Endophytic Microbes: Isolation, Identification, and Bioactive Potentials

Preface
Contents
Contributors
Part I: Cultivation Methods of Bacterial Endophytes
Chapter 1: Isolation of Endophytic Bacteria from Leaves, Stems, or Roots
1 Introduction
2 Materials
2.1 Isolation of Endophytic Bacteria from Tissues of Plant Leaves, Stems, and Roots
3 Methods
3.1 Isolation of Endophytic Bacteria from Tissues of Plant Leaves, Stems, and Roots
References
Chapter 2: Heat-Treatment Method for Isolation of Bacillus spp. from Plant Tissues
1 Introduction
2 Materials
2.1 Isolation of Bacillus from Roots
2.2 Isolation from the Cosmosphere, Microflora Associated with Corm Sheath
2.3 Isolation from the Solanaceous Phylloplane
2.4 Isolation from Cacao Leaf Tissue
2.5 Isolation from Cacao Pods
2.6 Isolation from Cacao Pods, Branch, and Flower
2.7 Isolation from White Asparagus
2.8 Isolation from the Spermosphere of Peas and Dwarf French Beans
3 Methods
3.1 Isolation of Bacillus from Roots
3.2 Isolation from the Cormosphere, Microflora Associated with Corm Sheath
3.3 Isolation from the Solanaceous Phylloplane
3.4 Isolation from Cacao Tissue
3.5 Isolation from Cocoa Pods, Branches, and Flowers
3.6 Isolation from Asparagus
3.7 Isolation from the Spermosphere of Peas and Dwarf French Beans
3.8 Isolation from Sugarcane Roots
References
Chapter 3: Isolation and Detection of Endophytic Streptomyces from Actinorhizal Casuarina Root Nodules
1 Introduction
2 Materials
2.1 Collection and Pre-treatment of Root Nodules
2.2 Isolation of Endophytic Streptomyces
2.3 Characterization of Streptomyces
2.4 Microscopic Analysis
2.5 Molecular Identification
3 Methods
3.1 Collection and Pre-treatment of Root Nodules
3.2 Isolation of Streptomyces from Root Nodules
3.3 Characterization of Streptomyces spp.
3.3.1 Morphological and Physiological Characteristic Analysis
3.3.2 Microscopic Analysis
3.3.3 Molecular Identification
References
Chapter 4: Isolation of Endophytic Fungi from Stems, Leaves, or Roots
1 Introduction
2 Materials
3 Methods
3.1 Surface Sterilization Technique [10]
3.2 Isolation of Endophytic Fungi
4 Note
References
Part II: Biochemical Methods for Identification of Endophytes
Chapter 5: Detection of Endophytes by Cultivation Method
1 Introduction
2 Materials
2.1 Sampling
2.2 Pre-treatment (Optional)
2.3 Disinfection
2.4 Disinfection Control
2.5 Culture Media (see Table 2)
2.6 Fragment Culture
3 Methods
3.1 Sampling
3.2 Pre-treatment (Optional for Roots)
3.3 Disinfection (Standard Procedure)
3.4 Control of Disinfection Procedure
3.5 Culture Media (see Table 2)
3.6 Fragment Culture
4 Notes
References
Chapter 6: Detection of Endophytes by Visualization Methods
1 Introduction
2 Materials
2.1 Sampling
2.2 Pre-treatment (Optional)
2.3 Visualization of Endophytes in Plant Cell Lines by FISH Technique
3 Methods
3.1 Sampling
3.2 Pre-treatment (Optional for Roots)
3.3 Disinfection
3.4 Light Microscope
3.5 Electron Microscope
3.6 Transmission Electron Microscope (TEM)
3.7 Fluorescence Microscope
3.8 Combined Confocal Microscopy and FISH (Fluorescence In Situ Hybridization)
3.9 Visualization of Endophytes in Plant Cell Lines by FISH Technique
References
Chapter 7: Detection of Endophytes by Immunological Methods
1 Introduction
2 Materials Required
2.1 ELISA Technique
2.1.1 Standard Solutions
2.1.2 Biotinylated Antibody
2.1.3 Avidin-Biotin-Peroxidase (ABC)
2.2 Indirect ELISA
2.3 Dot Blot Immunoassay (DBIA)
2.4 Tissue Print Immunoassay(TPIA)
2.5 Immunogold Labeling Technique
3 Methods
3.1 Isolation of Fungal Endophytes from Different Sources
3.2 Isolation of Bacterial Endophytes from Different Sources
3.3 Enzyme-Linked Immunosorbent Assay (ELISA)
4 Conclusion
References
Chapter 8: Detection of Endophytes by Electron Microscope
