (F(4, 90) =

3.20, p = 0.0165) against different concentrations and exposure times

of essential oils.

66

Table

5.4

Percentage reduction in buildup of population (Mean ± SE) of T.

castaneum (F(8, 90) = 4.35, p = 0.0182) and T. granarium (F(8,90) =

3.36, p = 0.00208) against different concentrations and exposure

times of essential oils.

67

Table

6.1

Percentage fumigant mortality (Mean ± SE) of T. castaneum (F(8,90)

= 7.84, p = 0.000), T. granarium (F(8, 90) = 1.80), p = 0.00562) and R.

dominica (F(8, 90) = 8.70, p = 0.0000) against exposure times of

essential oils

77

Table

6.2

Percentage fumigant mortality (Mean ± SE) of T. castaneum (F(8, 90)

= 9.59, p = 0.000) and T. granarium (F(8, 90) = 9.83, p = 0.000) against

different concentrations of essential oils.

78

Table. Percentage weight loss (Mean ± SE) of T. castaneum (F(8,90) = 7.84,

7.1 p = 0.000), T. granarium (F(8, 90) = 1.80), p = 0.00562) and R.

dominica (F(8, 90) = 8.70, p = 0.0000) against exposure times of essential oils. 92

Table. Percentage Feeding detterance index (Mean ± SE) of T. castaneum 7.2 (F(8,90) =

7.84, p = 0.000), T. granarium (F(8, 90) = 1.80), p = 0.00562) and R. dominica (F(8, 90) =

8.70, p = 0.0000) against

10

exposure times of essential oils. 93

Table Percentage weight loss (Mean ± SE) of T. castaneum (F(8,90) = 7.84,

7.3 p = 0.000), T. granarium (F(8, 90) = 1.80), p = 0.00562) and R.

dominica (F(8, 90) = 8.70, p = 0.0000) against concentrations of essential oils. 94

Table Percentage feeding detterance index (Mean ± SE) of T. castaneum 7.4 (F(8,90) =

7.84, p = 0.000), T. granarium (F(8, 90) = 1.80), p = 0.00562) and R. dominica (F(8, 90) =

8.70, p = 0.0000) against

different concentrations of essential oils. 95

Table Percentage weight loss (Mean ± SE) of T. castaneum (F(8,90) =

7.5 7.84, p = 0.000), T. granarium (F(8, 90) = 1.80), p = 0.00562) and R. dominica (F(8, 90) =

8.70, p = 0.0000) against concentrations

and exposure times of essential oils. 96

Table Percentage feeding detterance index (Mean ± SE) of T. castaneum

7.6 (F(8,90) = 7.84, p = 0.000), T. granarium (F(8, 90) = 1.80), p = 0.00562) and R. dominica

(F(8, 90) = 8.70, p = 0.0000) against different concentrations and exposure times of

essential oils. 97

Table Percentage larvae survived (Mean ± SE) of T. castaneum (F(12, 40)

8.1 = 12.21, p = 0.000), T. granarium (F(12, 40) = 14.35), p = 0.000)

and R. dominica (F(12, 40) = 7.05, p = 0.0000) against different concentrations of

essential oils 109

Table Percentage pupal transformation (Mean ± SE) of T. castaneum

8.2 (F(12, 40) = 10.92, p = 0.000), T. granarium (F(12, 40) = 14.45), p = 0.000) and R. dominica

(F(12, 40) = 12.94, p = 0.0000) against different concentrations of essential oils.

112

Table Percentag adult emergence (Mean ± SE) of T. castaneum (F(12, 40)

8.3 = 11.01, p = 0.000), T. granarium (F(12, 40) = 7.72), p = 0.000)

and R. dominica (F(12, 40) = 14.63, p = 0.0000) against different

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concentrations of essential oils. 115

List of Figures

Sr. No.

Fig. 4.1 Mean repellency (%) of essential oils of M. azadirachta, A. indica,

E. camaldulensis, N. tabacum and C. citrullus against T. castaneum,

T. granarium and R. dominica

45



T. granarium and R. dominica at different exposure times

46



T. granarium and R. dominica at different concentrations

46

Fig. 5.1 Mean contact mortality (%) of T.castaneum, T. granarium and R.

dominic against essential oils of M. azadirachta, A. indica, E.

camaldulensis, N. tabacum and C. citrullus

59

Fig. 5.2 Mean contact mortality (%) of T. castaneum, T. granarium and R.

dominica at different exposure times of essential oils of M.

azadirachta, A. indica, E. camaldulensis, N. tabacum and C. citrullus

60

Fig. 5.3 Mean contact mortality (%) of T. castaneum, T. granarium and R.

dominica at different concentrations of essential oils of M.


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Fig. 5.4 Mean reduction in population bulidup (%) of T.castaneum, T.

granarium and R. dominica against essential oils of M. azedarach,

A. indica, E. camaldulensis, N. tabacum and C. citrullus

62

Fig. 5.5 Mean reduction in population bulidup (%) of T.castaneum, T.

granarium and R. dominica against essential oils of M. azedarach, A.

indica, E. camaldulensis, N. tabacum and C. citrullus at different

concentrations

63

Title Page

No.

12

Fig. 6.1 Mean fumigant mortality (%) of T.castaneum, T. granarium and R.

dominic against essential oils of M. azadirachta, A. indica, E.


75


dominic at different exposure times of essential oils of M.


76


dominic at different concentrations of essential oils of M.


76

Fig. 7.1 Mean weight loss (%) of food commodities due to T. castaneum, T.

granarium and R. dominica essential oils of M. azadirachta, A.

indica, E. camaldulensis, N. tabacum and C. citrullus

88




exposure times

89




concentrations

89

Fig. 7.4 Mean feeding detterance index (%) of T.castaneum, T. granarium

and R. dominica essential oils of M. azadirachta, A. indica, E.


90


and R. dominic at different concentrations of essential oils of M.


91


and R. dominic at different exposure times of essential oils of M.


91

Fig. 8.1 Mean Larvae survived (%) in T. castaneum, T. granarium and R.

dominica against the essential oils of M. azadirachta, A. indica, E.


107

Fig. 8.2 Mean Larvae survived (%) in T. castaneum, T. granarium and R.


camaldulensis, N. tabacum and C. citrullus at different

concentrations

108

13

Fig. 8.3 Mean Pupae transformed (%) in T. castaneum, T. granarium and R.



110

Fig. 8.4 Mean Pupae transformed (%) in T. castaneum, T. granarium and R.



concentrations

111

Fig. 8.5 Mean adult emergance (%) in T. castaneum, T. granarium and R.



113

Fig. 8.6

Mean adult emargance (%) in T. castaneum, T. granarium and R.



concentrations

114

DECLARATION

I hereby declare that the contents of the thesis titled “Insecticidal, repellent, antifeedant and growth

regulatory influences of essential oil of indigenous medicinal plants against stored grain insect

pests” are product of my own research and no part has been copied from any published source

(except references, standard mathematical or genetic models /equations /formulate / protocols etc.).

I further declare that this work has not been submitted for the award of any other diploma/degree.

The University may take action if the information provided us found inaccurate at any stage, (in

case of any default the scholar will be proceeded against per HEC plagiarism policy).

SOHAIL AKHTAR

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Abstract

The present research work was conducted to find a noval, biodegradable, safe and environment

friendly replacement of conventional synthetic insecticide to control the insect pests in stored

commodities. The repellent, contact, fumigant, antifeedant and growth regulatory activities of

essential oils of Azadirachta indica, Melia azedarach, Colocynthis citrullus, Nicotiana tabacum

and Eucalyptus camaldulensis were checked against Tribolium castaneum, Rhyzopertha dominica

and Trogoderma granarium. Three concentrations of each essential oil (2, 4 and 6%) were tested

in each experiment. Findings of this research work revealed that for repellant bioactivities A. indica

was found most potent against T. castaneum and R. dominica while M. azidiractha was most active

against T. granarium. Overall effect of the concentration was highly significant and the maximum

repellency was obtained at 6% concentration followed by 4% and 2%. Results also showed that as

the duration of exposure increased repellency decreased. For contact and fumigant toxicities results

revealed that A. indica was found most toxic against T. castaneum and R. dominica while N.

tabacum was most effective against T. granarium. Results also showed that fumigant and contact

mortalities were maximum at highest level of concentration and exposure period. In case of

antifeedant and insect growth regulatory activities the most potent essential oil was found A. indica

followed by M. azedarach,C. citrullus N. tabacum and E. camaldulensis against T. castaneum,

T. granarium and R. dominica respectively. Antifeedant and growth inhibition effect was maximum

at 6% followed by 4% and 2% concentration.

15

CHAPTER 1

INTRODUCTION

1.1 Post-harvest losses due to infestation of stored grain insect pests

during storage

More than 20,000 species of field and storage insect pests have economically destroyed

approximately one-third of the worldwide food production. In developed countries postharvest

losses are up to 9 % while in developing countries of Africa and Asia the highest losses are up to

43% (Jacobson, 1982; Pimentel, 1991). About 600 species of beetle, 70 species of moths and about

355 species of mites attack stored products of agriculture and animal origin and cause quantitative

and qualitative losses and also reduce their dry weight and nutritional value (Rajendran, 2002; Sinha

and Watters, 1985). In stored grains, insect damage may account for 10-40% of loss worldwide

(Matthews, 1993; Papachristos and Stamopoulos, 2002; Raja et al., 2001). In temperate zones losses

due to insect pests during storage are 5-10 % while in tropical zone it is about 20- 30% (Haque et

al., 2000; Lohar, 2001) . In Pakistan the estimates of storage losses of food grains due to insects

have been reported to vary greatly; 4-10% (Huque et al., 1969), about 5.08% (Chaudhry, 1980), up

to 5% (Ahmad, 1984), 3.5 – 25.5% (Irshad and Balouch, 1985) about 2-6% by (Avesi, 1983) more

than 2.5% (Ahmad et al.,1992) more than 10-15 % (Jilani, 1980) The storage losses are mainly

caused by insect pests like Rhyzopertha dominica, Trogoderma granarium, Tribolium castaneum,

Tribolium confusum, Sitophilus oryzae, Sitophilus granarius, Sitotroga cerealella, Callosobruchus

chinesis, Callosobruchus analis and many others including rodents and birds (Ashfaq et al., 2001).

1.2 Major insect pests of stored grains

1.2.1 Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is

primary pest of flour and other milled products of cereals and a secondary pest of stored wheat

(LeCato, 1975; Hameed and Khattak, 1985; Irshad and Talpur 1993; Suresh and White 2001).

It has great economic importance among various stored grain insect pests and worldwide

distribution (Garcia et al., 2005). Both adult and larva of T. castaneum feed on cereals and their

products (Bagheri and Zenouz, 1995). It disperses from infested storage to fresh storage

16

(Campbell et al., 2002; Campbell and Arbogast, 2004) and infests stored products with their

larva layers and excrements which lead to lower the quality and quantity of stored products

greatly (Mondal, 1994). Besides the grain consumption, its attack also alters temperature and

moisture conditions that lead to an accelerated growth of molds, including toxigenic species

(Magan et al. 2003; Garcia et al., 2005; Mullen, 1992). In Pakistan the damage caused by T.

castaneum to various stored and food commodities like grain, flour and dried fruits are

recorded to be 15-20% (Khattak and Shafique, 1986). Members of genus Tribolium are

reported to secrete certain toxic quinones of carcinogenic nature in stored commodities thus

posing serious risks to human health (Ladisch, et al. 1967; El-Mofty et al. 1989) maulted skins

may cause dermatitis in people handling heavily infested grains (Pruthi and Singh, 1950).

1.2.2 Rhyzopertha dominica (Fab) (Coleoptera: Bostrichidae)

The lesser grain borer, Rhyzopertha dominica (Fabr.) (Coleoptera: Bostrichidae) is a major

insect pest of stored products and is cosmopolitan in distribution (Potter, 1935). It is mostly found

throughout warmer regions of the world and highly polyphagous in nature (Edde et al., 2005). It is

a strong flier, so that it can easily disperse from one infested storage facility to fresh storage and

create new infestations very quickly (Stejskal et al., 2003; Khan and Marwat, 2004; Campbell and

Arbogast, 2004; Gates, 1995). Females lay eggs on the surface of grain kernels, and upon hatching

the larva enters the kernel (Neethirajan et al., 2007; Ozkaya et al., 2009) and remains inside until

maturity (Chanbang et al., 2007). The developing larva feeds inside the kernel causing weight loss

and damage to the germ and endosperm (Gundu and Wilbur, 1957; Campbell and Sinha, 1976).

The adult emerges from the kernel by boring a large exit hole, producing what is commonly referred

to as an insectdamaged kernel (IDK) in wheat. Therefore, significant physical damage and weight

loss result from internal and external feeding by larvae and mature adults, respectively (Evans,

1981). R. dominica cause 40% loss in brown rice of initial weight over the 14 weeks through

infestation (Sittisuang and Imura, 1987). The average developmental time of R. dominica is 25 days

at 34.080C at 70% RH (Birch 1945a) and Optimum temperature for its development is 35.08C

(Birch 1945b). This species is well adapted to dry conditions (Emekci et al., 2004) can even survive

in grains with as low as 8% moisture content (Birch, 1945a,b).

17

1.2.3 Trogoderma granarium (Everts) (Coleoptera: Dermestidae)

The khapra beetle, Trogoderma granarium (Everts) (Coleoptera: Dermestidae) is one

of the most notorious primary insect pest of stored grains (Banks, 1977; Viljoen, 1990;

Sarmamy et al., 2011). It is among the 100 most invasive pests in the world (Lowe, et al.,

2005), mostly found in tropical and subtropical regions of Asia and Africa (Viljoen, 1990).

Khapra beetle is very common in geographical areas characterized by high temperature and

low humidity (Ghanem, 2007). T. granarium spread all over the world through cargo because

it is unable to fly (A Guide To The Beetles of Australia, 2010) and has been recognized as an

A2 quarantine organism for EPPO (OEPP/EPPO, 1981) Adult of T. granarium does not feed

but their larvae voraciously feed and cause heavy contamination to the stored product through

mass webbing and frass (Musa et al., 2010). Infestation of the khapra beetle is often followed

by colonization of secondary insect pests especially Ephestia cautella (Walker) and fungi

(Aspergillus flavus L.) which leads to deterioration of quality and loss the weight of food grains

as well (El-Nadi, 2001; Rahman et al., 1945). Feeding in maize kernels has been found to

adversely affect quality of minerals (Jood et al., 1992a), available carbohydrates (Jood et al.,

1993) protein and starch digestibility (Jood and Kapoor, 1992) and bioavailability of proteins

(Jood et al., 19921). Larvae consume an average of 3-12 mg of food during their development,

with females eating about double the amount as compared to males (Sohi, 1947; Karnavar,

1973). More food was consumed in constant darkness; however, constant light accelerated

development but reduced oviposition (Sohi, 1986). Losses caused by khapra beetle have been

reported to range from 0.2 to 2.9% over a period of 1-10.5 months, (Irshad and Iqbal1994).

The estimates of storage losses of food grains due to insects have been reported to range of 10-

18% (Hafiz and Hussain, 1961). In Pakistan weight loss is about 2.32% (Khan and Cheema,

1978)

1.3 Conventional chemicals used to control stored grain insect pests

From last many decades various types of fumigants and synthetic insecticides are used

as a quick and trusted control strategy against stored cereals insect pests. Methyl bromide as the

toxicant to insect pests was first time revealed by Le Goupil (1932), Later on methyl bromide

was found to be one of the main causes of ozone depletion and it was agreed to reduce its use

and to find its replacement (Bell et al., 1992; Butler and Rodriguez, 1996; Shaaya and

18

Kostyukovsky, 2006). Methyl bromide is scheduled for worldwide withdrawal from routine use

as a fumigant in 2015 under the directive of the Montreal Protocol on ozone-depleting

substances. Due to the internationally limited use of methyl bromide, the importance of

phosphine in controlling coleopteran stored insects has relatively grown (Zettler and Arthur,

2000). This situation increased the frequency of its applications and resulted in higher selection

pressure for phosphine resistance (Benhalima et al., 2004; Collins et al., 2002). Consequently,

since FAO (Food and Agriculture Organization) carried out globally phosphine resistance

between 1972 and 1973 years, there is a general increase in the frequency of resistant strains to

phosphine over time (Mills, 2001). Sulphuryl fluoride (Drinkall et al., 1996; Bell and Savvidou,

1999), high pressure CO2 (25 bar) (Nakakita and Kawashima, 1994; Ulrichs, 1994; Prozell et

al., 1997), Carbonyl sulphide (Desmarchelier, 1994) and ethyl formate (Hilton and Banks, 1997)

are also used as fumigants against stored product insect pests. Some other chemicals such as

bifenthrin, malathion and cypermethrin are used to achieve rapid control against stored product

insects but their high cost and adverse effects on non-target organisms and development of insect

resistance reduced their feasibility (Tsumura et al., 1994; Saxena and Sinha, 1995). The eco

toxicological effects of conventional synthetic pesticides embrace the environmental pollution

(Wright et al., 1993), have adverse effects on human health and non- target organisms, by direct

toxicity to beneficial insects, fishes and humans (Sighamony et al., 1986), development of

resistant strains (White, 1995) and residue in food grains (Fishwick, 1986) .

1.4 Plant derived insecticides: An alternative of conventional chemicals

Biopesticides of plant origin were reviewed recently (Regnault-Roger et al. 2002), and

it was concluded that botanicals have considerable market potential as reduced risk control

agents. Plant essential oils have been recognized as alternative sources for synthetic

insecticides because some are selective, degradable to nontoxic products, and have few harmful

effects on non-target organisms and the environment (Isman, 2000, 2001).

Essential oils (EOs) are secondary metabolites produced in plant metabolism that have

low toxicity to humans and wildlife and are environmentally safe (Katz, et al., 2008) They are

composed of volatile compounds that have a peculiar aromatic fragrance, and their lipophilic

nature helps them interfere with basic metabolic, biochemical, physiological and behavioral

functions of insects. (Mohamed and Abdelgalei, 2008; Nishimura, 2001) the active ingredients

19

of many EOs are reasonably priced and are commonly used as flavours and fragrances. Several

studies have shown that EOs possesses insecticidal and repellent properties, the ability to delay

development, adult emergence and fertility and deterrent effects on feeding behaviour against

many insect pests. (Batish, et al., 2008; Benzi, 2009) Moreover, because of the multiple sites

of action through which the essential oil can act, the probability of developing a resistant

population is very low. (Descamps, et al., 2011; Tripathi, 2009)

As the continuing program to isolate anti- insect control agents from plants, a large

number of essential oils were investigated as stored-product protectants as these essential oils

has contact toxicity, fumigant toxicity, repellant, antifeedant, growth inhibitor, ovicidal and

larvicidal properties. (Suthisut et al., 2011; Regnault-Roger et al., 2002, Prakash and Rao 1997,

Weaver and Subramanyam 2000, Shaaya et al., 1991; Sarac and Tunc 1995; Tunc et al., 2000;

Kim et al., 2003; Rozman et al., 2007) Botanicals used as insecticides presently constitute 1%

of the world insecticide market (Rozman et al., 2007). Derivatives from the neem tree,

Azadirachta indica (A. Juss) have received great attention by researchers. These derivatives

include leaves, seed extracts, kernel extracts, kernel powder and de-oiled cake. All of these

derivatives were proven effective for more than15 stored product species (Pereira and

Wohlgemuth, 1982; Dunkel et al., 1990; Makanjuola, 1989; Jilani, et al. 1988; Jilani and

Saxena, 1990; Xie, et al, 1995; Prakash and Rao,1997; Rahim, 1998; Weaver and

Subramanyam, 2000). Neem extracts contain several compounds that provide insecticidal

properties but the main active constituent is the triterpenoid azadirachtin (Butterworth and

Morgan, 1968). Azadirachtin has complex of behavioral and physiological modes of action; it

has an antifeedant, repellant, toxic, and growth regulating action on insects (Schmutterer 1988,

Mordue and Blackwell, 1993; Hou et al., 2004; Wong et al., 2005; Jacobson et al., 1983;

Senthil-Nathan et al., 2009). Melia Azadirachta powder has strong repellant action against R.

dominica (Khan and Marwat, 2004) and it has excellent feeding deterrence against stored grain

insect pests (Islam, 1983). The genus Eucalyptus (family Myrtaceae), represented by over 700

species distributed throughout the world is one of the most extensively cultivated pulpwoods

(Zobel, 1988). The insecticidal activity of Eucalyptus oils has been related to the presence of

components such as 1,8-cineole, citronellal, citronellol, citronellyl acetate, p-cymene,

eucamalol, limonene, linalool, α-pinene, γ-terpinene, αterpineol, alloocimene and

aromadendrene, (Batish et al., 2008). Among the various components of Eucalyptus oil, 1,8-

20

cineole is largely responsible for a variety of insecticidal properties (Duke, 2010). The

biological activity of essential oils and extracts of Eucalyptus species against stored grain pests,

elicited two types of toxicity a direct and indirect insecticidal effect, the direct insecticidal

effect has been reported to cause mortality to adults and larvae of T. granarium (Pal et al.,

1996). The indirect insecticidal effects are found by inhibiting growth and the cycle of

development of R. dominica (Singh et al., 1996). It also possesses repellent properties against

T. Castaneum (Verbel, et al. 2010). Eucalyptus camaldulensis has larvicidal, fumigant and

repellant properties against T. granarium and T. castaneum (Abbasipour et al., 2009;

Modarres- Najafabadi et al., 2006, Mohal 2006). Nicotiana tabacum is toxicant reduce the

population of stored grain insects and also possess the growth inhibitory properties (Idoko and

Adebayo, 2011) it also shows high mortality against Sitophilus zeamais (Danjumma et al.

2009). Nicotiana tabacum have the active ingredient nicotine which have stomach, contact and

respiratory poisoning properties against insect pests of stored grains (Lale, 2002). It also

exhibited the larvicidal and at high dose it shows feeding deterrence activities against T.

castaneum (Tiwari et al., 1995). Colocynthis Citrullus L. (Cucurbitaceae) is a medicinal plant

in Africa and Asia (Tavakkol-Afshari et al., 2005). Seed extracts of C. citrullus is toxicant and

inhibit the population T. castaneum at various concentrations (Nadeem et al., 2012).

Objectives of the study

Evaluate the bioactivities of locally grown plant essential oils as a replacement of

conventional insecticides against insect pests of stored products.

Calculate the effect of concentration and exposure time to obtain maximum repellent,

contact and fumigant insecticidal, phago-deterrent and growth regulatory activities of

essential oils.

CHAPTER 2

REVIEW OF LITERATURE

2.1 Repellency of essential oils of indigenous medicinal plants

Essential oils from plants belonging to numerous species have been widely investigated to

evaluate their repellent properties. Essential oils are volatile mixes of hydrocarbons; their repellent

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action has been connected to the occurrence of monoterpenes and sesquiterpenes. Though, in some

cases, these chemicals could work synergistically, improving their efficiency. Additionally,

utilization of other natural products in combination, such as vanillin, could increase the protection

time, potentiating the repellent effect of some essential oils. Amongst the plant families with

potential essential oils employed as repellents, Cymbopogon spp., Ocimum spp. and Eucalyptus

spp. are the most cited. Individual constituents present in these mixtures with high repellent action

include a-pinene, limonene, citronellol, citronellal, camphor and thymol. Finally, though from an

economical view point synthetic chemicals are still more often used as repellents than essential oils,

these natural products are promising to present efficient and safer repellents for humans as well as

environment (Nerio et al., 2010). The repellent effect of pea products against red flour beetle, rice

weevil and lesser grain borer was analyzed by Kumar et al. (2003) and illustrated that paddy grains

treated with a protein rich fraction obtained from peas (var. Bonneville) at 1% concentration were

repulsive to the adults. Findings of this study further depicted that red flour beetle was repelled

more rapidly pursued by rice weevil and lesser grain borer. In another study conducted by Dwivedi

and Shekhawat (2004) the repellent action of six aboriginal plants after screening against

Trogoderma granarium was determined using oflectometer. It was confirmed by oflactometer that

all the plants provide excellent repellency. Maximum repellency was obtained by acetone extract

of Emblica officinalis, while ether extract of Ziziphus jujube showed minimum repellency.

Likewise, Tapondjou et al. (2005) accomplished a study to find out the repellent action of the

essential oils extracted from the leaves of Cupressus sempervirens and Eucalyptus saligna through

GC-MS with cymol, which is one of the major components known for its repellency against maize

weevil and confused flour beetle, results indicated that both of these crude oil extracts generated a

strong repellent action in opposition to test insects as compared to cymol. Khanam et al. (2006)

examined the repellency of lignin obtained from sugarcane bagasse against stored grain insect pests

viz., Tribolium castaneum and Tribolium confusum and found that all the concentrations of the

product were found to be repellent to both the insect species. Strong repellent activity was observed

at concentrations of 471.57 and 628.76 μg.cm−2 to Tribolium confusum showing the repellency of

class V activity. Similarly, essential oils obtained from the leaf and fruit of Peruvian pepper were

found to have an insecticidal and insect repellent activity against Tribolium castaneum and

Trogoderma granarium in a study accomplished by Sattar et al. (2008).

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Repellent action of the mugwort essential oil against Tribolium castaneum was studied by

Wang et al. (2006) results indicated that mugwort oil had a very strong repellent activity to adults

and was significantly repellent at a 0.6 mL/mL (v/v). Khanam et al. (2008) investigated the repellent

action of various solvent extracts of Zingiber cassumunar leaf and rhizome against Tribolium

confusum and Tribolium castaneum. It was seen from the results of the study that both extracts

repelled the insect species. Findings of this study further demonstrated that maximum repellent

action was shown by the acetone extracts of the rhizome while all leaf extracts were found to have

a weak to moderate influence to Tribolium confusum. In the study carried out by Parugrug and

Roxas (2008) the insecticidal action of the five locally accessible plants: Neem, Lemon Grass,

Lantana, Basil and African marigold against Sitophilus zeamais (Motsch) was evaluated. It was

established from the results of the study that all the examined objects produced repellent activity

against Sitophilus zeamais. Findings of the study suggested that powdered leaves of lantana and

neem hold high repellant activity as compared to the powdered leaves of African marigold, lemon

grass and basil which exhibit a moderate repellency in opposition to Sitophilus zeamais in 96 hours

of disclosure. Kheradmand (2010) performed an experiment to examine the repellent act of

essential oils attained via steam distillation seeds of jojoba, Simmondasia chinensis (Link) against

two significant insects of stored products i.e., Oryzaephilus surinamensis and Callosobruchus

maculates. The results showed that the repellent activity of jojoba oil was 11.07± 4.01 and 21.41 ±

3.44 for Callosobruchus maculates and 12.74±.28 and 18.75±0.31 for Oryzaephilus surinamensis

by using Loschiavo methods and y-shape olfactometer, respectively. Similarly, Zapata and

Smagghea (2010) reported the repellent activity of four essential oils of the bark and leaves of

Drimys winteri and Laurelia sempervirens to control red flour beetle. It was found that after 4 hours

of exposure more than 90% repellent activity was attained against Laurelia sempervirens oil as

compared to Drimys winteri oil for which 3-10 times more concentration was required to obtain the

same activity.

