Dedication
Preface
Acknowledgments
Contents
Contributors
Part I: Plant Disease Diagnosis: Traditional Methods
Chapter 1: Obtaining and Maintaining Cultures of Pinewood Nematodes Bursaphelenchus xylophilus from Wild Dauers
1 Introduction
2 Materials
2.1 Obtaining B. xylophilus Dauers from Wild Populations
2.1.1 Trapping Monochamus spp.
2.1.2 Extracting Nematode Dauers from Monochamus spp.
2.2 Establishing and Maintaining Lab Populations from the Wild Dauers
3 Methods
3.1 Obtaining B. xylophilus Dauers from Wild Populations
3.1.1 Trapping Monochamus spp.
3.1.2 Extracting Nematodes from Monochamus spp.
3.2 Establishing and Maintaining Lab Populations from the Wild Dauers
3.2.1 Preparation of the Media
3.2.2 Obtaining and Maintaining Lab Cultures of B. xylophilus
4 Notes
References
Chapter 2: Survey and Monitoring of Phytophthora Species in Natural Ecosystems: Methods for Sampling, Isolation, Purification,...
1 Introduction
2 Materials
2.1 Sampling
2.1.1 Soil
2.1.2 Roots
2.1.3 Bleeding Bark Cankers
2.1.4 Naturally Fallen Leaves in Waterbodies or on Forest Ground
2.1.5 Water
2.2 Isolation
2.2.1 Soil Baiting with Leaves
2.2.2 Soil Baiting with Fruits
2.2.3 In Situ Baiting from Waterbodies
2.2.4 Agar Media for Phytophthora Isolations
2.3 Purification of Contaminated Cultures
2.3.1 Pancake Method
2.3.2 Green Apples
2.3.3 Purification of Mixed Cultures
2.4 Storage of Phytophthora
2.5 Pathogenicity Tests
2.5.1 Soil Infestation Trial
2.5.2 Under-Bark Inoculation Trial
3 Methods
3.1 Sampling
3.1.1 Soil
3.1.2 Roots
3.1.3 Bleeding Bark Cankers
3.1.4 Water
3.1.5 Naturally Fallen Leaves
3.1.6 Transport of Samples
3.2 Isolation
3.2.1 Leaf Baiting from Soil
3.2.2 Baiting from Soil with Green Apples
3.2.3 Direct Isolation from Roots Using PARPNH-Agar
3.2.4 Leaf Baiting from Roots
3.2.5 Isolation from Roots with Green Apples
3.2.6 Isolation from Filters with Green Apples (Modified After)
3.2.7 Direct Isolation from Filters Using PARPNH-Agar
3.2.8 Direct Isolation from Bleeding Bark Cankers Using PARPNH-Agar
3.2.9 Leaf Baiting from Bleeding Bark Cankers
3.2.10 Baiting from Bleeding Bark Cankers with Green Apples (Modified After)
3.2.11 In Situ Baiting from Waterbodies
3.2.12 Isolation from Baiting Leaves and Naturally Fallen Leaves Collected from Waterbodies or the Forest Ground
3.2.13 Isolation from Apple Baits
3.3 Cleaning of Contaminated Phytophthora Cultures
3.3.1 Cleaning of Bacteria-Contaminated Phytophthora Cultures Using the Pancake Method (Modified After)
3.3.2 Cleaning of Bacteria-Contaminated Phytophthora Cultures Using Green Apples (Modified After)
3.3.3 Purification of Phytophthora Cultures Mixed with Fungal Contaminants Using Selective PARPNH-Agar
3.3.4 Rebaiting from Phytophthora Cultures Mixed with Oomycete or Fungal Contaminants
3.4 Storage of Phytophthora
3.4.1 In Dry Agar Media (Method A)
3.4.2 In Dry Agar Media (Method B)
3.4.3 In Agar Media Under Liquid Solution
3.5 Pathogenicity Tests
3.5.1 Soil Infestation Trial: Production of Plants
3.5.2 Soil Infestation Trial: Production of Inoculum
3.5.3 Soil Infestation Trial: Infestation Technique and Flooding Regimes
3.5.4 Soil Infestation Trial: Assessment