1 Introduction
2 Materials
3 Methods
3.1 Sample Collection
3.2 Sample Sterilization
3.3 Isolation of Bacterial Endophytes
3.4 Isolation of Endophytic Fungi [13, 14 ]
3.5 Electron Microscopic Analysis
3.5.1 Fixation of Sample
3.5.2 Scanning Electron Microscopy
3.5.3 Transmission Electron Microscopy
References
Chapter 9: Detection of Endophytes by Reactive Oxygen Staining
1 Introduction
2 Materials
3 Methods
3.1 DAB Microscopic Method to Visualize Endophytes in Plant Tissues
3.1.1 Preparation of Staining Solution
3.1.2 Staining with DAB Solution
3.2 NBT Microscopic Method to Visualize Endophytes in Plant Tissues
3.2.1 Preparation of Staining Solution
3.2.2 Staining with NBT Solution
References
Part III: Molecular Methods for Identification of Endophytes
Chapter 10: Identification of Bacterial Endophytes by 16S rRNA
1 Introduction
2 Materials
3 Methods
3.1 Isolation of DNA from Endophytic Bacteria
3.2 16S Gene Amplification
3.3 Identification of Endophytic Bacteria [7]
References
Chapter 11: Identification of Fungal Endophytes by ITS rDNA Technique
1 Introduction
2 Materials
2.1 Isolation of Endophytic Fungi
2.2 Molecular Characterization
3 Methods
3.1 Isolation of Endophytic Fungi
3.2 Molecular Characterization of Endophytic Fungi
3.2.1 DNA Extraction
3.2.2 PCR Amplification
3.3 DNA Sequencing
3.4 Phylogenetic Analysis
References
Chapter 12: Identification of Fungal Endophytes by 28SrDNA Technique
1 Introduction
2 Materials
2.1 Sampling
2.2 Pre-treatment (Optional)
2.3 Disinfection
2.4 Materials for DNA Purification by Paper Slurry Method
2.5 Solutions Used for DNA Purification from Acrylamide
2.6 Other Reagents
3 Methods
3.1 Sampling and Isolation of Endophytes
3.2 DNA Extraction
3.3 Gel Purification Methods
3.3.1 Purification of DNA from Agarose Gels (Paper Slurry Method)
3.3.2 Gene-Kleen Protocol
3.3.3 Elution of DNA Fragments from Low-Melting Agarose Gel by Dr. Roe Method
3.3.4 Freezing Method
3.3.5 DNA Fragment Purification from Acrylamide
3.3.6 Rapid Elution of DNA from Agarose Gels
Glass Wool Spin Column Preparation
Eluting DNA from Agarose Gel Fragments
3.4 PCR Amplification of the Fungal 28S Regions
3.5 Construction of 28S rDNA Region Clone Library
3.6 Phylogenetic Analysis and Diversity Analysis
References
Chapter 13: Detection of Endophytes by Molecular Fingerprinting Techniques
1 Introduction
1.1 Molecular Fingerprinting
1.2 Data Collection and Analysis
2 Materials Required
2.1 RAPD
2.2 Diversity Analysis Using Denaturing Gradient Gel Electrophoresis (DGGE)
2.3 PCR-RFLP
2.4 Box-PCR Finger Printing
2.5 Its-RFLP
2.6 AFLP
3 Protocols of Different Fingerprinting Techniques
3.1 Fungal DNA Isolation
3.2 Molecular Characterization of Endophytic Fungi
3.2.1 ITS Region Amplification
3.2.2 TEF-1α Gene Amplification
4 Conclusion
References
Chapter 14: Metagenomics and Pyrosequencing Approach
1 Introduction
2 Materials
2.1 Estimation of DNA
2.2 PCR Amplification and Metabarcoding
2.3 OTU-Based Sequence Analysis
3 Methods
3.1 DNA Extraction
3.2 Estimation of DNA
3.3 PCR Amplification and Metabarcoding
3.4 OTU-Based Sequence Analysis
4 Note
References
Part IV: Methods to Proof Endophytism
Chapter 15: Cultivation-Based Studies of Endophytic Fungi in Plants
1 Introduction
2 Materials
2.1 Collection of Plant Material
2.2 Surface Sterilization of Plant Material
2.3 Endophytic Fungi Isolation and Cultivation
3 Materials and Methods
3.1 Collection of Plant Material
3.2 Surface Sterilization
3.3 Isolation and Cultivation of Endophytic Fungi
References
Chapter 16: Microscopy-Based Studies
1 Introduction
2 Materials
2.1 Materials for FISH (Fluorescence In Situ Hybridization)
2.2 Materials for GFP Tagging
2.3 Materials for GUS Staining