Insect repellency studies were conducted by other researchers Lu and Wu (2010) extracted

the essential oil by Soxhlet method with anhydrous diethyl ether from Ailanthus altissima (Swingle)

(Sapindales: Simaroubaceae) bark and investigated its repellent activities against Tribolium

castaneum, Oryzaephilus surinamensis, Sitophilus oryzae and Liposcelis paeta adults. A. altissima

bark oil significantly repelled all these insect pests with the repellency value reaching IV grade or

stronger during the whole exposure period. Further, in another insect repellency study Manzoor et

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al. (2011) investigated the effects of ethanolic extracts of five plants leaves Bakain, Mint, Habulas,

Lemongrass and Datura against three stored grain pests viz., Tribolium castaneum, Oryzaephilus

surinamensis and Collusobruchis chinesis disclosed that all treatments significantly repelled the

examined insects. Results of this study revealed that Lemon grass exhibited utmost repellent

activity of 39.75% against O. surinamensis extracts of Habulas exhibited highest repellent activity

of 64.05% against T. castaneum, and Datura extract exhibited utmost repellent activity of 31.67%

against C. chinesis. Iqbal et al. (2010) tested seven plant species for their repellent activity against

stored grain insects, out of these ethanol extract of Acorus calamus and Azadirachta indica

exhibited more than 40% average repellency over 8 weeks against T. castaneum at all application

rates. Curcuma longa and Peganum harmala showed this level at 1600 μg/cm2 and 800 μg/cm2

only whereas Saussurea lappa showed promising repellency only at 1600 μg/cm2. Repellency was

considerably higher during 1st week of testing. Azadirachta indica was comparatively persistent as

it remained effective throughout 8 weeks although showing relatively lower repellency than that of

Acorus calamus in the beginning. Likewise, laboratory bioassays were carried out by Auamcharoen

et al. (2012) to study the repellent activity of Duabanga grandiflora crude methanol extract against

Sitophilus oryzae. In this experiment repellent action was estimated by means of a choice test using

treated filter paper. Results depicted that the extract was having repellent action against Sitophilus

oryzae 3783% at 5 min to 2 hours following exposure and 60-100% repellent at 4 to 24 hours after

exposing. The findings of the study further confirmed that the exposure period emerged as the most

significant factor influencing the repellent action of methanol extract as compared to the tested

concentrations which exhibited no significant differences in the repellent activity. Satti et al. (2012)

conducted laboratory tests against Trogodarma granarium. The results showed that A. indica seeds

stored for 2, 3 and 4 years were better than the seeds stored for 1 or 5 years, in controlling the

studied pest. The newest seeds (1year old) seemed to exert more repellent effect than older seeds,

while diminishing of neem activities appeared to start after four years of storage when applied under

shade.

Benzi et al. (2009) extracted the essential oil from the leaves of Aloysia citriodora and A.

polystachya (Verbenaceae) and from fruits and leaves of Peruvian pepper and tested them for their

repellency in against the adults of Lesser Grain Borrer. Repellent assays showed that A. citriodora

was most effectual oil based on class scale. Likewise, Benzi et al. (2012) performed another insect

repellent study and exhibited the repellent action of the essential oils of leaves and fruits of Brazilian

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pepper tree in whole wheat. The results suggested that leaf essential oils have repellent action

towards insects at both concentrations (0.04 and 0.4% w/w) whereas essential oils obtained from

fruits lack repellent action towards insects. Verbal et al. (2010) tested the repellent activity of the

essential oils extracted from Eucalyptus citriodora (Hook) and Cymbopogon citrates using against

T. castaneum. The mean repellent dosage after 4 hours of exposing were 0.021 ml/L and 0.084ml/L

for Cymbopogon citrates and Eucalyptus citriodora, respectively which are lower as compared to

commercial products

These findings of these studies suggested that the repellent activity of both plant oil extracts prove

them potential agents as insect repellents. Descamps et al. (2011) isolated the essential oils from

fruits and leaves of Schinus areira (Anacardiaceae) and tested them for their repellent action against

Tribolium castaneum larvae and adults. A treated diet was also employed to assess the insect

repellency and a flour disk bioassay for the feeding deterrence activity and nutritional index

variation. The essential oils of leaves showed repellent actions, whereas that from the fruit was an

attractant. Moreover, both essential oils produced some alterations in nutritional index. In a research

accomplished by Stefanazzi et al. (2012) revealed that essential oils isolated from Elyonurus

muticus (Spreng), Tagetes terniflora (Kunth) and Cymbopogon citratus (Stapf) were found to be

repellent against larvae and adults of Triboilum castaneum and only on the adults of S. oryzae. The

repellent action of the essential oils extracted from palmarosa, lemongrass and wild oregano in

opposition to Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), using the area preference

was exhibited in a further study conducted by Gallardo et al. (2012) which illustrated that the

repellent activity of the oils declined in the order C. martinii >C. flexuosus>L. origanoides. The

findings of the study suggested that the essential oils assessed in this experiment might be valuable

in the repellent formulations against Tribolium castaneum. Further, in another study the repellent

action of Artemisia scoparia against the three stored products insects i.e., Bean beetles, Rice weevil

and Red flour beetle was evaluated. Results obtained from the study depicted that these oils were

significantly more repellent to Rice weevil and Red flour beetle than Bean beetles.

Kim et al. (2010) tested essential oil of Origanum vulgare for repellent action against

Tribolium castaneum adults. Results of the study showed that insect repellency depended on both

time as well as concentration. Ukeh (2008) investigated the repellent action of Aframonum

melegueta and Zingiber officinale in a four-armed airflow oflectometer and found that 10 ml of

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both crude oils significantly repelled R. dominica, Similarly Balachandra et al. (2011) investigated

the repellent action of Plectranthus zeylanicus plant against C. maculatus. It was established

through choice camber bioassays and olfactometer analysis that P. zeylanicus plant oil has

significantly repelled the insects. Aggarwal, et al (2001) conducted an insect repellency study and

found that Artemisia annwa essential oil contain an important component 1,8-cineole which was

proved to be effective against Callosobruchus maculatus, Rhyzopertha dominica and Sitophilus

oryzae . It was depicted from the results of the study that 1, 8-cineole has moderate repellency

against all three species with mean repellency of 65.74% in 1 hour at maximum dose rate of 4.0

µl/mL. Similarly, in one more insect repellency study the repellent activity of essential oil isolated

(by hydro-distillation method) from Litsea salicifolia was confirmed against Tribolium castaneum

and Sitophilus zeamais. The repellent action of the plant extracts of Aloysia polystachia (Griseb),

Solanum argentinum (Bitter) and Tillandsia recurvata (Bromeliaceae) in hexane, ethanol and

chloroform against Sitophilus oryzae was found in a research conducted by Viglianco et al. (2008).

In this research the repellency test was executed by filter paper circles cut in halves and results

obtained depicted that Aloysia polystachia and Solanum argentinum have a moderate repellent

effect against S. oryzae while hexane extract of Solanum argentinum was found to have a strong

repellent activity. Tunc and Erler (2003) declared in their insect repellency studies that the repellent

activity of different components of plant essential oils was dependent upon concentration.

Repellent activity declined steadily with increasing time. Among the eight essential oil components

tested in this study; 1,8-cineole Anethole, thymol and carvacrol were categorized as the most

promising repellents as they exhibited the maximum activity and more stability. Shah et al. (2008)

evaluated the repellant action of leaves of six indigenous plants viz., Typhonium trilobatum, Cleome

viscosa, Cassia occidentalis, Pongamia pinnata, Mesua ferrea, and Trewia nudiflora against

Oryzaephilus surinamensis (L.) at 2.5, 5.0, 7.5, and 10.0% concentrations. Extracts of water solvent

showed higher repellent effect than that of others except ethanol extract of M. ferrea. Considering

mean repellency rate, extracts of three solvents of all six plants were in the same repellency class

i.e. class II except water extract of P. pinnata (class III). It was found that the rate of repellency

increased with the increase of dose level. At 10.0% dose level all plant extracts showed the highest

repellency rate and were in repellency class III. Babarinde et al. (2011) investigated the bioactivity

of leaves powders of Piper guineense seeds and Moringa oleifera, which were applied alone or in

combination (with permiphos methyl), against larvae and adults Trogoderma granarium (Everts)

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in airtight pots The results of the study indicated that all the treatments were repellent to larvae of

tested insect species with 60% repellency by the treatment having mixture of plants, 50% repellency

in Piper guineense and 30% repellency in Moringa oleifera. Further, Conti et al. (2010) reported

the repellent activity of the essential oils of two tropical Lamiaceae, Hyptis spicigera (Lamarck)

and Hyptis suaveolens (Poitier) and of a Mediterranean one, Lavandula angustifolia (Miller),

against adults of the granary weevil, Sitophilus zeamais (Motschulsky). Results showed that the

three essential oils had repellent activity against S. zeamais adults. In another experiment

accomplished by Yao et al., 2008, (Z)-asarone was identified as a major active component from

ethanol extracts of Acorus calamus (L). rhizome through spectroscopic analysis. This component

was tested for its repellent action against adults of Sitophilus zeamais (Motschulsky) and results

indicated that this compound had strong repellency to S. zeamais. In repellency test, ethanol extracts

of Acorus calamus had 93.92% repellency at 629.08 µg/cm2 but only 71.38% at157.27 µg/cm2 12

hours after treatment. As a contrast, (Z)-asarone exhibited 84.50% repellency at 314.54 µg/cm2 and

77.02% at 78.63 µg/cm2 after12 hours subsequent to treatment. So, it was proposed that insecticidal

action of A. calamus extract might be owing to (Z)-asarone. The essential oil of Artemisia annua

L. was repellent activities against two economically important stored product insects: Tribolium

castaneum (Herbst) and Callosobruchus maculatus (L.) by (Tripathi et al., 2000). Adult beetles of

T. castaneum were repelled significantly by oil of A. annua at 1% concentration (vol:vol) and above

in filter paper arena test. It was revealed from results of study that oil from A. annua was largely

responsible for both repellent actions on both species of insect tested.

2.2 Contact Toxicity of essential oils of indigenous medicinal plants

Contact toxicity study of essential oils of Evodia lepta (Rutaceae) root barks against

the Sitophilus zeamais (Motsch.) and Tribolium castaneum (Herbst) was accomplished by

Jiang (2011) for the contact toxicity of the essential oil against S. zeamais and T. castaneum

adults with LD50 values of 125.21 and 166.94μg/adult, respectively. Zapata and Smagghea

(2010) applied topically four essential oils extracted from the leaves and bark of Laurelia

sempervirens and Drimys winteri against Tribolium castaneum. Results showed that LD50

values by topical application of L. sempervirens oils were from 39 to 44g/mg insect and for D.

winteri oils these were from 75 to 85g/mg insect. Similarly in another study, Lu and Wu (2010)

extracted the essential oil by Soxhlet method with anhydrous diethyl ether from Ailanthus

altissima (Swingle) bark. Results depicted that A. altissima bark oil possessed strong contact

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toxicity on S. oryzae adults which gradually increased with increase of exposure time and the

corrected percentage mortality reached 76.5% after 72 h treatment. Chu et al. (2011)

determined chemical composition and insecticidal activity of the essential oil of Artemisia

igniaria (Maxim.) (Asteraceae) aerial parts against Sitophilus zeamais (Motschulsky).

Essential oil of A. igniaria aerial parts was obtained by hydro-distillation and analyzed by gas

chromatography-mass spectrometry. Results of this study revealed that essential oil exhibited

strong contact toxicity against S. zeamais with an LD50 value of 14.41μg/adult. Tapondjou et

al. (2005) determined the contact toxicity of the essential oils extracted from Eucalyptus

saligna and Cupressus sempervirens leaves along with cymol against Sitophilus zeamais and

Tribolium confusum. Contact toxicity assayed by impregnation on filter paper discs or coating

onto maize grains showed that these chemicals caused significant mortality of the test insects.

Eucalyptus oil was more toxic than Cupressus oil to both insect species (LD50=0.36 ml/cm2

for S. zeamais and 0.48 ml/cm2 for T. confusum) on filter paper discs, and was more toxic to

S. zeamais on maize (LD50=38.05mL/40gg grain). Nenaah (2011) used glycoalkaloid fraction

and the two glycoalkaloids, a-chaconine and a-solanine of potato, Solanum tuberosum, for their

toxicity against the Trogoderma granarium (Everts). Results indicated considerable toxicity,

especially when adults were topically treated with the glycoalkaloids. The fraction was the

most toxic with LC50 of 16.7 and 11.9 mg/mg insect, 48 and 96 hours post treatment,

respectively. LC50 of a-chaconine and a-solanine 96 hours post treatment were 18.1 and 22.5

mg/mg insect, respectively. Moderate toxicities were recorded when insects were confined on

dry-film residues of botanicals with LC50’s ranging between 26.1 and 56.6, and 19.4 and 45.7,

mg/cm2 48 and 96 hours post treatment, respectively. Gallardo et al. (2012) conducted the

experiment to determine the contact toxicity of the essential oils isolated from Cymbopogon

martini, Cymbopogon flexuosus and Lippia origanoides against Tribolium castaneum (Herbst)

using the filter methods. Tested oils depicted low toxicity, showing less than 20% toxicity at

maximum tested concentration (1.2 mL/cm2) and exposure period (72 h).

Huang et al. (2000) determined contact toxicity of the essential oil of cardamom, Elletaria

cardamomum, against two stored-product insects, Sitophilus zeamais and Tribolium castaneum.

The adults of S. zeamais and T. castaneum were equally susceptible to the contact toxicity of the

oil at the LD50 level, with LD50 values of 56 and 52 mg/mg of insect respectively. However, S.

zeamais was more susceptible than T. castaneum at the LD95 level. Likewise, Kordali et al. (2006)

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reported in his studies that essential oils of aerial parts of three Artemisia species (A. absinthium,

A. santonicum and A. spicigera) were isolated by hydro-distillation method and tested for their

toxicity against to granary weevil, Sitophilus granarius (L.). All of the essential oils tested were

found to be toxic to adults of S. granarius. Results confirmed that oils showed about 80-90%

mortality of S. granarius at a dose of 9µL/L after 48 hours of exposure. Tripathi et al. (2002)

investigated the contact toxicity of essential oil extracted from the leaves of turmeric, Curcuma

longa against three storedproduct beetles, Rhyzopertha dominica, Sitophilus oryzae and Tribolium

castaneum. It was illustrated that the adults of R. dominica were highly susceptible to contact action

of C. longa leaf oil, with LD50 value of 36.71 µg/mg weight of insect. Benzi et al. (2009) extracted

the essential oil from leaves of Aloysia polystachya and Aloysi citriodora and from leaves and fruits

of Schinus molle and tested for contact toxicity studies. Results declared that A. polystachya was

the most toxic plant by filter paper assay (LC50 26.6 mg/cm2). Liu et al. (2010) reported that local

wild plants, Artemisia capillaris and Artemisia mongolica were found to possess insecticidal

activity against Sitophilus zeamais. The essential oils also show contact toxicity against S. zeamais

adults with LD50 values of 105.95 and 87.92µg/adult, respectively. Li et al. (2010) exhibited in his

studies that Murraya exotica possess insecticidal activity against the Sitophilus zeamais and

Tribolium castaneum. The essential oils also showed contact toxicity against S. zeamais and T.

castaneum adults with LD50 values of 11.41 and 20.94µg/adult, respectively. Chu et al. (2011)

evaluated that essential oils of Chenopodium ambrosioides possess strong insecticidal activities

against Sitophilus zeamais (Motsch.). Essential oil of C. ambrosioides was obtained by hydro-

distillation, and the constituents were determined by GC-MS analysis. The active compounds were

isolated and identified by bioassay-directed fractionation. Five active compounds [(Z)-ascaridole,

2carene, ρ-cymene, isoascaridole and α-terpinene] were isolated and identified from the essential

oil from Chinese C. ambrosioides. The LD₅₀ values (contact toxicity) of the crude essential oil and

(Z)-ascaridole against S. zeamais adults were 2.12 and 0.86 µg g⁻¹ body weight. Descamps et al.

(2011) extracted the essential oils from leaves and fruits of Schinus areira (Anacardiaceae) and

tested them for their contact toxicity by topical application against Tribolium castaneum

(Coleoptera: Tenebrionidae) larvae and adults. Results of this study confirmed that both oils caused

mortality against larvae in topical application. Balachandra et al. (2011) isolated essential oil from

Plectranthus zeylanicus plant and tested for contact toxicity against C. maculatus. The gas

chromatography studies of the essential oil of P. zeylanicus showed that ρ-cymene (3.5%), β-

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caryophyllene (0.2%), geranyl acetate (9.3%) and geraniol (7.2%) were the major constituents of

this essential oil. The adults of C. maculatus were susceptible to contact toxicity of P. zeylanicus

plant oil. LC50 values of (0.010 g L-1) were obtained for contact toxicity assays. In another study

conducted by Babarinde et al. (2011) the bioactivity of Piper guineense seeds and Moringa oleifera

leaf powders applied singly or in a mixture against larvae and adult of Trogoderma granarium

(Everts) evaluated in airtight containers. Adults were more susceptible to plant powders than larvae

and adult mortality recorded in P. guineense at 1.0 g, 0.5 g and M. oleifera at 1.0 g/20 g seeds were

not significantly different from the mortality observed with the recommended dose of Pirimiphos

methyl at five days after treatment. Larval mortality observed in a mixture of both plants (1:1; w/w)

caused significantly higher mortality (77.5%) than other treatments. Stefanazzi et al. (2012)

reported the bioactivity of essential oils from Tagetes terniflora (Kunth) Cymbopogon citratus

(Stapf.) and Elyonurus muticus (Spreng) against stored-grain pests. Contact toxicity was observed

with T. terniflora on adults of the both pests. Results depicted that in contact toxicity, this oil was

less toxic to Tribolium castaneum (Herbst).

Aggarwal et al. (2001) determined contact toxicity of 1,8-cineole, one of the components

of the essential oil of Artemisia annwa against three stored product coleopterans Callosobruchus

maculatus, Rhyzopertha dominica and Sitophilus oryzae. Contact toxicity assay revealed that LD50

values of 0.03, 0.04 and 0.04 µL/insect against C. maculatus, R. dominica and S. oryzae

respectively were found in the topical application assay. Likewise in another study Mondal and

Khalequzzaman (2006) investigated contact toxicity of the three essential oils, viz., Elletaria

cardamomum, Cinnamomum aromaticum and Syzygium aromaticum against Tribolium castaneum

(Herbst) larvae and adults. Three day old adults and 10 days old larvae were equally susceptible to

the contact toxicity of C. aromaticum oil, with LD50 values of 0.074 and 0.196 mg cm-2 respectively.

E. cardamomum oil provided higher toxicity to 14-day and 18- day old larvae having LD50 value

of 0.10mg cm-2. Mahfuz and Khanam (2007) reported the efficacy of seven different plants extracts

viz. Acorus calamus rhizome, leaves of Datura fastuosa, Datura stramonium and seeds of Datura

stramonium, Corchorus capsularis, Aphanamixis polystachea and Jatropha curcas on Tribolium

confusum adult. In this study, dose mortality experiments were conducted with three solvent

(petroleum ether, acetone and methanol) extracts separately but J. curcas seeds were tested with

petroleum ether extract only. Among three solvents, petroleum ether extract exhibited piquant toxic

effect against the beetle at all the intervals although D. fastuosa leaf produced no mortality at 24



30

hours of treatment. Acetone extract of A. calamus rhizome, D. fastuosa leaf, D. stramonium seed

and C. capsularis seed produces mortality at all the intervals but D. stramonium leaf and A.

polystachea seed did not show any toxic effect. It was confirmed by results of study that methanol

extract of C. capsularis seed showed toxicity at all the duration. Similarly, another study Mamun

et al. (2009) evaluated the toxicity of six botanicals, Bazna (Zanthoxylum rhetsa), Ghora-neem

(Melia sempervirens), Hijal (Barringtonia acutangula), Karanja (Pongamia pinnata), Mahogoni

(Swietenia mahagoni) and Neem (Azadirachta indica) against Tribolium castaneum Herbst. In this

study, leaf and seed extracts were prepared by using acetone, methanol and water as solvents. The

results showed that extracts of all the plants had direct toxic effect on red flour beetle. Among them,

Neem seed extract showed the highest toxic effect (mortality, 52.50%), whereas Hijal leaf extract

possessed the lowest toxic effect (mortality, 22.24%). Among the solvents, acetone extract showed

more toxic effect than other extracts. Seed extracts of respective plants were slightly more toxic

than leaf extract. It was suggested in accordance to results obtained from this study that the

effectiveness of most of the plant extracts increased proportionally with the increase of doses and

decreased with time.

Parugrug and Roxas (2008) evaluated the insecticidal action of five locally available

plants namely: Azadirachta indica (Neem), Cymbopogon citratus (Lemon Grass), Lantana

camara (Lantana), Ocimum basilicum (Basil) and Tagetes erecta (African marigold) against

maize weevil, Sitophilus zeamais (Motsch.) Results obtained from this study illustrated that

the corn grains treated with powdered leaves of lemon grass and basil exhibited a low mortality

of 5.33% and 0.66%, respectively. Likewise, Gandhi and Pillai (2011) evaluated the

insecticidal activities of pulverized leaves of Punica granatum (Pomegranate) and Murraya

koenigii (Curry tree) against Rhyzopertha dominica under laboratory conditions. Five different

concentrations of leaf powders ranging from 0.05-1 g/10g wheat grains and 10g beaten rice

(Poha) separately, were tested for their efficacy. Pulverized leaves of both plants produced high

incidence of mortality. The percentage mortality over controls ranged from 18-71% with P.

granatum and 18-65% with M. koenigii in wheat medium. However, it was 26-79% and 16-

74% correspondingly in beaten rice. Anita et al. (2012) tested the pulverized leaves of Annona

squamosa (L.), Moringa oleifera (Lam.) and Eucalyptus globulus (Labill.) for their insecticidal

activities, against Tribolium castaneum (Herbst). Different concentrations ranging from 0.05

to 2.0 g (0.05, 0.1, 0.15, 1.0 and 2.0 g) per 10.0 g wheat grains were tested against larvae and

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adults. When larvae were introduced to pulverized leaves of A. squamosa, M. oleifera and E.

globulus separately, mortality rate increased with increase in concentrations and resulted in

100% mortality within a short period of 8,9 and 10 days respectively with the highest

concentration. Boussaada et al. (2008) investigated the contact toxicity of sixteen aromatic

plant extracts from three species belonging to the Asteraceae family by using organic solvents

of increasing polarity against adults and larvae of Tribolium confusum. In this study all the

extracts showed higher larval mortality than adults. It reached respectively 83%, 77% by using

petroleum ether and methanol extracts of Rhaponticum acaule. These results suggested that

Myriangium duriaei and Rhaponticum acaule may be used in grain storage against insect pests.

Sarwar (2010) reported the contact toxcicity of leaf powders of Withania somnifera (L.),

Ocimum sanctum (L.), Albizia lebbeck (L.) and Dalbergia sisso (Roxb.) at 2% and 5% as

protectants against C. maculatus in seeds of stored chickpea. Results of this study revealed that

pest mortality was directly proportional to the concentration of the botanicals used.

Calamusenone (isolated from A. gramineus rhizome) was tested for its insecticidal

action against Sitophilus zeamais (Motsch.) and Rhizopertha dominica (Fab.), in an experiment

conducted by Huang et al. (2011) using dry film contact methods. In dry film contact

experiment, the maximum insecticidal action of calamusenone against Sitophilus zeamais and

Ryzopertha dominica adults were exhibited at 170.32 μg/cm2 following treatment for 72 hours,

with 96.2% and 98.7% mortalities, respectively. In another experiment accomplished by Yao

et al. (2008) (Z)-asarone was identified as a major active component from ethanol extracts of

Acorus calamus L. rhizome through spectroscopic analysis. This component was tested for its

contact toxicity against adults of Sitophilus zeamais (Motschulsky) and results indicated that

this compound has strong contact effects to S. zeamais. As (Z)-asarone brought about 15.56%

and 100.00% mortality at 15.73 µg/cm2 and 40.89 µg/cm2, respectively so is was proposed that

insecticidal action of A. calamus extract might be owing to (Z)-asarone. Two main active

components, diallyl trisulfide and methyl allyl disulfide were isolated from essential oil of

garlic by Huang et al. (2000) and further they were tested against Sitophilus zeamais

(Motschulsky) and Tribolium castaneum (Herbst) for their contact toxicity. The contact

toxicity of diallyl trisulfide was found to be greater as compared to that of allyl disulfide while

both of these compounds exhibited more toxicity against Triboilum castaneum adults than to

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Sitophillus zeamais adults.The results of study depicted that diallyl trisulfide is more potent

contact toxicant than methyl allyl disulfide.

