Mycology has an integral role to play in the development of the biotechnology and biomedical sectors. It has become a subject of increasing importance as new fungi and their associated biomolecules are identified. As this discipline comes to the forefront of research in these sectors, the requirement for a consolidation of available research approaches is required. The First Edition of this book has a few basic and applied protocols. With the Second Edition, this book provides consolidated information on recent developments and the most widely used mycological methods available in the fields of biochemistry, biotechnology and microbiology. The methods outlined offer clear and concise directions to the reader and covers both standard protocols and more applied mycological methods. This book provides useful information for undergraduates, post-graduates, and specialists and researchers studying fungal biology.
Author(s): Vijai Kumar Gupta, Maria Tuohy
Edition: 2
Publisher: Springer
Year: 2022
Language: English
Pages: 267
City: Singapore
Contents
1: Various Methods of Long-Term Preservation of Fungal Cultures in All-Russian Collection of Microorganisms (VKM)
1.1 Introduction
1.2 Cryopreservation of Filamentous Fungi
1.3 Freeze-Drying of Filamentous Fungi
1.4 Drying in Sterile Soil of Filamentous Fungi
1.5 Drying of Filamentous Fungi on Silica Gel
1.6 Protocols
1.7 Protocol of Drying on Silica Gel
1.7.1 Preparation of Sterile Silica Gel and Ampoules
1.7.2 Preparation of Cryoprotectant: 10% (v/v) Glycerol
1.7.3 Preparation of Cultures
1.7.4 Silica Gel Inoculation
1.7.5 Filling of Vials
1.7.6 Control of Viability
Annexies
Annex 1: Fields Attributes in the Table «Database Preservation Methods»
Annex 2: Maximal Preservation Times for VKM Fungal Species
References
Chapter 2: Sabouraud Agar and Other Fungal Growth Media
2.1 Sabouraud Dextrose Agar
2.1.1 History
2.1.2 Theory
2.1.3 Materials
2.1.4 Method of Sabouraud Agar Preparation
2.1.4.1 Standard Preparation
2.1.4.2 Method of Sabouraud Agar, Emmons Modification Preparation
2.1.4.3 Variations on Standard Sabouraud Agar
2.1.5 Methods of Inoculation and Incubation
2.1.6 Results
2.2 Potato Dextrose Agar
2.2.1 History
2.2.2 Theory
2.2.3 Methods of Preparation
2.2.4 Methods of Inoculation and Incubation
2.2.4.1 Quality Control
2.2.4.2 Inoculation and Incubation
2.2.5 Results
2.3 Bird Seed Agar
2.3.1 History
2.3.2 Theory
2.3.3 Methods of Preparation
2.3.3.1 Preparation of Bird Seed Agar
2.3.4 Method of Inoculation and Incubation
2.3.4.1 Quality Control
2.3.4.2 Inoculation and Incubation
2.3.5 Results
2.4 Dermatophyte Test Medium
2.4.1 History
2.4.2 Theory
2.4.3 Method of Preparation
2.4.4 Methods of Inoculation and Incubation
2.4.5 Results
2.5 Safety Notes
2.6 Additional Fungal Growth Media
References
Chapter 3: Fluorescence In Situ Hybridization of Uncultured Zoosporic Fungi
3.1 Introduction
3.2 Materials
3.3 Methods
3.3.1 Classical FISH Probing (See Note 3)
3.3.2 CARD-FISH Probing (See Note 7)
3.4 Notes
References
Chapter 4: Technique for Identifying and Counting Infective Chytrid Sporangia Using the Chitinaceous Fluorochrome Calcofluor W...
