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Development of Biocontrol Agents for the Control of Pests in Agriculture Using Chitin Metabolism As Target Pallavi B. Nahar Au

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Development of Biocontrol Agents for the Control of Pests in Agriculture Using Chitin Metabolism As Target Pallavi B. Nahar Au DEVELOPMENT OF BIOCONTROL AGENTS FOR THE CONTROL OF PESTS IN AGRICULTURE USING CHITIN METABOLISM AS TARGET PALLAVI B. NAHAR AUGUST 2004 DEVELOPMENT OF BIOCONTROL AGENTS FOR THE CONTROL OF PESTS IN AGRICULTURE USING CHITIN METABOLISM AS TARGET A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MICROBIOLOGY BY PALLAVI B. NAHAR DIVISION OF BIOCHEMICAL SCIENCES NATIONAL CHEMICAL LABORATORY PUNE-411008 (INDIA) AUGUST 2004 Affectionately dedicated to my beloved parents and sisters CONTENTS Declaration i Acknowledgement ii List of abbreviations iii Abstract iv-xi Chapter 1 Introduction 1-50 1.1 Origin 2 1.2 History 3 1.3 Pests in agriculture 3 1.4 Plant defense 6 1.5 Pest control strategies 12 1.5.1 Conventional pest control strategies 12 1.5.2 Chemical based pest control strategies 13 1.6 Pest control strategies based on biological control agents 18 1.6.1 Plants or plant parts for the control of plant diseases and insects 19 1.6.2 Importance of microorganisms in control of plant pathogenic fungi 20 and insects 1.6.2.1 Bacteria 20 1.6.2.2 Viruses 22 1.6.2.3 Fungi 24 1.6.2.3 A Mycoparasitism 25 1.6.2.3 B Entomopathogenesis 27 1.6.3 Use of mycolytic and cuticle degrading enzyme complex and their 32 inhibitors in pest control 1.6.3.1 Structure of fungal cell wall and insect cuticle 32 1.6.3.2 Enzymes involved in degradation of fungal cell wall 38 1.6.3.3 Enzymes involved in degradation of insect cuticle 39 1.6.3.4 Use of enzymes and enzyme inhibitors in biocontrol of pathogenic 40 fungi and insects 1.6.3.4A Importance of chitinases 40 1.6.3.4 B Importance of chitin deacetylase and chitosanase 42 1.6.3.4 C Use of non-enzymic components and chitin metabolism inhibitors 44 for the control of plant pathogenic fungi and insects 1.7 Trans-sectorial issues related to application of biocontrol agents 47 1.7.1 Researchers 47 1.7.2 Policy related studies 48 1.7.3 End users 48 1.7.3.1 Biosaftey 48 1.7.3.2 Bioethics 48 1.7.3.3 Socio-economics 49 1.8 Present investigations 50 Chapter II Materials and Methods 51-71 2.1 Materials 52 2.2 Organisms 53 2.2.1 Media 54 2.2.2 Maintenance 55 2.2.3 Isolation of entomopathogenic fungi 55 2.2.4 Mass production of conidia of entomopathogenic fungi by solid 56 state fermentation 2.2.5 Formulation studies of conidia of entomopathogenic fungi 56 2.2.6 Germination studies of conidia of entomopathogenic fungi in 57 presence of chickpea leaf extract 2.3 Enzyme production 57 2.3.1 Production of cuticle degrading enzyme complex of 57 entomopathogenic fungi 2.3.2 Extracellular constitutive and induced production chitin 57 metabolizing enzymes of M. anisopliae 2.3.3 Production of mycolytic enzyme complex of M. verrucaria 58 2.3.4 Inhibitor production 58 2.4 Enzyme assays 58 2.4.1 Chitinase assay 58 2.4.2 Chitosanase assay 59 2.4.3 Chitin deacetylase assay 60 2.4.4 N-acetylglucosaminidase activity 60 2.4.5 Protease assay 61 2.4.6 Lipase assay 61 2.4.7 Laminarinase assay 62 2.4.8 Mannanase assay 62 2.4.9 Chitin synthase assay 62 2.5 Hyphal tip bursting test 63 2.6 Biochemical techniques 63 2.6.1 Isolation of cell wall 63 2.6.2 Hydrolysis of chitin 64 2.6.3 Estimation of reducing sugars 64 2.6.4 Protein estimation 65 2.6.5 Solubilization of melanin 65 2.6.6 Ultrafiltration 65 2.6.7 Polyacrylamide gel electrophoresis (PAGE) 65 2.6.7.1 Detection of CDA activity after PAGE under native conditions 65 2.6.7.2 SDS-PAGE 66 2.6.7.3 Protein staining after electrophoresis 66 2.6.8 Determination of chitin and chitosan content in cell wall 67 2.6.9 Thin layer chromatography of cell wall synthesis inhibitor(s) 67 2.6.10 Microscopy 68 2.6.10.1 Detection of chitin deacetylase activity on insect (H. armigera ) 68 cuticle 2.6.10.2 FITC-WGA staining of the insect (H. armigera ) cuticle treated with 68 cuticle degrading enzyme complex of Myrothecium verrucaria 2.7 Application of biocontrol agent 69 2.7.1 Laboratory studies 69 2.7.1.1 Insect culture 70 2.7.1.2 Insect bioassay 70 2.7.2 Pot experiment 70 2.7.3 Field trials 71 Chapter III Use of Metarhizium anisopliae , Beauveria bassiana and 72-90 Nomuraea rileyi for the control of H. armigera on pigeon pea and chickpea crops 3.1 Introduction 73 3.2 Results and discussion 76 3.2.1 Isolation and selection of best strains of entomopathogenic fungi 76 3.2.2 Insect bioassays and cuticle degrading