2.4 Materials for Fluorogenic Dye Staining
3 Methods
3.1 Methods for FISH (Fluorescence In Situ Hybridization)
3.2 Methods for GFP Tagging
3.2.1 Transformation of Gram-Negative Bacteria by Electroporation
3.2.2 Bacteria Electro Transformation
3.2.3 Transformation of Bacteria by Conjugation
3.2.4 Plant Growth Conditions and GFP-Bacterial Inoculations
3.3 Methods for GUS Staining
3.3.1 Transformation of Bacteria by Conjugation (or by Electroporation as Described Earlier)
3.3.2 Detection of gusA-tagged Bacteria Activity in planta
3.4 Methods for Fluorogenic Dye Staining
3.4.1 DAPI/AO Staining
3.4.2 Calcofluor White/Propidium Iodide (PI) Staining
References
Chapter 17: Surrogate Host Test
1 Introduction
2 Materials
3 Protocol
References
Chapter 18: Hood and Shew Staining
1 Introduction
2 Materials
2.1 KOH-Aniline Blue Fluorescence Staining [1]
2.2 Preparation of Specimen for Live Staining
3 Methods
3.1 KOH-Aniline Blue Fluorescence Staining
3.2 Plate Method
3.3 Greenhouse Plant
4 Notes
References
Part V: Endophytes for Bioactive Metabolites
Chapter 19: Screening of Endophytes for Plant Growth-Promoting Metabolites
1 Introduction
2 Materials
2.1 Indole Acetic Acid (IAA) Detection
2.2 Phosphate Solubilization
2.3 Nitrogen Fixation Assay
2.4 Siderophores Production
2.5 ACC Deaminase Detection
2.6 HCN Production
2.7 Ammonium Production
3 Methods
3.1 Indole Acetic Acid (IAA) Detection [9]
3.1.1 Qualitative Method
3.1.2 IAA Standard Preparation
3.1.3 Estimation of IAA Production from Endophytic Bacteria
3.2 Phosphate Solubilization
3.3 Nitrogen Fixation Assay
3.4 Siderophore Production
3.5 ACC Deaminase Production
3.5.1 Qualitative Method
3.5.2 Quantitative Method
3.6 HCN Production
3.7 Ammonia Production
References
Chapter 20: Screening of Endophytes for Biocontrol Properties
1 Introduction
2 Materials
3 Methods
3.1 Dual Culture Method (Fungal Plant Pathogens)
3.2 Agar Well Diffusion Method (Bacterial Plant Pathogens)
4 Note
References
Chapter 21: Screening of Endophytes for Biocatalytic Tools
1 Introduction
2 Materials
2.1 General Procedure for the Isolation of Endophytes to Obtain New Biocatalysts
2.1.1 Sampling
2.1.2 Disinfection
2.1.3 Isolation of Endophytic Microorganisms
2.1.4 Conservation of Endophytic Microbial Strains
2.1.5 Identification of Bacteria Endophytic Strains
2.1.6 Identification of Fungal Endophytic Strains
2.2 Isolation of Endophytes with Biocatalytic Activity from Mentha pulegium
2.3 Isolation of Endophytes with Xylanase and Cellulase Activity
2.4 Isolation Endophytes with Carbonyl Reductase Activity
3 Methods
3.1 General Procedure for the Isolation of Endophytes to Obtain New Biocatalysts According to the Strategies Mentioned above
3.2 Particular Cases
3.2.1 Isolation of Endophytes with Biocatalytic Activity from Mentha pulegium
3.2.2 Isolation Endophytes with Xylanase and Cellulase Activity from Eucalyptus sp. Leaves, Bark, and Branches
3.2.3 Isolation Endophytes with Prelog Carbonyl Reductase Activity from Daucus carota, Curcubita maxima, and Foeniculum vulgare
References
Chapter 22: Screening of Endophytes for Biosurfactant Production
1 Introduction
2 Materials
3 Methods
3.1 Collection of Biosurfactant from Endophytes
3.2 Drop Collapse Assay
3.3 Oil Spread Assay
3.4 Emulsification Index (E24)
References
Chapter 23: Screening of Endophytes for Antibacterial Properties
1 Introduction
2 Materials
2.1 Screening of Endophytes for Antibacterial Property by Cross-Streak Method (Fig. 1)
2.2 Screening of Endophytes for Antibacterial Property Agar Plug Diffusion Method
3 Methodology
3.1 Screening of Endophytes for Antibacterial Property by Cross-Streak Method
3.1.1 Inoculation of Endophyte Culture by Single Streak
3.1.2 Inoculation of Target Bacterial Culture by Cross-Streak
