POTENTIAL FOR BIO-MANAGEMENT OF HELICOVERPA ARMIGERA HÜBNER ON TOMATO THROUGH COMBINATIONS OF MICROBIAL

AGENTS

By

MIRZA ABDUL QAYYUM

M.Sc. (Hons.) Entomology

2004-ag-1268

A thesis submitted in partial fulfilment of the requirements for the degree of

DOCTOR OF PHILOSOPHY (Ph.D.) in

ENTOMOLOGY

Department of Entomology

Faculty of Agriculture University of Agriculture, Faisalabad

Pakistan, Pakistan

2015

Declaration

I hereby declare that the contents of the thesis, entitled “Potential for bio-management of

Helicoverpa armigera Hübner on tomato through combinations of microbial agents” are

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

(except the references, standard mathematical and genetic models/equations/formula/

protocols etc.). I further declare that this work has not been submitted for award of any

other diploma/degree. The University may take action if the information provided is found

inaccurate at any stage (in case of any default, the scholar will be proceeded against as per HEC

plagiarism policy).

_______________________

Mirza Abdul Qayyum

2004-ag-1268

The Controller of Examinations,

University of Agriculture,

Faisalabad.

We, the Supervisory Committee, certify that the contents and format of thesis

submitted by Mirza Abdul Qayyum Regd. No. 2004-ag-1268 have been found satisfactory

and recommend that it be processed for evaluation by the External Examiner(s) for the

award of degree.

Supervisory Committee

Chairman

Dr. Waqas Wakil

Member

Prof. Dr. M. Jalal Arif

Member

Prof. Dr. Shahbaz Talib Sahi

Dedication

This piece of humble effort is being dedicated to

Prayers of my mother

Cares of my father

Inspiration of my sisters

Sincere concerns of my nephews and nieces

and

Guidence of my beloved teachers

Acknowledgements

All praise to ALMIGHTY ALLAH alone, the Most Merciful and the Most Compassionate and His Holy Prophet MUHAMMAD (Peace be upon him) the most perfect and exalted among and even of ever born on the surface of earth, who is, forever a torch of guidance and knowledge for the humanity as a whole.

The work presented in this manuscript was accomplished under the inspiring guidance, gorgeous assistance, constructive criticism, enlightened and kind supervision of Dr. Waqas Wakil, Assistant Professor Department of Entomology, University of Agriculture, Faisalabad. His efforts toward the inculcation of the spirit of constant work and the maintenance of professional integrity besides other invaluable words of advice will always serve as beacon of light throughout the course of my life. I take this humblest opportunity to my deepest sense of gratitude and thankfulness to him.

It is matter of great pleasure and honour for me to express my gratitude and appreciation to the members of my Supervisory Committee, Prof. Dr. M. Jalal Arif, Professor and Chairman, Department of Entomology, University of Agriculture, Faisalabad and Dr. Shahbaz Talib Sahi, Professor and Chairman, Department of Plant Pathology, University of Agriculture, Faisalabad, under whose kind and scholastic guidance, keen interest and constant encouragement, I completed my research.

Thanks are extended to my colleagues especially Dr. Kashif Ali, M. Yasin, M. Tahir, My precious friends Usman Tariq, Hafeez-ur-Rahman, Imran Ahmad, M. Saleem and Amjad Iqbal Amjad for their cooperation during the write-up of this dissertation.

This acknowledgment would be incomplete unless and until I offer my humble veneration to my affectionate Brothers Mirza M. Yousaf, Mirza M. Amin, Mirza M. Saleem, Mirza M. Naeem, Mirza M. Hanif and Mirza M. Umar and all my sisters, my fiancé, my nephews Mirza Salman Yousaf, Mirza Burhan Yousaf who always prayed for me, for their all inclusive support to carry myself through the noble ideas of my life.

Special mention goes to my enthusiastic approach of Higher Education Commission, Islamabad for providing financial assistance for the study. My PhD has been an amazing experience and I thank wholeheartedly, not only for the tremendous academic support, but also for giving me so many wonderful opportunities.

Mirza Abdul Qayyum

i

CHAPTER 1 Introduction ………………………………………………………………………………………………………………………………… 1-12

CHAPTER 2 Review of literature …………………………………………………………………………………………………………………….. 13-48 2.1 Insecticide Resistance ………………………………………………………………………………………………………. 13 2.1.1 Result oriented studies ……………………………………………………………………………………. 14

2.2 Entomopathogenic Fungi ………………………………………………………………………………………………….. 15-27 2.2.1 History …..………………………………………………………………………………………………………… 15 2.2.2 Geographical distribution ………………………………………………………………………………… 16 2.2.3 Application in pest management …………………………………………………………………….. 17 2.2.4 Classification of entomopathogenic fungi ……………………………………………………….. 18 2.2.5 Biology of entomopathogenic fungi ………………………………………………………………… 18 2.2.6 Mode of action ……………………………………………………………………………………………….. 19 2.2.7 Disease related virulence enzymes …………………………………………………………………. 20 2.2.8 Host range and specificity ……………………………………………………………………………….. 20 2.2.9 Safety to non-target organisms ………………………………………………………………………. 21 2.2.10 Combining entomopathogenic fungi with other microorganisms ……………………. 22 2.2.11 Entomopathogenic fungi as bio-pesticides ………………………………………………………. 23 2.2.12 Entomopathogenic fungi as endophyte …………………………………………………………… 23 2.2.13 Toxins and commercial products ……………………………..……………………………………… 24 2.2.14 Factors influencing the efficacy of entomopathogenic fungi …………………………… 25 2.2.15 Field application of entomopathogenic fungi ………………………………………………….. 26 2.2.16 Result oriented studies ……………………………………………………………………………………. 26 2.3 Entomopathogenic bacteria ……………………………………………………………………………………………… 28-35 2.3.1 History …………………………………………………………………………………………………………….. 28 2.3.2 Important entomopathogenic bacteria …………………………………………………………… 29 2.3.3 Bacillus thuringiensis ………………………………………………………………………………………. 29 2.3.3.1 Life cycle …………………………………………………………………………………………………………. 30 2.3.3.2 Ecology ……………………………………………………………………………………………………………. 30 2.3.3.3 Mechanism of action ………………………………………………………………………………………. 31 2.3.3.4 Commercial formulations ………………………………………………………………………………… 31 2.3.3.5 Methods of applications of Bacillus thuringiensis products ……………………………… 32 2.3.3.6 Superiority of Bt products over synthetic insecticides …………………………………….. 32 2.3.3.7 Concerns to use of Bt ………………………………………………………………………………………. 33 2.3.3.8 Interaction of Bt products and other toxins …………………………………………………….. 34 2.3.3.9 Effect of Bacillus thuringiensis on non-target invertebrates ……………………………. 34 2.3.3.10 Result oriented studies ……………………………………………………………………………………. 35 2.4 Entomopathogenic Viruses ………………………………………………………………………………………………. 36-41 2.4.1 Importance of entomopathogenic viruses …………………………………………………….... 36 2.4.2 History …………………………………………………………………………………………………………….. 37 2.4.3 Classification …………………………………………………………………………………………………… 38 2.4.4 Baculoviruses ………………………………………………………………………………………………….. 38 2.4.5 Nuclear polyhedrosis virus ………………………………………………………………………………. 39 2.4.6 Mode of action ……………………………………………………………………………………………….. 39 2.4.7 Ecology of baculoviruses …………………………………………………………………………………. 40 2.4.8 Role of NPV in agriculture ……………………………………………………………………………….. 40 2.4.9 Effect on non-target insects ……………………………………… ……………………………………. 40

Table of contents

ii

2.4.10 Result oriented studies ……………………………………………………………………………………. 41 2.5 Combined application of microbial agents ………………………………………………………………………… 43 2.6 Foliar persistence ……………………………………………………………………………………………………………… 45 2.7 Management practices.……………………………………………………………………………………………………. 46 2.8 References ……………………………………………………………………………………………………………………….. 48-81

CHAPTER 3 Multi-resistance against organophosphates, pyrethroids and newer chemistry insecticides in Pakistani populations of Helicoverpa armigera