2.4 Fumigant toxicity of essential oils of indigenous medicinal

Fumigant toxicity of the essential oil of cardamom, Elletaria cardamomum, against two

stored-product insects, Sitophilus zeamais and Tribolium castaneum, were examined by Huang

et al. (2000) who found that S. zeamais adults were more than twice as vulnerable as T.

castaneum adults at both LD50 and LD95 intensities. Lu and Wu (2010) extracted the essential

oil by Soxhlet method with anhydrous diethyl ether from Ailanthus altissima and investigated

its fumigant toxicity activities. Results revealed that A. altissima bark oil had high fumigant

activity against O. surinamensis and S. oryzae adults with the corrected percentage mortality

99.3 and 81.9% within 24 hours, respectively. Similarly in another study conducted by Rozman

et al. (2007) reported that compounds 1,8-cineole, camphor, eugenol, linalool, carvacrol,

thymol, borneol, bornyl acetate and linalyl acetate occur naturally in the essential oils of the

aromatic plants Lavandula angustifolia, Rosmarinus officinalis, Thymus vulgaris and Laurus

nobilis. These compounds were evaluated for fumigant activity against adults of Sitophilus

oryzae, Rhyzopertha dominica and Tribolium castaneum. The insecticidal activities varied with

insect species, compound and the exposure time. The most sensitive species was S. oryzae,

followed by R. dominica, T. castaneum against the tested compounds. 1,8-Cineole, borneol and

thymol were highly effective against S. oryzae when applied for 24 h at the lowest dose (0.1

ml/720 ml volume). For R. dominica camphor and linalool were highly effective and produced

100% mortality in the same conditions. Against T. castaneum no oil compounds achieved more

than 20% mortality after exposure for 24 h, even with the highest dose (100 ml/720 ml volume).

Findings of this study suggested that these compounds may be suitable as fumigants because

of their high volatility, effectiveness, and their safety. Suthisut et al. (2011) investigated the

fumigant toxicity of essential oils from rhizomes of Alpinia conchigera, Zingiber zerumbet,

Curcuma zedoaria and their major compounds; camphene, camphor, 1,8-cineole, a-humulene,

isoborneol, apinene, b-pinene and terpinen-4-ol was investigated against adults of Sitophilus

zeamais, Tribolium castaneum, Anisopteromalus calandrae and Trichogramma deion larvae.

The trial was evaluated at 0, 37, 74, 148, 296, 444, 593 µL/L in air after 12, 24 and 48 h for S.

zeamais, T. castaneum and A. calandrae, and 24 h for T. deion. Alpinia conchigera oils were

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toxic to S. zeamais, T. castaneum and T. deion, while the other two plant oils had low toxicity.

Adults of S. zeamais and T. castaneum were more susceptible to A. conchigera oils than their

eggs, larvae or pupae. Sitophilus zeamais adults (LC50 85 µ/L in air) were slightly more tolerant

of A. conchigera oils than T. castaneum (LC50 73 µ/L in air) after 48 h exposure. Synthetic

essential oils, a mixture of pure compounds in the same ratios of the extracted essential oils,

were tested with S. zeamais and T. castaneum adults. Synthetic essential oils were more toxic

than the extracted essential oils to both insects. Zingiber zerumbet oils (LC50 26 µL/L in air)

and C. zedoaria oils (LC50 25 µL/L in air) were significantly more toxic to adults of A.

calandrae than A. conchigera oils (LC50 37 µL/L in air) whereas T. deion larvae were more

sensitive to A. conchigera oils (LC50 62 µL/L in air) than Z. zerumbet and C. zedoaria oils

(LC50 > 593 µL/L in air). Hence, Tribolium castaneum was more susceptible than S. zeamais

to the eight pure compounds. Terpinen-4-ol was highly toxic to the both insects.

Zapata and Smagghea (2010) reported that oils of both Lonicera sempervirens and

Drimys winteri were found to be toxic towards T. castaneum when applied by fumigation. LC50

values for L. sempervirens oils were 1.6-1.7µL/L air, while these were 9.0-10.5µL/L air for D.

winteri oils. Additionally, with L. sempervirens oils 50% of the tested beetles were killed at

100 <µL/L air within 3.0-4.4 hours, while with D. winteri oils the LT50 values were 6.1-7.4

hours. Similarly, in another study, Kim et al. (2010) tested the insecticidal activities of

components of Origanum vulgare L. essential oil against red flour beetle, Tribolium castaneum

adults. All constituents were identified by GC-MS, and the main components were carvacrol

(67.2%), p-cymene (16.2%), γ- terpinene (5.5%), thymol (4.9%), and linalool (2.1%). In a

vapor phase fumigant assay, the origanum oil was more effective in closed conditions

(LD50=0.055 mg/cm3) than in open conditions (LD50 353 mg/cm3). This suggested that toxicity

is exerted largely in the vapor phase. Based on 24 hours LD50 values, the toxicity of

caryophyllene oxide (0.00018 mg/cm3) was comparable with that of dichlorvos (0.00007

mg/cm3). In addition, thymol, camphene, α-pinene, p-cymene, and γ-terpinene showed good

insecticidal activity (LD50=0.012–0.195 mg/cm3). Likewise, Michaelraj et al. (2008) exposed

the adults of Sitophilus oryzae and Rhyzopertha dominica and adults and eggs of Corcyra

cephalonica were exposed to essential oils of geranium (Geranium viscosissimum), lemongrass

(Cymbopogon flexuosus) and peppermint (Mentha piperita) in the fumigation chamber. After

48 hours exposure, complete mortality of adults of S. oryzae was recorded at 100 and 150

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µL/250 µL of peppermint oil. In case of R. dominica, 100% mortality was observed in all the

doses (50, 100, 150 and 200 µl/250 ml). C. cephalonica was not sensitive to peppermint at 5

µl/250 ml. Tripathi et al. (2002) investigated the fumigant toxicity of essential oil extracted

from the leaves of turmeric, Curcuma longa against three stored product beetles, Rhyzopertha

dominica, Sitophilus oryzae and Tribolium castaneum. Results revealed that adults of S. oryzae

were highly susceptible with LC50 value of 11.36 µg/L air. Further, in T. castaneum, the C.

longa oil reduced oviposition and egg hatching by 72 and 80%, respectively at the

concentration of 5.2 mg/cm2. At the concentration of 40.5 mg/g food, the oil totally suppressed

progeny production of all the three test insects. Nutritional indices indicate >81% anti-feeding

action of the oil against R. dominica, S. oryzae and T castaneum at the highest concentration

tested. In another research, Lolestani and Shayesteh, (2009) tested the insecticidal and ovicidal

effects of essential oil extracted from Ziziphora clinopodioides (Boiss.) (Lamiaceae) on adults

and eggs of Callosobruchus maculatus (Fab.) and Results showed that the oil had high

fumigant action against adults and eggs, the adults being more susceptible than the eggs. Benzi

et al. (2012) evaluated the fumigant toxicity of Brazilian pepper tree (Schinus molle L. var.

areira) and results showed that fumigant activity, neither of the essential oils was found to be

toxic. Liu et al. (2010) reported that local wild plants, Artemisia capillaris and Artemisia

mongolica were found to possess insecticidal activity against the maize weevil, Sitophilus

zeamais. The essential oils of aerial parts of the two plants were obtained by hydrodistillation

and were investigated by GC and GC-MS. The main components of A. capillaris essential oil

were 1,8-cineole (13.75%), germacrene D (10.41%), and camphor (8.57%) and the main

constituents of A. mongolica essential oil were alpha-pinene (12.68%), germacrene D (8.36%),

and gamma-terpinene (8.17%). Results of this study suggested that essential oils of A.

capillaris and A. mongolica possess fumigant toxicity against S. zeamais adults with LC50

values of 5.31 and 7.35 mg/L respectively. Moreover, Descamps et al. (2011) in his studies

extracted the essential oils from leaves and fruits of Schinus areira (Anacardiaceae) and tested

them for their fumigant toxicity by filter paper impregnation against Tribolium castaneum

(Coleoptera: Tenebrionidae) larvae and adults. Both oils produced mortality against larvae but

in case of adult insects fumigant toxicity was not found.

Balachandra et al. (2011) isolated the essential oil from Plectranthus zeylanicus plant

and tested for potential fumigant activity against C. maculatus. The gas chromatography studies



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of the essential oil of P. zeylanicus showed that ρ-cymene (3.5%), β-caryophyllene (0.2%),

geranyl acetate (9.3%) and geraniol (7.2%) were the major constituents. The adults of C.

maculatus were susceptible to fumigant toxicity of P. zeylanicus plant oil. Saroukolai et al.

(2012) tested the aromatic plant Thymus persicus for their fumigant toxicity against T.

castaneum and S. oryzae. The adult insects were exposed to the concentrations of 51.9,

111.1, 207.4 and 370.4μl/l air to estimate median lethal time (LT50) values. The fumigant toxicity

increased with increase in essential oil concentrations. The LT50 values at the lowest and the highest

concentrations tested were ranged from 28.09 to 13.47h for T. castaneum, and 3.86 to 2.30h for S.

oryzae. It was found that S. oryzae adults were much more susceptible to the oil than T. castaneum.

These results suggest that T. persicus essential oil merits further study as potential fumigant for the

management of these stored-product insects. Likewise, Ebadollahi et al. (2010) accomplished the

fumigant toxicity tests with the essential oil against adults of Tribolium castaneum (Herbst),

Lasioderma serricorne (F.) and Rhyzopertha dominica (F). At 27±1°C and 60±5% RH, it was

observed that L. serricorne (LC 50 = 3.835 µl/l) were significantly more susceptible than R.

dominica (LC 50 = 5.66 µl/l) and T. castaneum (LC 50 = 39.685 µl/l) 24 h after treatment. In all

cases, considerable differences in mortality of insects to essential oil vapor were observed with

different concentrations and times. Mortality increased as the doses of essential oils and exposure

period increased and after 72 hours fumigations, greatest percentages of mortality were obtained.

Negahban and Moharramipour (2007) reported that essential oils extracted from the species of the

family Myrtaceae found to have potent fumigant toxicity against three major stored-product insects:

Callosobruchus maculatus (Fab.), Sitophilus oryzae (L.) and Tribolium castaneum (Herbst). These

were the essential oils from Eucalyptus intertexta, Eucalyptus sargentii and Eucalyptus

camaldulensis. The mortality of 1-7-day-old adults of the insect pests increased with concentration

from 37 to 926 μL/L air and with exposure time from 3 to 24 hours. The LC50 values to the selected

essential oils were between 2.55 and 3.97 μL/L air for C. maculatus, 6.93 and 12.91μL/L for S.

oryzae and 11.59 and 33.50μL/L air for T. castaneum.

Essential oil of Eucalyptus pauciflora leaves was found to be the strongest toxicant in

a research conducted by Shukla et al. (2002). In this study, as a fumigant, killing time was

found to be 7 hours against R. dominica and S. oryzae while it was 5 hours against T.

granarium. The toxicity of the oil was found to be thermo stable and persisted up to 36 months.

Xie et al. (2010) proved that Horseradish essential oil is a biological fumigant which was

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extracted from Armoracia rusticana. In the absence of grain, a concentration of 2.25 ppm

horseradish essential oil could kill Sitophilus zeamais and Rhyzopertha dominica after 12 h at

25°C. 3 ppm of horseradish essential oil could kill all the S. zeamais and R. dominica on the

surface of maize, wheat and paddy during 72 h exposure at 25°C. The mortalities of S. zeamais

placed under maize, wheat and paddy were 100%, 100% and 98%, respectively, using 24 ppm

of horseradish essential oil at exposure for 72 h at 25°C. The mortalities of R. dominica were

100%, 93% and 86%, respectively, under the above conditions. This study also stated that

temperature did not result in significant differences on the fumigation efficacy of horseradish

essential oil and suggested that different types of stored grain had a significant influence on the

fumigation efficacy of horseradish essential oil. Correspondingly, Stefanazzi et al. (2012)

reported the bioactivity of essential oils from Tagetes terniflora (Kunth), Cymbopogon citratus

(Stapf.) and Elyonurus muticus (Spreng) against stored-grain pests. Results of this study

depicted that Fumigant toxicity was observed with T. terniflora on adults of both pests. Huang

et al. (2000) tested the major constituents of the essential oil of garlic, Allium sativum (L.),

methyl allyl disulfide and diallyl trisulfide against Sitophilus zeamais (Motschulsky) and

Tribolium castaneum (Herbst) for fumigant toxicity. The fumigant toxicity of diallyl trisulfide

was greater than that of methyl allyl disulfide to the adults of these two species of insects.

These two compounds were also more toxic to T. castaneum adults than to S. zeamais adults.

Younger larvae were more susceptible to the fumigant toxicity of these compounds. In a study

reported by Liska (2011) the potential fumigant effects of 1,8-cineole, essential oil component,

on the Tribolium castaneum pupae. The compound was tested in 6 doses; in two treatments

(fumigation without grain and with wheat grain), exposed for 48 hours, in 4 repetitions, for

each gender. The compound 1,8-cineole had lethal effect on the treated pupae at both genders

and in the both treatments. Total proportion of the normally developed beetles was decreased.

In addition, 1,8-cineole had also a growth regulator effect, producing adultoids and deformed

units, with males more susceptible. In the treatment with the grain there were significant lower

dead pupae, normally developed live male beetles and also deformed female units in the stage

2. Results of the proposed that, compound 1,8-cineole has multiple effect against T. castaneum

in pupal stage. Further in one more study conducted by Aggarwal et al. (2001) and determined

the fumigant toxicity of 1,8-cineole, one of the components of the essential oil of Artemisia

annwa, against three stored product coleopterans Callosobruchus maculatus F.(Coleoptera:



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Bruchidae), Rhyzopertha dominica F. (Coleoptera: Bostrychidae) and Sitophilus oryzae

L.(Coleoptera: Curculionidae). The compound was more effective as a fumigant and gave 93%

mortality against all three pest species at the dose of 1.0µL/L air under empty jar conditions as

compared to treatment of jars filled with grain (11.26% mortality).

Ebadollahi and Mahboubi (2011) used the essential oil of Azilia eryngioides (Pau)

against 1 to 7 days old Sitophilus granarius (L.) (Curculionidae) and Tribolium castaneum

(Herbst) (Tenebrionidae) adults. Fumigation bioassays conducted to estimate mean lethal time

LT50 values revealed that A. eryngioides oil had a strong insecticidal activity on adult test

insects that were exposed to 37.03, 74.07, 111.11, and 148.14µL/L. Mortality increased as

concentration and exposure time increased, and reached 100% at the 39 hours of exposure time

and concentrations higher than 111.11µL/L. Jiang (2011) evaluated the essential oil of Evodia

lepta root barks as fumigant against the maize weevil and red flour beetle. Results of this study

revealed that essential oil of E. lepta possessed strong fumigant toxicity against S. zeamais and

T. castaneum adults with LC50 values of 25.05 and 12.09 mg/L air, respectively. Tayoub et al.

(2012) evaluated the fumigants activity of essential oil vapours distilled from Eucalyptus

globulus and Origanum syriacum 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 312μL/L air and

187.5 μL/L air, respectively. The LC50 values of T. granarium larvae were 28.75 and

176.3μL/L air for oregano and eucalyptus oils, respectively. This study proved that the

essential oils from these two plants are effective against T. granarium larvae. In another

research, Talukder and Khanam (2011) observed the fumigant effect of emulsified petroleum

ether extract of Acorous calamus rhizome alone and three separated mixtures of different plants

materials (A. calamus rhizome + Corchorus capsularis seed, A. calamus + Thevetia neriifolia

seed and A. calamus + Zingiber cassumunar rhizome) against adults of Sitophilus oryzae (L.),

Callosobruchus chinensis (L.) and 13 days old larvae of Tribolium castaneum (Herbst.). In this

study, the fumigant toxicity of different combination of plant materials with A. calamus

extracts were assessed at four doses viz. 10, 20, 30 and 40 mg/100 mL volume and for A.

calamus alone was 5, 10, 20 and 30 mg/100 mL volume in space fumigation. Emulsified of A.

calamus rhizome extract was found to be the most effective against T. castaneum larvae and

C. chinensis adults at 24 and 48 hours. The LD50 values for T. castaneum larvae were 19.63

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and 4.08 mg/100 mL volume whereas in case of C. chinensis the LD50 values were 4.63 and

1.04 mg/100 mL volume at 24 and 48 hours, respectively. It was concluded from this study

that combination of A. calamus with Z. cassumunar extract was found to be the most effective

fumigant against S. oryzae adult (16.48 mg/100 ml volume) at 24 hours but it varied at 48 hours

where A. calamus with C. capsularis was the most effective against the beetle (4.08 mg/100

ml volume). Mahmoudvand et al. (2011) investigated the fumigant toxicity of some essential

oils extracted from Rosmarinus officinalis, Mentha pulegium, Zataria multiflora, and citrus

sinensis against adults of stored-product pests, including Tribolium castaneum, Sitophilus

granarius, Callosobruchus maculatus, and Plodia interpunctella. In this study, pure essential

oils were used in glass vials for the bioassay and LC50 values of Citrus sinensis var. hamlin

against T. castaneum, S. granarius, and C. maculatus were 391.28, 367.75, and 223.48μL/L

air after 24 hours, 362.40, 20.45, and 207.17 μL/L air after 48 hours of exposure, respectively.

Furthermore, LC50 values of the fumigant test of C. sinensis and M. pulegium essential oils

against S. granarius were 0.038 and 367.75μL/L air after 24 hours, 0.025 and 320.45 μL L-1

air after 48 h, respectively. On the other hand, LC50 values of R. officinalis and Z. multiflora

on P. interpunctella moths were 0.93 and 1.75 μL/ L after 24 hours. Results showed that among

tested essential oils, C. sinensis had good fumigant toxicity on T. castaneum, S. granarius, and

C. maculatus. In addition, M. pulegium essential oil was stronger than C. sinensis and S.

granarius. Results further indicated that both Z. multiflora and R. officinalis had fumigant

toxicity on P. interpunctella adults. In conclusion, it was indicated that these essential oils have

good fumigant toxicity against stored product pests. Manzoomi et al. (2010) studied the

fumigant toxicity of essential oils from Lavandula officinalis, Artemisia dracunculus and

Heracleum persicum against the adults of Callosobruchus maculates. The results of the study

indicated that the mortality of adults increased with increased concentration and exposure time.

LC50 values for oils from Lavandula officinalis, Artemisia dracunculus and Heracleum

persicum were 41.52, 210.61 and 337.58μL/L respectively. Toxicity of Lavandula officinalis

oil was more than other two plants (LC50 = 41.52 μL/L), but the essential oils from all three

plants were effective against this pest. Therefore, these essential oils were suggested to be used

for Callosobruchus maculates control in stores.

The fumigant toxicity of essential oil extracted from Litsea salicifolia against Sitophilus

zeamais and Tribolium castaneum was tested by Ko et al. (2010) who found that essential oils

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extracted by hydro-distillation method and then analyzed by GC-MS, exhibited fumigant

toxicity to these insects pests. Kumar et al. (2008) determined the potential of using essential

oil from leaves of Aegle marmelos to control insect infestation of stored gram from

Callosobruchus chinensis (L.) and wheat from Rhyzopertha dominica (F.), Sitophilus oryzae

(L.) ) and Tribolium castaneum (Herbst). The results emphasize the efficacy of A. marmelos

oil as fumigant against insect infestations of stored grains and strengthen the possibility of

using it as an alternative to synthetic chemicals for preserving stored grains. Likewise in

another research, Mondal and Khalequzzaman (2006) investigated fumigant toxicity of the

three essential oils, viz., Cardamom (Elletaria cardamomum), Cinnamon (Cinnamomum

aromaticum) and Clove (Syzygium aromaticum) against red flour beetle, Tribolium castaneum

(Herbst) larvae and adults. In fumigation bioassay cinnamon oil provided the highest toxicity

to adult and 10, 14 and 18 days old larvae, with LD50 values of 0.03, 0.05, 0.088 and 0.09

mg/cm respectively. Li et al. (2010) exhibited that Murraya exotica possess insecticidal activity

against the maize weevil, Sitophilus zeamais and red flour beetle, Tribolium castaneum. The

essential oil of aerial parts of M. exotica was obtained by hydrodistillation and investigated by

GC and GC-MS. The Results of the study depicted that essential oil of M. exotica possessed

fumigant toxicity against S. zeamais and T. castaneum adults with LC50 values of 8.29 and

6.84 mg/L respectively. Likewise, Khani and Asghari (2012) conducted an experiment in

which essential oils extracted from the foliage of Mentha longifolia (L.) and Pulicaria

gnaphalodes (Ventenat) and flowers of Achillea wilhelmsii (Koch) were tested in the laboratory

for volatile toxicity against two stored product insects, Tribolium castaneum (Herbst) and

Callosobruchus maculatus (F.). C. maculatus was found to be more vulnerable to the tested

plant products than T. castaneum. The oils of the three plants displayed the same insecticidal

activity against C. maculatus based on LC50 values (between 1.54 μl/L air in P. gnaphalodes,

and 2.65 μl/L air in A. wilhelmsii). Whereas, the oils of A. wilhelmsii and M. longifolia showed

the same strong insecticidal activity against T. castaneum (LC50= 10.02 and 13.05μL/L air,

respectively), the oil of P. gnaphalodes revealed poor activity against the insect (LC50 = 297.9

μl/L air). These results suggested that essential oils from the tested plants could be used as

potential control agents for stored-product insects.

Liu et al. (2010) proved that essential oil of Artemisia lavandulaefolia and Artemisia

sieversiana were found to possess insecticidal activity against the maize weevil Sitophilus zeamais.

40

The essential oils of A. lavandulaefolia and A. sieversiana possessed fumigant toxicity against S.

zeamais adults with LC50 values of 11.2 and 15.0 mg/L air, respectively. Likewise, Chu et al. (2011)

evaluated that essential oils of Chenopodium ambrosioides (L.) found to possess strong insecticidal

activities against the maize weevil Sitophilus zeamais (Motsch.) Essential oil of C. ambrosioides

was obtained by hydro-distillation, and the constituents were determined by GC-MS analysis. The

active compounds were isolated and identified by bioassay-directed fractionation. Five active

compounds [(Z)-ascaridole, 2carene, ρ-cymene, isoascaridole and α-terpinene] were isolated and

identified from the essential oil from Chinese C. ambrosioides. The LC₅₀ values (fumigation) of

the crude essential oils and the active compound (Z)-ascaridole against S. zeamais adults were 3.08

and 0.84 mg L⁻¹ air, respectively. Lim et al. (2011) derived essential oils from 18 species of

Myrtaceae family; further selected two oils to isolate main components of these oils and then tested

their fumigant action eggs and adult females of Tetranychus urticae at three different temperatures

of 5, 15 and 25°C. In this study two major components; piperitone and terpinene-4-ol were derived

from essential oils of Eucalyptus dives and Eucalyptus codonocarpa which exhibited elevated

fumigant action against female adults at 10µL/L at 15 and 25°C. The results obtained from this

study suggested that piperitone must be further explored as a potential fumigant in opposition to T.

urticae. It has been reported in the studies conducted by Sahaf et al. (2007) that essential oil derived

from dry seeds of Carum copticum has a strong insecticidal action and has a potential role as a

fumigant against stored product insects. In this study the chemical composition of C. copticum

essential oil was studied by GC and GC-MS and thymol, -terpinolene and -cymene were

identified as main active components of this essential oil. Results of this study depicted that the

mortalities of the insect species reached 100% at concentrations higher than 185.2 µL/L and 12

hours’ time.

Two main active components diallyl trisulfide and methyl allyl disulfide were isolated from

essential oil of garlic by Huang et al. (2000) and further were tested against Sitophilus zeamais

(Motschulsky) and Tribolium castaneum (Herbst) for their fumigant toxicity. The fumigant

toxicity of diallyl trisulfide was found to be greater as compared to that of allyl disulfide while

both of these compounds exhibited more toxicity against Triboilum castaneum adults than to

Sitophillus zeamais adults. Results exhibited that younger larvae were found to be more

susceptible to fumigant toxicity. The results of study depicted that diallyl trisulfide is more

potent fumigant than methyl allyl disulfide.

41

2.5 Anti-feedant effect of essential oils of indigenous medicinal plants

Feeding deterrence studies were conducted by Huang et al. (2000) to find out

antifeeding effect of the essential oil of Elletaria cardamomum against two stored product

insects, Tribolium castaneum and Sitophilus zeamais. Results of this study revealed that E.

cardamomum oil did not have any feeding deterrence influence on larvae or adults of Tribolium

castaneum. However, it considerably reduced all the nutritional indices for the adults of

Sitophilus zeamais, but with very minor antifeeding impact (27%) at a concentration of

1.44×104 ppm. Similarly in another study conducted by Rao et al. (2005) anti-feedant influence

of seed extract of custard apple in methanol, ethyl acetate and hexane against (7 days) old

larvae of the T. granarim and neonate was evaluated employing a feeding bioassay. The

findings of this study indicated that ethyl acetate extract has highest anti feedant effect whereas

comparatively less anti feeding activities were observed in case of methanol and hexane

extracts. Kumar et al. (2008) carried out a feeding deterrence study to find out the prospective

of employing essential oil from leaves of Aegle marmelos to control insect infestation of stored

gram from Callosobruchus chinensis (L.) and wheat from Rhyzopertha dominica (F.),

Sitophilus oryzae (L.) and Tribolium castaneum (Herbst). It was detected that grain damage

and weight loss was significantly decreased however, an increased antifeeding effect was

observed in fumigated wheat and gram samples infested with all insects excluding Tribolium

castaneum.