4.1 Introduction
4.2 Materials
4.3 Methods
4.3.1 Concentrations of Cells (See Note 1)
4.3.2 Preparation of Calcofluor Stock Solution
4.3.3 Staining and Visualization
4.4 Notes
References
Chapter 5: Assessment of Host Immune Responses to Fungal Pathogens
5.1 Introduction
5.2 Methods
5.2.1 Analysis of the Fungal Burden
5.2.1.1 CFU
5.2.1.2 qPCR13 Targeting the 18S rRNA Gene
5.2.2 Isolation of the Immune System Organs, Infected Organs, and Immune Cells
5.2.2.1 Thymus
5.2.2.2 Bone Marrow
5.2.2.3 Spleen
5.2.2.4 Lymph Nodes
5.2.2.5 Blood
5.2.2.6 Bronchoalveolar Lavage Fluid (BALF)
5.2.2.7 Infected Organs
5.2.2.8 Separation of Immune Cells and Erythrocyte Lysis
5.2.2.9 Histology
5.2.3 Detection and Quantification of Immune Cells
5.2.3.1 Cell Detection Using Flow Cytometry
5.2.3.2 Cell Counting
5.2.4 Detection and Quantification of Cytokines
5.2.4.1 Intracellular Cytokine Detection Based on Flow Cytometry
5.2.4.2 ELISA
5.2.4.3 Other Methods
5.3 Summary
References
Chapter 6: Recent Advances in Applications of Support Vector Machines in Fungal Biology
6.1 Introduction
6.1.1 Support Vector Machine
6.2 Domain Features
6.3 Review of Some Recent Fungal Bioinformatics Applications Using SVM
6.3.1 Haar Wavelets Features for Fungal Disease Detection in Maize Leaves
6.3.2 Quality Detection of Pomegranate Fruit Infected with Fungal Disease
6.3.3 Support Vector Machines Enabled Fungal Rust Disease Detection in Pea Plant (Pisam Sativam)
6.3.4 Volatomic Visualization for Fungal Infection Detection on Storage Jasmine
6.3.5 Hyperspectral Imaging Enabled Electronic Nose for Fungal Contamination Identification in Strawberries
6.3.6 Rapid Discrimination of Fungal Species by the Colony Fingerprinting
6.3.7 SVM Classifier Based Grape Leaf Disease Detection
6.3.8 Hyphae Detection in Fungal Keratitis Images
6.3.9 Plant Diseases Classification Using SVM and ANN (Artificial Neural Network)
6.3.10 A Hybrid Combination of Multiple SVM Classifiers for Automatic Recognition of the Damages and Symptoms on Plant Leaves
6.3.11 Machine Learning of Protein Interactions in Fungal Secretory Pathways
6.3.12 Fungal Adhesins and Adhesin Like Proteins Predictions
6.3.13 Computational Prediction of Antifungal Peptides
6.3.14 A Novel Method of Annotation of Antifungal Peptides Based on Distributed Representation of Protein Sequences
6.3.15 Identification of Antifungal Using Distributed Representation of Sequences
6.4 Illustration of Use of SVM in Fungal Bioinformatics
6.5 Concluding Remarks
References
Chapter 7: Real-Time Quantitative PCR Assay for the Assessment of Uncultured Zoosporic Fungi
7.1 Introduction
7.2 Materials
7.3 Methods
7.3.1 DNA Extraction and Purification
7.3.2 Real-Time qPCR Assays
7.4 Notes
References
Chapter 8: Assays for the Quantification of Antioxidant Enzymes in Fungi
8.1 Introduction
8.2 Materials and Methods
8.2.1 Materials
8.3 Methods
8.3.1 Fungal Tissue Homogenization
8.4 Protein Quantification
8.5 Antioxidant Enzymes Related to the Decomposition of ROS
8.5.1 Catalase (CAT) Activity
8.5.2 Peroxidases (Px) Activity
8.5.3 Superoxide Dismutase (SOD) Activity
8.6 Thiol Redox State Related Enzymes
8.6.1 Glutathione-S-Transferase (GST) Activity
8.6.2 Glutathione Reductase (GR) Activity
8.6.3 Glutathione Peroxidase (GP) Activity
8.6.4 Glucose-6-Phosphate Dehydrogenase (G6PD) Activity
8.7 Conclusions
References
Chapter 9: Cellulomics of Live Yeast by Advanced and Correlative Microscopy
9.1 Introduction
9.2 Materials
9.3 Experimental Methods
9.3.1 Preparing Coverslips
9.3.1.1 Cleaning Coverslips
9.3.1.2 Coating Coverslips
Poly-L-Lysine (PLL)
Cell-Tak
9.3.2 Sample Preparation
9.3.2.1 Fixed Samples