82-102

3.1 Introduction ……………………………………………………………………………………………………………………… 83 3.2 Material and methods ………………………………………………………………………………………………………. 84 3.2.1 Test insects ……………………………………………………………………………………………………… 84 3.2.1 Test chemicals …………………………………………………………………………………………………. 84 3.2.3 Bioassays ………………………………………………………………………………………………………… 84 3.2.4 Statistical analysis …………………………………………………………………………………………… 85 3.3 Results …………………………………………………………………………………………...………………………………… 86 3.3.1 Organophosphates ………………………………………………………………………………………….. 86 3.3.1.1 Profenofos ………………………………………………………………………………………………………. 86 3.3.1.2 Chlorpyrifos …………………………………………………………………………………………………….. 86 3.3.2 Pyrethroids ……………………………………………………………………………………………………… 86 3.3.2.1 Cypermethrin ………………………………………………………………………………………………….. 86 3.3.2.2 Deltamethrin …………………………………………………………………………………………………… 87 3.3.3 Newer chemistry insecticides ………………………………………………………………………….. 87 3.3.3.1 Chlorfenapyr …………………………………………………………………………………………………… 87 3.3.3.2 Spinosad …………………………………………………………………………………………………………. 87 3.3.3.3 Indoxacarb ………………………………………………………………………………………………………. 87 3.3.3.4 Abamectin and Emamectin benzoate ……………………………………………………………… 87 3.4 Discussion ………………………………………………………………………………………………………………………… 88 3.5 References ……………………………………………………………………………………………………………………….. 91

CHAPTER 4 Pathogenicity of tomato plants endophytically colonized with Beauveria bassiana against Helicoverpa armigera

103-127

4.1 Introduction ……………………………………………………………………………………………………………………… 104 4.2 Material and Methods ……………………………………………………………………………………………………… 105 4.2.1 Rearing of insects ……………………………………………………………………………………………. 105 4.2.2 Isolation of Endophytic fungi …………………………………………………………………………… 106 4.2.3 Fungal strains and culturing …………………………………………………………………………….. 106 4.2.4 Colonization of fungi in plants …….…………………………………………………………………… 106 4.2.5 Bioassays ………………………………………………………………………………………………………… 107 4.2.6 Plant growth characteristics and re-isolation of fungi ……………………………………… 108 4.2.7 Statistical analysis …………………………………………………………………………………………… 108 4.3 Results ……………………………………………………………………………………………………………………………… 108 4.3.1 Colonization of Beauveria bassiana …………………………………………………………………. 108 4.3.2 Pathogenicity of Beauveria bassiana ………………………………………………………………. 108 4.3.2.1 Effect of inoculation methods …………………………………………………………………………. 109 4.3.2.2 Effect of fungal isolates …………………………………………………………………………………… 110 4.3.3 Effect of inoculation methods of growth and development of tomato plants….. 111 4.4 Discussion ………………………………………………………………………………………………………………………… 112 4.5 References ……………………………………………………………………………………………………………………….. 116

iii

CHAPTER 5

Biological potential of Beauveria bassiana and Bacillus thuringiensis against Helicoverpa armigera: evaluation of toxicity, growth parameters and foliar persistence

128-155

5.1 Introduction ……………………………………………………………………………………………………………………… 129 5.2 Material and Methods ……………………………………………………………………………………………………… 131 5.2.1 Rearing of insects ……………………………………………………………………………………………. 131

5.2.2 Preparation of B. thuringiensis spore-crystal mixtures ……………………………………. 131

5.2.3 Preparation of fungal culture ………………………………………………………………………….. 131

5.2.4 Compatibility of B. bassiana and B. thuringiensis ……………………………………………. 132

5.2.5 Toxicity assays …………………………………………………………………………………………………. 132

5.2.5 Foliar persistence ……………………………………………………………………………………………. 133

5.2.6 Statistical analysis …………………………………………………………………………………………… 133 5.3 Results ……………………………………………………………………………………………………………………………… 132 5.3.1 Screening of B. thuringiensis …………………………………………………………………………… 134

5.3.2 Toxicity of microbial agents …………………………………………………………………………….. 134

5.3.3 Effect on the growth and development …………………………………………………………… 134

5.3.4 Effect on food consumption and frass production …………………………………………… 135

5.3.5 Foliar persistence ……………………………………………………………………………………………. 136 5.4 Discussion ………………………………………………………………………………………………………………………… 137 5.5 References ……………………………………………………………………………………………………………………….. 143

CHAPTER 6 Integration of Bacillus thuringiensis and nuclear polyhedrosis virus for the effective control of Helicoverpa armigera

156-181

6.1 Introduction ……………………………………………………………………………………………………………………… 157 6.2 Material and methods ………………………………………………………………………………………………………. 159 6.2.1 Bacillus thuringiensis toxin ……………………………………………………………………………… 159

6.2.2 Preparation of NPV …………………………………………………………………………………………. 160

6.2.3 Toxicity assays ………………………………………………………………………………………………… 160

6.2.4 Foliar persistence ……………………………………………………………………………………………. 160

6.2.5 Statistical analysis …………………………………………………………………………………………… 161 6.3 Results ……………………………………………………………………………………………………………………………… 161 6.3.1 Toxicity of microbial agents …………………………………………………………………………….. 161

6.3.2 Effect on the growth and development …………………………………………………………… 162

6.3.3 Effect on food consumption and frass production …………………………………………… 163

6.3.4 Foliar persistence ……………………………………………………………………………………………. 164 6.4 Discussion ………………………………………………………………………………………………………………………… 164 6.5 References ……………………………………………………………………………………………………………………….. 169

CHAPTER 7 Field application of microbial agents for the control of tomato fruitworm and economics of application

182-210

7.1 Introduction ……………………………………………………………………………………………………………………… 183 7.2 Material and methods ………………………………………………………………………………………………………. 186 7.2.1 Experimental design ………………………………………………………………………………………… 186

7.2.2 Fungal strains and culturing …………………………………………………………………………….. 186

7.2.3 Preparation of B. thuringiensis solid formulation ……………………………………………. 186

iv

7.2.4 NPV suspension preparation …………………………………………………………………………… 187

7.2.5 Treatment application …………………………………………………………………………………….. 187 7.2.6 Data recording ………………………………………………………………………………………………… 187 7.2.7 Statistical analysis …………………………………………………………………………………………… 188 7.3 Results ……………………………………………………………………………………………………………………………… 188 7.3.1 Fruit infestation ………………………………………………………………………………………………. 189

7.3.2 Reduction in larval density and fruit infestation ……………………………………………… 189 7.3.3 Natural enemies ……………………………………………………………………………………………… 190 7.3.4 Fruit yield and cost-benefit ratio ……………………………………………………………………… 191 7.4 Discussion ………………………………………………………………………………………………………………………… 192 7.5 References ……………………………………………………………………………………………………………………….. 197

Summary 215-216

v

Table No. Title Page Table 3.1 Geographical characteristics of the localities from where H. armigera populations were

collected in Punjab, Pakistan 96

Table 3.2 Toxicity of organophosphates against different populations of H. armigera from various localities in Punjab, Pakistan

99

Table 3.3 Toxicity of synthetic pyrethroids against different populations of H. armigera from various localities in Punjab, Pakistan

100

Table 3.4 Toxicity of new chemistry insecticides against different populations of H. armigera from various localities in Punjab, Pakistan

101

Table 4.1 Factorial analysis of mortality, pupation and adult emergence of H. armigera exposed to B. bassiana inoculated tomato plants

121

Table 4.2 Mortality (%±SE) of second and fourth instar H. armigera larvae fed on fungal inoculated tomato plants at different weeks

122

Table 4.3 Pupation (%±SE) of second and fourth instar H. armigera larvae fed on fungal inoculated tomato plants at different weeks

123

Table 4.4 Adult emergence (%±SE) of second and fourth instar H. armigera larvae fed on fungal inoculated tomato plants at different intervals

124

Table 4.5 Factorial analysis of growth parameters of tomato plants when inoculated with B. bassiana

125

Table 5.1 Median lethal concentrations (μg g-1) and biological index of 10 different isolates of B. thuringiensis

151

Table 5.2 Factorial analysis of mortality, pupation, adult emergence and egg eclosion of H. armigera exposed to B. bassiana and B. thuringiensis

151

Table 5.3 Mean mortality (%±SE) of second and forth instar H. armigera larvae treated with B. bassiana and B. thuringiensis

152

Table 5.4 Pupation, adult emergence and eclosion (%±SE) of second and forth instar H. armigera larvae treated with B. thuringiensis and B. bassiana

152

Table 5.5 Effect of B. thuringiensis and B. bassiana on the development of H. armigera 153

Table 5.6 Analysis of co-variance for second and fourth instar larvae of H. armigera regarding weight gain and frass production at a given level of diet consumption when treated with B. bassiana and B. thuringiensis alone and in combination. Initial weight of larvae and diet consumption were taken as covariate

