Advanced Techniques in Soil Microbiology

By: Ajit Varma (Editor), Ralf OelmÃ¼ller (Editor)

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Hardcover | 25 June 2007

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Hardcover
456 Pages

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23.39 x 15.6 x 2.54

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"Advanced Techniques in Soil Microbiology" presents a wide range of biotechnological methods for application in soil microbiology analysis. These include all essential methods involving molecular biology, immunology, microbiology, and structural biology, such as transcriptome analysis, RNAi technology, molecular matchmaking, RAPD, T-RFLP and FT/MS.

The techniques and procedures have been selected with the aim of offering practical guides for immediate use in the laboratory. The systems investigated range from individual molecules and cells to entire eukaryotic organisms, with a focus on bacteria, fungi, mycorrhiza, and higher plants. This volume of state-of-the-art, practice oriented methods will be of great use both to the first-timer and to the experienced scientist.

Industry Reviews

Aus den Rezensionen: "... Advanced Techniques in Soil Microbiology wendet sich an den Praktiker, der aktuelle biochemische Methoden direkt im Labor anwenden mochte. Dieses Ziel wird dadurch erreicht, dass die Methoden in Listenform Schritt fur Schritt erklart werden. Besonders hervorgehoben werden sollte hierbei, dass auch kleinere Tipps ... in dem Buch ... eine Erwahnung finden. ... Diese ... Tipps werden ... kurz erklart, wodurch das Studium der Protokolle auch fur den erfahrenen ... Wissenschaftler durchaus Neues zu bieten hat ..." (Jost Liebich, in: GDCh Mitteilungsblatt Gesellschaft Deutscher Chemiker, 2008, Issue 1, S. 17 f.)

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Non-Fiction Science Biology, Life Sciences Microbiology excluding Medical Earth Sciences, Geography, Environment, Planning Earth Sciences Geology & The Lithosphere Soil Science Engineering & Technology Biochemical Engineering Biotechnology Botany & Plant Sciences

The Environment Conservation of The Environment Agriculture & Farming Agricultural Science Stratigraphy