The anti-feedant impact of 16 aromatic plant extracts (in organic solvents) of 3 species

belonging to the Asteraceae family in opposition to larvae and adults of Confused flour beetle

was analyzed by Boussaada et al. (2008) and Scorzonera undulata Vahl was found to be more

effective against flour beetle. Likewise, Viglianco et al. (2008) reported the antifeeding impact

(employing wheat wafer disks) of plant extracts of Aloysia polystachia (Griseb), Solanum

argentinum (Bitter et Lillo) and Tillandsia recurvata (L.) in hexane, ethanol and chloroform

against Sitophilus oryzae (L.). The study demonstrated that a stronger anti feeding impact was

shown by Aloysia polystachia compared to Tillandsia recurvata and Solanum argentinum

extracts. The findings of this study also revealed that amongst the extracts evaluated, maximum

antifeeding impact was shown by the chloroform extract of Aloysia polystachia while hexane

extract of Solanum argentinum resulted in strongest repellent impact. In another study Sarwar

42

(2010) reported the anti-feedant effect of leaf powders of Withania somnifera, Ocimum

sanctum, Albizia lebbeck, and Dalbergia sisso at both concentrations i.e. 2% and 5% as

protectants against C. maculatus in seeds of stored chickpea. Promising results were obtained

on chickpea grain samples stored for three months with 2% and 5% concentrations of dried

grounded leaves. Results of this study revealed that grains treated with W. somnifera leaves

showed only 10.00 and 5.00 % weight loss, whereas grains stored with crushed leaves of O.

sanctum, A. lebbeck and D. sisso showed 17.66 and 15.66, 36.66 and 34.66, and 42. 00 and

41.33% weight loss, respectively, with respect to control 46.00 to 48.00%. These findings

suggest that maximum control in chickpea grain losses was achieved in storage with W.

somnifera, O. sanctum and A. lebbeck as compared to control. Du et al. (2011) screened

numerous Chinese mangrove plants for feeding deterrent and illustrated that the ethanol

extracts of Ceriops tagal twigs and stems hold considerable antifeeding activity against red

flour beetle . From the ethanol extract, 3 feeding deterrent diterpenoid compounds (categorized

as tagalsin A, B, and H) were isolated by bioassayguided fractionation and these compounds

were found to have a powerful feeding deterrent activity against Tribolium castaneum adult.

Another study scrutinized that Euphorbia fischeriana roots possess significant anti feeding

activity against two stored product insects i.e., red flour beetle and maize weevil (Geng et al.,

2011). Nenaah (2011) accomplished a study to explore the anti-feedant activity of the glycol-

alkaloid fraction and the two glycolalkaloids i.e., a-solanine and a-chaconine of Solanum

tuberosum, for their antifeedant activity against Trogoderma granarium (Everts). Nutritional

assessments employing the flour disc bioassay were also conducted by Nenaah (2011) which

exposed significant diminution in the growth rate, food utilization and food consumption rate

by Khapra beetle (T. granarium) at concentrations varying between 20 and 30 mg/g food with

feeding deterrent index reaching 82.40%. Similarly, Benzi et al. (2012) used Brazilian pepper

tree (Schinus molle L.) for its nutritional index variation and anti-feedant activity employing a

flour disk bioassay and found that essential oils of this fruit have a powerful antifeeding activity

(62%) whereas leaves have a minor impact (40.60%). Descamps et al. (2011) extracted

essential oils from fruits and leaves of Schinus areira (Anacardiaceae) and tested them for their

feeding deterrence against Tribolium castaneum larvae and adults. A flour disk bioassay for

the antifeeding activity and nutritional index variation was also conducted. Findings of the

study showed that the essential oils produced some changes in nutritional index.





43

Huang et al. (2000) tested the main components of the essential oil of Allium sativum,

methyl allyl disulfide and diallyl trisulfide against Sitophilus zeamais and Tribolium castaneum

for antifeeding effect and found that the growth rate, food utilization and food consumption of

adults of both insect species were significantly decreased by the application of methyl allyl

disulfide. It was reported by Stefanazzi et al. (2012) that essential oils obtained from Tagetes

terniflora (Kunth), Cymbopogon citratus (Stapf.) and Elyonurus muticus (Spreng) have an

antifeeding effect on stored grain pests. The results of this study indicated that Cymbopogon

citratus reduced the competence of alteration of consumed food and comparative growth rate

in Tribolium castaneum larvae. Similarly in another study Ebadollahi (2011) used the essential

oils obtained from Lavandula stoechas and Eucalyptus globules for their antifeedant activity

against adults of Red floor beetle and found that both oils significantly reduced the insect

feeding. It was depicted from the results of the study that these oils represented a dose

dependent anti-feeding action and owing to the enhancement of oil quantity, feeding of

Tribolium castaneum was reduced leading to a conclusion that both of these essential oils have

prospective agents against T. castaneum and perhaps further insect pests.

Lozowicka and Kaczynski (2013) proved in their feeding deterrence studies that

derivatives of a novel compound alpha-asarone have a strong antifeedant activity against stored

products pests. In this study a total of 23 constituents of alpha-asarone were categorized and

synthesized of which 10 compounds exhibited a strong anti-feedant activity against Sitophilus

granarius, Trogoderma granarium. and Tribolium confusum. The results of this study

suggested that occurrence of double bond within side chain and three methoxy groups are

important for anti-feedant action. Two main active components diallyl trisulfide L. and methyl

allyl disulfide were isolated from essential oil of Allium sativum by (Huang et al., 2000) and

further they were tested against Sitophilus zeamais(Motschulsky) and Tribolium castaneum

(Herbst) for their anti-feedant action. Feeding deterrence indices of 27 and 51% were obtained

in Sitophilus zeamais adults and Tribolium castaneum larvae, respectively, at the concentration

of 2.98 mg/g food, whereas feeding deterrence of 85% was achieved in T. castaneum adults at

a much lower concentration of 0.75 mg/g food. The results of study depicted that diallyl

trisulfide is a more potent feeding deterrent than methyl allyl disulfide as it decreased all the

nutritional indices in all of the insects tested.






44

2.6 Growth regulatory influences of essential oils of indigenous medicinal

plants

Growth regulatory effect of plant essential oils was studied by different scientist. In a trial

conducted by Tripathi et al. (2001) the compound1,8-ciineole was applied to filter paper at a

concentration of 3.22-16.10mg/cm2. Results showed that there is significant reduction in the

hatching of T. castaneum eggs and the subsequent survival rate of the larvae. Adult emergence was

also reduced by 1,8-cineole. In another research Tripathi et al. (2001) extracted essential oil from

the leaves of turmeric, Curcuma longa L., and applied for progeny production in three stored-

product beetles, Rhyzopertha dominica, Sitophilus oryzae and Tribolium castaneum. Results

showed that in T. castaneum, the C. longa oil reduced oviposition and egg hatching by 72 and 80%,

respectively at the concentration of 5.2mg/cm2. At the concentration of 40.5mg/g food, the oil

totally suppressed progeny production of all the three test insects. The insecticidal effect of the

azadirachtin-based insecticide, Neem Azal, was examined against adults of Rhyzopertha dominica,

Sitophilus oryzae, and Tribolium confusum on whole oats, and peeled oats by Athanassiou, et al.

(2005). The insecticide was applied at three dose rates, which were equivalent to 50, 100, and 200

ppm of azadirachtin. Adults of the above-mentioned species were exposed to the treated grains at

25C and 65% RH, and mortality was assessed after 24 h, 48 h, 7 d, and 14 d of exposure. Then,

all adults were removed, and the treated substrate remained at the same conditions for an

additional 45 d. After this interval, the grains were checked for progeny production. For all

species, significantly less progeny was recorded in the treated grains than in the untreated

grains, with the exception of T. confusum on oats where offspring were significantly reduced

only at the highest rate. Similarly in another study conducted by Nukenine et al. (2011) in

which they tested the Calneem oil from Ghana and local neem oils from two localities in

northern Cameroon (Garoua and Maroua) were tested at 0 (untreated control), 2, 4, 6, 8 and

12ml/kg, on the adult and immature stages of the maize weevil Sitophilus zeamais for

reproduction inhibition. The neem oils from Cameroon were extracted using the traditional

kneading method and a hydraulic press in the laboratory (refined). Maize grains were coated

with the five neem seed oils (Calneem, Garoua traditional and refined, and Maroua traditional

and refined, respectively). Results exhibited that the oils arrested the development of the hidden

eggs and immature stages in the maize grains. Azadirachtin was also incorporated in wheat

45

flour by Mukherjee and Ramachandran, (1989), which led to reduced growth and survival of

T. castaneum larvae at 1 ppm and above. Fecundity was not affected when fed on azadirachtin

treated flour. Topical application at 1, 2 and 5 ppm on eggs and larvae did not have any adverse

effects but reduced emergence of normal adults when applied on less than 6 h old pupae. In

another study the efficacy of 1, 8 cineole as grain protectant against S. granarius, S. zeamis, T.

castaneum and P. truncates was investigated in the laboratory using grain treatment. 1,8

cineole applied on whole wheat as well as maize grains. Development of eggs and immature

stages within grain kernels as well as progeny emergence were completely inhibited in treated

grain Obeng-Ofori et al. (1997). Similarly Reddy and singh (1998) isolated neem seed volatiles

from need seed oil and evaluated for their bioactivity against eggs, 2- and 15-day-old grubs

and adults of pulse beetle, Callosobruchus maculatus. All stages were found susceptible to

different doses of volatiles. Eggs when exposed to 100200 µl dose for 3 days failed to hatch.

At a 50 µl dose with a 5-day exposure period the eggs that hatched failed to reach adult stage

and terminated as grubs. The adults were found most susceptible followed by grubs and eggs.

The effects of the volatiles were time-dependent. Similar kind of investigations were made by

Tariq (2013) in which they tested the Fine Neem Seed Powder (NSP) in three dozes, viz. 0.5%,

1.0% and 2.0% (w/w) for determination of its toxicity and detrimental effects on life stages of

red flour beetle, Tribolium castaneum. NSP served as an Insect Growth Regulator (IGR). It

was observed that at 0.5% dose, the number of larvae was not very different to control but the

weight of larvae was lesser. At 1.0% dose, the number and weight of larvae were significantly

reduced. At 2.0% dose, both the number and weight of larvae, pupae and adults were reduced

remarkably. The insect growth inhibition was increased by increasing the dose of NSP.

Obeng-Ofori,1995 tested plant oils (cottonseed, soybean, corn, groundnut and palm) at

different dosages in the laboratory for their ability to suppress the populations of Cryptolestes

pusillus and Rhyzopertha dominica in maize and sorghum. A dose of 5 ml/kg of each oil

significantly decreased the progeny produced by R. dominica. In another trial conducted by

Saidana et al. (2010) where several extracts of three Tunisian halophytic plants for an eventual

growth inhibition activity against the serious pest of stored products Trogoderma granarium

responses varied with plant material, extract type, and exposure time. Similarly Mhemed,

(2011) the seed powders of four plants; harmal (Harmal peganum), black pepper (Piper

nigrum), radish (Raphanus sativus) and celery (Apinum graveolens) were tested at the

46

concentrations 2%, 4% and 6% to evaluate their effects on some biological aspects and

mortality of Trogoderma granarium Everts Seed powders had also significant effect on

decreasing average number of F1 progeny. The number of F1 progeny found in celery

treatment at concentration of 6% was zero, followed by pepper 23, radish 31 and harmal 56.

Whereas in the control was 78. The seed powders disrupted the life cycle of the insect resulting

in prolonging the period of F1 adult appearance; the periods were 20 days for control, 26 days

for harmal and radish, no adult appearance in pepper and celery even after 34 days

Upadhyay and Jaiswal (2007) calculated effective concentration (EC50) of P.

nigrum essential oil. It was observed that the concentration to lessen the number of T.

castaneum larvae transformed to pupae to 50 percent was 6.919 μl. The percentage of test insect

larvae transferred to pupae stage and percentage of pupae transferred to adult stage was

significantly decreased with the increase in concentration when compared to the control. Chaubey

(2007) selected essential oils from fruits of Nigella sativa, Anethum graveolens and

Trachyspermum ammi to examine developmental inhibitory activities against T. castaneum.

Essential oils significantly reduced the development of larvae. Median effective concentrations of

essential oil (EC50) that is required to stop the transformation of half of the larval population to

pupae were 5.62, 7.86 and 6.70µl for N. sativa, A. graveolens and T. ammi, respectively. A similar

study was conducted by Ilesanmi and Gungula (2010) to evaluate the growth regulatory effects of

essential oils extracted from the seeds of Moringa oleifera and Azadirachta indica against

Callosobruchus maculatus. Stored cowpea was treated with concentrations of pure essential oil (0.5

to 1.5 ml per 200g of cowpea) and their mixtures. Essential oils significantly reduced oviposition

potential of test insect i.e., application of pure A. indica reduced egg laying potential from 6513

(control) to 8 eggs at 0.5ml/200 g concentration. Total weevil population was recorded even zero

in the treated samples as compared to 4988 in the control.

47

CHAPTER 3

Materials and Methods

3.2.1 Plant Materials

Plant parts from the locally grown medicinal plants Azadirachta indica, Melia

azedarach, Colocynthis citrullus, Nicotiana tabacum and Eucalyptus camaldulensis were

collected from different locations of Faisalabad, Punjab, Pakistan (Longitude 73°74 East;

Latitude 30°31.5 North; Altitude: 184 m). These fresh plant parts were brought to laboratory

of Grain Research, Training and Storage Management Cell, Department of Entomology,

University of Agriculture Faisalabad and dried in shade at room temperature. Dried plant parts

were ground to powder using stone electric grinder (Machine No. 20069, Pascall

Engineering Co. Ltd.). This powder was then sieved through 40 mesh sieve

48

3.2.2 Extraction

Soxhelt extraction apparatus (Model WHM12295, DAIHAN Scientific Co., Ltd.) was

used to prepare essential oils. Soxhelt thimble was filled with 50 g of fine botanical powder

and placed in flask. Acetone was used as solvent in bottom flask. This process of extraction oil

from all plant powders was repeated to achieve enough quantity of essential oil based on the

nature of plant material. Extracted essential oil was then purified by evaporating solvent by

using electric rotary evaporator. Pure extracted essential oils were preserved in glass vials at 4

ºC to prepare the concentrations of 2, 4 and 6% by mixing acetone as solvent. These

concentrations were used for subsequent experiments as given under different chapters

3.2.3 Test Insects

Infested grain samples from Faisalabad Punjab, Pakistan (Longitude 73°74 East;

Latitude 30°31.5 North; Altitude: 184 m), were collected and brought to laboratory. Test insect

population consisted of Rhyzopertha dominica, Tribolium castaneum and Trogoderma

granarium. Collected insects were kept in the jars of 9.5cm diameter and covered with the

muslin cloths. Rearing of the insects was performed in the laboratory for three months to

achieve the uniform populations. Insects were regularly checked for their growth and sieved

and transferred to jars (J) half filled with uninfested wheat grain or wheat flour diet.

Temperature at 30 ± 2 ºC and relative humidity at 65 ± 5% was maintained for insect maximum

growth by using incubator (Model MIR-254, SANYO).

49

CHAPTER 4

REPELLENY OF ESSENTIAL OIL OF FIVE INDIGENOUS MEDICINAL PLANTS

AGAINST THREE STORED GRAIN INSECT PESTS.

ABSTRACT

The present research work was conducted to evaluate the repellent efficiencies of essential oils of

Azadirachta indica, Melia azedarach, Colocynthis citrullus, Nicotiana tabacum and Eucalyptus

camaldulensis against Tribolium castaneum, Rhyzopertha dominica and Trogoderma granarium.

Three concentrations of each essential oil (2, 4 and 6%) were tested by following filter paper area

preference method at ambient temperature of 30 ± 2 °C and 65 ± 5% relative humidity. All essential

oils demonstrated their repellent potential against all test insects. A. indica was found to be the

potent against R. dominica and T. castaneum with highest repellency of 69.62 and 66.66%

respectively while M. azidiractha was most active against T. granarium. Overall effect of the

50

concentration was highly significant and the maximum repellency (66.66, 62.22 and 45.22%) was

observed at 6% concentration followed by 4 and 2% against R. dominica and T. castaneum and T.

granarium respectively. Overall effect of time was also significant but repellency decreased as the

duration of exposure increased maximum repellency (68.88, 62.88 and 48.44%) was observed after

24 hours against R. dominica, T. castaneum and T. granarium respectively.

4.1 Introduction

In Pakistan estimates of storage losses of food grains due to insects have been reported

to vary greatly; 4-10% (Huque et al., 1969), about 5.08% (Chaudhry, 1980), up to 5% (Ahmad,

1984), 3.5 – 25.5% (Irshad and Balouch, 1985) about 2-6% by (Avesi, 1983) more than 2.5%

(Ahmad et al.,1992) more than 10-15 % (Jilani, 1980) The storage losses are mainly caused by

insect pests like Rhizopertha dominica, Trogoderma granarium, Tribolium castaneum, T.

confusum, Sitophilus oryzae, S. granarius, Sitotroga cerealella, Callosobruchus chinesis, C.

analis and many others including rodents and birds (Ashfaq et al., 2001).

The control of insect pests is primarily dependent upon continuous application of

insecticides and fumigants. Excessive use of pesticides has given rise to many serious problems

to the environment and the development of resistance (Champ and Dye, 1977; Irshad and

Gillani, 1989, 1991; Irshad et al., 1992; Irshad and Iqbal, 1994). The problems caused by

pesticides and their residues have increased the need for effective biodegradable pesticides

51

with greater selectivity. Essential oils having compounds monoterpenoids, offer promising

alternatives to classical repellents (Saim and Meloan, 1986; Ndungu et al., 1995).

Plants provide potential alternatives to currently used insect control agents. In the past,

few indigenous plants of Pakistan were studied for repellent effects on red flour beetle (Jilani

et al., 1989, 1991, 1993) and for their repellent and feeding deterrent effects against lesser grain

borer (Jilani and Saxena, 1990). Chemicals that prevent insect damage to plants or animals by

rendering them unattractive or offensive are called repellents. There are two broad types of

repellents. Physical repellents produce repellency by physical means and are of contact stimuli,

auditory, barrier, visual, excitatory and anti-feeding actions. Chemical repellents affect tactile,

olfactory or gustatory receptors of insects and could be of plant origin or synthetic (Mahulikar

and Chavan, 2007).

The present investigation has been carried out with a view to assess the chemical repellent

effect of plant species viz. Azadirachta indica, Melia azedarach, Colocynthis citrullus, Nicotiana

tabacum and Eucalyptus camaldulensis against Tribolium castaneum, Rhyzopertha dominica and

Trogoderma granarium. These plants are aboriginal, abundantly available and possess medicinal

properties. Some of these plants have also been found effective as repellent against stored grain

insect pests. (Khan and Marwat, 2004) Mordue and Blackwell, 1993; Abbasipour et al., 2009

(Tiwari et al., 1995)

4.2 Materials and Methods.

The procedure for collection and rearing of test insects and extraction of essential oils is as given

in chapter 3

4.2.1 Bioassay

Area Preference Method was followed for the evaluation of repellant activities of the essential oils

of Azadirachta indica, Melia azedarach, Colocynthis citrullus, Nicotiana tabacum and Eucalyptus

camaldulensis (Tapondjou et al., 2005; Olivero et al., 2009; Olivero-verbel et al., 2010; Caballero-

gallardo et al., 2011). Filter papers of 9 cm (Whatman No.1) were used as test area after cutting

into two equal halves. One half of the filter paper was treated as uniform as possible with 0.5ml of

each concentration (2,4 and 6 %) of essential oil and second half was treated with acetone alone

(0.5ml) as control. The filter papers were allowed to air dry for 10 minutes to evaporate the solvent.

52

Both the half filter papers of acetone and essential oil were rejoined to make a full disc using

adhesive tape. These treated and re-attached halves were placed in 9 cm glass petri dish. 20 test

insect from mixed-sex population of Rhyzopertha dominica (adult), Tribolium castaneum (adult)

and Ttogoderma granarium (larvae) were released at the center of each filter paper disc and covered

the petri dishes with lid to overcome the escape of insect. Then petri dishes were kept in incubator

at 30 ± 2 °C and at 65 ± 5 % R.H. 3 replications were performed for each concentration of essential

oils. The numbers of test insects present on the untreated and treated portions of the filter paper

disc halves in each concentration were recorded after 24, 48 and 72 hours of exposure.

The following formula for the calculation of percentage repellency (PR) was used as described

by Asawalam et al. (2006):

PR =

Nc = number of insects on the control (untreated) area.

Nt = number of insects on the treated area.

4.2.2 Statistical Analysis

The collected data were subjected to the Analysis of Variance (ANOVA) using Statistical

software (Stat Soft, 8.0). Significant differences among the treatments were determined by

Tukey’s HSD tests (p ≤ 0.05).

53

4.3 Results

All essential oils showed significant repellency against T. castaneum, R. dominica and T.

granarium. Results regarding to the mean repellency of essential of essential oils against T.

castaneum showed that A. indica (66.66%) has the strongest repellant properties followed by

M. azedarach (61.11%), C. citrullus (41.11%), N. tabacum (36.85%) and E. camaldulensis

(32.96%), respectively. While in case of R. dominica, it was observed that A. indica (69.62%)

has the strongest repellant properties followed by M. azedarach (66.29%), N. tabacum

(53.33%), C. citrullus (49.62%) and E. camaldulensis (32.96%). Results regarding to the

repellency of plant essential oils against T. granarium, gave the clear picture that M. azedarach

(52.22%) has the strongest repellant properties followed by A. indica (49.62%), E.

camaldulensis (28.88%), C. citrullus (28.51%) and N. tabacum (18.41%) (Fig.4.1). Overall

effect of time was highly significant. Results showed that with the passage of time repellency

decreased. Repellency was highest after 24 hours (66.88, 62.88 and

48.44%) followed by 48 hours (53.11, 47.44 and 32.44%) and 72 hours (43.11, 32.88 and

25.11%) against R. dominica, T. castaneum and T. granarium respectively (Fig.4.2). Overall

effect of concentration for all plants was highly significant. It was observed that there is

positive correlation between dose rate of toxicants and repellency of insect i.e. increased with

increase of doses. Highest repellency was obtained at 6% (66.66, 61.22 and 45.55%) followed

by 4% (54, 46.44 and 34.88%) and 2% (44.22, 35.55 and 26%) against R. dominica, T.

castaneum and T. granarium respectively (Fig. 4.3). Data given in table 4.1 described

interaction of plant and time. Results showed that in case of T. castaneum after 24 hours of

treatment highest repellency was found against M. azedarach (82.22%) which was statistically

similar with A. indica (74.44%) least repellency was observed against E. camaldulensis

(17.77%) after 72 hours of treatment which was statistically similar with C. citrullus (21.11%)

and N. tabacum (22.22%). After 48 hours of treatment highest repellency was shown by M.

azedarach (64.64%) which was statistically at par with A. indica while repellency of N.

tabacum (38.33%) was at par with E. camaldulensis (32.22%) and C. citrullus (43.33%). In

case of R. dominica results showed that after the 24 hours of treatment same highest repellency

(80%) was observed in M. azedarach and A. indica which was statistically different from rest

of the plant essential oils while repellency C. citrullus

54

(64.44%) and N. tabacum (58.88%) was statistically at par with each other but different from

E. camaldulensis (46.66%). Least repellency was observed in E. camaldulensis (21.11%) after 72

hours of treatment which was statistically different with all other plant essential oils. After 48 hours

of treatment highest repellency was shown by A. indica (67.70%) which was statistically at par M.

azedarach (65.55%)while repellency of N. tabacum (47.77%) was at par with C. citrullus (53.33%).

Repellency of E. camaldulensis (25.55%) was significantly different from rest of the plant essential

oil in this treatment. Effect of plant and time against T. granarium showed that highest repellency

was found in case of M. azedarach (63.33) which was statistically similar with A. indica (62.22)

after 24 hours of treatment followed by C. citrullus (44.44%) and E. camaldulensis (41.11%) which

were statistically similar while least repellent effect was found against N. tabacum (31.11) which

was statistically different with all other plants. After 72 hours of the treatment least repellency was

observed against N. tabacum (8.88%) which was statistically different from all other plants at all

exposure intervals. Data given in table 4.2 shows the interaction of plant essential oil and

concentration. Results showed against T. castaneum that highest repellency was observed at 6%

concentration against A. indica (83.33%) followed by M. azedarach (73.33%), C. citrullus

(55.55%), N. tabacum (50.55%) and E. camaldulensis (43.33%). Repellency at 6 % concentration

of A. indica was significantly different from M. azedarach and all other plants, while repellency of

C. citrullus and N. tabacum was statistically at par with each other. Least repellency was observed

against E. camaldulensis (24.24%) at 2% concentration which was significantly different from all

other plants. In case of R. dominica results showed that highest repellent activities (81.11%)

observed at 6% were statistically similar in A. indica and M. azedarach followed by C. citrullus

(63.33%), N. tabacum (62.22%) which were statistically at par and the repellency was least against

E. camaldulensis (45.55%) was statistically different from other plants. Least repellency was

observed against E. camaldulensis (20%) at 2% concentration which was significantly different

from all other plants.

Data indicate in table 4.3 described the interaction of time and concentration of plant essential oil

against T. castaneum. In general, it was observed that there is positive relation between repellency

and doses, i.e., repellency increased with increase of doses; however, a negative correlation was

found between repellency and time, i.e., repellency decreased with increase of exposure period in

all essential oils. Results showed that at highest level of concentration i.e., 6% and minimum time

55

period i.e., 24 hours repellency (73.33%) was maximum, while minimum repellency (23.33%) was

observed at lowest level of concentration i.e., 2% and maximum exposure duration i.e., 72 hours.

4.4 Discussion

From the results, it was observed that the oils from A. indica, M. azedarach, C.

citrullus, N. tabacum and E. camaldulensis showed different repellent activity against T.

castaneum, R. dominica and T. granarium. This may because of the active compounds of the

plants and the sensitivity of the insect to the plant oils. This was supported by Ngamo et al.

(2007), who reported that the essential oils of different plants did not have same efficiency on

all the pests considered in their study and the insects had different sensitivity according to the

oils. The obtained results expressed that A. indica proved to be most efficient toxicant against

R. dominica and T. castaneum are in agreement with the research conducted by Mohiuddin et

al. (1993) who found that among the 12 plant essential oils tested against T.castaneum, oil of

A. indica showed 80-100 percent repellent activity.

According to the susceptibility of test insects, pattern observed was R. dominica > T. castaneum

> T. granarium. Similar conclusion was drawn by Ukeh (2008) who investigated the repellent

action of Aframonum melegueta and Zingiber officinale in a four-armed airflow oflectometer

and found that 10 microliter of both crude oil significantly repelled R. dominica. These results

are contradictory to those of studies conducted by Kumar et al. (2003) who illustrated protein

rich fraction obtained from peas (var. Bonneville) at 1% concentration depicted that T.

castaneum was repelled more rapidly pursued S. oryzae and R. dominica

The findings of this research suggested that essential oils remarkably repel the stored

grain insect pests. This make them useful stored product insect pest protectant however many

factors influence their efficiency i.e. concentration and exposure time were major factors.