153

Table 6.1 Factorial analysis of mortality, pupation, adult emergence and egg eclosion of H. armigera exposed to HaNPV and B. thuringiensis

176

Table 6.2 Mean mortality (%±SE) of second and forth instar H. armigera larvae treated with HaNPV and B. thuringiensis

176

Table 6.3 Pupation, adult emergence and eclosion (%±SE) of second and forth instar H. armigera larvae treated with B. thuringiensis and HaNPV

177

Table 6.4 Effect of B. thuringiensis and HaNPV on the development of H. armigera 178

Table 6.5 Analysis of co-variance for second and fourth instar larvae of H. armigera regarding weight gain and frass production at a given level of diet consumption when treated with HaNPV and B. thuringiensis alone and in combination. Initial weight of larvae and diet consumption were taken as covariate

179

List of Tables

vi

Table 7.1 Analysis of variance table for larval population of H. armigera and tomato fruit infestation during 2013 and 2014

207

Table 7.2 Larval density of H. armigera in tomato plants during 2013and 2014 after application of five weekly sprays of microbial agents

208

Table 7.3 Means proportion tomato fruitworm infested fruits in Faisalabad during 2013 and 2014 after application of five weekly sprays of microbial agents

209

Table 7.4 Economics and cost-benefit ratio of application of microbial agents against H. armigera during 2013 and 2014

214

vii

Figure No. Title Page Figure 3.1 Location of fifteen sampling sites for H. armigera field populations in Punjab Province 97 Figure 3.2 Resistance ratio (RR) of organophosphate, pyrethroid and newer chemistry insecticides

against populations of H. armigera from various localities in the Pakistan Punjab 98

Figure 4.1 Colonization (%±SE) of tomato plants with different B. bassiana isolates using different methods, five weeks post incoluation

126

Figure 4.2 Growth response of tomato plants (a: plant height (cm), b: leaf area (cm2), c: fresh shjoot weighy, d: dry shoot weight (mg), e: fruit weight (mg), f: number of leaves) with different methods of inoculation using B. bassiana isolates

127

Figure 5.1 Weight gain (a), frass production (b) and diet consumption (c) in second and fourth instar larvae of H. armigera when treated with B. bassiana and B. thuringiensis

154

Figure 5.2 Foliar persistence of B. bassiana and B. thuringiensis on second (a) and fourth instar (b) larvae of H. armigera at different intervals under greenhouse conditions

155

Figure 6.1 Weight gain (a), frass production (b) and diet consumption (c) in second and fourth instar larvae of H. armigera when treated with HaNPV and B. thuringiensis

180

Figure 6.2 Foliar persistence of HaNPV and B. thuringiensis on second (a) and fourth instar (b) larvae of H. armigera at different intervals under greenhouse conditions

181

Figure 7.1 Reduction in larval density over control (%±SE) after field application of microbial agents during 2013(a) and 2014 (b)

210

Figure 7.2 Reduction in fruit infestation over control (%±SE) in tomato after field application of microbial agents during 2013(a) and 2014 (b)

211

Figure 7.3 Population levels of natural enemies (No.±SE) persisted after field application of microbial agents during 2013

212

Figure 7.4 Population levels of natural enemies (No.±SE) persisted after field application of microbial agents during 2014

213

List of Figures

viii

ANOVA Analysis of variance Bb Beauveria bassiana BI Biological index Bt Bacillus thuringiensis BV Budded viron CBR Cost-benefit ratio CDE Cuticle degrading enzymes CFU Colony forming unit CPV Cytoplasmic polyhedrosis virus CTF Co-toxicity factor DAM Direct application method DF Degree of freedom DNA Deoxyribonucleic acid EC Emulsifiable concentrate EPF Entomopathogenic fungi FAO Food and Agriculture Organization GABA Gamma-aminobutyric acid GDP Gross domestic product GoP Government of Pakistan GR Germination GV Grannulovirus HSD Honestly significant difference IM Injection method IPM Integrated Pest Management IRAC Insecticide Resistance Action

Committee IRM Insecticide Resistane Management LC Lethal conccentration LD Lethal dose

LT Lethal time MRR Marginal rate of return NPV Nucleopolyhedrosis Virus/ Nuclear polyhedrosis Virus NSKE Neem seed kernel extract OB Occlusion bodies ODV Occlusion derived viron OPs Organophosphates Pbo piperonyl butoxide PCR Polymerase chain reaction PDA Potato dextrose agar POB Polyhedral occlusion bodies RCBD Randomized complete block RDM Root dip method RR Resistance ratio SC Soluble concentrate SDA Sabouraud dextrose agar SDW Sterile distilled water SE Standard error SP Sporulation SSM Solid substrate method TB Tuberclosis TFW Tomato fruitoworm ULV Ultra low volume USDA United States Department of

Agriculture UV Ultra violet VG Vegetative growth Vip Vegetative insecticidal protein WG Wettable granule

List of Abbreviations

ix

Abstract

The present study was aimed at evaluating the insecticidal efficacy of different microbial agents

against Helicoverpa armigera, the voracious pest of tomato. The study comprised of the series of

experiments conducted to determine the extent of field evolved resistance in H. armigera against

conventional and new chemistry insecticides, to evaluate the endophytic capacity of Beauveria

bassiana into tomato plants, to evaluate the lethal action of B. bassiana, B. thuringiensis and HaNPV

under laboratory, green house and field conditions against H. armigera. Finally the effect of microbial

agents was determined on the survival of natural allies, and the economics of application of microbial

agents was calculated from yield harvested. Endophytic colonization of B. bassiana not only lowered

the damage infestation of H. armigera but also improved the plant health. Synergistic effect (CTF≥20)

on the mortality was observed when larvae were exposed to simultaneous application of higher

concentration of B. bassiana and lower concentration of B. thuringiensis both in case of second and

fourth instar H. armigera larvae. Lower concentration of B. bassiana yielded additive effect in

combination with Bt. Higher concentration of NPV also integrates synergistically with lower

concentration of Bt. Lower concentration of NPV works independently with higher and lower

concentration of Bt. Percent pupation, adult emergence and egg eclosion from surviving individuals

was found inversely correlated to toxic level of microbial agents. Increase in larval and pupal duration

while decrease in pupal weight and adult duration was recorded depending upon the lethal action of the

applied agent. The toxic nature of microbial agents also influenced the weight gain, frass production

and diet consumption. Foliar application of B. bassiana and B. thuringiensis was found significantly

persistent up to 12 days and mortality of second and fourth instar larvae was decreased with the time.

Microbial agents in simultaneous application are proved to be effective in lowering the larval density

of H. armigera and hence lowering the yield losses. Microbial agents are relatively safe to natural

enemies of H. armigera and hence proved to be eco-safe agents. Maximum marginal return was

obtained in combined application of microbial agents than their individual application

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INTRODUCTION

Pakistan being a developing nation with agricultural background is an important

country in Asian continent. Major portion of the population is associated with agriculture for

earning their livelihood directly or indirectly. Agriculture is the single largest sector and is

considered as mainstay and back bone of Pakistan’s economy. As an industry, it shares about

21% to Pakistan's gross domestic product (GDP) (GoP, 2014). Agriculture sector in Pakistan is

characterized by small land holders with low productivity and inefficient use of crop inputs.

Presently, agriculture sector, the backbone of economy has encountered a number of problems,

particularly low crop productivity, price hike for inputs, power shortage, water scarcity and

low export value in international trade hubs (Amjad, 2012). The situation got even worse due

to lack of supports for agricultural supplies, unpredicted pricing system, occasional scarcity of

agricultural inputs, unavailability or lack of access to information, non-technical policies of

government, reduced marginal return and shortage of irrigation facilities are gigantic issues

threatening agriculture and ultimately farming community (Amjad, 2012; Anonymous, 2013).

Agriculture is inevitable to Pakistan as much of the country’s export (>55 percent)

earning is associated to agro-based raw and processed products (Hayee, 2005). Under the

umbrella of agriculture, the role of crop sub-sector is comprehensively vital. For the last sixty

years, its involvement to agricultural GDP persisted as premier followed by livestock, fishing

and forestry. It is a source of living for more than 66 percent of population, sharing about 21

percent in GDP and engaging 43.4 percent of employed labor force (Ahmad et al., 2008).

Irrespective of political and global financial crisis and environmental changes, agriculture has

its impact on country’s growth performance serving enormous section of the population

(Mustafa, 2004).