3.5.5 Under-Bark Inoculation Trial
4 Notes
References
Chapter 3: Field and Laboratory Procedures for Fusarium circinatum Identification and Diagnosis
1 Introduction
2 Materials
2.1 Soil or Roots Collection
2.2 Collection of Branches, Needles, Cones, Canker, or Seeds
2.3 Spore Trapping
2.4 Insect VectorΒ΄s Trapping
2.5 Isolation and Identification of F. circinatum
2.6 DNA Extraction from Pure Culture and Spores
2.7 Multiplex Conventional PCR
2.8 Real-Time PCR
2.9 Dual-Labelled Probe Real-Time PCR
2.10 Sanger Sequencing and Interpretation
2.11 EF1 Gene Partial Sequencing
2.12 Maintenance and Conservation of F. circinatum Isolates
3 Methods and Protocols
3.1 Sampling in Nurseries
3.2 Sampling in Forests and Plantations
3.2.1 Soil
3.2.2 Cones and Seeds
3.2.3 Twigs and Branches
3.2.4 Cankers
3.2.5 Roots
3.2.6 Spore Trapping
3.2.7 Insect Vectors of F. circinatum
3.3 Sample Processing in the Laboratory
3.3.1 Isolation of F. circinatum from Plant Tissue
3.3.2 Isolation of F. circinatum from Seedlings
3.3.3 Isolation of F. circinatum from Seeds
3.3.4 Isolation of F. circinatum from Soil Samples
3.3.5 Isolation of F. circinatum from Insect Samples
Squashing Specimens in Selective Media
Sonication of Specimens
3.4 Morphological Identification of F. circinatum
3.4.1 Sexual Stage of F. circinatum
3.4.2 Asexual Stage of F. circinatum
3.5 Molecular Methods for F. circinatum Detection
3.5.1 DNA Extraction from Pure Culture
3.5.2 Multiplex Conventional PCR
3.5.3 Real-Time PCR
3.5.4 Dual-Labelled Probe Real-Time PCR
3.5.5 F. circinatum Detection by Sanger Sequencing of PCR Products
3.5.6 EF1 Gene Partial Sequencing
3.6 Maintenance and Conservation of F. circinatum Isolates
4 Notes
References
Part II: DNA Extraction Protocols
Chapter 4: Rapid Extraction of Plant Nucleic Acids by Microneedle Patch for In-Field Detection of Plant Pathogens
1 Introduction
2 Materials
2.1 Microneedle Patch Fabrication
2.2 MN Patch-Based Nucleic Acid Extraction
2.3 Real-Time PCR and RT-PCR Amplification of MN-Extracted Nucleic Acid Samples
2.4 Real-Time LAMP and RT-LAMP Amplification of MN-Extracted Nucleic Acid Samples
3 Methods
3.1 Microneedle Patch Fabrication
3.2 MN Patch-Based Nucleic Acid Extraction
3.3 Real-Time PCR and RT-PCR Amplification of MN-Extracted Nucleic Acid Samples
3.3.1 Reverse Transcription (RT) Reaction (Required Only for RNA Viruses Such as TSWV)
3.3.2 PCR Amplification
3.4 Real-Time LAMP and RT-LAMP Amplification of MN-Extracted Nucleic Acid Samples
4 Notes
References
Chapter 5: DNA Extraction Methods to Obtain High DNA Quality from Different Plant Tissues
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 DNA Extraction and Purification
3 Methods
3.1 Sample Collection
3.2 DNA Extraction in CTAB Buffer
3.2.1 Bark Woody Flakes, Woody Frass, and Xylem Shavings (See Note 7)
3.2.2 Woody Parts (Twigs and Leaves) from Challenging Matrices
3.3 DNA Extraction by Using Maxwell RSC PureFood GMO and Authentication Kit
4 Notes
References
Chapter 6: A Rapid Method for Extracting High-Quality DNA from Soils
1 Introduction
2 Materials
2.1 Sample Collection
2.2 DNA Extraction from Soil and Analysis
2.3 Solutions
3 Methods
3.1 Sample Collection
3.2 DNA Extraction