These factors have been indicated and pointed out in some earlier studies (Tunc and Erler,2003;

Ogendo et al., 2008; Sattar et al., 2010 and Mishra and Tripathi, 2011).

Treatments with low concentrations resulted in less repellency 2% concentration

exhibited less repellency 42.44, 35.55 and 26.00% which significantly increased to 66.66,

62.22 and 45.22% at 6 % essential oil concentration against R. dominica, T. castaneum and T.

granarium respectively (Fig.2.3). Similar trend was observed by Pugazhvendan et al. (2012)

who reported more repellency with 5 % concentration than 2.5 %. These results are also in

56

accordance with the study conducted by Shah et al. (2008) who evaluated the repellant action

of leaves of six indigenous plants at 2.5, 5.0, 7.5, and 10.0% concentrations he found that the

rate of repellency increased with the increase of dose level. At 10.0% dose level all plant

extracts showed the highest repellency rate and were in repellency class III.

Exposure time also significantly affected percentage repellency. Repellency was the highest after

24 hours (68.88, 62.88 and 48.44%) followed by 48 hours (53.11, 47.44 and 32.44%) and 72 hours

(43.11, 32.88 and 25.55%) against R. dominica, T. castaneum and T. granarium respectively

(Fig.4.2). This result confirms by the findings of Tunc and Erler (2003) who declared in their insect

repellency studies that repellent activity declined steadily with increasing time. These results are

also in accordance with the trial conducted Trongtokit et al. (2005) who evaluated that the

protective effectiveness of essential oils dissipates rapidly with the passage of time which may be

because of their high volatility. These results are contradictory to some extent with those of studied

by Manzoor et al. (2011) who evaluated repellency of D. stramonium against T.castaneum and

found no significant difference at 24 (73.33%) and 48hr (73.33%) however repellency was

decreased to 63.33% after 72hr of treatment.

57

Insects

Fig.4.1 Mean repellency (%) of essential oils of M. azedarach, A. indica, E. camaldulensis,

N. tabacum and C. citrullus against T. castaneum, T. granarium and R. dominica

b b

a

b

b

a

a

b

a a

c

c

c

c

c

0

20

40

60

80

100

A. indica M. azedarach E. camaldulensis N. tabacum C. citrullus

Plants

T. castaneum R. dominica T. granarium

58

Exposure time

Fig. 4.2 Mean repellency (%) of essential oils of M. azedarach, A. indica, E. camaldulensis,

N. tabacum and C. citrullus against T. castaneum, T. granarium and R. dominica at different

exposure times

Concentrations

2% 4% 6%

T. castaneum R. dominica … T. granarium

Fig. 4.3 Mean repellency (%) of essential oils of M. azedarach, A. indica, E. camaldulensis, N.

tabacum and C. citrullus against T. castaneum, T. granarium and R. dominica at different

concentrations

c

c

c

b

b

b

a

a

a

0

20

40

60

80

c

c

c

b b

b

a a

a

0

10

20

30

40

50

60

70

80


Insects

72 48 24

Table 4 Percentage repellency (Mean ± SE) of

59

.1 T. castaneum (F(8,90) = 84.4, p = 0.00003),

T. granarium (F(8,90) = 54.3, p = 0.010059) and R. dominica (F(8,90) = 6.21, p = 0.00002) against

different exposure times of essential oils.

Plants Time

(hours)

Repellency (%)


A. indica 24 74.44 ± 4.12 b 80.00 ± 4.44 a 62.22 ± 3.64 a

48 58.88 ± 4.40 cd 67.77 ± 2.77 b 46.66 ± 3.33 bc

72 53.33 ± 4.08 de 64.44 ± 3.33 c 40.00 ± 2.88 c

M. azedarach 24 82.22 ± 3.51 a 80.00 ± 5.00 a 63.33 ± 3.33 a

48 64.44 ± 2.88 c 65.55 ± 4.44 bc 53.33 ± 3.33 b

72 50.00 ± 5.47 e 53.33 ± 4.08 de 40.00 ± 2.88 c

E. camadulensis

24

48

48.88 ± 3.09 ef

32.22 ± 2.22 h

46.66 ± 5.00 e

31.11 ± 3.51 f

41.11 ± 2.60 c

25.55 ± 2.93 de

72 17.77 ± 3.64 i 21.11 ± 3.09 g 20.00 ± 2.88 ef

N. tabacum

24

48

50.00 ± 4.08 e

38.33 ± 4.56 hi

58.88 ± 3.09 cd

53.33 ± 2.77de

31.11 ± 3.51 d

14.44 ± 2.93 fg

72 22.22 ± 3.64 i 47.77 ± 4.08 e 8.88 ± 3.09 g

C. citrullus

24

48

58.88 ± 4.54 cd

43.33 ± 4.40 gh

64.44 ± 4.44 bc

47.77 ± 2.77 e

44.44 ± 4.44 c

22.22 ± 2.22 e

72 21.11 ± 3.51 i 36.66 ± 4.08 f 18.88 ± 3.51 ef

Means within the same column followed by the same letter are not significantly different. ANOVA, HSD (P < 0.05)


60

.2 T. castaneum (F(8,90) = 3.66, p = 0.000983)

and R. dominica (F(8,90) = 41.3, p = 0.039443) against different concentrations of essential

oils.

Plants Conc. (%) Repellency(%)

T. castaneum R. dominica

A. indica 2 52.22 ± 3.64 ef 58.88 ± 3.51 cd

4 64.44 ± 5.03 c 68.88 ± 4.23 b

6 83.33 ± 4.40 a 81.11 ± 5.12 a

M. azedarach 2 48.88 ± 3.09 fg 51.11 ± 3.51 ef

4 61.11 ± 3.51 cd 66.66 ± 4.08 b

6 73.33 ± 4.40 b 81.11 ± 4.54 a

E. camadulensis

2

4

24.44 ± 5.03 l

35.55 ± 4.54 ij

20.00 ± 2.88 i

33.33 ± 3.72 h

6 43.33 ± 4.40 gh 45.55 ± 5.03 ef

N. tabacum

2

4

24.44 ± 4.12 l

35.55 ± 4.12 ij

44.44 ± 1.75 fg

52.22 ± 2.22 de

6 50.55 ±4.74 ef 63.33 ± 2.35 bc

C. citrullus

2

4

27.77 ± 4.93 kl

40.00 ± 5.77 ih

37.77 ± 4.00 gh

48.88 ± 4.23 ef

6 55.55 ± 6.03 ed 62.22 ± 4.64 cb



61

.3 T. castaneum (F(4,90) = 45.2, p = 0.015339) and R. dominica (F(4, 90) = 77.90, p =

0.004799) against different concentrations and exposure times of essential oils.

Time

(hours)

Conc. (%) Repellency (%)

T. castaneum R. dominica

24

2

4

48.66 ± 3.06 f

62.66 ± 3.83 e

52.66 ± 3.71 e

66.00 ± 3.87 c

6 77.33 ± 4.19 a 82.00 ± 3.92 c

48

2

4

36.66 ± 3.18 b

44.66 ± 3.50 f

42.66 ± 3.58 d

52.66 ± 3.83 e

6 61.00 ± 3.65 e 64.00 ± 3.75 c

72

2

4

21.33 ± 4.12 d

32.00 ± 4.04 c

32.00 ± 3.67 b

43.33 ± 3.47 d

6 45.33 ± 4.45 f 54.00 ± 3.75 e



62

63

CHAPTER 5

CONTACT TOXICITY OF ESSENTIAL OIL OF FIVE INDIGENOUS MEDICINAL

PLANTS AGAINST THREE STORED GRAIN INSECT PESTS.

ABSTRACT

The present research work was conducted to evaluate the contact toxicity of essential oils of

Azadirachta indica, Melia azedarach, Colocynthis citrullus, Nicotiana tabacum and

Eucalyptus camaldulensis against three stored grain insect pests i.e., Tribolium castaneum,

Rhyzopertha dominica and Trogoderma granarium. Three concentrations of each essential oil

(2, 4 and 6%) were tested after 48,72 and 96 hours of exposure to check the contact mortality

at ambient temperature of 30 ± 2 °C and 65 ± 5% relative humidity. All essential oil

demonstrated toxicities against all test insects. A. indica proved most effective against R.

dominica and T. castaneum with the highest mortality 66.13 and 58.08% respectively, however

N. tabacum was most effective against T. granarium with maximum mortality 67.8%. Overall

effect of concentration was highly significant. At 6% concentration the mortality (63.10, 56.07

and 56.16%) was maximum against R. dominica, T. granarium and T. castaneum respectively.

Similarly longest exposure period of 96 hours showed highest mortality followed by 72 and 48

hours against all test insects

64

5.1 Introduction

Insects are a problem in stored grain throughout the world because they reduce the

quantity and quality of grain. The stored pest red flour beetle, Tribolium castaneum (Herbst) is

a major pest in anthropogenic structures used for the processing and storage of grain based

products (e.g. flour mills, warehouses, retail stores). This species has a long association with

human stored food and has been found in association with a wide range of commodities

including grain, flour, peas, beans, nuts, dried fruits and spices (Pugazhvendan et al., 2009).

The Khapra beetle, Trogoderma granarium (Coleoptera; Dermestidae) is considered to be one

of the most serious pests of stored grain products, various leguminous crops, rice, oat, barley,

and rye throughout the world (Lowe et al., 2000). Losses caused by Trogoderma granarium

have been reported to range from 0.2 to 2.9% over a period of 1 to10.5 months (Irshad et al.,

1988). Rhyzopertha dominica (F.) (Coleoptera; Bostrichidae) is one of the main pests

associated with stored grains due to its high biotic potential and to its broad host range since it

can attack wheat, barley, rice and oat (Lorini et al., 2002) what increase the need for efficient

control.

Control of stored-product insect populations is primarily dependent upon continued

applications of insecticides (White and Leesch, 1995). In spite of its efficacy, their repeated

use for several decades has disrupted biological control system by natural enemies and led to

outbreaks of insect pests, widespread development of resistance, undesirable effects on

nontarget organisms, and environmental and human health concerns (White and Leesch, 1995;

Viljoen,1990; Subramanyam and Hagstrum, 1995; Ghanem and Shamma, 1995).

These problems have highlighted the need for the development of new types of

selective insect-control alternatives. Plants may provide potential alternative to currently used

insect-control agents because they constitute a rich source of bioactive chemicals (Wink,

1993). Since these are often active against a limited number of species including specific target

insect, they are often biodegradable to nontoxic products, potentially suitable for use in

integrated pest management, and they could lead to the development of new classes of safer

insect-control agents. Much effort has, therefore, been focused on plant-derived materials for

potentially useful products as commercial insect-control agents. Little work has been done to

65

manage stored-product insects by using aromatic medicinal plants despite their excellent

pharmacological actions.( Tang and Eisenbrand, 1992; Namba, 1993)

This work was carried out to evaluate the insecticidal potential of essential oils of

indigenous medicinal plant species viz. Azadirachta indica, Melia azedarach, Colocynthis

citrullus, Nicotiana tabacum and Eucalyptus camaldulensis against three stored grain insect

pests i.e., Tribolium castaneum, Rhyzopertha dominica and Trogoderma granarium These

plants are aboriginal, abundantly from under laboratory conditions.

5.2 Materials and Methods

The procedure for collection and rearing of insects and extraction of essential oils is as given

earlier in chapter 3

5.2.1 Bioassay

The contact bioassay of essential oils was evaluated by using Filter paper method. Aliquots of

the solutions of the essential oil were applied on the Whatman No 1 Filter papers. 2,4 and 6%

of concentrations of essential oil were used. Control was treated with acetone alone. Treated

filter papers were be kept in air and allowed to evaporate the solvent (acetone) for 10 min.

Treated filter papers were placed in glass Petri dishes and 20 test insects were released in each

Petri dish. These Petri dishes were closed with glass lid and sealed with plastic tape to prevent

the insects from escape. The Petri dishes were kept in incubator (Model MIR-254, SANYO) at

25 ± 2 °C and 65 ± 5 % relative humidity. Number of dead insects was recorded in each

concentration after 48, 72 and 96 hours of treatment.

5.2.3 Post treatment buildup of population

To assess the population buildup effect the survivor specimens from contact toxicity after 96

hours of exposure were released in glass jars for 60 days (volume: 25 cc) onto 50 g of the same

food commodity used in the rearing of insect pests. Three replications for each of the three

tested concentrations and for control treatments were carried out. In order to record the F1 and

F2 generations growth, the population build up levels (i.e., the number of living specimens

creating the colonies) was recorded after 30 and 60 days, respectively. This experiment was

66

conducted in the same environmental conditions under which the toxicity trials were carried

out.


The data were arranged in tabulated form. The mortality (%) was corrected by

Abbots’s formula:

Corrected Mortality

where, Mo = Observed mortality, Mc = Control mortality

The collected data was subjected to Analysis of Variance (ANOVA) using Statistica software

(Stat Soft, 8.0). Means are separated by the Tukey's multiple range test when ANOVA was

significant (p < 0.05).

5.3 Results

Contact toxicity of five plant essential oils was evaluated against T. castaneum, R.

dominica and T. granarium. Result exhibited that in this experiment all the essential oils of

indigenous medicinal plants showed significant contact toxicity against T. castaneum. Highest

mean mortality (58.04 %) was observed against A. indica followed by M. azedarach (47.1%),

C. citrullus (43.18%), N. tabacum (42.72%), and E. camaldulensis (35.11%). Mean contact

mortality of all plant essential oils was statistically different from each other. In case of R.

dominica result exhibited A. indica was proved most toxic plant with highest mortality (67.8%)

followed by M. azedarach (56.22%), C. citrullus (50.28%), N. tabacum (45.32%) and E.

camaldulensis (43.79%). In case of T. granarium result exhibited that all the essential oils of

indigenous medicinal plants showed significant contact toxicity. N. tabacum proved most toxic

plant with highest mortality (67.8% %) followed by A. indica (51.22%), C. citrullus (41.33%),

M. azedarach (40.57%) and E. camaldulensis (31.06%). Mean contact mortality of all plant

essential oils was statistically different from each other (Fig 5.1). Effect of exposure time was

67

also observed which revealed that as the duration of exposure increase the mortality of test

insect was also increased. After 96 hours mortality was (63.33, 57.41 and

55.44%) followed by 72 hours (51.22, 46.23 and 44.63%) and 48 hours (41.29, 35.85, 35.24%)

against R. dominica, T. granarium and T. castaneum respectively (Fig.5.2). Effect of

concentration was highly significant with positive correlation i.e. as the dose rate of essential

oil increases the toxicity towards test insects was also increased. Mortality of test insect pests

was maximum at highest concentration and it was (63.10, 56.07 and 56.16%), (52.71, 47.71

and 45.20%) and (40.04, 35.61 and 34.43%) against R. dominica, T. castaneum and T.

granarium at 6, 4 and 2 % concentration of essential oils respectively. Toxic effect was

significantly different at all concentrations (Fig.5.3). Data given in (Table 5.1) revealed the

interaction of plant essential oil and exposure time. Results showed that in case of T.

castaneum, A. indica proved most lethal after 96 hours of the treatment with mortality (63.21%)

which was statistically different from M. azedarach (59.19%) while mortality against N.

tabacum (52.30%) was statistically at par with C. citrullus (54.54%), and E. camaldulensis

(49.90%). Least mortality was recorded against E. camaldulensis (23.15%) after 24 hours of

treatment which was statistically different from all other treatments while highest mortality at

this time interval was against A. indica (52.30%) almost similar in case of C. citrullus (32.38%)

and N. tabacum (32.26%) which was statistically at par with M. azedarach (36.20). The

interaction of plant essential oil exposure time for the mortality of R. dominica showed that A.

indica was most lethal after 96 hours of the treatment with mortality (72.10%) which was

statistically different from M. azedarach (66.60%) while mortality against N. tabacum

(61.00%) was statistically at par with C. citrullus (60.57%), and E. camaldulensis (54.40%).

Least mortality was found against N. tabacum (31.75%) after 24 hours of treatment which was

statistically different from all other treatments while highest significant mortality at this time

interval was in A. indica (55.60%). In case of T. granarium results showed that N. tabacum

was proved most lethal after 96 hours of the treatment with mortality (79.54%) followed by A.

indica (61.49%) which was statistically different from each other and all other toxicants while

M. azedarach (52.57%) and C. citrullus (51.43%) exhibited statistically similar toxicities,

mortality was least in E. camaldulensis (42.04%) and significantly different. After 24 hours

of exposure time least mortality was found in E. camaldulensis (23.15%) which was

statistically different from all other treatments while highest mortality observed at this time

68

interval was against A. indica (61.49%). Data tabulated in (Table 5.2) described the effect of

plant and concentration on the mortality of T. castaneum. Results exhibited that maximum

mortality (70.11%) was obtained against A. indica at 6% concentration which was significantly

different from all other plant essential oils while mortality was same against C. citrullus

(54.54%) and N. tabacum (54.03%) these both mortalities are statistically at par with M.

azedarach (56.32%) and E. camaldulensis (45.35%). At 2 % concentration least mortality was

found in E. camaldulensis (25.43%) which was statistically different from all other toxicants.

In case of T. granarium effect of plant and concentration on the mortality showed that

maximum mortality (70.11%) was obtained against N. tabacum at 6% concentration which was

significantly different from A. indica (63.21%) while mortality was statistically at par against

C. citrullus (51.43%) and azidirach (48.57%) while E. camaldulensis (38.06%) was found least

toxic. At 2 % concentration least mortality was found against E. camaldulensis (22.72%) which

was statistically different from all other toxicants. Data given in (Table 5.3) described the effect

of time and concentration. Results revealed that there is direct relationship between exposure

time and concentration with toxicity as the exposure time and concentration of toxicant

increases the mortality of test insect increases and vice versa. Mortality was highest (67.44%)

at 6 % and 96 hours exposure duration while least (45.88%) at 2 % concentration and 24 hours

exposure time

Highest reduction in buildup of population in case of T. castaneum (65 %) was observed

against A. indica followed by M. azedarach (60.55%), C. citrullus (55.55%), E. camaldulensis

(53.88%) and N. tabacum (51.11%), . Mean reduction in population buildup of all plant

essential oils was statistically different from each other. In case of R. dominica result exhibited

M. azedarach was proved most toxic plant with highest reduction in population buildup

(64.44%) followed by N. tabacum (62.22%), C. citrullus (61.66%), A. indica (60.55%) and E.

camaldulensis (53.88%). In case of T. granarium result exhibited that all the essential oils of

indigenous medicinal plants showed significant reduction in population buildup N. tabacum

proved most toxic plant with reduction in population buildup (80%) followed by M. azedarach

(62.77%), A. indica (53.88%), C. citrullus (52.77%), and E. camaldulensis (43.33%) (Fig 5.4).

Effect of concentration was significant as the dose rate of essential oil increases the reduction

in buildup of population towards test insects was also increased. Reduction in buildup of

population of test insect pests was maximum at highest concentration and it was (71, 68.66 and

69

73.33%), (62, 60.33 and 57%) and (50, 47.33 and 47%) against R. dominica, T. granarium and

T. castaneum at 6, 4 and 2 % concentration of essential oils respectively. Toxic effect was

significantly different at all concentrations (Fig.5.4).

5.4 Discussion

This experiment was conducted to evaluate contact insecticidal bioactivities of A.

indica, M. azedarach, C. citrullus, N. tabacum and E. camaldulensis against T. castaneum, R.

dominica and T. granarium. All the essential oils tested were found significantly lethal to test

insects at all exposure times and concentration levels. A. indica was proved most efficient

toxicant with high mortality (67.8 and 58.04 %) followed by M. azedarach (56.22, 47.1%),

C. citrullus (50.28, 43.18%), N. tabacum (45.32, 42.72%), and E. camaldulensis (43.79,

35.11%) against R. dominica and T. castaneum respectively (Fig.3.1). This study was in

agreement with the findings of Mamun et al. (2009) evaluated the toxicity of six botanicals,

Bazna (Zanthoxylum rhetsa), Ghora-neem (Melia sempervirens), Hijal (Barringtonia

acutangula), Karanja (Pongamia pinnata), Mahogoni (Swietenia mahagoni) and Neem

(Azadirachta indica) against Tribolium castaneum Herbst. Among them, A. indica seed extract

showed the highest toxic effect (mortality, 52.50%), similar results were documented by a trail

conducted by Tripathi et al. (2002) in which they investigated the contact toxicity of essential

oil extracted from the leaves of turmeric, Curcuma longa L., against three storedproduct

beetles, Rhyzopertha dominica F. (lesser grain borer), Sitophilus oryzae L. (rice weevil), and

Tribolium castaneum Herbst (red flour beetle). It was illustrated that the adults of R. dominica

were highly susceptible to contact action of C. longa leaf oil, with LD50 value of 36.71

microg/mg weight of insect. In case of T. granarium the order of mortality 67.8, 51.22, 41.33,

40.57 and 31.06% against N. tabacum, A. indica, C. citrullus, M. azedarach, and E.

camaldulensis respectively (Fig. 5.1). Almost similar mortality against T. granarium was

obtained in the research performed by Al-Moajel who prepared powders from parts of different

plant species and found 62% mortality at 6% concentration. These results obtained in this

experiment was strongly supported by the findings of Negahban et al. (2006); Ayvaz et al.

(2010); Alzogaray et al. (2011) they suggested that insecticidal activity of the essential oils

usually varies depending on the stage of the insect, the species, and the plant origin of the

essential oil that would be attributed to the diverse chemical composition of the oils and the



70

interactions that may occur between the individual components of the mixture. The insecticidal

activities of N. tabacum, A. indica, C. citrullus, M. azedarach, and E. camaldulensis was

confirmed by Mohal (2006); Weaver and Subramanyam, (2000); Nadeem et al., 2012; Khan

and Marwat, (2004); Izakmehri et al (2012) respectively.

Mortality of T. castaneum, R. dominica and T. granarium significantly increased with

the dose rate of essential oils. Mortality of test insect pests was maximum at the highest level

of concentration. Mortality was (63.10, 56.07 and 56.16%), (52.71, 47.71 and 45.20%) and

(40.04, 35.61 and 34.43%) at 6, 4 and 2 % concentration of essential oils against R. dominica,

T. granarium and T. castaneum respectively (Fig. 5.3) Similar results were obtained in the

experiment performed by Gallardo et al. (2012) they studied contact toxicity of Cymbopogon

martini, Cymbopogon flexuosus and Lippia origanoides against T. castaneum by using filter

paper methods. Maximum 20% mortality was documented at highest concentration (1.2

mL/cm2). In most recent studies Zia et al. (2013) found efficacy of essential oils extracted from

peel of various citrus species at four concentrations of each oil; 2, 4, 6 and 8% the results

revealed that the essential oil vapors showed variable toxicity to insects depending on

concentration and exposure duration. Similar effect of concentration on the mortality of stored

grain insect pests was confirmed by Omar et al. (2012) their findings proved that ethanol plant

extracts increased the mortality rate by increasing concentration.

Percentage of mortality recorded was significantly high when insects were kept in

contact with essential oil for longer period of time. Longest exposure time of 96 hours showed

highest mortality followed by 72 and 48 hours against all test insects (Fig. 5.2) these results

were similar with the studies conducted by Lu and Wu (2010) they demonstrated that contact

toxicity of Ailanthus altissima (Swingle) (Sapindales: Simaroubaceae) bark essential oil

against S. oryzae adults enhanced with increased exposure time and the corrected percentage

mortality reached 76.5% after 72 hours of treatment. Similar effect of exposure time on the

mortality of stored grain insect pests was confirmed by Omar et al., (2012) their findings

proved that ethanol plant extracts showed that the mortality rate increased with increasing

concentration as well as the length of exposure time. Zia et al. (2013) found efficacy of essential

oils extracted from peel of various citrus species at four exposure durations of 24, 48, 120 and

71

168 hours were assayed for their insecticidal activity. The results revealed that the essential oil

vapors showed variable toxicity to insects depending on concentration and exposure duration.

72

insects

Tribolium castaneum Rhyzopertha dominica Trogoderma granarium

A. indica M. azedarach E. camaldulensis

N. tabacum C. citrullus

Plants

Fig. 5.1 Mean contact mortality (%) of T.castaneum, T. granarium and R. dominica against

essential oils of M. azedarach, A. indica, E. camaldulensis, N. tabacum and C. citrullus

b

b

b

c

b

a

a

a

b

a

c

c

c

a

c

0

20

40

60

80

73

Exposure times

Fig. 5.2 Mean contact mortality (%) of T. castaneum, T. granarium and R. dominica at different

exposure times of essential oils of M. azedarach, A. indica, E. camaldulensis, N. tabacum and

C. citrullus

Concentrations

c

c c

b

b

b

a

a a

0

20

40

60

80


insects

48 72 96

c

c

c

b

b

b

a

a a

0

20

40

60

80


insects

2 % % 4 6 %

74

Fig. 5.3 Mean contact mortality (%) of T. castaneum, T. granarium and R. dominica at different

concentrations of essential oils of M. azedarach, A. indica, E. camaldulensis, N. tabacum and

C. citrullus

Insects

Fig. 5.3 Mean reduction in population buildup (%) of T.castaneum, T. granarium and R.

dominica against essential oils of M. azedarach, A. indica, E. camaldulensis, N. tabacum and

C. citrullus

a b

b

c

b

b

a a

b

a

c

b

c

a

c

0

10

20

30

40

50

60

70

80

90

A. indica M. azedarach E. camaldulensis N. tabacum C. citrullus

Plants


75

Concentration

Fig. 5.4 Mean reduction in population buildup (%) of T.castaneum, T. granarium and R.

dominica against essential oils of M. azedarach, A. indica, E. camaldulensis, N. tabacum and

C. citrullus at different concentrations

c

c

c

b

b

b

a

a

a

0

10

20

30

40

50

60

70

80


Insects

2 % 4 % 6 %

76

Table 5

77

.1 Percentage contact mortality (Mean ± SE) of T. castaneum (F(8,90) = 15.98, p =

0.000), T. granarium (F(90, 8) = 3.36, p = 0.00562) and R. dominica (F(8,90) = 8.70, p = 0.000)

against of essential oils indigenous medicinal plants.