The sustainability achieved through reforms in agriculture sector would fulfil all

economic targets through its closest concern and association to other sectors. Agriculture in

Pakistan needs to be on sound footing via technological and economic revolution if it has to

sustain its role towards the improvement of livelihoods as well as macroeconomic welfare and

Chapter 1

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prosperity. At national level legislation, agriculture and food security concerns are hot cake

issue and policy agenda. Recently the concerted actions of the government have left an

inspiring sign with growth rate of 2.1 percent during 2013-14 (GoP, 2014). A rapid agricultural

enhancement would help in restructuring economics and ultimately the living standards of

population sector involved with it. The fortune of a major fragment of public relies mainly on

revolutionized agriculture with timely provision of low cost inputs, improved methods of

farming, ensured prices of the produce and sustained market shape.

Globalization offers opportunities to improve farm incomes manifold if

competitiveness, quality and other targets of standards be achieved. Nature has endowed

Pakistan with varied climate and soil conditions, meticulous men power, gigantic irrigation

and agricultural research system, diverse market, multi-range consumers that would offer an

opportunity to compete in international market (Khan, 2011). A look at the agriculture sector

regarding productivity, consumption, exports, expansion and worth of produce helps to

reveal that yet after sixty five years of independence, we are far behind many of the

developing nations.

The majority of farmers are unable to make efficient use of the resources and

technology due to unskilled approach, no access to modern scientific innovations and

strictness to conventional methodologies. Agriculture industry must have to ensure a rapid

growth rate (>5 percent) to guarantee a swift growth of national income, macroeconomic

permanence, enhanced employment, safeguarding self suffiency in food and a reduction in

rural poverty in Pakistan (Naqvi et al., 1992, 1994).

Provision and flow of information about latest techniques and discoveries is one of

the hurdles for the slow progress. Further the laggardness in adopting a technology also

leaves many flaws. Revolution in agriculture sector could only be brought by the

technological changes as a consequence of research and development, rapid flow of

information, revised and improved infrastructure and brainstorming of farming community

(Iqbal and Ahmad, 2006). Empirical data about the rate of growth of agriculture in many

developed and developing countries highlight the importance of research and development

through its direct significance on productivity (Evenson, 2002).

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Statistical figures of growth in sub-sectors of agriculture in Pakistan present even

alarming situation. In contrast to several developed and developing nations having similar

resources, Pakistan appears far behind this race. In future, this would even worsen the

situation to compete in global markets if this sector left unattended (Iqbal and Ahmad, 2006).

Agriculture sector in Pakistan is dominated by crops and livestock with major value added

share. In crop sub-sector, major and minor crops contribute about 40 and 15 percent value

added share. There has been a wide fluctuation in share of crops productivity in agriculture

over time. Thus, stability in GDP share and income output is the demand of time. Although

the major crops are the more influencing than the minor ones, yet there could have enhanced

potential for vegetables, fruits, and flowers sub-sectors (Iqbal and Ahmad, 2006).

Vegetable production in Pakistan is recently on a fast track of progress, yet many

gaps are still there to be bridged. Low production rate and inferior quality are one of the

hurdles for attracting international merchant’s interest for export earnings. Low standard

nursery seedlings, lack of disease free seeds, conventional cultivation practices and severe

postharvest losses are factors limiting the production of vegetables. The growers willing to

procure improved quality planting material often remain desperate. For horticultural crops,

the extent of postharvest losses appears to be more than 30-40 percent with careful loss

estimate of over Rs.49 billion every year (Iqbal and Ahmed, 2006). In order to attain and

sustain self-sufficiency in food, agriculture is being reshaped with increased emphasis on

small and medium farming, easy farms-to-market supplies, subsidies for electricity based

inputs and control of water logging and salinity (Anonymous, 2008).

Vegetable production is the economically best part of cropping sector which under

ideal conditions bears maximum marginal returns for the investment. Vegetables are the short

span crops bearing maximum yield with little extra attention using same resources as required

for other economic or cash crops. Pakistan owes a rich diversity of vegetables covering 365

days of the year. Among wide list of vegetables, tomato (Solanum lycopersicum) is the most

common and profitable vegetable quite suited to Pakistani climate. It is an important plant fruit

of which is used in almost every dish. It is an important vegetable crop belonging to

Solanaceae (night shade family) and is grown throughout the year in Pakistan. It is grown in a

wide range of climate in the open, under protection in the green houses, and in heated green

Page | 4

houses. It is grown by most home gardeners and by commercial gardeners. It can be eaten

either fresh or processed into many different products (Anonymous, 2014).

The history of long evolution shows that tomato is native to South America. Andean

zone is likely to be the center of origin of wild tomato. Genetic record clarifies that the

progenitors of tomato were herbaceous green plants bearing small green fruit and the center of

this diversity located in the highlands of Peru. Prehistoric humans of Mexico used to grow one

of the famous species, Lycopersicon esculentum. This part of the world, Indian Subcontinent

was introduced to tomato cultivation when English traders of East India Company during 1822

stepped here for trade (Anonymous, 2009).

Tomato (Solanum lycopersicon) is among the widely used and remunerative vegetable

crop which is mostly cultivated for fresh market sale and processed products (Ravi et al.,

2008). After potato and sweet potato, it is regarded as world's most used vegetable crop after

potato and sweet potato, but among the list of canned vegetables, it is at the top (Olaniyi et al.,

2010). The annual production of tomato during 2012 at the world level was 161.79 m tones

while in Pakistan tomato was cultivated on about 0.05 m ha with the production of 0.56 m

tones ranking at 35th position over the globe. Average production of tomato in Pakistan is 10.1

tones per hectare (FAO, 2014) which is quite low as compared to other major tomato

producing countries of the world.

In Punjab, Gujranwala and Nankana Saheb produce 22 percent of the province's total

production. Both the districts are in close vicinity of three large urban centers of Lahore,

Faisalabad and Gujranwala. Muzaffargarh is the third largest tomato producing district of the

Punjab with a share of 9.2 per cent. The district is next to Multan, the biggest urban center in

southern Punjab. Muzaffargarh and Multan jointly host the province's 7.2 percent urban

population (Anonymous, 2008).

Tomato crop, being suited to all sort of climates, harbors rich diversity of insect pests.

The attraction and perishability of the fruit invites several insects and diseases and about 200

species of insects have been found damaging this crop worldwide (Lange and Bronson, 1981).

The entire structure of the plant and texture of the fruit provides food, housing, and

reproduction sites for the pests. The damage to the crop occurs by scarring, boring and tissue

destruction that ultimately leads to abnormalities in shape or color making the fruits

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undesirable for fresh market sales. Fruits get contaminated by whole insects, insect faecal

pallets, sheded skins and eggs that in turn lower its acceptability by the users.

Survival of crop from these notorious pests completely relays on repeated use of

pesticides which have created health hazard problems not only to human and animals but also

aided to speed up environmental pollutions, appearance of new pests, outbreak of secondary

pests and development of resistance against insecticides (Ricky, 2010).

Tomato is with round stem which is soft, rittle and hairy when young, but becomes

angular, hard and almost woody when old. The leaves are alternate 5-10 inches long, odd-

pinnate, with 7-9 short stemmed leaflets. The flowers are borne in clusters located on the stem

between the nodes. The fruit is a many celled berry having a fleshy placenta and many small

kidney shaped seeds. Fruit skin has deposition of cutin, varying from cultivar to cultivar

(Rudich and Atherton, 1986). Tomato is a juicy and fleshy fruit. The red and yellow

pigmentations in it are due to ‘lycopen’ and ‘carotene’. The plant, due to extreme

unpalatability is not relished by any cattle. The plant does well in temperate areas that have

frost-free conditions. Out sandy, loamy friable soils with adequate humus, good drainage, and a

pH of 5.7 to 7.7 are best for its cultivation. Tomato is produced throughout the country in the

plains and in hilly tracts (Anonymous, 2005).

Nutritional status of tomato is remarkable as recent studies have revealed that people

who eat large amount of tomato or tomato products are at a lower probable risk of cancer,

specifically to lungs, stomach colon, rectal (La Vecchia, 1998) and prostate cancer

(Giovannucci, 1999; Giovannucci et al., 2002). Presence of lycopene (Fuhrman et al., 1997;

Rao and Agarwal, 1998; Chopra et al., 2000; Maruyama et al., 2001) imparts additional

properties to confer defense against oxidative damage to vulnerable membrane lipids,

exclusively in conjunction with vitamin E (Fuhrman et al., 1997). The uncooked fresh use of

tomatoes is supposed to power the heart and other organs. Additionally the skin's ability to

protect against harmful UV rays gets magnified by the presence of lycopene. One pound of

fresh tomato has been reported to contain protein 4.0g, fats 1.2g, iron 2.4mg, thiamine 0.24mg,

riboflavin 0.16mg, niacin2.5mg, ascorbic acid 93mg and food energy 91 calories (Thompson

and Kelly, 1985).