enzyme activities of 78 entomopathogenic fungi 3.2.3 Solid state fermentation 81 3.2.4 Formulation studies 82 3.2.5 Field evaluation 85 3.2.5.1 Pigeon pea 85 3.2.5.2 Chickpea 87 Chapter IV Biochemical studies of chitin metabolizing enzymes of 90- Metarhizium anisopliae 104 A. Cuticle degrading enzyme complex of M. anisopliae isolates B. Importance of extracellularly produced constitutive chitin deacetylase and chitosanase in biocontrol of insect pest, H. armigera 4.1 Introduction 91 4.2 Results and discussion 92 4.2 A Cuticle degrading enzyme complex of Metarhizium anisopliae 92 isolates 4.2.1 A Time course for constitutive and induced production of cuticle 92 degrading enzymes of M. anisopliae 4.2 B Importance of extracellularly produced chitin deacetylase and 96 chitosanase in biocontrol of insect pest, H. armigera 4.2.1 B Bioassay with H. armigera 96 4.2.2 B Detection of chitin deacetylase activity after PAGE under native 96 conditions 4.2.3 B Biochemical characteristics of chitin deacetylase from M. anisopliae 97 4.2.4 B Hydrolysis of chitin 100 4.2.5 B Detection of CDA activity on insect (H. armigera ) cuticle 101 4.2.6 B Cell wall analysis 102 Chapter V Use of chitin metabolizing enzymes and chitin synthesis 104- inhibitors , either singly or in combination to arrest the chitin 113 metabolism in insects and fungi 5.1 Introduction 105 5.2 Results and discussion 106 5.2.1 Mycolytic enzyme activities on M. verrucaria NCIM 903 106 5.2.2 Screening of micro-organisms for cell wall synthesis inhibitors 106 5.2.3 Isolation of chitin metabolism inhibitor(s) 108 5.2.4 Effect of CDE complex of M. verrucaria on H. armigera 109 5.2.5 Pot experiment 112 Chapter VI Conclusion 114- 117 Chapter VII References 118- 134 List of publications / Presentations 135- 137 List of Tables No. Title Page no. 1.1 Loss in yield due to insect pests and fungal pathogens 4 1.2 Effect of various fungal / plant secondary metabolites 8 on infection-related morphogenesis in phytopathogenic fungi 1.3 The crop-wise consumption of pesticides in India 13 1.4 Market status for pesticides 17 1.5 Examples of mycoparasitic fungi reported in India 25 1.6 Examples of entomopathogenic fungi reported in India 28 1.7 Characteristic properties of mycopesticide in 31 production and use 1.8 Inhibition of chitin biosynthesis 45 2.1 Source of chemicals and materials 52 2.2 List of organisms used 53 2.3 Media 54 3.1 Comparative analysis of insect bioassay and 80 biochemical characteristics of M. anisopliae, B. bassiana and N. rileyi isolates 3.2 Effect of different oil formulations on the germination 82 of conidia of selected fungal isolates 3.3 Effect of chickpea leaf extract on the conidial 84 germination of M. anisopliae M 34412, B. bassiana B 3301, and N. rileyi N 812 3.4 Efficacy, pod damage and yield of different treatments 85 against H. armigera infestation on pigeon pea crop under field conditions 3.5 Efficacy, pod damage and yield of different treatments 88 against H. armigera infestation on chickpea crop under field conditions 4.1 Constitutive chitin deacetylase and induced chitinase 95 activities in different M. anisopliae isolates 4.2 Substrate specificity for extracellular chitin 97 deacetylase of Metarhizium anisopliae isolates 4.3 The enzymatic hydrolysis of chitin with Metarhizium 100 anisopliae culture filtrates 4.4 Chitin and chitosan contents of cell wall of M. 102 anisopliae mycelium 5.1 Hyphal tip bursting test of extracellular broth of 107 microbial cultures 5.2 Extraction of chitinase inhibitor (s) of Bacillus sp. 102 108 5.3 Effect of M. verrucaria cuticle degrading enzyme 110 preparation on 3 rd instar H. armigera larvae 5.4 Cuticle degradation of H. armigera using cuticle 111 degrading enzyme preparation List of Figures No. Title Page no. 1.1 Insects and fungi share a common bond 33 1.2 Chitin synthesis 35 3.1 Life cycle of Helicoverpa armigera 74 3.2 Morphology of M. anisopliae, B. bassiana, and N. 77 rileyi and their pathogenecity towards H. armigera 3.3 Percent mortality of Helicoverpa armigera with 84 different oil formulations of entomopathogenic fungi 3.4 Persistence of insect pathogenic fungi after first spray 87 in terms of % mortality of H. armigera in the field 4.1 Time course for constitutive production of chitin 93 metabolizing enzymes of M. anisopliae in YPG medium 4.2 Time course for induced production of chitin 94 metabolizing enzymes of M. anisopliae in chitin medium 4.3 Visualization of chitin deacetylase activity on a 7.5 % 96 PAGE gel 4.4 SDS-PAGE 99 4.5 The detection of chitosan formed on the cuticle due to 101 the germinating M.
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