Plants Time

(hours)

Mortality (%)


A. indica 48 52.30 ± 3.53 cd 55.60 ± 3.52 e 41.95 ± 3.31 e

72 58.62 ± 3.85 b 64.70 ± 3.35 bc 51.72 ± 3.65 d

96 63.21 ± 4.08 a 72.10 ±3.51 a 61.49 ± 3.05 c

M. azedarach 48 36.20 ± 2.58 g 45.66 ± 3.50 g 30.86 ± 2.28 f

72 45.97 ± 2.87 ef 56.41 ±3.99 de 38.29 ±2.06 e

96 59.19 ± 3.04 b 66.60 ±3.51 b 52.57 ± 2.86 d

E. camadulensis

48

72

23.15 ± 3.30 i

32.26 ± 3.28 gh

33.46 ± 3.01 h

41.49 ± 3.10 gh

20.45 ± 2.10 g

30.68 ± 2.57 f

96 49.90 ± 2.38 de 56.40 ± 3.34 de 42.04 ± 2.25 e

N. tabacum

48

72

32.18 ± 3.60 h

43.68 ± 2.78 f

31.75 ±3.30 i

43.22 ±4.03 gh

55.75 ± 3.27 d

68.75 ± 3.85 b

96 52.30 ± 3.08 cd 61.00 ±2.99 c 79.54 ± 4.58 a

C. citrullus

48

72

32.38 ± 3.04 gh

46.61 ± 3.27 f

40.00 ± 3.53 h

50.28 ±3.09 f

30.86 ± 2.85 f

41.71 ± 3.31e

96 54.54 ± 3.55 c 60.57 ±3.83 cd 51.43 ± 3.10 d


Table 5

78

.2 Percentage contact mortality (Mean ± SE) of T. castaneum (F(8, 90) = 4.35, p =

0.0182) and T. granarium (F(8,90) = 3.36, p = 0.00208) against different concentrations and

exposure times of essential oils.

Plants Conc. (%) Mortality (%)

T. castaneum T. granarium

A. indica 2 44.25 ± 1.46 de 38.50 ±2.35 f

4 59.77 ± 1.88 b 53.44 ± 3.33d

6 70.11 ± 1.88 a 63.21 ± 3.04c

M. azedarach 2 37.35 ± 3.04 f 32.57 ± 3.36 g

4 47.70 ± 3.60 d 40.57 ± 3.44 f

6 56.32 ± 3.56 cd 48.57 ± 2.96 e

E. camadulensis

2

4

25.43 ± 4.36 h

34.54 ± 4.33 fg

22.72 ± 3.00 h

32.38 ±3.27 g

6 45.35 ± 3.30 df 38.06 ± 3.23 f

N. tabacum

2

4

32.76 ± 3.33 g

41.38 ± 2.85 e

53.41 ± 2.57 d

70.45 ± 4.06 b

6 54.02 ± 2.78 c 79.54 ± 3.99 a

C. citrullus

2

4

32.38 ± 3.27 g

42.61 ± 2.79 d

30.86 ± 3.10 g

41.71 ±2.84 f

6 54.54 ± 3.75 c 51.43 ± 3.33 de


.3 Percentage contact toxicity (Mean ± SE) of T. granarium (F(4, 90) = 3.20, p =

Table 5

79

0.0165) against different concentrations and exposure times of essential oils.

Time Concentrations

2% 4% 6%

24 hours 26.37 ± 2.80 d 36.30 ± 3.14 g 44.87 ± 3.64 f

48hours 34.58 ± 2.97 g 56.11 ± 3.73 c 34.58 ± 2.97 g

48 hours 45.88 ± 2.59 f 56.11 ± 3.73 c 67.44 ± 4.07 a


Table 5

80

81

Table 5.4 Percentage reduction in buildup of population (Mean ± SE) of T. castaneum (F(8, 90)

= 4.35, p = 0.0182) and T. granarium (F(8,90) = 3.36, p = 0.00208) against different

concentrations and exposure times of essential oils.

Time Reduction in buildup of population (%) Plants

(hours) T. castaneum T. granarium

A. indica 48 50.00±1.66ab 46.33±1.66gfi

72 61.66±1.66cd 55.66±1.66eh

96 70.00±1.66hgf 60.00±1.66e

M. azedarach 48 51.66±1.66a 48.66±1.66hi

72 66.66±1.66bc 65.33±1.66c

96 76.66±1.66efg 75.66±1.66d

E. camadulensis

48

72

45.00±1.66gh

51.66±1.66efg

43.33±1.66i

51.66±1.66df

96 65.00±1.66bcd 63.33±1.66g

N. tabacum

48

72

43.33±1.66h

51.00±1.66de

63.33±1.66d

83.33±1.66b

96 58.66±1.66efg 93.33±1.66a

C. citrullus

48

72

45.00±1.66hg

53.33±1.66ef

45.33±1.66i

51.66±1.66gf

96 68.33±1.66bc 63.33±1.66d


82

CHAPTER 6

FUMIGANT TOXICITY OF ESSENTIAL OILS OF FIVE INDIGENOUS MEDICINAL

PLANTS AGAINST THREE STORED GRAIN INSECT PESTS.

ABSTRACT

The present research work was conducted to evaluate the fumigant toxicity of essential oils of

Azadirachta indica, Melia azedarach, Colocynthis citrullus, Nicotiana tabacum and Eucalyptus

camaldulensis against Tribolium castaneum, Rhyzopertha dominica and Trogoderma granarium.

Three concentrations of each essential oil (2, 4 and 6µL/L air) after 24, 48 and 72 hours of exposure

were tested to find the contact mortality at ambient temperature of 30 ± 2 °C and 65 ± 5% relative

humidity. All essential oil demonstrated toxicities against all test insects. A. indica proved most

effective against R. dominica and T. castaneum with the highest mortality 66.13 and 58.08%

respectively, while N. tabacum was most effective against T. granarium with maximum mortality

67.8%. Overall effect of concentration was highly significant for mortality. At 6 µL concentration

the maximum mortality was 63.10, 56.07 and 56.16% against R. dominica, T. granarium and T.

castaneum respectively. Similarly the longest exposure time of 72 hours showed the highest

mortality followed by 48 and 24 hours against all test insects.

83

6.1 Introduction

Insects always remained one of the major problems causing economic loss Dubey et al.

(2008) by damaging stored commodities qualitatively and quantitatively, throughout the world

(Madrid et al., 1990; Sinha and Watters, 1985). In Pakistan, these losses were recorded from

3.5 to 25% during storage by Irshad and Baloch, (1985). Tribolium castaneum, Rhyzopertha

dominica and Trogoderma granarium are major damage causing insect pests observed in

stored commodities (Danahaye et al., 2007; Anon, 2009; Haines, 1991; Lale, 2002).

Pest control in many storage systems depends on fumigation with either methyl

bromide or phosphine. The use of methyl bromide is mainly restricted because of its potential

to damage the ozone layer (Butler and Rodriguez, 1996; MBTOC, 1998). The future use of

phosphine could be threatened by the further development of resistant strains (Bell and Wilson,

1995; Daglish and Collins, 1999). Weaver and Subramanyam (2000) suggested that fumigant

activity in botanicals could have a greater potential use chemical than grain protectants in future

on the basis of their efficacy, economic value and use in large-scale storages. More current

research showed that essential oils and their constituents may have potential as alternative

compounds to currently used fumigants (Singh et al., 1989; Shaaya et al. 1991, 1997; Regnault-

Roger et al. 1993; Dunkel and Sears 1998; Huang and Ho 1998; Huang et al. 2000; Tunc et al.

2000; Lee et al. 2001). Major constituents from aromatic plants, mainly monoterpenes, are of

special interest to industrial markets because of other potent biological activities in addition to

their toxicity to insects (Kubo et al. 1994; Isman 2000; Weinzierl 2000).

The present study was carried out to evaluate the fumigant potential of essential oils of

indigenous medicinal plant species viz. Azadirachta indica, Melia azedarach, Colocynthis

citrullus, Nicotiana tabacum and Eucalyptus camaldulensis against Tribolium castaneum,

Rhyzopertha dominica and Trogoderma granarium.


The procedure for collection and rearing of insects and extraction of essential oils is given earlier

in chapter 3

84

6.2.1 Bioassay

Space fumigation method (Shaaya et al., 1991) was used for the evaluation of fumigant

toxicities of essential oils. 0.5-L flat-bottom air tight plastic space fumigation chambers with a

small amount of food were used for experiment. Twenty adults of each test insect species were

introduced in each test chamber. Essential oils were applied separately on small pieces of

Whatman No. 1 filter paper to provide dosages of 2, 4 and 6µL/L in air. Control was treated

with acetone alone. Treated filter papers were be kept in air and allowed to evaporate the

solvent (acetone) for 10 min. Fumigation chambers were sealed to make them air tight after

suspending the treated filter papers in the middle of chamber below the lid to avoid physical

contact and to assure uniform distribution of fumes. Data for adult mortality for each

concentration was recorded after 24, 48 and 72 hours of the treatment


The data were arranged in tabulated form. The mortality (%) was corrected by

Abbot’s formula:

Corrected Mortality

where, Mo = Observed mortality, Mc = Control mortality

The collected data was subjected to Analysis of Variance using Statistica software

(8.0, Stat Soft, Inc. 1984-2008). Means of significant treatments were separated using

TukeyHSD test at α =5 %

6.3 Results

Result exhibited that all the essential oils of indigenous medicinal plants showed significant

fumigant toxicity against test insects. In case of T. castaneum, A. indica was proved most toxic

85

essential oil with highest mean mortality (75.67 %) followed by M. azedarach (72.6%), C. citrullus

(61.39%), N. tabacum (55.36%) and E. camaldulensis (47.25%). In case of R. dominica essential

oils of indigenous medicinal plants showed significant fumigant toxicity. A. indica was proved most

toxic essential oil with highest mean mortality (64.13%) followed by M. azedarach (56.22%), C.

citrullus (50.28%), N. tabacum (45.32%), and E. camaldulensis (31.06%). Fumigant toxicity of

essential oils of indigenous medicinal plants against T. granarium showed that N. tabacum was

proved most toxic essential oil with highest mean mortality (61.36%) followed by A. indica

(51.72%), C. citrullus (41.33%), M. azedarach (40.57%) and E. camaldulensis (31.06%). Mean

fumigant mortality of all plant essential oils was statistically different from each other (Fig 6.1).

Effect of exposure time intervals was also observed; results revealed that as the duration of exposure

increase the mortality of test insect was also increased. Mortality was (74.15, 63.33 and 55.99%),

(62.36, 51.22 and 44.75%) and (51.16, 41.29 and 34.94%) against T. castaneum, R. dominica and

T. granarium after 96, 72 and 48 hours respectively (Fig.6.2). Effect of concentration was highly

significant as the dose rate of essential oil increases the toxicity towards test insects was also

increased. Highest mortality (73.92, 63.10 and 55.03%) was recorded at 6µL which is the highest

level of concentration followed by 4 µL/L air (62.03, 52.10 and 40.04%) and 2 µL/L air (51.73,

40.04 and 34.70%) against T. castaneum, R. dominica and T. granarium respectively (Fig.6.3).

Data packed in (Table 6.1) revealed the interaction of plant essential oil with their exposure time.

Results showed that in case of T. castaneum, A. indica was proved most lethal after 96 hours of the

treatment with mortality (86.78%) which was statistically similar with M. azedarach (83.90%) but

statistically different from C. citrullus (73.29%), N. tabacum (65.51%) and E. camaldulensis

(61.29%) mortalities of C. citrullus, N. tabacum and E. camaldulensis was also statistically

different from each other. After 24 hours of treatment least mortality was found in E. camaldulensis

(35.10%) which was statistically different from all other treatments while highest mortality at this

time interval was against A. indica (65.51%) at par with M. azedarach (62.64%) while mortality

against C. citrullus (47.16%) and N. tabacum (45.40%) was statistically similar. In case of R.

dominica results showed that A. indica was proved most lethal after 96 hours of the treatment with

mortality (72.10%) which was statistically different from M. azedarach (66.60%) while mortalities

against C. citrullus (60.57%) were statistically at par with N. tabacum (61.00%) and E.

camaldulensis (56.40%). After 24 hours of treatment least mortality was found against N. tabacum

(31.75%) and E. camaldulensis (33.46%) which was statistically different from all other treatments

86

while mortality was highest against A. indica (55.60%). In case of T. granarium results exhibited

that N. tabacum was proved most lethal after 96 hours of the treatment with mortality (72.15%)

followed by A. indica (61.49%), M. azedarach (52.57%), C. citrullus (51.43%) E. camaldulensis

(42.04%) toxicities among all plant essential oil were statistically different from each other. After

24 hours of exposure time least mortality was found in E. camaldulensis (20.45%) which was

statistically different from all other treatments while highest mortality observed at this time interval

was against N. tabacum (50.56%). Data tabulated in (Table 6.2) described the effect of plant and

concentration on the mortality of T. castaneum. Results exhibited that maximum mortality

(83.90%) was obtained in A. indica at 6 µL/L air concentration which was statistically similar with

M. azedarach (82.18%) but different from C. citrullus (74.43%), N. tabacum (67.24%) and E.

camaldulensis (61.08%) while mortalities of C. citrullus, N. tabacum and E. camaldulensis was

statistically different from each other. At 2 µL/L air concentration the least mortality was found

against E. camaldulensis (33.97%) which was statistically different from all other toxicants at the

same concentration. Effect of plant and concentration on the mortality of T. granarium exhibited

that maximum mortality (73.86%) was obtained against N. tabacum at 6% followed by A. indica

(63.21%), C. citrullus (51.43%), M. azedarach (48.57%), E. camaldulensis (38.06%) toxicities

among all plant essential oil were statistically different from each other. At 2 % concentration least

mortality was found in E. camaldulensis (22.72%) which was statistically different from all other

toxicants.

6.4 Discussion

This experiment was designed to check the fumigant insecticidal bioactivities of A.

indica, M. azedarach, C. citrullus, N. tabacum and E. camaldulensis against T. castaneum, R.

dominica and T. granarium. Results obtained from this study give the clear picture that T.

castaneum was found most susceptible insect against the fumigant toxicity of A. indica, M.

azedarach, C. citrullus and E. camaldulensis followed by R. dominica at the least susceptible was

T. granarium, while results are slightly different when N. tabacum was used as toxicant where T.

granarium was found more susceptible followed by T. castaneum and R. dominica (Fig.6.1). in

other studies some contradictory results were found in a trail conducted by Rozman et al. (2007)

they evaluated essential oils of the aromatic plants Lavandula angustifolia, Rosmarinus officinalis,

Thymus vulgaris and Laurus nobilis for fumigant activity against adults of Sitophilus oryzae,

87

Rhyzopertha dominica and Tribolium castaneum. The insecticidal activities varied with insect

species, the most sensitive species was S. oryzae, followed by Rhyzopertha dominica and Tribolium

castaneum, no oil compounds achieved more than 20% mortality after exposure for 24 h, against

T. castaneum even with the highest dose (100 ml/720 ml volume). Results presented by Benzi et

al. (2012) were also contradictory to this study they evaluated the fumigant toxicity of Brazilian

pepper tree ( Schinus molle L. var. areira (L.) and found that fumigant activity, neither of the

essential oils was found to be toxic. Similar results were obtained by Ali et al. (2012) they evaluated

insecticidal efficiencies of Datura alba and found T. granarium more tolerant than S. oryzae with

33.5 and 45 % mortality, respectively. This difference in the toxicity of different essential oils

against different species of insect pest was justified by the findings of Negahban et al. (2006);

Ayvaz et al. (2010); Alzogaray et al. (2011) they suggested that insecticidal activity of the essential

oils usually varies depending on the stage of the insect, the species, and the plant origin of the

essential oil that would be attributed to the diverse chemical composition of the oils and the

interactions that may occur between the individual components of the mixture. Fumigant toxicity

of the essential oil was justified by the study of Tripathi et al. (2002); Tripathi et al. (2003a); Ayvaz

et al. (2010) they said due to their high volatility under ambient temperatures, essential oils have

fumigant activity that might be important for controlling stored-product insect pests.

Fumigant toxicity of T. castaneum, R. dominica and T. granarium significantly increased

with the dose rate of essential oils. Mortality of test insect pests was maximum at the highest level

of concentration. Mortality was(73.92, 63.10 and 55.03%), (62.03, 52.10 and 40.04%) and (51.73,

40.04 and 34.70%) at 6, 4 and 2 µL/L air concentration of essential oils against R. dominica, T.

granarium and T. castaneum respectively (Fig.6.3) results reported by Negahban and

Moharramipour (2007) are in accordance with the current studies the essential oils Eucalyptus

intertexta R.T. Baker, Eucalyptus sargentii Maiden and Eucalyptus camaldulensis Dehnh. found

toxicant against stored grain insect pests and the mortality of 1-7-day-old adults of the insect pests

increased with concentration from 37 to 926 μL/L.

Fumigant toxicity of T. castaneum, R. dominica and T. granarium significantly increased

with the increase of exposure time of essential oils. Mortality of test insect pests was maximum at

the longest exposure of 72 hours. Mortality was (5.16, 62.36 and 76.15 %), (41.29, 51.22 and 63.33

%) and (34.94, 44.74 and 55.94%) after 24, 48 and 72 hours against T. castaneum R. dominica, and

T. granarium respectively (Fig.6.3) similar results were obtained in pervious findings that mortality







88

increased as the doses of essential oils and exposure period increased and after 72 hours

fumigations, greatest percentages of mortality were obtained in a trail conducted by Ebadollahi et

al. (2010). The mortality of 1-7-day-old adults increased with exposure time from 3 to 24 hours

Negahban and Moharramipour (2007). Similar trend was observed by Liska et al. (2010) who

recorded 5.0 % fumigant toxicity of camphor at 2hr and 13.2% after 4 hr. of treatment against T.

castaneum. These findings were also in accordance with previous studies carried out by Shukla et

al. (2002), Lee et al. (2004), Rozman et al. (2006) and Xie et al. (2010).

Insects








89

Fig.6.1 Mean fumigant mortality (%) of T.castaneum, T. granarium and R. dominica against


Exposure times

c c

c

b b

b

a a

a

0

20

40

60

80

100


Insects

24 48 72

90

Fig.6.2 Mean fumigant mortality (%) of T.castaneum, T. granarium and R. dominica at different

exposure times of essential oils of M. azedarach, A. indica, E. camaldulensis, N.

tabacum and C. citrullus

Fig.6.3 Mean fumigant mortality (%) of T.castaneum, T. granarium and R. dominica at different

exposure times of essential oils of M. azedarach, A. indica, E. camaldulensis, N.


Table 6.1 Percentage fumigant mortality (Mean ± SE) of T. castaneum (F(8,90) = 7.84, p = 0.000),

T. granarium (F(8, 90) = 1.80), p = 0.00562) and R. dominica (F(8, 90) = 8.70, p =

0.0000) against exposure times of essential oils.

Plants Time

(hours)

Mortality (%)


A. indica 24 65.51 ± 1.92 c 55.60 ± 3.52 e 41.95 ± 3.31 d

48 74.71 ± 2.35 b 64.70 ± 3.3 bc 51.72 ± 3.65 c

72 86.78 ± 2.87 a 72.10 ± 3.31 a 61.49 ± 4.05 b

M. azedarach 24 62.64 ± 2.8 cd 45.66 ± 3.50 g 30.86 ± 2.28 e

c

c c

b b

b

a a

a

0

20

40

60

80


Concentration

2 % 4 % 6 %

91

48 71.26 ± 2.87 c 56.41 ± 3.9 de 38.29 ± 2.84 d

72 83.90 ± 2.78 a 66.60 ± 3.51 b 52.57 ± 2.06 c

E. camadulensis

24

48

35.10 ± 3.52 f

45.35 ± 4.26 g

33.46 ± 3.03 i

41.49 ± 3.1 gh

20.45 ± 2.10 f

30.68 ± 2.10 e

72 61.29 ± 4.52 d 56.40 ± 3.3 de 42.04 ± 2.57 d

N. tabacum

24

48

45.40 ± 3.76 g

55.17 ± 3.83 e

31.75 ± 3.30 i

43.22 ± 4.0 gh

50.56 ± 3.51 c

61.36 ± 4.00 b

72 65.51 ± 3.33 c 61.00 ± 2.99 c 72.15 ± 3.52 a

C. citrullus

24

48

47.16 ±3.61 f

65.34 ± 3.7 de

40.00 ± 3.53 h

50.28 ± 3.83 f

30.86 ± 2.85 e

41.71 ± 3.31 d

72 73.29 ± 3.27 b 60.57 ± 3.0 cd 51.43 ± 3.10 c


Table 6.2 Percentage fumigant mortality (Mean ± SE) of T. castaneum (F(8, 90) = 9.59, p =

0.000) and T. granarium (F(8, 90) = 9.83, p = 0.000) against different concentrations of essential

oils.

Mortality (%) Plants Conc. (µL/L air)

T. castaneum T. granarium

A. indica 2 68.39 ± 2.30 cd 38.50 ± 2.35 e

4 74.71 ± 3.23 b 53.44 ± 3.33 c

6 83.90 ± 2.89 a 63.21 ± 3.04 b

92

M. azedarach 2 63.21 ± 2.78 ef 32.57 ± 3.36 f

4 72.41 ± 2.22 bc 40.57 ± 3.44 e

6 82.18 ± 1.89 a 48.57 ± 2.96 d

E. camadulensis

2

4

33.97 ± 2.77 j

45.92 ± 2.77 i

22.72 ± 3.00 g

32.38 ± 3.27 f

6 61.86 ± 3.23 g 38.06 ± 3.23 e

N. tabacum

2

4

42.53 ± 3.08 i

56.32 ± 2.84 g

48.86 ± 3.30 d

61.36 ± 3.08 b

6 67.24 ± 3.44 de 73.86 ± 3.28 a

C. citrullus

2

4

50.56 ± 2.35 h

60.79 ± 3.36 f

30.86 ± 3.10 f

41.71 ± 2.84 e

6 74.43 ± 3.04 b 51.43 ± 3.33 cd


CHAPTER 7

ANTIFEEDANT EFFACT OF ESSENTIAL OILS OF FIVE INDIGENOUS MEDICINAL

PLANTS AGAINST THREE STORED GRAIN INSECT PESTS

ABSTRACT

The present research work was carried out to assess the ant feeding effect of Azadirachta indica,

Melia azedarach, Colocynthis citrullus, Nicotiana tabacum and Eucalyptus camaldulensis in

comparison with untreated samples of control against Tribolium castaneum, Ryzopertha dominica

and Trogoderma granarium. All the essential oils showed the prominent feeding deterrence

93

activities. The most potent essential oil against all insect pests was found A. indica, with maximum

reduction in weight loss and feeding deterrence index (0.56, 1.02, 1.69%) (75.44, 54.57 and

39.21%) followed by M. azedarach (0.63, 1.05 and

1.76%) (67.59, 50.85 and 34.92%) C. citrullus (0.65, 1.17 and 1.76%) (65.35, 43.57 and

33.94%) N. tabacum (0.7, 1.22 and 1.84) (58.43, 38.87 and 30.28%) and E. camaldulensis

(0.84, 1.32 and 1.97%) (45.11, 38.98 and 23.18) against T. castaneum, T. granarium and R.

dominica respectively. Overall effect of concentration on weight loss and feeding deterrence

index was also studied. Results give the clear picture as the dose rate of toxicant increase its

potency goes increases. Weight loss was (0.37, 0.85 and1.50%) (0.48, 0.96 and 1.61%,) (1.26,

1.09 and 1.74%) and (1.72 and 2.39%) at the dose rate of 6%, 4%, 2% and controls against T.

castaneum, T. granarium and R. dominica respectively. Feeding deterrence index also

increases with the concentration of tested essential oil. Feeding deterrence index was (71.74,

50.41 and 37.22%), (62.83, 44.18 and 32.54%) and (52.59, 36.65 and 27.17%) at 6%, 4%, 2%

concentration of toxicant

7.1 Introduction

Food safety and security is a key issue now a days, especially in the circumstances of day by

day rapidly increasing population of the world (Tubiello et al., 2007). Pakistan has a population

of 177 million which is increasing with 2.07% growth rate. Saving the produced grains at

harvesting can be helpful to meet the food requirements of increasing population. In stored

grains, insect damage may account for 10-40% of loss worldwide (Matthews, 1993;

Papachristos and Stamopoulos, 2002; Raja et al., 2001). In Pakistan the damage caused T.

castaneum to various stored and food commodities like grain, flour and dried fruits is recorded

to be 15-20% (Khattak and Shafique, 1986). The weight loss caused by T. granarium in

94

Pakistan is about 2.32% (Khan and Cheema, 1978) The lesser grain borer, Rhyzopertha

dominica (Fabr.) (Coleoptera: Bostrichidae), is a threatening pest of stored products and is

cosmopolitan in distribution (Potter, 1935). It mostly found throughout warmer regions of the

world and highly polyphagous in nature (Edde et al., 2005).

Attempts to control stored grain pests mostly relied on synthetic insecticides and

fumigants such as methyl bromide (Chakrabarti, 1996), Phosphine (Pimentel et al., 2007) and

Sulphuryl fluoride (Bell and Savvidou, 1999). However drawbacks associated with the use of

conventional control strategies (synthetic insecticides) such as effect on non-target organisms

(Rajendran and Sriranjini, 2008), human health concerns (Isman, 2006), environmental

pollution (Ogendo et al., 2003), effects on ozone depletion (Shaaya and Kostyukovsky, 2006)

and development of insect resistance and pest resurgence (Sousa et al., 2009) have necessitated

to find out some biodegradable, safe and environmental friendly sources of pesticides.