3.3 DNA Quality and Quantity
4 Notes
References
Part III: Molecular Methods to Detect Plant Pathogens
Chapter 7: Invasive Alien Plant Pathogens: The Need of New Detection Methods
1 Introduction
2 Key Challenges for Invasive Species
3 Innovative Methods for Biosurveillance
4 Airborne Spore Trapping
5 Molecular Promising Approaches
References
Chapter 8: Detection of Airborne Inoculum of Hymenoscyphus fraxineus: The Causal Agent of Ash Dieback
1 Introduction
2 Materials
2.1 Field Sampling
2.2 DNA Extraction
2.3 Real-Time Quantitative PCR (qPCR)
3 Methods
3.1 Ascospore Sampling by Rotorods
3.2 DNA Extraction of H. fraxineus Spores from Tapes
3.3 Ascospore Suspension
3.4 qPCR Detection and Quantification
4 Notes
References
Chapter 9: Molecular Detection of Wheat Blast Pathogen in Seeds
1 Introduction
2 Materials
2.1 Biological Enrichment
2.2 Grinding of Seeds
2.3 Extraction of Total DNA
2.4 Amplification of DNA by Conventional or Real-Time PCR or by LAMP
3 Methods
3.1 Seed Sampling and Biological Enrichment
3.2 Grinding of Seeds and DNA Extraction
3.3 Total DNA Extraction
3.4 Testing by Conventional PCR
3.5 Testing by Conventional PCR
3.6 Testing by Real-Time PCR
3.7 Testing by Real-Time PCR
3.8 Testing by Real-Time PCR
3.9 Testing by LAMP
3.10 Testing by LAMP
4 Notes
References
Chapter 10: Detection and Identification of the Causal Agents of Dothistroma Needle Blight
1 Introduction
2 Materials
2.1 Microscopy and Fungal Isolation
2.2 DNA Extraction
2.3 PCR
2.4 Gel Electrophoresis
3 Methods
3.1 Microscopical Examination
3.2 Single Spore (SS) Isolation From Needles
3.3 Molecular Identification of Isolates
3.3.1 DNA Extraction
3.3.2 ITS PCR and Sequencing
3.3.3 Mating-Type PCR
3.3.4 Conventional PCR: Species Identification Using Species-Specific Primers
3.3.5 Gel Electrophoresis
4 Notes
References
Chapter 11: The Use of qPCR to Detect Cryphonectria parasitica in Plants
1 Introduction
2 Materials
2.1 Sample Acquisition and Handling
2.2 DNA Extraction From Bark and Mycelium
2.3 Preparation of the PCR Controls
2.4 Duplex qPCR
3 Method
3.1 Sample Acquisition and Handling
3.2 DNA Extraction From Chestnut Bark and Mycelium
3.2.1 Chestnut Bark
3.2.2 Mycelium
3.3 Preparation of the PCR Controls
3.4 Duplex qPCR
3.5 Results Interpretation
3.5.1 Experimental Controls
3.5.2 Samples
4 Notes
References
Chapter 12: Rapid Molecular Diagnostics in the Field and Laboratory to Detect Plant Pathogen DNA in Potential Insect Vectors
1 Introduction
2 Materials
2.1 Sample Collection
2.2 Nucleic Acid Extraction Using FTA Matrix Cards
2.3 Nucleic Acid Amplification Using PCR
2.4 DNA Amplification Using RPA
2.4.1 RPA Product Detection Using Agarose Gel Electrophoresis
2.4.2 RPA Product Detection Using a Real-Time PCR Instrument
2.5 DNA Amplification Using LAMP
3 Methods
3.1 Sample Collection
3.2 Nucleic Acid Extraction Using FTA Matrix Cards
3.3 Conventional PCR Targeting Insect CO1 and X. fastidiosa rpoD
3.4 Nucleic Acid Amplification Using RPA
3.4.1 RPA Product Detection Using Agarose Gel Electrophoresis
3.4.2 RPA Product Detection Using a Real-Time PCR Instrument
3.5 Nucleic Acid Amplification Using LAMP
4 Notes
References
