Microbial-Based Biopesticides: Methods and Protocols (Methods in Molecular Biology, 1477)

✍ Scribed by Travis R. Glare (editor), Maria E. Moran-Diez (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 215
• Category: Library

✦ Synopsis

This volume focuses on the developmental areas of biopesticides: production, formulation, application and field efficacy. Chapters guide readers through methods and techniques on environmental, mammalian, safety, and registration. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Microbial-Based Biopesticides aims to ensure successful results in the further study of this vital field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: What are Microbial-based Biopesticides?
1 Integrated Pest Management in Sustainable Agriculture
2 What Are Microbial-Based Biopesticides?
3 From the Field to the Bench and Back
4 Methods in Biopesticide Research
References
Part I: Screening, Isolation and Identification of Potential Biological Control Agents
Chapter 2: Isolation and Mass Production of Trichoderma
1 Introduction
2 Materials
2.1 General Media
2.2 Isolation
2.3 Mass Production of Trichoderma Inoculum on Brown Rice and Wheat Grain
3 Methods
3.1 Trichoderma Isolation from Plant Roots
3.2 Trichoderma Isolation from Rhizosphere and Bulk Soil Samples
3.3 Trichoderma Conidia Production on Plates
3.4 Strain Purification: Isolation of Colonies Derived from a Single Conidium
3.5 Mass Production of Trichoderma Inoculum on Brown Rice
3.6 Mass Production of Trichoderma Inoculum on Wheat Grain
4 Notes
References
Part II: Evaluating Mode of Action of Microorganisms
Chapter 3: Methods for the Evaluation of the Bioactivity and Biocontrol Potential of Species of Trichoderma
1 Introduction
2 Materials
2.1 General Media
2.2 Nonvolatile Metabolites Production
2.3 Rhizospheric and Endophytic Colonization of Maize. Modification: Growth Promotion and Systemic Resistance
2.4 Detached Strawberry Necrotic Leaf Assay
2.5 Biocontrol Activity Against B. cinerea in Grapes
2.6 Wheat/Root Lesion Nematode Bioassay
3 Methods
3.1 Dual Culture Plate Assay (Mycoparasitism)
3.2 Production of Volatile Compounds
3.3 Production of Nonvolatile Metabolites
3.4 Rhizospheric and Endophytic Colonization of Maize. Modification: Growth Promotion and Systemic Resistance
3.5 Detached Strawberry Necrotic Leaf Assay
3.6 Biocontrol Activity Against B. cinerea in Grapes
3.7 Wheat/Root Lesion Nematode Bioassay to Screen Beneficial Trichoderma Isolates
4 Notes
References
Part III: Mass Production and Formulations: Bacteria
Chapter 4: Purification of the Yersinia entomophaga Yen-TC Toxin Complex Using Size Exclusion Chromatography
1 Introduction
2 Materials
2.1 Items Required for Culturing
2.2 Yen-TC Purification
2.3 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Components
3 Methods
3.1 Culture and Yen-TC Production
3.2 Purification of Yen-TC
3.3 SDS-PAGE
3.4 Protein Silver Stain for SDS-­PAGE Gels
3.5 Protein Quantification Using a Bradford Microtiter Plate Assay
4 Notes
References
Chapter 5: Coated Solid Substrate Microbe Formulations: Pseudomonas spp. and Zeolite
1 Introduction
1.1 Factors Reducing Viability
1.2 Adsorption on Solid Surfaces
1.3 Other Coating Techniques
2 Materials
3 Methods
3.1 Strains Maintenance and Growth
3.2 Coating Zeolite (See Note 1)
3.3 Evaluating Efficacy
4 Notes
References
Part IV: Mass Production and Formulations: Fungi
Chapter 6: Production of Conidia by the Fungus Metarhizium anisopliae Using Solid-State Fermentation
1 Introduction
2 Materials
2.1 Organisms
2.2 Culture Media
2.3 Conidia Production in Plastic Bag
2.4 Conidia Production in Tubular Bioreactor
3 Methods
3.1 Metarhizium Anisopliae Propagation
3.2 M. Anisopliae Reactivation in Tenebrio Molitor
3.3 Strain Conservation
3.4 Inocula Production
3.5 Conidia Production in Plastic Bags
3.6 Conidia Production in Tubular Bioreactors