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Epidemiologic studies has suggests to improve the antioxidant capacity by the

consuming different tomato products that ultimately decreases the probability of onset of

diseases associated with oxidative stress (Burney et al., 1989; Franceschi et al., 1994; Parfitt

et al., 1994; Giovannucci et al., 1995). Presence of active compounds viz a viz carotenoids,

flavonoids and vitamin C make tomatoes rich source of antioxidant properties. One of the

main carotenoid constituent, ‘lycopene’ exhibits the greatest reducing action of singlet

oxygen that helps to protect blood cells from NO2 radical damage (Bohm et al., 1995).

Rickets suffering in children can be cured by tomato juice which is a casual practice at

homes. Tomatoes are, however, not advised in case of respiratory troubles, kidney problems

and for the TB patients (Anonymous, 2005). Tomato is used both as green and ripe for edible

purposes. It is eaten with ‘Salads’ or mixed with meat, pulse, and vegetable dishes. Slices of

red tomato are used for garnishing. It provides raw material to the country’s expanding

canned food industry by producing juice, soups, paste, puree, and ketchups. Green tomatoes

are filled in pies, cookies, mincemeat, sandwich spread, in preserves and pickles

(Anonymous, 2005).

Seasonal variation does have no influence on tomato consumption in Pakistan as it is

pre-requisite of cooking and no recipe seems to be completed without it. This results in a

strong, inelastic and year-round demand for this agricultural produce and no factor that can

cut down its demand at any time of the year. At special events or festivals like Eid, its intake

surpasses the average for a few days when the entire population indulges in festive cooking.

Inspite of all these benefits, per capita consumption of tomatoes stands at 33 kg in the world

while Pakistanis consume less than 3 kg in a year (GoP, 2007).

Tomatoes are active ingredient in ketchup, pickles, puree, salad, soup, sauces and

other countless recipes. It is widely used in salad as well as for culinary purposes. Its fruit are

rich source of vitamins A, C, band minerals like Calcium, Phosphorus and Iron (Cheema and

Dhaliwal, 2005). Tomatoes, technically a fruit but not a vegetable, are naturally enriched

with all health benefits. The richness of health benefits and ease in cooking makes them an

evitable component of daily diet. It is used as raw in sandwiches, salads, etc. It ranks first in

processed products and its main products are paste, sauces and ketchup.

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Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a polyphagous

cosmopolitan pest and a serious threat to many crops of economic importance (Wakil et al.,

2012). It is the key pest damaging cotton, tomato, chickpea, pigeon pea, maize, okra, potato,

sunflower, tobacco and cabbage in Pakistan (Talekar et al., 2006) which occasionally results

in complete crop failure (Ahmad et al., 2003). Heavy infestation rate of this pest on cash

crops and vegetables, particularly on tomato divert the attention of keen growers to secure

their crop productivity from its damage (Ahmad et al., 2008).

The goal of increased productivity and premium quality of the tomato

crop is greatly hampered and restricted by the tomato fruitworm, H. armigera which causes

damage to the developing fruits and results in yield loss ranging from 20 to 60 percent (Lal

and Lal, 1996; Ravi et al., 2008). Estimate of yield loses in Pakistan during 2009-10 were

ranged from 14.7 to 32.6% in various districts of Punjab (Wakil et al., 2010). It is wide

spread in distribution throughout Asia, Africa, Australia and the Mediterranean Europe

(Sharma, 2005). Being cosmopolitan in nature, H. armigera attacks more than 200 plant

species including cash crops, vegetables, fruit crops and trees (Manjunath et al., 1989; Fitt,

1991). About 60 cultivated crops from 14 families and 67 other plant species belonging to 39

families have been reported in Punjab (Saleem and Younas, 1982). Based on its host range, it

has been named differently as tomato fruitworm, gram caterpillar or pod borer, cotton

bollworm and corn earworm (Nasreen and Mustafa, 2000). Although H. armigera has several

host crops, and many crops support egg and larval densities, yet it prefers tomato crop as

suitable host for oviposition. High ovoposition rates have been reported in cotton, okra,

sunflower and tomato crop (Ravi et al., 2005).The repeated, non-judicial and over dose use

of various insecticides by Pakistani farmers had resulted in the broad spectrum resistance

development in indigenous population of H. armigera to almost all the insecticides available

for its control such as pyrethroids (Ahmad et al., 1997, 1998), organophosphates (OPs)

(Ahmad et al., 1999) and carbamates (Ahmad et al., 2001) and endosulfan (Ahmad et al.,

1995, 1998).

Combining several approaches like chemical and biological control in integrated pest

management (IPM) may help to efficiently combat target pests which suits specific climate.

Integration of chemical and biological tactics could be eco-safe and specific in action

(Simberloff and Stiling, 1996; Gentz et al., 2010).

Page | 8

To manage this insect pest in the long run of time, reduced risk insecticides are the

dire need of time and are pragmatically suggested options. Among the biological control

agents used as an alternative to synthetic insecticides in vegetables pests, there is a

preference for entomopathogenic fungi, B. thuringiensis and Nuclear Polyhedrosis Virus

(NPV). The combined use of microbial formulations (HaNPV) and B. thuringiensis has

attained greater repute among agricultural community as a successful tool in integrated pest

management (IPM) strategies as the value of individual application of HaNPV could suffer

seriously if insect could be able to develop widespread resistance against it. Viral insecticidal

formulations are powerful agent in biological control of agricultural and forest pest because

they are convenient and safe in use and are simply manipulated products. Nuclear

polyhedrosis virus (NPV), are arthropod-specific viruses. Integrated use of these compounds

cause hazards to insect’s physiology and behavior particularly feeding deterrence deformed

molting and other sublethal consequences which delay potential selection for resistance in

targeted pests (Michereff-Filho, 2008).

Fungal pathogens have proved their worth as a potential agent regulating insect

population as an effective mycopesticide (Vega et al., 2012). Entomopathogens of fungal

origin exist as natural endophyte and in response to artificial plant inoculation by several

methods (Vega, 2008). Beauveria bassiana sensu lato (Bals-Criv) Vuillemin (Ascomycota:

Hypocreales) is a familiar entomopathogenic fungus reported worldwide in its distribution. It

is considered as an anamorph of Cordyceps bassiana, a teleomorph in the ascomycetous

family Clavicipitaceae (Sung et al., 2007). Members belonging to this cryptic family inhabit

varied habitats and include diversity of endophyte, entomo and plant pathogens, parasites of

fungi and slime molds. A key characteristic of this family is the production of secondary

metabolites of toxic nature (White et al., 2003). In spite of performing duty of parasitizing

various agriculturally important insect pests, additionally it has been found endophytically

colonizing various plants (Vega, 2008), and providing protection to plant against pathogens.

As for as the pathogenecity of entomophaghous fungus to various targeted insect is

concerned, it depends upon various factors where along with abiotic factors (temperature,

relative humidity, light and dark period etc), the specific host species, host pathogen

interaction, and application time of this fungal insecticide is very important. Mode of action

Page | 9

of different formulations of entomopathogenic fungi is different to infect the host organism

(Subramanyam and Roesli, 2000). Spores get penetrated into insect body either through

cuticle or it thrives in to their alimentary canal; additional drastic effects occur on targeted

insects in the lateral case (Broom et al., 1976). Fungal invasion through cuticle has been

observed in most cases (Akbar et al., 2004) as certain long chain hydrocarbons of insect

cuticle facilitate the conidial attachment through hydrophobic interaction in conidial cell wall

(Boucias et al., 1991). A carbon-energy source works effectively necessitated by

entomopathogenic fungi (Smith and Grula, 1981) and ample provision of carbon in insect

cuticle stimulates the conidial growth on epicuticle (Napotilano and Juarez, 1997).

Hyphomycetous fungi follow certain enzymatic processes to make a way in to insect cuticle,

for example M. anisopliae secretes exoproteases which tend to degrade the insect cuticle. In

the similar way, release of endoproteases, esterases, lipases, chitobiases and chitinases is

facilitated by certain entomopathogenic fungi (Boucias and Pendland, 1998; Butt et al.,

1998) in order to ensure their spore infiltration.