9.3.2.2 Live Samples
Physical Immobilisation of Cells with PDMS Stamps
Biochemical Trapping of Cells with Cell-Tak
9.3.3 Pros and Cons of Immobilisation Techniques
9.4 Imaging Modes
9.4.1 Imaging Fixed Cells in QI Mode
9.4.2 Imaging Live Cells in QI Mode
9.4.3 Tracking Dynamic Processes in QI Mode
9.4.4 Correlative AFM-QI-LSCM Live Cell Imaging
9.5 AFM QI Mode Image/Data Processing
9.5.1 AFM Image Processing and Overlay
References
Chapter 10: Molecular Taxonomy and Multigene Phylogeny of Filamentous Fungi
10.1 Introduction
10.2 Fungal Genomic DNA Extraction
10.2.1 Materials
10.2.2 Method
10.3 Agarose Gel Electrophoresis
10.3.1 Material
10.3.2 Method
10.4 Selection of Gene Target and Their Amplification
10.4.1 Materials
10.4.2 Method
10.5 PCR Product Purification and Quantification
10.6 DNA Sequencing (Sanger Method)
10.6.1 Materials
10.6.2 Method
10.7 Perform Sequencing PCR in Thermal Cycler Using Following Conditions
10.7.1 Purification of Sequencing PCR Product
10.8 Sequence Editing, Analysis, and Identification
10.8.1 Basic Local Alignment Search Tool (BLAST)
10.8.2 MycoBank Database Search
10.8.3 UNITE Database Search
10.8.4 Method
10.9 Molecular Phylogeny and Phylogenetic Tree
10.9.1 Phylogenetic Tree Construction
10.9.2 Phylogenetic Tree Construction Using MEGA Software
References
Chapter 11: Fluorochrome-Based Methods for Fungal Sample Examination
11.1 Introduction
11.2 Sample Preparation to Observe Fungi Nuclei and Chromosome
11.3 DAPI Methods
11.3.1 Materials
11.3.2 Methods
11.3.3 Culture Block on Inverted Microscope
11.3.4 Slide and Coverslip Preparation
11.4 Culture Block on Inverted Microscope and Propidium Iodide in Nuclei Marking
11.4.1 Materials
11.4.2 Method
11.5 Sample Preparation to Observe Fungi Chromosome
11.5.1 Materials
11.5.2 Method
11.6 Sample Preparation to Observe Fungi Cell-Wall
11.7 Calcofluor White Staining
11.7.1 Materials
11.8 AlexaFluor 488 WGA Conjugate Staining
11.8.1 Materials
11.8.2 Method
11.9 Reactive Oxygen Species (ROS) in Fungi
11.9.1 Materials
11.9.2 Methods
11.9.2.1 Conidia Suspension
11.10 Fungi Cellular Death Studies with Propidium Iodide
11.10.1 Materials
11.10.2 Methods
11.10.3 Conidia or Yeast Suspension
11.11 Live-Dead Test for Fungi Cells
11.11.1 Materials
11.11.2 Method
11.12 Sample Preparation to Observe Fungi-Plant Interactions
11.12.1 Materials
11.12.2 Methods
11.13 Important Plant Structures Defence Against Phytopathogenic Fungi
11.14 Callose Deposition
11.14.1 Materials
11.14.2 Method
11.15 Lignin Localization in Plant-Fungi Interactions
11.15.1 Materials
11.15.2 Method
11.16 Sample Preparation to Observe Autofluorescent Fungi and Specific Structures
11.17 Autofluorescent Rust Fungi
11.17.1 Materials
11.17.2 Method
11.18 Autofluorescent Cercosporin
11.18.1 Materials
11.18.2 Method
References
Chapter 12: Yeast Isolation Methods from Specialized Habitats
12.1 Introduction
12.2 Common Materials
12.3 Methods
12.4 Yeast Isolation Protocols from Natural Habitats
12.4.1 Rumen Fluid/Digesta
12.4.1.1 Materials
12.4.1.2 Protocol
12.4.2 Insect Gut (Termite/Beetle)
12.4.2.1 Materials
12.4.2.2 Protocol
12.4.3 Hot Spring
12.4.3.1 Materials
12.4.3.2 Protocol
12.4.4 Flower
12.4.4.1 Materials
12.4.4.2 Protocol
12.4.5 Nectar
12.4.5.1 Materials
12.4.5.2 Protocol
12.4.6 Rotten Wood
12.4.6.1 Materials
12.4.6.2 Protocol
12.5 Yeast Isolation Protocols from Anthropogenic Habitats
12.5.1 Compost
12.5.1.1 Materials
12.5.1.2 Protocol
12.5.2 Molasses
12.5.2.1 Materials
12.5.2.2 Protocol
12.5.3 Press Mud
12.5.3.1 Materials
12.5.3.2 Protocol
12.5.4 Distillery Effluent/Spent Wash
12.5.4.1 Materials
12.5.4.2 Protocol
References
Index