Detection and Diversity of Fungi from Environmental Samples: Traditional Versus Molecular Approaches	p. 1
Introduction	p. 1
Microscopy and Culture-Based Methods	p. 2
Molecular-Based Methods	p. 4
The Nuclear-Encoded Ribosomal DNA Gene: Phylogenetic and Systematic Value	p. 5
Denaturing Gradient Gel Electrophoresis: Applicability, Usefulness and Bias	p. 7
Conclusions and Future Directions	p. 11
References	p. 11
Functional Genomic Approaches for Mycorrhizal Research	p. 17
Introduction	p. 17
Yeast Two Hybrid: An Approach for Understanding Signaling Pathways	p. 18
Agrobacterium-Mediated Transformation in Laccaria bicolor	p. 22
Materials and Methods	p. 24
Interaction Studies of Laccaria bicolor with Aspen (Populus tremuloides) Seedlings	p. 24
Yeast Two-Hybrid Protocol	p. 26
Agrobacterium-Mediated Transformation in Laccaria bicolor	p. 28
References	p. 30
Automated Fluoroscence Sequencing and Troubleshooting	p. 35
Introduction	p. 35
Evolution of the Method	p. 36
Manual Sequencing	p. 36
Automated Sequencing	p. 37
Pyrosequencing	p. 39
Methods	p. 39
Template Preparation	p. 39
Reaction Setup (BigDye Terminator Cycle Sequencing)	p. 40
Performing Cycle Sequencing	p. 41
Preparing Extension Products for Electrophoresis	p. 42
Trouble Shooting	p. 43
Problem: Flat Line or "Dead On Analysis"	p. 43
Problem: Noisy Data (Background)	p. 45
Problem: Reading Near the Primer	p. 46
Problem: Strong Terminator Peaks	p. 46
Problem: Low Intensity of Shorter Products	p. 48
Problem: Longer Fragments Missing	p. 49
Problem: Presence of Spikes	p. 49
Problem: Weaker Signals	p. 49
References	p. 50
mRNA Quantitation Using Real Time PCR	p. 53
Introduction	p. 53
Methods	p. 54
Chemistry and Primer/Probe Design	p. 54
RNA Isolation from the Sample	p. 57
Reverse Transcription	p. 58
Real Time PCR Set Up	p. 59
Instrumentation	p. 61
Data Analysis	p. 67
Notes	p. 68
References	p. 71
Laboratory Practice for the Production of Polyclonal and Monoclonal Antibodies	p. 73
Introduction	p. 73
Production of Polyclonal Antibodies	p. 74
Choice of Animal and Method of Immunization	p. 74
Preparation for Immunization	p. 75
Production of Polyclonal Antibodies	p. 76
Materials and Equipment	p. 77
Production of Monoclonal Antibodies	p. 77
Immunization of Mice or Rats	p. 77
Myeloma Cell Culture	p. 78
Setup for Fusion of Myeloma with Spleen Cells	p. 79
Selection and Cloning of Hybridoma	p. 79
Production of Monoclonal Antibodies	p. 80
Materials and Equipment	p. 81
Purification of Antibody	p. 82
Purification of IgG by Precipitation with Ammonium Sulfate	p. 83
Purification of IgG by DEAE-Sepharose Chromatography	p. 83
Purification of IgG Using Immobilized Protein A	p. 84
Analysis of Purity of IgG by Electrophoresis	p. 86
Materials and Equipment	p. 88
Enzyme-Linked Immunosorbent Assay	p. 88
Materials and Equipment	p. 90
Conclusion	p. 90
References	p. 90
Modern Techniques for Analyzing Immunological Responses	p. 93
Introduction	p. 93
Type of Immune Responses	p. 93
Innate Immune Response	p. 94
Adaptive Immune Response	p. 94
Adaptive Immune System	p. 94
Humoral Immune System	p. 95
Cellular Immune System	p. 96
Different Assay Systems to Study the Adaptive Immune Response	p. 96
Mixed Lymphocyte Proliferation Assays	p. 96
Detection of Type of T Helper Responses (Th1/Th2)	p. 98
Cytotoxic T Lymphocyte Activity	p. 99
Flow Cytometric Analysis of Immune Cells	p. 102
Materials and Equipment	p. 105
Magnetic Activated Cell Sorting	p. 105
Materials and Equipment	p. 107
Isolation of Mononuclear Cells from Peripheral Blood	p. 107
Materials and Equipment	p. 108
Conclusions	p. 108
References	p. 108
Transcriptome Analysis	p. 111
Introduction	p. 111
RNA Preparation	p. 113
Northern Analysis	p. 113
Principle	p. 113
Procedure	p. 114
Applications	p. 116
In Situ Hybridization	p. 116
Principle	p. 116
Procedure	p. 117
Applications	p. 119
Dot Blot and Slot Blot	p. 120
Principle	p. 120
Procedure	p. 120
Applications	p. 123
Reverse Transcriptase-Polymerase Chain Reaction	p. 123
Principle	p. 123
Procedure	p. 124