Hyphal bodies are produced on reaching to haemocoel by fungus which at first

attempt degrades the fat tissues and after the gut tissues; the malphigian tubules are the last

body tissue which undergoes the depletion process. Thus the multiple action of the fungal

spore inside the insect body leads to the death of target species. On entrance in to host body,

these entomopathogenic fungi cast their toxic effect with various toxic substances which they

produce e.g. Beauveria bassiana generates beauvericin (Zizka and Weiser, 1993),

bassianolide and Oosporein (Eyal et al., 1994) which affect the respiratory chain enzymes. In

the same way some of them cause tetanic paralysis (Dumas et al., 1996) whereas other may

suppress the immunity system of an insect (Cerenius et al., 1990). Some bacteria may also

invade on insects primarily infected with fungi (Poprawski et al., 1997) as fungal abrasion

may provide the way for such microbes.

Mostly the members of fungi from entomopathogenic texa are host specific but some

of them are now considered as the most diverse group with a variety of genotypes (Inglis et

al., 2001), which ensures their effective use against field crop insect pests and for the insect

pests of stored grain as well. As the need of biological protectant of plants and stored

commodities has been increased, the entomophagous fungi are receiving much attention

(Hluchy and Samsinakova, 1989) The first species which was being studied in this regards is

Page | 10

B. bassiana (Balsomo) Vuillemin (Ascomycota: Hyphomycetes) (Moino et al., 1998; Dal-

Bello et al., 2001; Akbar et al., 2004; Vassilakos et al., 2006), however, another species

Metarhizium anisopliae (Metschnikoff) Sorokin is also very prominent in controlling field as

well as stored insect pests (Dal-Bello, 2001; Batta, 2003, 2004; Michalaki et al., 2006). B.

bassiana (Balsamo-Crivelli) Vuillemin has a very diverse and widespread host range

mycoinsecticides based on B. bassiana are used against agricultural, veterinary and medical

insect pests (Devi et al., 2008). Role of environmental conditions and susceptibility of the

insect population is vital to affect successful fungal toxicity. The scale of difference between

the two responses can be estimated by keeping the environmental factors constant and

comparing virulence of an isolate to different insect species and different populations within

an insect species. The isolate can be considered as exhibiting specific preference if

differences in virulence of an isolate to different insect species are greater than the difference

in virulence to different insect populations within an insect species.

Microbial pesticides prepared from entomopathogenic bacterium are successful agent

for managing many important agricultural pests. It is often an integral part of products used

in biological control strategies worldwide and about 95% microbial pesticides being used

globally are bacterial in origin with annual sale of about $100 million (Federici et al., 2006).

Bacillus thuringiensis (Berliner) is a gram-positive soil bacterium, spore forming mesophile

having ability to produce proteinaceous parasporal inclusions during sporulation. It can

produce δ-endotoxin which exhibits insecticidal nature by working as intestinal toxin. The

crystalline inclusions along with spores have the great potential to suppress many insect pests

(Vidyarthi et al., 2002). The unique insecticidal characteristics of Bt strains have proved its

worth in pest management applications (Schnepf et al., 1998). In some non-insecticidal B.

thuringiensis, property of killing human cancer cells have been discovered in parasporal

inclusion proteins (Mizuki et al., 1999).

Bt toxins have been found to be safe for mammals and environment due to their

specific mechanism of action (Höfte and Whiteley, 1989; Schnepf et al., 1998), and for this

reason Bt-spore crystal mixture have been in use as bio-pesticide against coleopteran,

dipteran and lepidopteran insects (Feitelson et al., 1992; Schnepf et al., 1998). The success of

Bt toxins have verified them as alternatives to synthetic chemicals in agriculture (Schnepf et

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al., 1998). Insecticidal potency and viability of Bt strain varies considerably against

lepidopterous insect pests (Dulmage and Collaborators, 1981). The δ-endotoxins of Bt have

proved their worth as potential alternative to synthetic chemicals in the field of agriculture

(Schnepf et al., 1998).

Microbial insecticides especially bacterial formulations made from B. thuringiensis

are quite effective against number of economically important insect pests because they are

not harmful to humans, other mammals, or non-target species. Insect resistance to Bt-

formulations reported till now seems to be developed less quickly and less strongly as

compared to resistance to chemical insecticides (Tabashnik et al., 1994; Ives et al., 2008)

because Bt-toxins are quite complex in action mechanism against target insects (Tabashnik,

1992; Tabashnik, 2005; Tabashnik et al., 2013) and for insects to develop resistance against

Bt-toxins requires several important mutations (Carlton and Gonzalez, 1986).

Viral insecticidal formulations are powerful agent in biological control of agricultural

and forest pest because of their appropriateness and safety as they are simply formulated and

deployed to target the particular host. Mainly the susceptible hosts for nuclear polyhedrosis

viruses (NPVs) are arthropod and preferably lepidopterous insect pests (Liu et al., 2006).

Insecticides kill target insects instantly after contact as it acts as a highly specific

stomach poisons but NPV is fairly slow acting and takes several days or weeks to kill an

insect and during that time insect continues to feed. The midgut cells are principal initial

target tissues which are basis for replication of the nuclear polyhedrosis virus are attacked

after ingestion by a susceptible host, followed by transmission of infection from cell to cell.

Against H. armigera, either microbial products i.e. B. thuringiensis and NPV can interact

either synergistically or additively depending upon differences in their modes of action

(Wraight et al., 2005; Marzban et al., 2009). The toxicity of pathogenic combinations may

depend on several factors such as concentration or bacteria or design of bioassay.

Considering the extent of damage encountered to tomato crop by H. armigera, it was

considered worthwhile to exploit microbial agents to evaluate their insecticidal activity under

laboratory and field conditions. The present study was conducted to meet the following

objectives;

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1. To find out the extent of field evolved resistance in H. armigera collected from

various localities against conventional and new chemistry insecticides.

2. To isolate endophytic fungi and then colonize entomopathogenic fungi in tomato

plants for their toxic potential to H. armigera through inoculation and direct

application procedure.

3. To screen different commercial Bt isolates, find their compatibility to

entomopathogenic fungi, evaluate their lethal action in laboratory and determine

foliar persistence in green house against H. armigera.

4. To integrate the virulent Bt isolate with NPV, find their action on the survival and

development of H. armigera under laboratory conditions and determine their foliar

persistence in green house.

5. To evaluate the toxicity of microbial agents under field conditions against H.

armigera and their effect on natural allies, yield and cost-benefit ratio of application.

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REVIEW OF LITERATURE

2.1 Insecticide Resistance

History is evident that the development of insecticidal resistance in insects is the

major and a limiting factor for efficient chemical control throughout the world (Whalon et

al., 2008). The resistance in H. armigera in various populations was suspected when the

insecticides failed to provide effective control in field where they provided before.

Insecticides belonging to pyrethroids group affect the sodium-potassium channel of target

insect leading to its death (Ahmad et al., 2007a, b) and involvement of pyrethroid

hydrolyzing action of esterases is responsible for the detoxifying action of pyrethroids

(Kranthi et al., 1997) which render these insecticides ineffective. Presence of these esterase

isozymes in excess is the reason for varied level of pyrethroid resistance in many pests

(Byrne et al., 2000). An insect population exhibiting resistance ratio of >10 is generally

considered to be resistant against any insecticide (Valles et al., 1997).

Chemical analysis reveals that in H. armigera, esterases linked to pyrethroid

resistance can be inhibited by the use of organophosphorus insecticides like profenofos and

chlorpyrifos (Gunning et al., 1999) which detoxify their effect by binding to active site of the

enzymes involves. Decreased sensitivity of CNS is the reason behind pyrethroid resistance in

S. littoralis (Gammon, 1980). Overproduction of esterase isozymes seizes the ester bonds of

pyrethroids thus rendering them less efficient against target pests (Young et al., 2006).

Use of the same type of insecticides for years had enabled insect pests to thrive well

with various mechanisms of resistance evolved in them. Several new chemistry insecticides

now are available in market with novel model of action. The repeated, non-judicial and over

dose use of various insecticides by Pakistani farmers had resulted in the broad spectrum

resistance development in indigenous population of H. armigera to almost all the insecticides

available for its control such as endosulfan (Ahmad et al., 1995, 1998), organophosphates

(OPs) (Ahmad et al., 1999), pyrethroids (Ahmad et al., 1997, 1998), and carbamates (Ahmad

et al., 2001).