Chapter 13: A Panel of Real-Time PCR Assays for the Direct Detection of All of the Xylella fastidiosa Subspecies
1 Introduction
2 Materials
2.1 Sampling Plant Tissue
2.2 Sampling From Woody Hosts
2.3 DNA Extraction
2.4 DNA Extraction From Woody Hosts
2.5 DNA Purification
2.6 qPCR
2.7 Equipment and General Lab Supplies
3 Methods
3.1 Sampling Plant Tissue
3.2 Sampling From Woody Hosts
3.3 DNA Extraction
3.4 DNA Extraction From Woody Hosts
3.5 DNA Purification
3.6 Setting up qPCR Reactions and Plates
3.7 Perform the qPCR Run
3.8 Analyze Data
4 Notes
References
Chapter 14: Selective Quantification of Erwinia amylovora Live Cells in Pome Fruit Tree Cankers by Viability Digital PCR
1 Introduction
2 Materials
2.1 Sample Collection and Preservation
2.2 Physical Sample Processing
2.3 PMAxxTM Dye Treatment
2.4 DNA Extraction
2.5 Digital PCR
3 Methods
3.1 Canker Sample Collection and Cryopreservation
3.2 Sample Processing
3.3 PMAxx Dye Treatment
3.4 DNA Extraction
3.5 Digital PCR
4 Notes
References
Chapter 15: Innovative Detection of the Quarantine Plant Pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens, Causal Age...
1 Introduction
2 Materials
2.1 Bacterial Cultures
2.2 DNA Template Extraction
2.3 Agarose Gel Preparation
2.4 End-Point, Real-Time, and LAMP PCR
3 Methods
3.1 Bacterial Culture Preparation
3.2 DNA Template Extraction
3.2.1 Thermal Lysis
3.2.2 Commercial Kits
3.3 End-Point PCR for Cff
3.4 Real-Time PCR for Cff
3.5 LAMP PCR for Cff
3.6 Electrophoresis of PCR Products on Agarose Gel
4 Notes
References
Chapter 16: Specific Detection of Pseudomonas savastanoi Pathovars: From End-Point PCR to Real-Time PCR and HRMA
1 Introduction
2 Materials
2.1 Bacterial Growth Media
2.2 DNA Extraction
2.3 Gel Electrophoresis Reagents
2.4 End-Point PCR Materials
2.5 Real-Time PCR Materials
2.6 Multiplex-HRM Assay Materials
3 Methods
3.1 Bacterial Cultures and Growth Conditions
3.2 Bacterial Genomic DNA Extraction
3.3 End-Point PCR
3.4 Real-Time PCR
3.4.1 SYBR Green Real-Time PCR
3.4.2 TaqMan Real-Time PCR
3.5 Multiplex-HRM Assay
4 Notes
References
Part IV: High-Throughput Sequencing
Chapter 17: Computational Analysis of HTS Data and Its Application in Plant Pathology
1 Introduction
2 Materials
3 Methods
3.1 Genome Assembly and Annotation
3.2 Transcriptome Analysis
3.3 Analysis of Metabarcoding Data with QIIME2
3.4 Analysis of sRNA Sequencing Data for miRNA Prediction
4 Notes
References
Chapter 18: Biomonitoring of Fungal and Oomycete Plant Pathogens by Using Metabarcoding
1 Introduction
1.1 Databases (Public Versus Curated), Genic Regions, Their Strength and Weaknesses, and Their Importance
1.2 Material for Biomonitoring and DNA Extraction
1.3 Popular Sequencing Platforms
1.4 Validation of Findings
2 Materials
2.1 Instruments Needed
2.2 Reagents for Ion Torrent and Sequencing Library Preparation
2.3 Other Solution or Reagents
3 Methods
3.1 gDNA Purification Using QuickPick
3.2 Quantification of Purified gDNA Using a Qubit Fluorometer (See Note 7)
3.3 Target Region PCR Amplification with Fusion Primers Technology
3.4 Electrophoresis on PCR Products (i.e., Indexed Amplicons)
3.5 Sequentially Purify PCR Products at Two Different Concentrations