3.2 Screening of Endophytes for Antibacterial Property Agar Plug Diffusion Method (Fig. 2)
3.2.1 Preparation of Agar Plug
3.2.2 Preparation of 0.5McFarland Standard Target Bacterial Inoculum
3.2.3 Inoculation of MHA Plate
3.2.4 Transfer of Agar Plug
4 Observations
4.1 Screening of Endophytes for Antibacterial Properties by Cross-Streak Method
4.2 Screening of Endophytes for Antibacterial Properties by Agar Plug Diffusion Method
References
Chapter 24: Screening of Endophytes for Nematicidal Activities
1 Introduction
2 Materials
2.1 Isolation of Endophytic Microbes from Roots, Shoots, and Leaves of Plant Samples
2.2 Nematicidal Efficacy of Endophytic Microbes In Vitro
2.2.1 Preparation of Fungal Culture Filtrates (FCF)
2.3 Screening of Endophytic Fungi for Nematicidal Activities (In Vivo)
3 Methods
3.1 Isolation of Endophytic Microbes from Roots, Shoots, and Leaves of Plant Samples
3.2 Nematicidal Efficacy of Endophytic Microbes In Vitro
3.3 For Endophytic Bacteria
3.4 For Endophytic Fungi
3.5 Preparation of FCF
3.6 Screening of Endophytic Fungi for Nematicidal Activities (In Vivo)
3.7 Efficacy Test and Colonization Ratio of Selected Fungal Isolates
4 Observation
References
Chapter 25: Screening of Endophytes for Virucidal Activity
1 Mechanism of Endophytes in Antiviral Activity
2 Isolation of Endophytes
2.1 Ribonuclease Activity Estimation Method Using Endophytic Bacteria
2.2 Screening of Endophytes for Virucidal Activity by Sap Transmission Method
2.2.1 Materials Required
2.2.2 Protocol for Screening of Endophytic Microbes for Virucidal Activity
2.3 Screening of Endophytic Microbes for Virucidal Activity Using Insect Vector
References
Chapter 26: Screening of Endophytes for Fungicidal Activities
1 Introduction
2 Materials
3 Methodology
3.1 Preparation of Potato Dextrose Agar Medium Plate (PDAMP)
3.2 Preparation of Yeast Malt Agar Medium Plate (YMAMP)
3.3 Preparation of Endophytic Mycelial Disk (Fungus & Actinomycetes)
3.4 Preparation of Target Fungal Mycelial Disk
3.5 Inoculation of Dual Culture (Figs. 1 and 2)
3.5.1 Inoculation of Target Fungus and Endophytes (Fungus & Actinomycetes)
3.5.2 Inoculation of Endophytic Bacteria
3.6 Incubation
4 Observations
References
Chapter 27: Screening of Endophytes for Anticancer Compounds
1 Introduction
2 Materials
2.1 Obtaining Active Fungal Colonies
2.2 Obtaining Fungal Colonies in Liquid Medium
2.3 Extraction of Metabolites from Endophytic Fungi
2.4 Processing of Extracts and Verification of Metabolites
2.5 Cancer Cell Lines Culture, Reagent, and Equipment
2.6 Cytotoxicity Assays in Cancer Cell Lines
2.6.1 Trypan Blue Protocol
2.6.2 MTT Colorimetric Assay
2.6.3 LDH
2.6.4 Alamar Blue
2.6.5 Colony Formation Assay
2.6.6 Electron Microscopic Analysis
2.6.7 Colony Formation Assay
2.6.8 Annexin V-FITC-propidium Iodide (PI) Assay
2.6.9 Analysis of Mitochondrial Transmembrane Potential (ΔΨm)
2.6.10 Cell Cycle Analysis
2.6.11 Western Blotting
2.6.12 Caspase-3 Activity
3 Methods
3.1 Obtaining Active Fungal Colonies
3.2 Obtaining Fungal Colonies in Liquid Medium
3.3 Extraction of Metabolites from Endophytic Fungi
3.4 Processing of Extracts and Verification of Metabolites
3.5 Anticancer Analysis of Bioactive Compounds from Endophytes
3.5.1 Cancer Cell Lines
3.5.2 Perform Bioactive Compounds Treatment
3.5.3 Trypan Blue Assay Protocol
3.5.4 MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) Assay
3.5.5 LDH Assay
3.5.6 Alamar Blue Assay Method
3.5.7 Colony Formation Assay
3.5.8 Annexin V-FITC-Propidium Iodide (PI) Assay
3.5.9 Analysis of Mitochondrial Transmembrane Potential (ΔΨm)
3.5.10 Transmission Electron Microscopy (TEM) Analysis
3.5.11 Cell Cycle Analysis
3.5.12 Western Blotting
3.5.13 Caspase Activity Assay
References
Index