Application of RT-PCR	p. 125
DNA Microarray	p. 126
Principle	p. 126
Procedure	p. 127
Applications	p. 129
Conclusions	p. 129
References	p. 130
RNAi Technology: a Tool for Functional Validation of Novel Genes	p. 133
Introduction	p. 133
Machinery Involved in RNAi	p. 134
Inducer	p. 135
Dicer	p. 135
RNA-Dependent RNA Polymerase	p. 135
RNA-Induced Silencing Complex	p. 135
miRNA and siRNA	p. 136
RNAi as a Tool of Functional Genomics	p. 136
Production of dsRNA	p. 137
Constitutive and Inducible RNAi	p. 138
Antisense RNA and RNAi	p. 140
Potential Areas of Application	p. 140
Conclusions	p. 142
References	p. 142
Molecular Matchmaking: Techniques for Biomolecular Interactions	p. 145
Introduction	p. 145
Tools for the Study of Protein-Protein Interactions	p. 145
The Two-Hybrid System	p. 147
The Split-Ubiquitin System	p. 148
Reverse Two-Hybrid System	p. 148
Sos Recruitment System (Cyto Trap Yeast Two-Hybrid System)	p. 148
Yeast One-Hybrid System	p. 149
Double Interaction Screen	p. 149
Yeast Three-Hybrid or Tri-Hybrid System	p. 150
Procedure	p. 150
Reagents, Materials, and Equipment	p. 151
Notes and Points to Watch	p. 152
References	p. 152
Environmental Proteomics: Extraction and Identification of Protein in Soil	p. 155
Introduction	p. 155
Sample Preparations	p. 156
Protocols for Protein Extraction from Soil	p. 157
Extraction of Extracellular Protein	p. 157
Extraction of Whole-Cell Protein	p. 157
Protein Loading	p. 158
Protein Expression Analyses	p. 158
SDS-PAGE	p. 158
Two-Dimension SDS-PAGE Analysis	p. 158
Gel Staining	p. 161
Coomassie Brilliant Blue Staining Protocol (For Mini Gels)	p. 162
Silver Staining Protocol	p. 162
Image Analysis	p. 163
Spot Cut	p. 163
Protein Digestion	p. 163
Mass Spectrometry Analysis	p. 164
Spectral Analysis	p. 164
N-Terminal Amino Acid Sequencing	p. 165
Conclusions	p. 165
References	p. 166
DGGE and RISA Protocols for Microbial Community Analysis in Soil	p. 167
Introduction	p. 167
Soil DNA Extraction	p. 168
Equipment	p. 168
Chemicals	p. 168
DNA Extraction Protocol	p. 168
Polymerase Chain Reaction Protocol for DGGE	p. 170
First Round PCR	p. 170
GC Clamp 16S PCR (Second Round PCR)	p. 171
DGGE Techniques	p. 172
Equipment	p. 172
Chemicals	p. 173
Assembling the Gel Chamber	p. 173
Casting the Gel	p. 174
Loading of the Samples	p. 175
Staining and Imaging of the Gels	p. 175
Ribosomal Intergenic Spacer Analysis	p. 175
Equipment	p. 176
Chemicals	p. 176
PCR Protocol	p. 176
Gel Preparation and Loading	p. 177
Gel Running	p. 178
Staining and Imaging of the Gels	p. 178
Data Analysis	p. 178
Conclusions	p. 179
References	p. 179
Soil Microbial Community Structureand Function Assessed by FAME, PLFA and DGGE - Advantages and Limitations	p. 181
Introduction	p. 181
Microbial Community Structure Based on Fatty Acid Patterns	p. 182
FAME Extraction and Data Analysis	p. 183
PLFA Analysis	p. 185
Advantages and Limitations of Fatty Acid Patterns	p. 188
Denaturing Gradient Gel Electrophoresis	p. 189
DNA Extraction from Soil	p. 189
Polymerase Chain Reaction	p. 190
DGGE Procedures	p. 192
Conclusions	p. 196
References	p. 197
Measurement of Microbial Biomass and Activity in Soil	p. 201
Introduction	p. 201
Protocols for Microbial Biomass Determination	p. 202
Chloroform Fumigation-Extraction Method for Microbial Biomass C and N	p. 202
Hexanol Extraction Method for Microbial P	p. 205
Protocol for Total Microbial Activity Determination	p. 207
Equipment	p. 207
Reagents	p. 207
Protocol for Extraction	p. 208
Protocol for Soil Dehydrogenase Enzyme Analysis	p. 208
Equipment	p. 209
Reagents	p. 209
Protocol for Extraction	p. 209
Conclusions	p. 209
References	p. 210
Immuno-Technology for the Localization of Acid Phosphatase Using Native Gel Bands in Piriformospora indica and Other Soil Microorganisms	p. 213
Introduction	p. 213
Taxonomic Status	p. 213
Phosphatases	p. 214
Immunotechnology for the Detection and Localization of Acid Phosphatase in P. indica	p. 216
Extraction of Protein and Enzyme Assay	p. 216
Purification of Protein by Column Chromatography	p. 217