3.6 Quantify Bidirectional Barcoded Sample Library by qPCR, Dilute and Pool Your Library
3.7 Plan Run and Prepare Template (Ion Chef)
3.8 Ion Chef Protocol
3.9 Ion S5 Initialization and Sequencing Protocol
3.10 Downstream Data Analysis Using Bioinformatics
4 Notes
References
Chapter 19: Phytobiome Metabarcoding: A Tool to Help Identify Prokaryotic and Eukaryotic Causal Agents of Undiagnosed Tree Dis...
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 PCR Amplification
2.3 Gel Electrophoresis
2.4 Illumina Library Preparation and Sequencing
2.5 Bioinformatic Processing
3 Methods
3.1 DNA Extraction
3.2 Bacterial 16S Amplification
3.3 Phytoplasma 16S Nested PCR
3.4 Fungal ITS PCR
3.5 Nematode 18S Semi-Nested PCR
3.6 Illumina Library Preparation and Sequencing
3.7 Quality Control and Taxonomic Assignments in QIIME2
3.8 Conduct Diversity Analyses Using R
3.9 Differential Abundance Analysis in RStudio
4 Notes
References
Part V: Plant-Pathogen Interactions
Chapter 20: Plant-Fungal Interactions: Laser Microdissection as a Tool to Study Cell Specificity
1 Introduction
2 Materials
2.1 Major Equipment
2.2 LMD Supplies
3 Methods
3.1 Fixation and Embedding in Paraffin
3.2 Laser Microdissection (LMD)
3.3 RNA Extraction, DNase Treatment, and RT-PCR/RT-qPCR
4 Notes
References
Chapter 21: Somatic Embryogenesis as a Tool for Studying Grapevine-Virus Interaction
1 Introduction
2 Materials
2.1 Tissue Culture
2.2 RNA Extraction
2.3 RT-PCR
3 Methods
3.1 Plant Regeneration From Somatic Embryogenesis
3.2 RNA Extraction
3.3 Detection of Viruses and Viroids
3.4 Virus Inoculation in Healthy Grapevine Plants
4 Notes
References
Chapter 22: A Quantitative Luminol-Based Assay for ROS Burst Detection in Potato Leaves in Response to Biotic Stimuli
1 Introduction
2 Materials
2.1 Sample Preparation and Plant Material
2.2 ROS Assay
3 Methods
3.1 Preparation of Stock Solutions
3.2 Collection of Leaf Discs from Potato Plants
3.3 ROS Measurement
3.4 Representation of Data
3.4.1 ROS Production in Response to Different Elicitors
4 Notes
References
Part VI: Population Genomics in Plant Pathology
Chapter 23: Genetic Analysis of Plant Pathogens Natural Populations
1 Introduction to Genetic Analysis of Natural Populations
1.1 Population Genetics of Plant Pathogens
1.2 Basic Concepts in Population Genetics
1.3 Evolutionary Forces Affecting Plant Pathogen Populations
1.3.1 Mutations
1.3.2 Recombination and Reproductive Strategies
1.3.3 Population Size and Genetic Drift
1.3.4 Gene Flow
1.3.5 Selection
1.4 From Population Genetics to Population Genomics: The Rising of NGS
2 Genetic Analysis Workflow
2.1 Sampling
2.2 Genotyping
2.3 Data Analysis
2.3.1 Genetic Diversity
2.3.2 Genetic Distance and Structure
2.3.3 Linkage Disequilibrium and Estimation of Mode of Reproduction
References
Chapter 24: Microsatellite Genotyping in the Chestnut Blight Fungus Cryphonectria parasitica
1 Introduction
2 Material
2.1 DNA Extraction
2.2 PCR
2.3 Fragment Analysis
3 Methods
3.1 DNA Extraction
3.2 PCR
3.3 Fragment Analysis
3.4 Data Analysis
4 Notes
References
Chapter 25: A Multiplex PCR Approach to Determine Vegetative Incompatibility Genotypes and Mating Type in Cryphonectria parasi...