Chapter 2

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New chemistry insecticides are unique in their mode of action and environmental

profile than old ones and also help to combat the resistance developed previously. The

differences of LC50 values for different populations may be caused by diversity of genetics,

geographic environments, host plant varieties, agro practices or other factors in sampling

sites (Zheng et al., 2011). Development of resistance in various crop pests of economic

importance is a great threat to Pakistani farmers particularly in H. armigera (Ahmad et al.,

2003), Bemisia tabaci (Ahmad et al., 2002), Plutella xylostella (Khaliq et al., 2007), S. litura

(Saleem et al., 2008), Earias vittella (Ahmad and Arif, 2009) and S. exigua (Ahmad and

Arif, 2010).

2.1.1 Result oriented studies

Armes et al. (1996) surveyed Pakistan, India and Nepal from 1991 to 1995 to

determine the status of resistance in H. armigera to conventional insecticides. Findings of the

studies suggested that field evolved resistance is ubiquitous against pyrethroids tested in

subcontinent during study period. Significant level of resistance was recorded for

cypermethrin and fenvalerate which ranged from 5-6500 and 16-3200 fold respectively.

Resistance level was much higher in areas where cotton and pulses were grown intensively

especially in central and southern India. Pre-treatment application of metabolic synergist,

piperonyl butoxide (pbo) significantly suppressed the resistance developed against

pyrethroids but complete elimination of resistance was not noted. Most of the resistance

found against carbamate, organophosphate and pyrethroid insecticides in the Indian

subcontinent apparently seem due to an inherited or inducible mixed function oxidase

complex.

Ahmad et al. (1999) reported the pattern of resistance to organophosphate insecticides

in Pakistan's field collected populations of H. armigera during 1994 to 1997 using IRAC

leaf-dip method and found medium to high level resistance against ethion, dithiophosphorate,

monocrotophos and orthophosphorate. During 1994, status of evolved resistance was lower

against conventionally being used insecticides like chlorpyrifos and thiophosphorates but it

tend to rise up from 1996 to 1997. Resistance ratio remained lower against thiophosphorates

like methamidophos, parathion-methyl, quinalphos, and triazophos. Low level of resistance

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against such insecticides signified their low intrinsic efficacy when tested against H.

armigera.

Ahmad et al. (2003) observed the susceptibility of H. armigera to new chemistry

insecticides like abamectin and emamectin benzoate, chlorfenapyr, fipronil, indoxacarb and

spinosad. IRAC leaf-dip method was used to estimate the level of development of resistance

in H. armigera field populations which were found to be resistant to conventional

insecticides. Most of the populations showed no sign of developed resistance and found quite

susceptible to reference population but some of them were found with slight level of

resistance. The reason behind that strange fact could be attributed to phenomena of cross

resistance to conventional insecticides.

Aggarwal et al. (2006) devised a management strategy for H. armigera in cotton in

Punjab (India) during 2003-04. Following Integrated Resistance Management (IRM) strategy

resulted in significantly lowering the resistance and increased population of beneficial

insects. Adoption of IRM strategy lowered the development of insecticide resistance in H.

armigera (1-2.9 folds) to insecticides viz. cypermethrin, chlorpyriphos, endosulfan,

fenvalerate, methomyl and quinalphos. A significant increase as a result of it was found

(28.7%) showing the superiority of IRM strategy over the traditional strategies.

Ghafoor et al. (2011) assessed the resistance level of H. armigera collected from

cotton fields in Faisalabad against mixture of Indoxacarb and Profenofos. Survival rate of

larvae decreased with increase of dose rate. Findings of the study suggested that combined

concentration with increased dose rate was indirectly proportional to survival rate of H.

armigera.

2.2 Entomopathogenic Fungi

2.2.1 History

Entomopathogenic fungi bear a pleasant history of recognition from ancient times.

Their descriptions are even more evident in drawings centuries ago. Japanese paintings of

Beauveria bassiana in silkworm infections and Cordyceps spp. in insect infections date back

to 19th century (Samson et al., 1988), showing their brightly colored and prominent fruiting

bodies. Now these fungi have a vast discipline of study under the umbrella of “insect

Page | 16

pathology”. Fighting against several diseases of honey bees and silkworm also provide strong

historic blue prints (Steinhaus, 1956; 1975). Earliest success stories of use of

entomopathogenic fungi to manage insect come from the pioneer works of Louis Pasteur,

Elie Metchnikoff and Agostino Bassi in the field of invertebrate pathology (Steinhaus, 1956,

1975).

Entomopathogenic fungi are scattered widely in nature with specific as well as broad

host ranges against different arthropods insects and plant pathogens. They had an important

role in regulating the insect populations. Fungi predominantly belonging to four classes bear

a rich diversity that infects insects from more than 700 species from 90 genera

(Khachatourians and Sohail, 2008). Earlier studies regarding the use of entomopathogenic

fungi were conducted in 1800s aiming to develop control tactics for managing muscardine

disease that led the silk worm industry towards decline (Steinhaus, 1975). Bassi in1835

(Steinhaus, 1975) proposed the germ theory using silkworm and fungus found invading the

disease was later named Beauveria bassiana in Bassi’s honor.

2.2.2 Geographical distribution

Entomopathogenic fungi are cosmopolitan in distribution with no boundary of

continents yet a more diverse occurrence limited to terrestrial ecosystems. Even the places

with no arthropods or host insects are invaded by such fungi thriving solitary or in complex

forms. Some species of genus Beauveria can be found almost across the world even in harsh

climatic conditions of tropical rainforests (Aung et al., 2008) and in far north of Canada up to

latitude of 75° (Widden and Parkinson, 1979). The harsh climate of north of the Arctic Circle

bear many of such species like B. bassiana, Tolypocladium cylindrosporum and M.

anisopliae reported in Norway (Klingen et al., 2002), Isaria farinose, B. bassiana and M.

anisopliae in Finland (Vänninen, 1995). Some reports of rare occurrence from Antarctica and

Arctic Greenland (Eilenberg et al., 2007) are also present showing their hard style of

survival. In Intactic region, researchers isolated Paecilomyces antarctica from the Antarctic

springtail from the peninsular Antarctic (Bridge et al., 2005).

Some species of entomopathogenic fungi are universal in distribution like

Lecanicillium, Beauveria, Neozygites and Conidiobolus and also have been found on

Antarctic region even without presence of any of their arthropod hosts (Bridge et al., 2005).

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Quesada-Moraga et al. (2007) reported that altitude has no effect on occurrence of

entomopathogenic fungi up to range of 1608 m, beyond that limit, probability of occurrence

of B. bassiana decreases. Greater diversity of insect pathogenic fungi appears up to altitudes

(>5200 m) (Sun and Liu, 2008).

Regarding habitat, a greater variation has been reported and they can be found in the

soil or in the over ground habitat, forest soil litter and upper surface of the soil found rich

source of Hypocreales. Underwood and below the canopy area of trees exhibit greater

diversity of Entomophthorales, rush communities and meadow species of spider pathogenic

fungi were dominant (Sosnowska et al., 2004). Humid tropical forests were found rich

sources of vast array of fungal fauna dominating by Cordyceps (Evans, 1982; Aung et al.,

2008).

Greater diversity of member of Hypocreales like Isaria, Beauveria, and Metarhizium

has been isolated from soil invading insects (Samson et al., 1988; Keller and Zimmerman,

1989). Both B. bassiana and M. anisopliae almost coexist in the common habitat

everywhere, but this is accepted that B. bassiana exhibit sensitivity towards disturbed or

cultivated soil and thus confined to the natural habitats. While capability of M. anisopliae to

persist in disturbed soils is well documented (Rath et al., 1992; Vänninen, 1995; Quesada-

Moraga et al., 2007; Sánchez-Peña et al., 2011).

2.2.3 Application in pest management

The primary idea for the use of entomopathogens to control insect pests came from

investigation on silk worm disease. However, the invention of synthetic insecticides over

rules this idea for using EPFs as microbial control agents. During late 1950s the efforts to use

EPFs for management of insect resurfaced and this field got revolutionized by many

discoveries. To date, there are many commercial products available worldwide; based on a

number of fungal species (Shah and Goettel, 1999; Copping, 2001), but there is yet a lot of

room for the many.

Recently various entomopathogens are under practical implementation for the control

of insect pests in field and green house crops, orchards, turf and lawn, ornamentals, range

lands, stored products, forest pests and insect vectors of medical and veterinary importance

(Burges, 1981; Tanada and Kaya, 1993; Lacey and Kaya, 2000). The only constraint that

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makes entomopathogens a second choice after conventional insecticides is their cost- benefit

ratio and time to death. However, the increased concerns of pesticide residues, safety to

human belongings environmental pollution and effect on non-target organisms make

entomopathogens a dire need of the time. They are easy to apply using conventional

equipment simple to grow on artificial media and safe to preserve for later use. The host

specific nature and long term persistence enable them to provide long term protection.