3.7 Evaluation of Conidia Quality
4 Notes
References
Chapter 7: Liquid Culture Production of Fungal Microsclerotia
1 Introduction
2 Materials
2.1 Culture Maintenance and Growth
2.2 Microsclerotia Harvesting and Drying
2.3 Measurements
3 Methods
3.1 Stock Cultures and Inoculum Development
3.2 Shake Flask Microsclerotia Production
3.3 Microsclerotia Counting, Harvesting, and Drying
3.4 Microsclerotia Evaluation
4 Notes
References
Chapter 8: Isolation and Assessment of Stability of Six Formulations of Entomopathogenic Beauveria bassiana
1 Introduction
2 Materials
2.1 Galleria Diet
2.2 Quarter-Strength Potato Dextrose Agar
2.3 Agar slants
3 Methods
3.1 Isolation of Beauveria bassiana from the Soil
3.2 Single Conidium Suspension
3.3 Beauveria bassiana Identification
3.4 Culture Maintenance
3.5 Cultivation of Beauveria bassiana
3.6 Barley/Rice/Wheat Bran Substrate Formulation
3.7 Clay/Kaolin/Peat Formulations of Beauveria bassiana
3.8 Determination of Stability of Formulations
4 Notes
References
Part V: Mass Production and Formulations: Viruses
Chapter 9: Cell Culture for Production of Insecticidal Viruses
1 Introduction
2 Materials
2.1 Production of Insect Viruses in Suspension Cultures: Shake Flasks, Spinner Flasks, Stirred Tank, and Airlift Bioreactors
2.1.1 Cell Line and Virus
2.1.2 General Cell Culture Equipment and Consumables
2.1.3 Culture Medium and Supplements (See Note 1)
2.1.4 Bioreactors
2.1.5 Cell Counts/Sampling
2.1.6 Cell Cryopreservation Equipment
2.2 Production of Cells and Virus in Adherent Cell Cultures: T-Flasks, Roller Bottles, Microcarriers
2.2.1 Cell Line and Virus
2.2.2 Culture Medium, Solutions, and Microcarriers
2.2.3 Bioreactors
3 Methods
3.1 Production of Insect Viruses in Suspension Cultures
3.1.1 Cell Density and Cell Viability Enumeration
3.1.2 Cell Cryopreservation: Freezing/Thawing
3.1.3 Aseptic Technique in the Biological Safety Cabinet (BSC)
3.1.4 Culture of HearNPV in Shaker Flasks
3.1.5 ODV Extraction to Initiate HearNPV Infections in Suspension Culture
3.1.6 OB Count
3.1.7 Culture of AgMNPV in Spinner Flask Bioreactors
3.1.8 Culture of HearNPV in Stirred Tank Bioreactors
3.1.9 Culture of AgMNPV in Airlift Bioreactors
3.2 Production of Insect Viruses Using Adherent Cultures: DSIR-HA-1179-­OrNV System as an Example
3.2.1 Preparation of Culture Medium
3.2.2 Preparation of Cell Inoculum
3.2.3 OrNV Production in T-Flasks
3.2.4 OrNV Production in the Roller Bottle System
3.2.5 OrNV Production in Microcarriers
3.2.6 Quantification of Infectious Virus Titer by Endpoint Dilution (See Note 19)
4 Notes
References
Part VI: Mass Production and Formulations: Nematodes
Chapter 10: Formulation of Nematodes
1 Introduction
2 Materials
2.1 Production of Nematodes on Galleria mellonella
2.2 Formulation Outside Insect Cadavers
2.3 Establishing Nematode Concentration
2.4 Choosing a Binder for Wettable Powder Formulations
2.5 Producing Nematode-
2.6 Produce a Matrix for a Nematode Bait Station
2.7 Packaging
2.8 Evaluating Formulations
3 Methods
3.1 Production of Infected Cadavers of the Greater Wax Moth, Galleria mellonella (See Refs. 11, 16)
3.2 Formulation Outside Insect Cadavers
3.3 Separating Living from Dead Nematodes
3.4 Establishing Nematode Concentration
3.5 Choosing a Binder for Wettable Powder Formulations
3.6 Slow Release Formulations
3.7 Producing Nematode-­Containing Alginate Beads (See Fig. 3)
3.8 Produce a Matrix for a Nematode Bait Station
3.9 Packaging
3.10 Evaluating Formulations
3.10.1 Assessing Number and Infectivity of Formulated Entomopathogenic Nematodes (See Note 5)
3.10.2 Infectivity Test (See Ref. 29)
4 Notes
References
Chapter 11: In Vivo Production of Entomopathogenic Nematodes
1 Introduction
2 Materials
2.1 Basic Methods (White Trap, Culture, and Strain Maintenance)
2.2 Optimization and Scale-Up
2.3 Advanced/Automated Methods
2.3.1 Improved Insect Production