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 PCR
2.3 Fragment Length Analysis
3 Methods
3.1 DNA Extraction
3.2 PCR
3.3 Fragment Length Analysis
3.4 Data Analysis
3.5 Nonfluorescent Duplex- or Monoplex-PCR
4 Notes
References
Part VII: Biocontrol
Chapter 26: A Detached Leaf Assay for Rapidly Screening Plant Pathogen-Biological Control Agents
1 Introduction
2 Materials
2.1 Pathogen Inoculum
2.2 Bacterial Suspension
2.3 Detached Leaf Assay (DLA)
3 Methods
3.1 Phytophthora cactorum Inoculum/Zoospore Production
3.2 Bacterial Suspension
3.3 Detached Leaf Assay (DLA)
3.4 Image Analysis
4 Notes
References
Chapter 27: Spray-Induced Gene Silencing to Study Gene Function in Phytophthora
1 Introduction
2 Materials
2.1 Propagation of P. infestans
2.2 Plant Material
2.3 dsRNA Synthesis
2.4 Equipment
3 Methods
3.1 Phytophthora infestans - Culture
3.2 Plant Material
3.3 Phytophthora infestans DNA Extraction
3.4 dsRNA Synthesis: In Vitro Transcription
3.5 dsRNA Synthesis: Bacterial Cloning
3.5.1 PCR for Inserts
3.5.2 Plasmid Extraction
3.5.3 Restriction Digestion
3.5.4 Ligation
3.5.5 Transformation of E. coli
3.5.6 Verification of Target Insertion
3.5.7 dsRNA Synthesis
3.6 DLA
3.6.1 Trypan Blue Staining
3.7 Confocal Microscopy
3.8 RNA Extraction
3.9 cDNA Synthesis
3.10 qRT-PCR
3.10.1 qRT-PCR Analysis
4 Notes
References
Chapter 28: Plant Sampling for Production of Essential Oil and Evaluation of Its Antimicrobial Activity In Vitro
1 Introduction
2 Materials
2.1 Plant Sample Collection
2.2 Distillation of EOs
2.3 In Vitro Test: Determination of the Antimicrobial Activity of EOs
2.4 In Vitro Test: Determination of the Minimum Inhibitory Concentration (MIC)
2.5 Production of Γ-Cyclodextrin-EO Complex
3 Methods
3.1 Sampling Method of Plant Tissues for EO Production
3.1.1 Factors to Consider in Sample Collection: Plant Age
3.1.2 Factors to Consider in Sample Collection: Tissue Age and Tissue Source
3.1.3 Factors to Consider in Sample Collection: Season
3.2 Steam Distillation of EOs
3.3 In Vitro Test: Determination of the Antimicrobial Activity of EOs
3.3.1 Protocol for Fungi
3.3.2 Protocol for Bacteria
3.4 In Vitro Test: Determination of the Minimum Inhibitory Concentration (MIC)
3.4.1 Protocol for Fungi
3.4.2 Protocol for Bacteria
3.5 Γ-Cyclodextrin-EO Complex
4 Notes
References
Index