Purification of Protein by Ion Exchange Chromatography	p. 217
Native Polyacrylamide Gel Electrophoresis	p. 219
Detection of Enzyme in Native PAGE	p. 220
Isolation of Acid Phosphatase for Raising Antibody	p. 222
Production of Antibodies using Acid Phosphatase in Native Gel	p. 222
Antiserum Preparation	p. 224
Purification of Immunoglobulin from Serum	p. 225
Western Blot	p. 226
Immuno-Fluorescence	p. 228
Localization of ACPase by Immunogold Technique	p. 228
Troubleshooting	p. 232
Conclusions	p. 232
References	p. 232
Use of Short Oligonucleotide Primers in Random Amplified Polymorphic DNA Techniques for Species Identification	p. 237
Introduction	p. 237
Polymorphism between Piriformospora indica and Sebacina vermifera, Members of the Order Sebacinales	p. 239
General Protocol for RAPD Technique to Show Polymorphism	p. 241
Experimental Procedures	p. 242
Troubleshooting	p. 244
Conclusions	p. 244
References	p. 245
Co-Cultivation with Sebacinales	p. 247
Introduction	p. 247
Sebacinaceae - Novel Fungi	p. 248
Host Spectrum	p. 249
Functions of the Sebacinaceae	p. 251
Eco-Functional Identity	p. 252
Axenic Co-Cultivation of Sebacinaceae	p. 254
Procedure	p. 254
Protocol	p. 256
Media Compositions	p. 256
Seed Surface Sterilization and Germination	p. 261
Protocol for Seed Surface Sterilization	p. 262
Inoculum Placement in the Pots	p. 262
Results	p. 262
Comparative Study on Plant Growth with Treated Endosymbionts	p. 264
In Vivo Co-Cultivation of Sebacinales	p. 264
Conclusions	p. 266
References	p. 267
Quantitative Histochemistry: a Forgotten Tool with New Applications	p. 271
Introduction	p. 271
Sample Preparation and Handling	p. 272
Microphotometry	p. 274
Biochemical Analysis: Real Time Microassays	p. 276
Spatial Resolution of Basic Steps of Fungal Trehalose Metabolism in Symbiosis	p. 277
References	p. 279
Ion Cyclotron Resonance Fourier Transform Mass Spectrometry for Non-Targeted Metabolomics of Molecular Interactions in the Rhizosphere	p. 281
Introduction	p. 281
The Chemical Biology Approach	p. 282
Complementary Analytical Approaches	p. 283
Targeted Analysis	p. 284
Metabolite Profiling	p. 285
Non-Targeted Analysis	p. 285
Resolving Structural Information from Molecular Complexity with ICR-FT/MS	p. 286
Top-Down Approach: From ICR-FT/MS-Profiling Analysis to Structural Hypothesis	p. 288
Complementary Analytical Tools	p. 290
Bottom-Up Approach: From Hypothesis-Driven Experiments Upwards to ICR-FT/MS	p. 290
Conclusion	p. 292
References	p. 292
Application of Terminal-Restriction Fragment Length Polymorphism for Molecular Analysis of Soil Bacterial Communities	p. 295
Introduction	p. 295
A General Protocol for Taxonomic T-RFLP Profiling of Soil Bacterial Communities	p. 297
Materials	p. 297
Experimental Procedure	p. 298
Troubleshooting	p. 299
Standardization of T-RFLP Profiles	p. 299
Other Applications of T-RFLP to Soil Bacterial Communities	p. 301
Conclusions	p. 302
References	p. 302
Molecular Symbiotic Analysis Between Arabiopsis thaliana and Piriformospora indica	p. 307
Introduction	p. 307
Beneficial Interaction Between Plants and Fungi: Piriformospora indica and Arabidopsis thaliana as a Model System	p. 308
Co-Cultivation of P. indica and Arabidopsis under Standardized Growth Conditions	p. 309
Map-Based Cloning of a Mutated Gene	p. 312
Rapid DNA Extraction	p. 313
Confirmation of a Mutated Phenotype of an EMS Mutant by the Analysis of an Independent T-DNA Insertion Line	p. 313
Differntial Display to Identify Genes which are Regulated in Response to P. indica	p. 314
Activation Tagged Lines	p. 315
Identification of Biochemical Pathways in A. thaliana which are Regulated by P. indica	p. 317
References	p. 317
Biophysical Phenomics Reveals Functional Building Blocks of Plants Systems Biology: a Case Study for the Evaluation of the Impact of Mycorrhization with Piriformospora indica	p. 319
Introduction	p. 319
Biophysical Phenomics of the Fast Fluorescence Rise O-J-I-P	p. 320
The Energy Cascade in the Photosynthetic Apparatus	p. 320