Numerous types of insecticidal activities of essential oils have been documented

(Papachristos and Stamopoulos, 2002). Along with feeding deterrent effects of various plant

essential oil was confirmed (Suthisut et al., 2011; Ko et al., 2010) Melia Azadirachta powder has

excellent feeding deterrence against stored grain insect pests (Islam, 1983). Nicotiana tabacum

exhibited feeding deterrence activities against T. castaneum (Tiwari et al., 1995). Citrullus

colocynthis L. (Cucurbitaceae) is a medicinal plant in Africa and Asia (TavakkolAfshari et al.,

2005). Seed extracts of C. citrullus inhibit the population of T. castaneum at various concentrations

(Nadeem et al., 2012) Azadirachtin has a unique complex of behavioral and physiological modes

of action; it has an antifeedant, action on insects (Schmutterer 1988) whereas Eucalyptus

camaldulensis has repellant properties against T. granarium and T. castaneum (Abbasipour et al.,

2009)


The procedure for collection and rearing of insect and extraction of essential oils is given earlier

in chapter 3

7.2.1 Bioassay

Weight loss method was adopted for the evaluation of antifeedant activities of essential

oils. Weighed amount (5g) of grain (for Trogoderma granarium and Rhyzopertha dominica)





95

and wheat flour for Tribolium castaneum) was treated with 2 ml of the essential oil and 2, 4

and 6% of concentration for each essential oil. Control was treated with acetone alone. Treated

diet was kept in air and allowed to evaporate the solvent (acetone) for 10 min. 20 test insects

were released in each jar on treated diet. Jars were kept in incubator (Model MIR-254,

SANYO) at 25 ± 2 °C and 65 ± 5% relative humidity after covering with a cloth to avoid insect

escape. Four replications were performed for each concentration of the essential oils. Data

regarding seed damage and weight loss were recorded after 15, 30 and 45 days of treatment.

Count and weight method was followed for calculation of the seed weight loss by T. granarium

and R. dominica (Boxall, 1986).

Weight loss (%) =

where,

Wu = Undamaged seeds (weight)

Nu = Undamaged seeds (number)

Wd = Damaged seeds (weight)

Nd = Damaged seeds (number)

The weight loss (%) of wheat flour by T. castaneum in the treated and control sets was calculated

by using the formula suggested by Parkin (1956).

Weight loss (%) =

where,

Wi=Weight of wheat flour before the experiment W

=Weight of wheat flour after the experiment.

Feeding deterrence was calculated by using the feeding deterrent index following Isman et al.

(1990).

FDI (%) =

96

Where,

C = Weight loss in the control diet.

T = Weight loss in the treated diet.



(Stat Soft, 8.0). Means were separated by the Tukey's multiple range test when results was

significant (p < 0.05)

7.3 Results

7.3.1 Antifeedant effect of essential oils against T. castaneum

Assessment of weight loss and feeding deterrence index of essential oils of indigenous

medicinal plants against T. castaneum, and R. dominica in wheat flour and T. granarium in

whole wheat treated with the essential oils, in comparison with untreated samples of control

was calculated. All plant essential oils showed significant reduction in weight loss against . A.

indica proved most efficient toxicant where weight loss (0.56, 1.02 and 1.69%) was minimum

followed by M. azedarach (0.63, 1.05 and 1.76%), C. citrullus (0.65 and 1.78%), N. tabacum

(0.71, 1.22 and 1.84%) E. camaldulensis (0.84, 1.32 and 1.97%) against T. castaneum, T.

granarium and R. dominica respectively (Fig. 7.1). Overall effect of time duration on weight

loss was also analyzed. Results showed that the weight loss by T. castaneum and R. dominica

increased as the time period increased. Weight loss was (0.89 and 1.60%), (0.68 and 1.81%)

and (0.47 and 2.02%) against T. castaneum and R. dominica after 45, 30 and 15 days

97

respectively while in case of T. granarium results showed that weight loss increased from 15

to 30 days but after that there was no significant increase in weight loss because only larval

stage of this insect cause damage and within 30 days all larvae were converted into pupae or

adults. Weight loss was 1.24, 1.22 and 1.00% after 45, 30 and 15 days respectively (Fig.7.2).

Overall effect of concentration on weight loss was also studied. Results gave the clear picture

as the dose rate of toxicant increased its potency increased. Weight loss was (0.37, 0.85

and1.50%) (0.48, 0.96 and 1.61%,) (1.26, 1.09 and 1.74%) against T. castaneum, T. granarium

and R. dominica at the dose rate of 6,4 and 2% respectively (Fig.7.3). Effect of plant essential

oil on the feeding deterrence index showed that the feeding deterrence index was higher against

A. indica (75.44, 54.47 and 39.21%) followed by M. azedarach (67.59, 56.39 and 50.85%), C.

citrullus (65.37, 43.57 and 33.94%), N. tabacum (58.43, 38.87 and 30.28%) E. camaldulensis

(45.11, 30.98 and 23.18%) against T. castaneum, T. granarium and R. dominica respectively

(Fig.7.4). Overall effect of time duration on feeding deterrence index revealed that as the

duration of exposure increases feeding deterrence index decreases accordingly. Feeding

deterrence index was (73.98 and 35.54%), (59.41 and 31.20%) and (53.55 and 30.19%) against

T. castaneum and R. dominica after 15, 30 and 45 days respectively. In case of T. granarium

overall effect of time duration on feeding deterrence index revealed that as the time duration

increase from 15 to 30 days, feeding deterrence index decreased but after 30 to 45 days there

was no significant decrease in feeding deterrence index because only larval stage of this insect

cause damage and within 30 days all larvae were converted into pupae or adults and feeding

deterrence index was 48.61%, 41.85% and 40.78% after 15, 30 and 45 days respectively (Fig

7.5). Overall effect of concentration on the feeding deterrence index was also increases with

respect to the concentration of tested essential oil. Feeding deterrence index was (71.74, 50.41

and 37.22%), (62.83, 44.18 and 32.54%) and (52.59, 36.65 and 27.17%) against T. castaneum,

T. granarium and R. dominica at 6%, 4%, 2% concentration of toxicant respectively. (Fig.7.6).

Data given in (Table 7.1) describes the effect of plant essential oils on the weight loss of T.

castaneum. Feeding inhibition potential was maximum against A. indica (0.37%) after 15 days

of treatment of wheat flour followed by M. azedarach (0.40%), C. citrullus (0.47%), N.

tabacum (0.50%), E. camaldulensis (0.58%), weight loss in all toxicants were significantly

different from each other. Maximum weight loss was found against E. camaldulensis after 45

days of treatment (1.07%) while minimum weight loss after 45 days was found against A.

98

indica (0.77%) which was statistically different from each other and all other plant essential

oils. Effect of plant essential oils on the weight loss of R. dominica at different exposure times

showed that feeding inhibition potential was maximum against A. indica (1.56%) after 15 days

of treatment of wheat flour followed by M. azedarach (1.53%), C. citrullus (1.60%), N.

tabacum (1.63%), E. camaldulensis (1.71%), weight loss in all toxicants were significantly

different from each other. Maximum weight loss was found against E. camaldulensis after 45

days of treatment (2.20%) while minimum weight loss after 45 days was found against A.

indica (1.90%) which was statistically different from each other and all other plant essential

oils. Effect of plant essential oils on the weight loss of T. granarium revealed that feeding

inhibition potential was maximum against A. indica (0.90%) after 15 days of treatment of wheat

flour followed by M. azedarach (0.95%), which was statistically different from each other and

all other plants while weight loss in C. citrullus (1.057%) and N. tabacum (1.057%) were

statistically similar, weight loss in E. camaldulensis (1.11%) which was higher and statistically

different from all other toxicants. Maximum weight loss (1.40) was found against E.

camaldulensis after 45 days of treatment while minimum weight loss after 45 days was found

against A. indica (1.90%) which was statistically different from each other and all other plant

essential oils. Data tabulated in (Table 7.2) represent the effect of plant and time on feeding

deterrence index against T. castaneum. Results revealed that feeding deterrence index was

maximum against A. indica (86.05%) after 15 days of treatment of wheat flour followed by M.

azedarach (82.18%), C. citrullus (73.16%), N. tabacum (63.39%), E. camaldulensis (59.11%)

and feeding deterrence index in all toxicants were significantly different from each other. In

case of R. dominica results revealed that feeding deterrence index was maximum against A.

indica (41.54%) after 15 days of treatment of wheat flour followed by M. azedarach (39.50%),

C. citrullus (35.11%), N. tabacum (33.28%), E. camaldulensis (28.28%) and feeding

deterrence index in all toxicants were significantly different from each other. In case of T.

granarium feeding deterrence index was maximum against A. indica (57.26%) after 15 days of

treatment of wheat flour was statistically similar with M. azedarach (56.39%) but different

from C. citrullus (45.49%) and N. tabacum (44.86%) was statistically similar with each other

while weight loss against E. camaldulensis (39.02%) was statistically different from rest of the

toxicants. Data packed in (Table 7.3) gave understanding about the effect of plant essential oils

and concentration. For T. castaneum results showed that maximum reduction in weight loss

99

was found against A. indica (0.20%) at 6% concentration followed by M. azedarach (0.31%),

C. citrullus (0.33%), N. tabacum (0.41%) and E. camaldulensis (0.59%). Weight loss in all

toxicants was significantly different from each other. Maximum weight loss (1.26%) was found

where no toxicant was applied and flour is just treated with acetone. While in case of R.

dominica result showed that maximum reduction in weight loss was found against A. indica

(1.58%) at 6% concentration was statistically different from all other toxicants followed by M.

azedarach (1.68%) and C. citrullus (1.69%) which were statistically at par with each other but

different from rest of the toxicants, reduction in weight against N. tabacum (1.78%) and E.

camaldulensis (1.96%) were significantly different from each other. Maximum weight loss

(2.39%) was found where no toxicant was applied and flour is just treated with acetone. In case

of T. granarium result showed that maximum reduction in weight loss was found against A.

indica (0.67%) at 6% concentration followed by M. azedarach (0.0.72%), C. citrullus (0.87%),

N. tabacum (0.93%), E. camaldulensis (1.07%) weight loss in all toxicants were significantly

different from each other. Maximum weight loss (1.72%) was found where no toxicant was

applied and flour is just treated with acetone Data tabulated in (Table 7.4) represent effect of

plants and concentration on feeding deterrence index. Results revealed that feeding deterrence

index of T. castaneum was maximum against A. indica (84.92%) at 6% followed by M.

azedarach (76.57%), C. citrullus (74.82%), N. tabacum (67.83%), E. camaldulensis (54.58%)

and feeding deterrence index in all toxicants were significantly different from each other.

Results showed that that feeding deterrence index in case of R. dominica was maximum against

A. indica (39.76%) at 6% followed by M. azedarach (39.64%) which was at par with C.

citrullus (38.89%) but different from N. tabacum (35.20%), E. camaldulensis (28.14%) and

feeding deterrence index in all toxicants were significantly different from each other. In case

of T. granarium results revealed that feeding deterrence index was maximum against A. indica

(61.3%) at 6% followed by M. azedarach (57.86%), C. citrullus (49.54%), N. tabacum

(45.60%), E. camaldulensis (37.93%) and feeding deterrence index in all toxicants were

significantly different from each other.

7.4 Discussion

The present research work was carried out to assess the ant feeding effect of A.

100

indica, M. azedarach, C. citrullus, N. tabacum and E. camaldulensis in comparison with

untreated samples of control against T. castaneum, R. dominica and T. granarium. All the

essential oils showed the prominent feeding deterrence activities. The most potent essential oil

against all insect pests was found A. indica, with maximum reduction in weight loss and FDI

(0.56, 1.02, 1.69%) (75.44, 54.57 and 39.21%) followed by M. azedarach (0.63, 1.05 and

1.76%) (67.59, 50.85 and 34.92%) ,C. citrullus (0.65, 1.17 and 1.76%) (65.35, 43.57 and

33.94%) N. tabacum (0.7, 1.22 and 1.84) (58.43, 38.87 and 30.28%) and E. camaldulensis

(0.84, 1.32 and 1.97%) (45.11, 38.98 and 23.18) against T. castaneum, T. granarium and R.

dominica respectively. Similar investigations were made by Morgan (2009) who confirmed the

feeding deterrence activities of Azadirachtin, a triterpene isolated from Azadirachta indica, is

a very active antifeedant against 90 % of the more than 600 tested herbivorous and stored

product insect species. Contradictory results were obtained in the studies carried out by Huang

et al. (1997) they proved changes in feeding behavior of T. castaneum adults when subjected

to nutmeg oil (Myristica fragrans) but feeding deterrent effect was more prominent in S.

zeamais as compared to T. castaneum adults this may be due to the fact that different essential

oil has different efficiencies against different insect pests. In another investigation made by Ko

et al. (2010) results obtained from this experiment showed that antifeedant effect of Litsea

salicifolia at different concentrations of 0, 4, 6, 8 and 10% against these two insect pests and

calculated FDI (Feeding Deterrent Index) showed T. castaneum more susceptible (FDI=

84.62% at 10% concentration) than S. zeamais (FDI= 29.63% at 10% concentration) these

results are somewhat similar to the present study in which highest FDI against T.

castaneum was 75.44 % (Fig.7.4) this difference in FDI’s value was may be due to difference

in the concentration and nature of toxicants. Feeding action of glycol-alkaloid fraction TGA

fraction of Solanum tuberosum against (T. granarium) was reported by Nenaah (2011) who

exposed significant diminution in the food utilization and food consumption rate by khapra

beetle at concentrations varying between 20 and 30 mg g-1 food with feeding deterrent index

reaching 82.40% .

Overall effect of concentration on weight loss and feeding deterrence index was also

studied. Results give the clear picture as the dose rate of toxicant increase its potency goes increases.

Weight loss was (0.37, 0.85 and1.50%) (0.48, 0.96 and 1.61%,) (1.26, 1.09 and 1.74%) and (1.72

101

and 2.39%) at the dose rate of 6%, 4%, 2% and controls against T. castaneum, T. granarium and R.

dominica respectively (Fig. 7.3). Feeding deterrence index also increased with respect to the

concentration of tested essential oil. Feeding deterrence index was (71.74, 50.41 and 37.22%),

(62.83, 44.18 and 32.54%) and (52.59, 36.65 and 27.17%) at 6%, 4%, 2% concentration of toxicant

(Fig.7.6) These findings are in accordance with earlier reports made with essential oil of L.

salicifolia against S. zeamais and T. castaneum (Ko et al., 2010). Feeding deterrence index at the

lowest concentration (4%) of L. salicifolia against T. castaneum was documented 53.58% which

increased to 84.62% with the application of the highest (10%) concentration of essential oil. Similar

trend was also observed by Abbasipour et al. (2011) who reported Feeding Deterrence Index of D.

stramonium against T. castaneum increased from 34.93% to 97.21% with the increase in

concentration from 947 to 3007 mg /l. Recent investigation carried out by Jaya et al. (2012) to

determine feeding deterrence index of Coleus aromaticus against T. castaneum resulted in the same

effect of concentration i.e., FDI at 250 ppm was 56.39 % witch increased to 72.31 % at 500 ppm

and achieved 100% FDI at 1000 ppm concentration. These results are also in agreement with that

of Sarwar (2010), Geng et al. (2011) and Nenaah (2011).

Insects

102

Fig.7.1 Mean weight loss (%) of food commodities due to T. castaneum, T. granarium and R.

dominica essential oils of M. azedarach, A. indica, E. camaldulensis, N. tabacum and C.

citrullus

Fig. 7.2 Mean weight loss (%) of food commodities due to T. castaneum, T. granarium and R.


citrullus at different exposure times

c

a

c b

b

b a

a

b

0

0.5

1

1.5

2

2.5


Insects

days 15 30 days 45 days

103

Insects

Fig. 7.3 Mean weight loss (%) of food commodities due to T. castaneum, T. granarium and R.


citrullus at different concentrations

Insects

c

c

c

c

a

b

b

b

b

a

a

a

0

0.5

1

1.5

2

2.5

3

6 % % 4 2 % control

Concentrations


a

a

a

a

a

b

b

b

b

b

c

c

c

c

c

0

10

20

30

40

50

60

70

80

90

E. camaldulensis N. tabacum C. citrullus M. azedarach A. indica

Plants


104

Fig.7.4 Mean feeding deterrence index (%) of T.castaneum, T. granarium and R. dominica


Concentrations

Fig. 7.5 Mean feeding deterrence index (%) of T.castaneum, T. granarium and R. dominic at

different concentrations of essential oils of M. azedarach, A. indica, E. camaldulensis, N.


c

a

c

b

b

b

a

c

a

0

10

20

30

40

50

60

70

80


Insects

2 % 4 % 6 %

105

Fig.7.6 Mean feeding deterrence index (%) of T.castaneum, T. granarium and R. dominic at

different exposure times of essential oils of M. azedarach, A. indica, E. camaldulensis, N.


c

c

b

b

b

b

a

a

a

0

10

20

30

40

50

60

70

80


Insects

days 45 days 30 days 15

Percentage weight loss (Mean ± SE) of T. castaneum (F(8,90) = 7.84, p =


106

Table 7.1

0.000),

0.0000) against exposure times of essential oils.

Plants Time (Days) Weight loss (%)

T. Castaneum R. dominica T. granarium

A. indica

15

30

0.375±0.12 a

0.535±0.12 e

1.505±0.12 a

1.665±0.12 e

0.905±0.12 a

1.065±0.12 b

45 0.770±0.11 i 1.900±0.12 i 1.095±0.12 c

M. azedarach

15

30

0.405±0.53 b

0.628±0.11 g

1.535±0.11 b

1.763±0.11 g

0.905±0.11 a

1.128±0.11de

45 0.877±0.10 k 2.007±0.10 k 1.138±0.11 e

E. camaldulensis

15

30

0.589±0.08 f

0.877±0.07 k

1.719±0.08 f

2.007±0.07 k

1.119±0.08 d

1.407±0.07 h

45 1.074±0.07 l 2.204±0.07 l 1.447±0.07 i

N. tabacum

15

30

0.507±0.09 d

0.752±0.08 h

1.637±0.09 d

1.882±0.08 h

1.057±0.09 b

1.302±0.08 g

45 0.892±0.10 k 2.022±0.10 k 1.317±0.08 g

C. citrullus

15

30

0.477±0.10 c

0.641±0.10 g

1.607±0.10 c

1.771±0.10 g

1.057±0.10 b

1.221±0.10 f

45 0.835±0.11 j 1.965±0.11 h 1.23±0.107 f


T. castaneum (F(8,90) =

= 0.000), R. dominica (F(8, 90) = 8.70, p

107

Table 7.2 Percentage Feeding deterrence index (Mean ± SE) of

7.84, p T. granarium (F(8, 90) = 1.80), p = 0.00562) and = 0.0000)

against exposure times of essential oils.

Plants Time

(Days)

(%) FDI


A. indica

15

30

86.05±2.42a

75.98±3.48c

41.54±1.178a

39.75±1.82b

57.26±1.62a

53.65±2.41b

45 64.35±2.86f 36.35±1.63c 52.50±2.39b

M. azedarach 15 82.18±2.75b 39.50±1.33b 56.39±1.846a

30 65.94±3.27f 34.50±1.71de 48.36±2.275c

45 54.66±2.18h 30.82±1.24g 47.80±2.275c

E. camaldulensis

15

30

59.11±3.040g

39.15±3.056k

28.28±1.47h

20.48±1.59i

39.02±2.039f

28.08±2.122h

45 37.06±2.47j 20.77±1.41h 25.84±2.122i

N. tabacum

15

30

69.39±3.145e

52.59±2.62i

33.28±1.52f

27.51±1.37h

44.86±2.102d

36.29±1.82g

45 53.31±2.46hi 30.05±1.40g 35.45±1.82g

C. citrullus

15

30

73.16±3.28d

73.16±2.24f

35.11±1.59d

33.75±1.17ef

45.49±2.19d

42.88±1.55e

45 58.38±2.57g 32.95±1.47f 42.32±1.55e



= 0.000), R. dominica (F(8, 90) = 8.70, p

108

Table 7.3

0.000),

0.0000) against concentrations of essential oils.

Plants Conc.

(%)

(%) Weight loss


A. indica

2

4

0.458±0.0043 d

0.312±0.0043 b

1.588±0.065 d

1.442±0.054 b

0.924±0.041 f

0.766±0.021 c

6 0.205±0.0043 a 1.335±0.049 a 0.672±0.022 a

0 1.262±0.0043 k 2.392±0.062 k 1.72±0.0345 l

M. azedarach

2

4

0.551±0.004 ef

0.418±0.0043 c

1.681±0.071 ef

1.548±0.071 c

0.980±0.046 g

0.836±0.041 d

6 0.3166±0.004 b 1.446±0.068 b 0.726±0.032 b

0 1.262±0.0043 k 2.392±0.061 k 1.686±0.032 l

E. camaldulensis

2

4

0.834±0.0043 j

0.701±0.0043 i

1.964±0.075 j

1.831±0.075 i

1.317±0.059 k

1.183±0.058 j

6 0.590±0.0043 g 1.720±0.070 g 1.071±0.053 h

0 1.262±0.0043 k 2.392±0.061 k 1.72±0.035 l

N. tabacum

2

4

0.654±0.0043 h

0.458±0.0043 e

1.784±0.056 h

1.664±0.055 e

1.165±0.043 i

1.060±0.048 h

6 0.418±0.0043 c 1.5488±0.05 c 0.939±0.084 f

0 1.262±0.0043 k 2.392±0.061 k 1.72±0.0330 l

C. citrullus

2

4

0.561±0.0043 f

0.453±0.0043 d

1.691±0.0499 f

1.583±0.050 d

1.076±0.024 h

0.967±0.024 g

6 0.330±0.0043 b 1.460±0.046 b 0.870±0.032 e


= 0.000), R. dominica (F(8, 90) = 8.70, p

109

0 1.262±0.0043 k 2.392±0.067 k 1.766±0.032 l


Control = 0 %

Table 7.4 Percentage feeding deterrence index (Mean ± SE) of

7.84, p T. granarium (F(8, 90) = 1.80), p = 0.00562) and = 0.0000)

against different concentrations of essential oils.

Plants Conc.

(%)

(%) FDI


A. indica

2

4

65.00±3.512817d

76.46±3.021443b

33.68±1.071748d

44.20±0.764779b

46.65±1.367543f

55.63±0.417887c

6 84.92±3.045104a 39.76±0.642062a 61.13±0.579150a

M. azedarach 2 57.76±3.723916ef 29.80±1.229634ef 43.19±1.612817g

4 68.44±4.166158c 35.37±1.341895c 51.51±1.484307d

6 76.57±4.178960b 39.64±1.274443b 57.86±1.101065b

E. camaldulensis

2

4

35.00±3.287571j

45.75±3.686259i

17.89±1.275911j

23.50±1.360537i

23.58±2.050049j

31.43±2.146775i

6 54.58±3.652470g 28.14±1.246432g 37.93±1.975496h

N. tabacum

2

4

48.92±2.322418h

58.55±2.916931e

25.30±0.698680h

30.34±0.961597e

32.43±1.248208c

38.57±1.699552h

6 67.83±3.023049c 35.20±0.868503c 45.60±1.589265f

C. citrullus

2

4

56.27±1.760908fg

64.97±2.204307d

29.19±0.257803g

33.73±0.407484d

37.40±0.252781h

43.75±0.413710g

6 74.82±2.601387b 38.89±0.517168b 49.54±0.967156e



= 0.000), R. dominica (F(8, 90) = 8.70, p

110

Table 7.5

0.000),

0.0000) against concentrations and exposure times of essential oils.

Time (Days) Conc. (%) T. Castaneum R. dominica T. granarium

15

2

4

0.384±0.029743

0.271±0.026347

1.514±0.029743

1.401±0.026347

0.922±0.032637

0.809±0.029752

6 0.171±0.025707 1.301±0.025707 0.709±0.027971

0 1.056±0.002520 2.186±0.002520 1.594±0.007488

30

2

4

0.629±0.039958

0.491±0.044901

1.759±0.039958

1.621±0.044901

1.167±0.040113

1.029±0.045903

6 0.380±0.042026 1.510±0.042026 0.918±0.043631

0 1.246±0.001260 2.376±0.001260 1.784±0.007163

45

2

4

0.822±0.035203

0.689±0.036299

1.952±0.035203

1.819±0.036299

1.188±0.041744

1.054±0.047060

6 0.564±0.037833 1.694±0.037833 0.939±0.044805

0 1.484±0.003217 2.614±0.003207 1.805±0.007202


Control = 0


= 0.000), R. dominica (F(8, 90) = 8.70, p

111

Table 7.6 Percentage feeding deterrence index (Mean ± SE) of

7.84, p T. granarium (F(8, 90) = 1.80), p = 0.00562) and

= 0.0000) against different concentrations and exposure times of essential oils.

Time (Days) Conc. (%) T. castaneum R. dominica

15

2

4

63.71±2.805926d

74.40±2.485556b

30.55±1.376882e

35.75±1.218988d

6 83.83±2.425144a 40.33±1.188782a

30

2

4

49.24±3.222412h

60.37±3.621016f

25.76±1.685988f

31.58±1.894540c

6 69.30±3.389169c 36.25±1.773236d

45

2

4

44.83±2.362605i

53.73±2.436147g

25.21±1.348767f

30.29±1.390750e

6 62.10±2.539097e 35.07±1.449523b



= 0.000), R. dominica (F(8, 90) = 8.70, p

112


= 0.000), R. dominica (F(8, 90) = 8.70, p

113

CHAPTER 8

INSECT GROWTH REGULATORY ACTIVITIES OF ESSENTIAL OILS OF FIVE

INDIGENOUS MEDICINAL PLANTS AGAINST THREE STORED GRAIN INSECT

PESTS.

ABSTRACT

The present research work was conducted to evaluate the insect growth regulatory activities of

essential oils of Azadirachta indica, Melia azedarach, Colocynthis citrullus, Nicotiana tabacum

and Eucalyptus camaldulensis against Tribolium castaneum, Rhyzopertha dominica and

Trogoderma granarium. Three concentrations of each essential oil (2, 4 and 6%) were tested

by following filter paper area preference method at ambient temperature of 30 ± 2 °C and 65 ±

5% relative humidity. All essential oil demonstrated growth inhibition effect against all test

insects. A. indica was found to be the potent against R. dominica and T. castaneum, T.

granarium respectively. Minimum larvae, pupae and adults were found at 6% concentration

followed by 4% and 2% against R. dominica, T. castaneum and T. granarium respectively.