2.2.4 Classification of entomopathogenic fungi

Most of the fungi with insect pathogenic nature are grouped under divisions;

Ascomycota, Zygomycota and Deuteromycota (Samson et al., 1988), as well as

Chytridiomycota and Oomycota. Many genera of interest in entomopathogenic fungi

presently belong either to the class Hyphomycetes in the Deuteromycota or to the class

Entomophthorales in the Zygomycota. A surprising fact about the pathogenicity of

entomopathogenic fungi is that not all of them infect insect, but some of them reported to

infect non target individuals like Gibellula species infect spiders and Cordyceps and Erynia

infect ants.

2.2.5 Biology of entomopathogenic fungi

Life cycle of entomopathogenic fungi is governed by a number of important factors

of nature of host, life stage, nutrition, temperature and relative humidity. Sometimes they

behave in an odd way; even isolates from the same species can behave differentially to the

same target host just because of difference in requirements of biotic and abiotic factors

(Sierotzki et al., 2000; Pell et al., 2001; Shaw et al., 2002). In general practice, opportunistic

fungi infect several species of insect orders, producing toxins to overcome the host defense

mechanisms to ultimately kill it (Roberts, 1981; Samson et al., 1988). Higher fungi with

parasitic nature like Entomophthorales spread infection by colonizing the host tissue with no

or negligible use of toxins (Humber, 1984). Entomophthorales reveal bio-trophic associations

with insect host with very little or no saprophytic association on host death, while,

Hyphomycetes may be hemi-biotrophic on insect host and exhibit saprophytic association

after hosts death.

The uniqueness of fungal infection process is that they do not restrict the feeding and

movement of host, conidia on the other hand breach, discharge and disperse into the

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haemocoel over a long period of time prior to death (Pell et al., 2001). The

entomopathogenic mode of life may have started from a common saprophyte inhabiting into

the leaf litter and soil (Humber, 1984; Samson, et al., 1988; Evans, 1989; Spatafora and

Blackwell, 1993). Recently all genera of Hyphomycetes have been proved teleomorphs in the

Clavicipitales, and availability of plenty of hosts has made life stages of these fungi simplest

in agricultural conditions (Evans 2003).

2.2.6 Mode of action

The fungal infection process is rather complex, encompassing chemical and physical

actions from earlier attachment of spore till the host death. The infection process may involve

following these steps; (1) spores getting attached to the host cuticle; (2) germinating on the

host; (3) penetrating within the host cuticle; (4) overcoming the host immune system; (5)

proliferating hyphal bodies into the hemocoel; (6) saprophytic outgrowth from the dead host,

producing and releasing new conidia to repeat the cycle. Hydophobic nature of spores play a

key role in fungal attachment, sometime the hydrophobicity of the cuticular surface is even

involved. Presence of hydophobin like proteins within exterior surface of B. bassiana conidia

makes this species a successful tool in agriculture.

Many a factors influence the germination and further infection process particularly

susceptible host stage, humidity, optimum temperature and cuticular lipid contents such as

aldehydes, ketones, short-chain fatty acids, wax, esters and alcohols which may exhibit

antimicrobial activity. Generally, fungal spore prefer to breach through the non-sclerotised

areas of the cuticle such as joints, between segments or the mouthparts. The conidial

germination starts after 10 h of attachment and may complete by 20 h at 20-25 ˚C. Before

infection process, germ tube produce appressorium or penetration pegs which is accompanied

by mechanical and chemical processes by the production of several enzymes (proteases,

chitinases and lipases).

Several intermediate steps are involved in between host immune system and

penetrating fungus (Vilcinskas and Götz 1999). For example Beauveria species produce

proteolytic enzymes and toxins, whereas hosts in return initiate their cellular and humoral

defence system by the production of anti-fungal compounds, proteins and inducible protease

inhibitors to detoxify the fungal toxins. After successful penetration, the fungul spore

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produces hyphal bodies (yeast like cells), disperse passively in the hemolymph and

permitting the fungal spores to invade adjacent tissues by vegetative growth and toxins

production.

The fungal incubation period again depends on biotic and abiotic factors like; insect

host, host stage, fungal virulence, fungus strain, temperature and humidity. After depleting

the nutrients from the host body, fungus start producing conidia on the exterior surface of the

cadaver under humid conditions, while under dry conditions, it persists in hyphal stage inside

the cadaver. During incubation period, the host shows feeding and behavioural changes,

reduction in body weight, fecundity, behavioural fever and malformations (Müller-Kögler

1965; Ekesi 2001; Ouedraogo et al. 2003).

2.2.7 Disease related virulence enzymes

Studies with modern instrumentation and facilitation have enabled man to know the

wide array of virulence enzymes liable for successful interaction with the host and

environment (Khachatourians and Qazi, 2008). The enzymes responsible for pathogenesis are

generally grouped in to chitinases, lipases, peptidases and proteases.

2.2.8 Host range and specificity

Fungal infections encompass hosts belonging to all insect orders with no

discrimination of life stages. In most pathological studies, some preference for infection to

immature holomatbolous insects have been more commonly documented (McCoy et al.,

1988; Tanada and Kaya, 1993). Among different species of entomopathogenic fungi, the host

range may differ significantly among different species and difference even occur among

strains of a given species. In case of obligate pathogens, some sort of specification has even

recorded showing narrow host range and complex life cycles linked to their hosts. For

instance S. castrans is restricted to anthomyiid flies (Eilenberg and Michelsen, 1999) and

entomophthorans, Massospora spp. remain restricted to a particular genus of cicadas (Soper,

1974). In contrast, deuteromycetes, particularly B. bassiana, have wide host range including

numerous genera of insects (McCoy et al., 1988).

It becomes worthwhile to know that reports about the specificity of host range mainly

rely on in vitro studies which do not clearly draw the true picture in nature. Fungal pathogen

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biology, ecology and insect host could reduce the chance of encounter between host and

pathogen. Additionally, typical conditions for bioassay like high dose rate, temperature and

optimal relative humidity do not represent the true picture in applicable under field

conditions. Factors that regulate an insect fungus interaction for the successful infection are

same that mediate specificity from attachment (Zebold et al., 1979), to germination on the

host cuticle (Altre et al., 1999; Wraight et al., 1990), to successful dodging of the host

defenses (Hung and Boucias, 1992; Pendland and Boucias, 1993). Furthermore, specificity

also depends on the coexistent pathogen and potential host’s natural environmental context;

spatial and temporal factors and physiological interactions determine the true biological host

range (Carruthers et al., 1997; Hajek et al., 1996a).

2.2.9 Safety to non-target organisms

Among long list of microbial entomopathogens, entomopathogenic fungi shows

widest spectrum of host range which sometimes magnifies concerns over their safety to non-

target organisms. While register a bio-pesticide, one must have to consider the long and short

term reaction to other invertebrates tested through series of laboratory bioassays (Hall et al.,

1982). This seems to be one of the constraints for isolates that show high potential to target

host but also found fatal to non-target hosts. Several species of EPFs have extended host

ranges that even may infect the humans. Such fungi are often not included as a part of

microbial control agents. A number of factors determine specificity and host range of fungal

pathogen such as fungal strain, physiological state of the host, defense mechanisms, nutrition,

nature and kind of cuticle and epicuticular layers (McCoy et al., 1988).

The phenomenon of infection is also governed by the environment as well as the

dispersion and density of pathogen and host populations (Carruthers and Soper, 1987). In

inoculative approach, the fungus is introduced with increased abilities of developing

infection in the pest population while in inundative approach; epizootics are induced by

supplementing the density of pathogen.

In regard of safety and specificity, the entomopathogenic fungi can be categorized

into two groups, (i) highly specific fungi, (ii) broad spectrum fungi. Highly specific fungi

pose a minimal threat to the non-target organisms, such as Entomophaga maimaiga,

restricted to many lepidopteran species, Aschersonia aleyrodis confined to several

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homopteran species in the family Aleyrodiae, Entomophaga grylli restricted to Orthoptera

and Pandora neoaphidis restricted to aphids (MacLeod, 1963). Whilst, broad spectrum fungi

have extended host ranges, which have raised safety concerns, like, B. bassiana epizootics

has been recorded in more than 700 species of arthropods (Li, 1988) and other species

including Paecilomyces farinosus, M. anisopliae and Zoophthora radicans. Regardless of

thousands of publications on entomopath