Microstates - Functional Building Blocks of Photosynthesis	p. 320
Measuring Fluorescence Transients with PEA, Handy-PEA and FIM- Fluorimeters	p. 322
How Fluorescence Kinetics Provide an Insight to the Microstates - Functional Blocks of PSII	p. 323
Case Study	p. 332
Mycorrhization and the Advantages of Piriformospora indica, an Emerging Growth Booster	p. 332
Phenomics of the O-J-I-P Fluorescence Transient for the Study of Cadmium Stress on Chick Peas (Cicer arietinum L. Chafa variety) With and Without Symbiosis With Glomus mosseae, G. caledonium and Piriformospora indica	p. 333
Correlation of Physiological with Biophysical Parameters	p. 337
Conclusions	p. 338
References	p. 338
Analysis of the Plant Protective Potential of the Root Endophytic Fungus Piriformospora indica in Cereals	p. 343
Introduction	p. 343
Plant Responses and Resistance to Pathogens	p. 344
Local Reactions	p. 344
Systemic Reactions and Resistance in Cereals	p. 344
Beneficial Microbial Endophytes Protecting Cereals from Pathogens	p. 345
Interaction of P. indica with Cereals	p. 345
P. indica Colonizes Root Cortical Cells in Barley	p. 346
P. indica Enhances Biomass and Yield in Barley	p. 346
Approaches to Study the Mechanism of P. indica-Induced Pathogen Resistance	p. 347
P. indica Induces Disease Resistance Against Root Pathogens	p. 347
P. indica Induces Systemic Disease Resistance	p. 348
Assessment of the Antioxidant Capacity of P. indica-Infested Roots	p. 350
Gene Expression Induced by P. indica in Barley Leaves	p. 351
Conclusions	p. 351
References	p. 352
Members of Sebacinales Confer Resistance Against Heavy Metal Stress in Plants	p. 355
Introduction	p. 355
Scientific Background	p. 355
Differential Display to Understand Cd²⁺ Resistance Mediated by Endophytic Fungi	p. 357
Studies on Protein Level	p. 357
Two-Dimensional Gel Electrophoresis, Preparation of Proteins	p. 359
Mass Spectrometry, Preparation of Samples by Tryptic Digestion	p. 359
References	p. 360
Screening of Plant Growth-Promoting Rhizobacteria	p. 363
Introduction	p. 363
Candidature for Being a Rhizobacteria	p. 364
Screening Methods	p. 365
Criteria for Screening	p. 365
Selection of Screening Methods	p. 365
Classic Methods	p. 366
Modern Methods	p. 368
Molecular Methods	p. 370
Metagenomics	p. 371
Tracking of GEMs	p. 372
Conclusions	p. 372
References	p. 373
Research Methods in Arbuscular Mycorrhizal Fungi	p. 377
Introduction	p. 377
Assessment of AM Fungal Propagules in Soil	p. 378
Soil Sampling	p. 378
Spore Extraction	p. 378
Quantification of Spore Numbers	p. 379
Infectivity Assays	p. 379
Identification of AM Fungi	p. 380
Use of Fatty Acids for Identification of AM Fungi	p. 381
Quantification of AM Fungal Root Colonization in Root	p. 382
Clearing and Staining Roots	p. 382
Modifications of Staining Procedure	p. 383
Measurement of Root Colonization by AM Fungi	p. 384
Extraction and Quantification of Extra-Radical Mycelium of AM Fungi in Soils	p. 384
Assessment of Growth Response of Effective Isolates	p. 385
Inoculum Production of AM Fungi	p. 386
On-Farm Production of AM Fungi	p. 386
Traditional Culture Methods	p. 387
AM Fungal Culture Using Aeroponic and Hydroponic Culture	p. 388
Monoaxenic Culture of AM Fungi	p. 389
Storage of AM Fungal Inoculum	p. 390
Conclusions	p. 390
References	p. 391
Field Trials of Bioinoculants	p. 397
Introduction	p. 397
Effect of Mycorrhizal Infection on Nutrient Uptake	p. 398
Effect of Soil Fumigation and Mycorrhizal Inoculation on Plant Growth Under Field Conditions	p. 399
Effect of Mycorrhizal Inoculation on Plant Growth and Nutrient Uptake under Non-Sterile Field Conditions	p. 403
Soil and Crop Management System	p. 408
Inoculation Techniques	p. 409
Conclusion	p. 411
References	p. 412
Subject Index	p. 415
Table of Contents provided by Publisher. All Rights Reserved.

Advanced Techniques in Soil Microbiology

At a Glance

Hardcover

Industry Reviews

Shipping

How to return your order

Defective items

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