= 0.000), R. dominica (F(8, 90) = 8.70, p

114

8.1 Introduction

Agriculture stored products are heavily attacked by stored grain insect pests throughout the

world and these pests cause 10 to 40 % annual loss (Rajashekar et al., 2010). High level of

insect pest infestation makes the stored product quantitatively and qualitatively unfit for human

consumption not only by feeding on it but also contaminate it by leaving exuviae, casting skins,

webbing and producing toxic materials (Arlian, 2002; Rajashekar et al., 2012). T. granarium

(Irshad et al., 1988; Khan and Kulachi, 2002) as primary whereas T. castaneum (Mondal, 1994;

Moino et al., 1998) and C. ferrugineus (Tuff and Telford, 1964; Anonymous, 2009) as

secondary pests cause considerable damage to stored commodities.

Currently deltamethrin, chlorpyrifos, bifenthrin and malathion are applied as contact

synthetic insecticide to protect stored products but their high cost, adverse effects on nontarget

organisms and development of insect resistance reduced their feasibility (Georghiou, 1990;

Saxena and Sinha, 1995). One of the most effective methods is fumigation. Phosphine and

methyl bromide have long been established fumigant but the development of insect resistance

against phosphine (Leelaja et al., 2007; Rajashekar et al., 2006) and contribution of methyl

bromide toward ozone depletion (WMO, 1995; Shaaya and Kostyukovsky, 2006) made them

unfit for application as stored product protectant.

This situation requires the development of some novel, biodegradable, environmental

friendly and target specific insecticide. Essential oils extracted from higher plants have been

suggested as replacement for these synthetic conventional insecticides (Bakkali et al ., 2008;

Isman and Machial, 2006).

Plant derived essential oils have demonstrated insect growth regulatory actions along

with various insecticidal activities against insect pests of stored products. They have been

proved for their potential in killing insect eggs and affecting the growth of larvae and pupae of

stored product insects. Su et al. (1972) reported that the nonvolatile portion of Citrus oil, 1% by

weight protected peas from C. maculatus infestation when applied to black eyed pea surfaces.

Bioactive compounds of plants origin having ovicidal and growth regulatory action is being

used as grains’ guard agents against beetles in storage (Babu et al., 1989; Khaire, 1992; Pacheco

et al., 1995; Guillaume et al., 2005; Adugna, 2006). Ho et al. (1997) determined that when seeds

treated with steam distilled oil from fresh garlic (Allium sativum), the eggs of T. castaneum and


= 0.000), R. dominica (F(8, 90) = 8.70, p

115

S. zeamais failed to produce F1 progeny by the application of the concentration of >2000ppm

in rice and > 5000 ppm in wheat, respectively.

Growth regulatory effects of Artemisia annua L. (Astraceae) were studied by

Haghighian et al. (2008) against T. confusum. It was observed that 1600 ¼L/L dose of essential

oil significantly affected the weight of adults (1.31 ± 0.015), larvae (0.48 ± 0.003) and pupae

(2.17 ±0.02). Moreover, essential oil showed ovicidal effect and reduced the number of adults

in F1 progeny as compared to control. Similar studies to estimate growth regulatory effects of

Ajuga. iva, Peganum harmala, Raphanus vaphanistrum and Aristolochia baetica against T.

castaneum were conducted by Jbilou et al. (2006). Due to the effect of P. harmala and R.

vaphanistrum, last larval period was observed to prolong from 7.1 days (control) to 8.2 and 8.3

days, respectively. On the other hand larval period was decreased from 7.1 (control) to 6.6 days

when test insect was treated with A. iva.

Main constituents of the essential oil of garlic (methyl allyl disulfide, diallyl trisulfide

and Allium sativum L.) were examined against T. castaneum and S. zeamais by Huang et al.

(2000) and they found Methyl allyl disulfide the most effective as it decreased insect growth

rate and food utilization in both test insects.




= 0.000), R. dominica (F(8, 90) = 8.70, p

116


The procedure for collection and rearing of insects and extraction of essential oils is given

earlier in chapter 3

8.2.1 Bioassay

Wheat grain for Trogoderma granarium and wheat flour for Tribolium castaneum and

Rhyzopertha dominica was treated with 2 ml of different concentrations (2, 4 and 6%) of each

essential oil. Acetone alone was used as control. Treated samples were allowed to evaporate

solvent for 10 min by keeping them in dry air. Twenty (20) larvae of the each test insect were

released in each jar on treated diet. Jars were kept in incubator (Model MIR-254, SANYO) at

25 ± 2 °C and 65 ± 5 % relative humidity. Data for larval emergence, pupation and adult

emergence from pupae were recorded after 7, 15, 21, 30, 45 day after removing adults from

jars. Experiment was carried out with 3 replications.



(Stat Soft, 8.0). Means were separated by the Tukey's multiple range test when ANOVA was

significant (p < 0.05)

8.3 Results:

Insect growth regulatory activities of A. indica, M. azedarach, C. citrullus, N. tabacum and E.

camaldulensis were determined against T. castaneum, T. granarium and R. dominica. For this

purpose number of larval survived, pupae transformation from larvae and adult emergence from

pupae of T. castaneum, T. granarium and R. dominica were checked.

8.3.1 Larva survival:

All essential oils applied were found to have significant toxic effect on number of larvae against

T. castaneum, T. granarium and R. dominica. Number of larvae was minimum in R. dominica,

T. castaneum and T. granarium against A. indica (49.16, 57.50 and 60.41%) followed by M.

azedarach (55.83, 64.58 and 67.08%) C. citrullus (57.91, 67.50 and 67.91%),


= 0.000), R. dominica (F(8, 90) = 8.70, p

117

N. tabacum (62.91, 72.91and 72.91%) and E. camaldulensis (73.75, 77.50 and 82.5%)

respectively. (Fig. 8.1). Overall effect of concentration on the larva survival was found to be

significant. As the concentration of essential oil increased the number of larvae decreased

significantly. Number of larvae was minimum (33.66, 47 and 48.66%) at 6% concentration

followed by 4% (45.66, 58.333 and 60.66%), 2 % (61.66, 70.66 and 76.66%) and control

treatment (96, 98 and 94%) in R. dominica, T. castaneum and T. granarium respectively (Fig.

8.2) Data given in Table 8.1 exhibited the interaction of plant essential oil against the larva

survival of R. dominica, T. castaneum and T. granarium. Results showed that minimum larvae

was survived (21.66, 33.66 and 36.66) against A. indica at 6% concentration of essential oil

which were statistically at par with M. azedarach (28.33, 41.33 and 43.33%) while survived

larva against M. azedarach was also at par with C. citrullus (31.33, 45.00 and 48.33%) while

C. citrullus was at par with N. tabacum (38.83, 53.33, 53.66%) while maximum larvae was

survived against E. camaldulensis (48, 61.66 and 63.66%) at this concentration in R. dominica,

T. castaneum and T. granarium respectively. At 2 % concentration minimum larvae was found

against A. indica (45.00, 56.33 and 60.00%) was statistically different from all essential oils

while maximum larvae was observed against E. camaldulensis (71.33, 83.33 and 91.00%) was

also statistically different from all other plant essential oils against R. dominica, T. castaneum

and T. granarium.

8.3.2 Pupal transformation

All essential oils applied were found to have significant toxic effect on pupal transformation

against T. castaneum, T. granarium and R. dominica. Number of pupae transformation was

minimum in R. dominica, T. castaneum and T. granarium against A. indica (41.66, 45.83and

52.08 %) followed by M. azedarach (48.33, 53.33 and 59.58 %) C. citrullus (50.41, 55.83 and

60.83%), N. tabacum (55.41, 60.83 and 65.41 %) and E. camaldulensis (63.75, 66.66 and

74.16%) respectively (Fig. 8.3). Overall effect of concentration on the pupae transformation

was found to be significant. As the concentration of essential oil increase the pupae

transformation decreased significantly. Number of pupae was minimum (23.66, 32 and 38.66%)

at 6% concentration followed by 4% (35.66, 43.33 and 50.66%), 2% (51.66, 55.66 and 66.66%)

and control treatment (95, 96 and 93%) in R. dominica, T. castaneum and T. granarium


= 0.000), R. dominica (F(8, 90) = 8.70, p

118

respectively (Fig. 8.4). Data given in table 8.1 exhibited the interaction of plant essential oil

against the pupae transformation of R. dominica, T. castaneum and T. granarium. Results

showed that minimum pupae were emerged in R. dominica, T. castaneum and T. granarium

(11.66, 13.33 and 26.66) against A. indica at 6% concentration of essential oils which were

statistically different from all other plat essential oils in case of R. dominica, T. castaneum while

statistically at par with M. azedarach in case of T. granarium. The percentage of pupal

transformation in case of M. azedarach (18.33, 26.66 and 33.33%) against R. dominica, T.

castaneum and T. granarium was statistically at par with C. citrullus (21.66, 30.33 and 36.33%),

pupae transformation was maximum against E. camaldulensis (38.33, 46.66 and53.33%) in R.

dominica, T. castaneum and T. granarium respectively. At 2 % concentration minimum pupae

was found against A. indica (35.00, 41.66 and 50.00%) was statistically different from all

essential oils while maximum pupae was observed against E. camaldulensis (64.33, 68.33 and

78.33%) was also statistically different from all other plant essential oils against R. dominica,

T. castaneum and T. granarium respectively.

8.3.3 Adult emergence

All essential oils applied were found to have significant toxic effect on adult emergence against

T. castaneum, T. granarium and R. dominica. Number of adult emerged was minimum in R.

dominica, T. castaneum and T. granarium against A. indica (31.66, 33.75 and

44.58%) followed by M. azedarach (39.58, 40.83 and 51.25%) C. citrullus (40.41, 43.75 and

51.25%), N. tabacum (45.41, 48.75 and 56.25%) and E. camaldulensis (53.75, 55 and 66.66%)

respectively (Fig. 8.5) Overall effect of concentration on the adult emergence was found to be

significant. As the concentration of essential oil increase the adult emergence decreased

significantly. Number of adults were minimum (13.66, 17and 32%) at 6% concentration

followed by 4% (25.66, 28.33 and 40.66%), 2% (40.66, 41.44 and 53.33%) and control

treatment (90, 91 and 87%) in R. dominica, T. castaneum and T. granarium respectively (Fig.

8.6) Data given in table 8.3 exhibited the interaction of plant essential oil against the adult

emergence of R. dominica, T. castaneum and T. granarium. Results showed that minimum

adults was emerged in R. dominica, T. castaneum and T. granarium (1.66, 3.3 and 18.33) was

statistically at par with M. azedarach (8.33, 11.33 and 28.66%) was statistically at par with C.

citrullus (11.00, 15.66 and 31.33%) at par with N. tabacum (18.33,


= 0.000), R. dominica (F(8, 90) = 8.70, p

119

23.00 and 35.00) against R. dominica, T. castaneum and T. granarium respectively while

maximum adults were emerged against E. camaldulensis (28.33, 31.66 and 48.66%) in R.

dominica, T. castaneum and T. granarium respectively was statistically different from all other

plant essential oils. At 2 % concentration minimum adults were found against A. indica (23.00,

25.00 and 38.33%) was statistically different from all essential oils while maximum adults was

observed against E. camaldulensis (53.00, 58.33 and 71.33%) was also statistically different

from all other plant essential oils in T. castaneum , R. dominica and T. granarium respectively

8.4 Discussion

This experiment was conducted to check out the growth inhibition potential of essential oils of

A. indica, M. azedarach, C. citrullus, N. tabacum and E. camaldulensis were determined against

T. castaneum, T. granarium and R. dominica. For this purpose number of larval survived, pupae

transformation from larvae and adult emergence from pupae of T. castaneum, T. granarium and

R. dominica were recorded. All the plant essential oil found significant in arresting the growth

of test insects these results are in accordance with the pervious studies conducted by Obeng-

Ofori, (1995) in which plant oils (cottonseed, soybean, corn, groundnut and palm) at different

dosages were evaluated in the laboratory for their ability to suppress the populations of

Cryptolestes pusillus and Rhyzopertha dominica in maize and sorghum. A dose of 5 ml/kg of

each oil significantly decreased the progeny produced by R. dominica. Similarly Chaubey

(2007) selected essential oils from fruits of Nigella sativa, Anethum graveolens and

Trachyspermum ammi to examine developmental inhibitory activities against T. castaneum.

Essential oils significantly reduced the development of larvae. However, contradictory results

were obtained by a study presented by Mukherjee and Ramachandran, (1989), Fecundity was

not affected when fed on azadirachtin treated flour. Topical application at 1, 2 and 5 ppm on

eggs and larvae did not have any adverse effects on T. castaneum, this may be due to different

environmental conditions

Effect of concentration on the survival of larva, transformation of pupa and emergence

of the adults was significant number of larvae was minimum (33.66, 47 and 48.66%) at 6%

concentration followed by 4% (45.66, 58.333 and 60.66%), 2 % (61.66, 70.66 and 76.66%) and

control treatment (96, 98 and 94%) similarly Number of pupae was minimum (23.66, 32 and

38.66%) at 6% concentration followed by 4% (35.66, 43.33 and 50.66%), 2% (51.66, 55.66 and


= 0.000), R. dominica (F(8, 90) = 8.70, p

120

66.66%) and control treatment (95, 96 and 93%) and the number of adults was minimum (23.66,

32 and 38.66%) at 6% concentration followed by 4% (35.66, 43.33 and 50.66%), 2% (51.66,

55.66 and 66.66%) and control treatment (95, 96 and 93%) in R. dominica, T. castaneum and

T. granarium respectively. Results clearly indicated that as the concentration of the essential oil

increased the development of the insect in all stages reduced similar results were obtained by

the studies reported by different scientists. Tripathi et al. (2001) extracted essential oil from the

leaves of turmeric, Curcuma longa L., and applied for progeny production in three stored-

product beetles, Rhyzopertha dominica, Sitophilus oryzae and Tribolium castaneum. Results

showed that in T. castaneum, the C. longa oil reduced oviposition and egg hatching by 72 and

80%, respectively at the concentration of 5.2mg/cm2. At the concentration of 40.5mg/g food,

the oil totally suppressed progeny production of all the three test insects similarly Tariq (2013)

tested the Fine Neem Seed Powder (NSP) in three dozes, viz. 0.5%, 1.0% and 2.0% (w/w) for

determination of its toxicity and detrimental effects on life stages of red flour beetle, Tribolium

castaneum. NSP served as an Insect Growth Regulator (IGR). It was observed that at 0.5%

dose, the number of larvae was not very different to control but the weight of larvae was lesser.

At 1.0% dose, the number and weight of larvae were significantly reduced. At 2.0% dose, both

the number and weight of larvae, pupae and adults were reduced remarkably. The insect growth

inhibition was increased by increasing the dose of NSP. Similarly Mhemed, (2011) the seed

powders of four plants; harmal (Harmal peganum), black pepper (Piper nigrum), radish

(Raphanus sativus) and celery (Apinum graveolens) were tested at the concentrations 2%, 4%

and 6% to evaluate their effects on some biological aspects and mortality of Trogoderma

granarium Everts Seed powders had also significant effect on decreasing average number of F1

progeny. The number of F1 progeny found in celery treatment at concentration of 6% was zero,

followed by pepper 23, radish 31 and harmal 56. Whereas in the control was 78. The seed

powders disrupted the life cycle of the insect resulting in prolonging the period of F1 adult

appearance; the periods were 20 days for control, 26 days for harmal and radish, no adult

appearance in pepper and celery even after 34 days. Similar kind of results were found by a

research presented by Upadhyay and Jaiswal (2007) in which the effective concentration

(EC50) of P. nigrum essential oil. It was observed that the concentration to lessen the number

of T. castaneum larvae transformed to pupae to 50 percent was 6.919 μl. The percentage of test


= 0.000), R. dominica (F(8, 90) = 8.70, p

121

insect larvae transferred to pupae stage and percentage of pupae transferred to adult stage was

significantly decreased with the increase in concentration when compared to the control.


= 0.000), R. dominica (F(8, 90) = 8.70, p

122

Fig 8.1 Mean Larvae survived (%) in T. castaneum, T. granarium and R. dominica against the


b

b

a

a

b

c

c

b

b

c

a

a

a

a

a

0

10

20

30

40

50

60

70

80

90

100

A. indica M. azedarach C. citrullus N. tabacum E. camaldulensis

Plants



= 0.000), R. dominica (F(8, 90) = 8.70, p

123

Fig 8.2 Mean Larvae survived (%) in T. castaneum, T. granarium and R. dominica against the

essential oils of M. azedarach, A. indica, E. camaldulensis, N. tabacum and C. citrullus at

different concentrations

Table 8.1 Percentage larvae survived (Mean ± SE) of T. castaneum (F(12, 40) = 12.21, p = 0.000),


0.0000) against different concentrations of essential oils.

Plants Conc. (%) (%) Larvae survived

b

b

b

b

a

a

a

b

c

c

c

a

0

20

40

60

80

100

120

6 % 4 % 2 % 0 %

Concentration



= 0.000), R. dominica (F(8, 90) = 8.70, p

124


A. indica

2

4

56.33±1.66de

43.66±1.66b

45.00±1.66 ef

33.33±1.66bcd

60.00±1.66 ef

48.33±1.66bcd

6 33.66±1.66a 21.66±1.66 a 36.66±1.66 a

0 98.33±1.66 j 96.66±1.66 k 96.66±1.66 k

M. azedarach

2

4

65.00±1.66efg

53.33±1.66cd

48.33±1.66 gh

41.66±1.66 ef

71.66±1.66 gh

56.66±1.66def

6 41.33±1.66ab 28.33±1.66 ab 43.33±1.66 ab

0 96.66±1.66 j 96.66±1.66 k 96.66±1.66 k

E. camaldulensis

2

4

83.33±1.66i

71.66±1.66gh

71.33±1.66jk

68.66±1.66hi

91.00±1.66jk

76.33±1.66hi

6 61.66±1.66def 48.00±1.66fg 63.66±1.66fg

0 98.33±1.66 j 96.66±1.66 k 96.00±1.66 k

N. tabacum

2

4

76.33±1.66hi

63.66±1.66fg

63.33±1.66hi

48.33±1.66fg

83.33±1.66 hi

63.33±1.66 fg

6 53.66±1.66cd 38.33±1.66cde 53.33±1.66cde

0 98.33±1.66 j 96.66±1.66 k 96.66±1.66 k

C. citrullus

2

4

71.66±1.66gh

56.00±1.66de

60.00±1.66hi

43.66±1.66ef

75.00±1.66hi

56.66±1.66ef

6 45.00±1.66bc 31.33±1.66bc 48.33±1.66bc

0 98.33±1.66 j 96.66±1.66 k 96.66±1.66 k


= 0.000), R. dominica (F(8, 90) = 8.70, p

125

Fig 8.3 Mean Pupae transformed (%) in T. castaneum, T. granarium and R. dominica against

the essential oils of M. azedarach, A. indica, E. camaldulensis, N. tabacum and C. citrullus

b

b

b

b

b

c

c

c

c

c

a

a

a

a

a

0

10

20

30

40

50

60

70

80

90


Plants



= 0.000), R. dominica (F(8, 90) = 8.70, p

126

Fig 8.4 Mean Pupae transformed (%) in T. castaneum, T. granarium and R. dominica against

the essential oils of M. azedarach, A. indica, E. camaldulensis, N. tabacum and C. citrullus at


Table 8.2 Percentage pupal transformation (Mean ± SE) of T. castaneum (F(12, 40) = 10.92, p =

0.000), T. granarium (F(12, 40) = 14.45), p = 0.000) and R. dominica (F(12, 40) = 12.94, p =


0

20

40

60

80

100

120

6 % 4 % 2 % 0 %

Concentration



= 0.000), R. dominica (F(8, 90) = 8.70, p

127

Plants Conc.

(%)

(%) Pupal Emergence


A. indica

2

4

41.66±1.66ef

28.33±1.66 b

35.00±1.66ef

23.33±1.66bcd

50.00±1.66ef

38.33±1.66bcd

6 18.33±1.66 a 11.66±1.66 a 26.66±1.66 a

0 95.00±1.66 j 96.66±1.66 k 93.33±1.66 k

M. azedarach

2

4

50.0±1.6ef

38.33±1.66de

18.33±1.66ab

31.66±1.66def

61.66±1.66gh

46.66±1.66def

6 26.66±2.18bc 18.33±1.66bc 33.33±1.66ab

0 98.33 ±1.66j 96.66±1.66 k 96.66±1.66 k

E. camaldulensis

2

4

68.33±1.66 i

56.66±1.66gh

68.33±1.66 j

51.66±1.66hi

78.33±1.66 j

66.66±1.66hi

6 46.66±2.8def 38.33±1.66fg 53.33±1.66fg

0 95.00±1.66 j 96.66±1.66 k 93.93±1.66 k

N. tabacum

2

4

61.66±1.66 hi

51.66±1.66fg

58.33±1.66i

38.33±1.66fg

73.33±1.66i

53.33±1.66fg

6 38.33±1.66cd 28.33±1.66def 43.33±1.66cde

0 98.33±1.66 j 96.66±1.66 k 91.66±1.66jk

C. citrullus

2

4

30.33bc±1.66

41.66de±2.88

50.00±1.66hi

33.33±1.66ef

65.00±1.66hi

48.33±1.66ef

6 56.66±±2.88gh 21.66±1.66bc 36.33±1.66bc

0 95.00gh±1.66 96.66±1.66 k 93.33±1.66 k


= 0.000), R. dominica (F(8, 90) = 8.70, p

128

Fig 8.5 Mean adult emergence (%) in T. castaneum, T. granarium and R. dominica against the


b

b

b

b

b

c

c

c

c

c

a

a

a

a

a

0

10

20

30

40

50

60

70

80


Plants



= 0.000), R. dominica (F(8, 90) = 8.70, p

129

Concentration

Fig 8.6 Mean adult emergence (%) in T. castaneum, T. granarium and R. dominica against the

essential oils of M. azedarach, A. indica, E. camaldulensis, N. tabacum and C. citrullus at


Table 8.3 Percentage adult emergence (Mean ± SE) of T. castaneum (F(12, 40) = 11.01, p = 0.000),



Plants Conc.

(%)

(%) adult emergence


b

b

c

a

c

c

b

b

a

a

a

c

0

20

40

60

80

100

120

6 % 4 % 2 % % 0

m T. castaneu R. dominica T. granarium


= 0.000), R. dominica (F(8, 90) = 8.70, p

130

A. indica

2

4

23.33±1.66de

13.33±1.66 b

25.00±1.66 ef

13.33±1.66bcd

38.3±1.6bcde

31.66±1.66bc

6 3.33±1.66 a 1.66±1.66 a 18.33±3.33 a

0 88.66±1.66 j 86.66±1.66 k 86.66±1.66 i

M. azedarach

2

4

35.33±1.66 def

23.66±1.66cd

36.66±1.66gh

21.66±1.66def

46.66±1.66efg

33.33±1.66bcd

6 11.33±1.66 ab 8.33±1.66ab 31.66±1.66bc

0 88.66±1.66 j 86.66±1.66k 88.66±2.88 i

E. camaldulensis

2

4

53.00±1.66def

41.33±1.66gh

58.33±1.66 j

41.66±1.66hi

71.0±1.66 h

58.33±1.66 g

6 31.66±1.66 i 28.33±1.66fg 45.66±2.88 def

0 91.33±1.66 j 86.66±1.66 k 90.33±3.33 i

N. tabacum

2

4

46.33±1.66efg

36.66±1.66hi

48.66±1.66i

28.00±1.66fg

58.33±4.40 g

43.33±1.66cdef

6 23.00±1.66cd 18.33±1.66cde 35.00±3.33bcde

0 91.66±1.66 j 91.33±1.66 k 91.33±1.66 i

C. citrullus

2

4

41.00±1.66gh

26.33±1.66de

40.66±1.66bc

23.33±1.66def

51.66±1.66fg

38.33±1.66bcde

6 15.66±1.66bc 11.00±1.66hi 28.33±3.33ab

0 93.00±1.66 j 86.66±1.66 k 91.33±1.66i

Summary

Post-harvest losses are key issue which is damaging Pakistan as well as world’s economy

seriously. Agricultural stored commodities, cereals and their products face heavy quantitative

and qualitative damage and loss due to insect pest infestation. Trogoderma granarium,


= 0.000), R. dominica (F(8, 90) = 8.70, p

131

Tribolium castaneum and Rhyzopertha dominica are the major insect pests responsible for this

economic loss to stored agriculture products in Pakistan. To ensure the grain safety during

storage by minimizing insect pest infestation, different conventional control strategies are used

which have some drawbacks. Increasing insect pest resistance and resurgence against traditional

insecticides, environmental and human health hazards by residual effect, of currently used

insecticides, necessitates development of more efficient, human and environment friendly

biorational control strategies. In present studies, essential oils and ozone were evaluated as a

replacement for these conventional control techniques because of their salient features i.e., no

residual effects on stored commodity, safe for environment, human and non-target organisms,

no handling, storage and dispose of hazards, economical, easy to use and having strong

insecticidal properties even against resistant strains of stored grain insect pests.

The repellent, contact, fumigant, antifeedant and growth regulatory activities of

essential oils of Azadirachta indica, Melia azedarach, Colocynthis citrullus, Nicotiana

tabacum and Eucalyptus camaldulensis were checked against Tribolium castaneum,

Rhyzopertha dominica and Trogoderma granarium. Three concentrations of each essential oil

(2, 4 and 6%) were tested in each experiment. Findings of this research work revealed that for

repellant bioactivities A. indica was found most potent against T. castaneum and R. dominica

while M. azidiractha was most active against T. granarium. Overall effect of the concentration

was highly significant and the maximum repellency was obtained at 6% concentration followed

by 4% and 2%. Results also showed that as the duration of exposure increased repellency

decreased. For contact and fumigant toxicities results revealed that A. indica was found most

toxic against T. castaneum and R. dominica while N. tabacum was most effective against T.

granarium. Results also showed that fumigant and contact mortalities were maximum at highest

level of concentration and exposure period. In case of antifeedant and insect growth regulatory

activities the most potent essential oil was found A. indica followed by M. azedarach,C.

citrullus N. tabacum and E. camaldulensis against T. castaneum, T. granarium and R.

dominica respectively. Antifeedant and growth inhibition effect was maximum at 6% followed

by 4% and 2% concentration.


= 0.000), R. dominica (F(8, 90) = 8.70, p

132

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