2.3.2 Automated Inoculation and Harvest
2.3.3 Production and Formulation of Cadavers
3 Methods
3.1 Basic Methods (White trap, Culture, and Strain Maintenance)
3.2 Optimization and Scale-Up
3.3 Advanced/Automated Methods
3.3.1 Improved Insect Production
3.3.2 Tenebrio Molitor Rearing
3.3.3 Diet Supplements
3.3.4 Automated Inoculation and Harvest
3.3.5 Production and Formulation of Cadavers
4 Notes
References
Part VII: Monitoring of Applied Microbes
Chapter 12: Detection and Quantification of the Entomopathogenic Fungal Endophyte Beauveria bassiana in Plants by Nested and Quantitative PCR
1 Introduction
2 Materials
2.1 Disinfection of Plant Material
2.2 Components for DNA Plant Extraction
2.3 Two-Steps Nested-PCR Amplification
2.4 Agarose Gel Components
2.5 Real-Time PCR Quantification
3 Methods
3.1 Disinfection of Plant Material
3.2 DNA Plant Extraction
3.3 Two-Step Nested-PCR Amplification
3.4 Agarose Gel Electrophoresis
3.5 Real-Time PCR Quantification
4 Notes
References
Chapter 13: Plant Tissue Preparation for the Detection of an Endophytic Fungus In Planta
1 Introduction
2 Materials
3 Methods
3.1 Assessing Surface Sterilization Efficacy
3.2 Surface Sterilization of Inoculated Plant Tissue
3.3 Protocol for Treating Plant Samples with PMA for PCR
4 Notes
References
Part VIII: Quality Control, Safety, and Registration
Chapter 15: Analytical Methods for Secondary Metabolite Detection
1 Introduction
2 Materials
2.1 Beauveria brongniartii
2.1.1 Materials for Submerged Culture
2.1.2 Fungal Growth on Barley Kernels
2.1.3 Buffer Preparation
2.1.4 Extraction of Melocont™-WP, Melocont™-Pilzgerste, and Biological Samples
2.1.5 Preparation of Stock Solutions and Solvents
2.1.6 HPLC–DAD Conditions
2.1.7 HPLC-DAD Assay Validation: Submerged Culture Broth and Biocontrol Formulations
2.1.8 HPLC–DAD Assay Validation: Potato Tubers
2.2 Metarhizium brunneum
2.2.1 Cultivation of Metarhizium brunneum
2.2.2 Extract Preparation
2.2.3 Thin Layer Chromatography for Fraction Analysis
2.2.4 HPLC–DAD and HPLC–DAD–MS/MS Conditions for Fraction Analysis
2.2.5 Chromatography on Sephadex LH-20 Material
2.2.6 High-Speed Counter-Current Chromatography (HSCCC)
2.2.7 Sample Preparation from Culture Filtrate
2.2.8 UHPLC-DAD-
2.2.9 Assay Validation
3 Methods
3.1 Beauveria brongniartii
3.1.1 Submerged Culture of B. brongniartii
3.1.2 Fungal Growth on Barley Kernels
3.1.3 Extraction from Culture Filtrate
3.1.4 Extraction from Barley Kernels or Melocont™-Pilzgerste
3.1.5 Extraction from Melocont™-WP
3.1.6 Extraction of Biological Samples: Potato Tubers
3.1.7 HPLC–DAD Assay for the Analysis and Quantification of Oosporein
3.1.8 HPLC–DAD Assay Validation: Submerged Culture Broth and Biocontrol Formulations
3.1.9 HPLC–DAD Assay Validation: Potato Tubers
3.2 Metarhizium brunneum
3.2.1 Cultivation of Metarhizium brunneum
3.2.2 Preparation of Crude Extract
3.2.3 Chromatography on Sephadex LH-20 Material
3.2.4 High-Speed Counter-Current Chromatography (HSCCC)
3.2.5 Fraction Analysis by Thin Layer Chromatography
3.2.6 Fraction Analysis by HPLC–DAD– MS/MS
3.2.7 Sample Preparation from Culture Filtrate
3.2.8 UHPLC–DAD Method for Destruxin Quantification from Culture Filtrate
3.2.9 UHPLC–DAD–QTOF–MS/MS Method for Destruxin Identification from Culture Filtrate
3.2.10 Assay Validation
4 Notes
References
Chapter 16: Development of Biopesticides and Future Opportunities
1 Introduction
1.1 The Need
1.2 Increasing Use of Microbial-­Based Biopesticides
2 Regulation
2.1 Data Requirements for Registration
3 Areas of Potential Improvement in Biopesticides
3.1 Improving Regulation
3.2 Strain Selection
3.3 Production and Formulation
3.4 Application and Monitoring
3.5 Quality Control
3.6 Environmental and Mammalian Safety
4 Innovative Approaches
4.1 Endophytes
4.2 Bioactives
5 Integrated Pest Management (IPM)
6 Summary and Future Directions
References
ERRATUM TO
Index