ATLAS OF SOIL ASCOMYCETES
DETECTION AND DIVERSITY OF FUNGI FROM ENVIRONMENTAL SAMPLES: TRADITIONAL VERSUS MOLECULAR APPROACHES INTRODUCTION MICROSCOPY AND CULTURE-BASED METHODS MOLECULAR-BASED METHODS THE NUCLEAR-ENCODED RIBOSOMAL DNA GENE: PHYLOGENETIC AND SYSTEMATIC VALUE DENATURING GRADIENT GEL ELECTROPHORESIS: APPLICABILITY, USEFULNESS AND BIAS CONCLUSIONS AND FUTURE DIRECTIONS REFERENCES FUNCTIONAL GENOMIC APPROACHES FOR MYCORRHIZAL RESEARCH YEAST TWO HYBRID: AN APPROACH FOR UNDERSTANDING SIGNALING PATHWAYS AGROBACTERIUM-MEDIATED TRANSFORMATION IN LACCARIA BICOLOR MATERIALS AND METHODS INTERACTION STUDIES OF LACCARIA BICOLOR WITH ASPEN (POPULUS TREMULOIDES) SEEDLINGS YEAST TWO-HYBRID PROTOCOL AUTOMATED FLUOROSCENCE SEQUENCING AND TROUBLESHOOTING EVOLUTION OF THE METHOD MANUAL SEQUENCING AUTOMATED SEQUENCING PYROSEQUENCING METHODS TEMPLATE PREPARATION 3 3 2 REACTION SETUP (BIGDYE TERMINATOR CYCLE SEQUENCING) PERFORMING CYCLE SEQUENCING PREPARING EXTENSION PRODUCTS FOR ELECTROPHORESIS TROUBLE SHOOTING PROBLEM: FLAT LINE OR “DEAD ON ANALYSIS” PROBLEM: NOISY DATA (BACKGROUND) PROBLEM: READING NEAR THE PRIMER PROBLEM: STRONG TERMINATOR PEAKS PROBLEM: LOW INTENSITY OF SHORTER PRODUCTS PROBLEM: LONGER FRAGMENTS MISSING PROBLEM: PRESENCE OF SPIKES PROBLEM: WEAKER SIGNALS MRNA QUANTITATION USING REAL TIME PCR CHEMISTRY AND PRIMER/PROBE DESIGN RNA ISOLATION FROM THE SAMPLE REVERSE TRANSCRIPTION REAL TIME PCR SET UP INSTRUMENTATION DATA ANALYSIS NOTES LABORATORY PRACTICE FOR THE PRODUCTION OF POLYCLONAL AND MONOCLONAL ANTIBODIES PRODUCTION OF POLYCLONAL ANTIBODIES CHOICE OF ANIMAL AND METHOD OF IMMUNIZATION PREPARATION FOR IMMUNIZATION MATERIALS AND EQUIPMENT PRODUCTION OF MONOCLONAL ANTIBODIES IMMUNIZATION OF MICE OR RATS MYELOMA CELL CULTURE SETUP FOR FUSION OF MYELOMA WITH SPLEEN CELLS SELECTION AND CLONING OF HYBRIDOMA PURIFICATION OF ANTIBODY PURIFICATION OF IGG BY PRECIPITATION WITH AMMONIUM SULFATE PURIFICATION OF IGG BY DEAE-SEPHAROSE CHROMATOGRAPHY PURIFICATION OF IGG USING IMMOBILIZED PROTEIN A ANALYSIS OF PURITY OF IGG BY ELECTROPHORESIS ENZYME-LINKED IMMUNOSORBENT ASSAY CONCLUSION MODERN TECHNIQUES FOR ANALYZING IMMUNOLOGICAL RESPONSES TYPE OF IMMUNE RESPONSES INNATE IMMUNE RESPONSE ADAPTIVE IMMUNE RESPONSE ADAPTIVE IMMUNE SYSTEM HUMORAL IMMUNE SYSTEM CELLULAR IMMUNE SYSTEM DIFFERENT ASSAY SYSTEMS TO STUDY THE ADAPTIVE IMMUNE RESPONSE MIXED LYMPHOCYTE PROLIFERATION ASSAYS DETECTION OF TYPE OF T HELPER RESPONSES (TH1/TH2) CYTOTOXIC T LYMPHOCYTE ACTIVITY FLOW CYTOMETRIC ANALYSIS OF IMMUNE CELLS MAGNETIC ACTIVATED CELL SORTING ISOLATION OF MONONUCLEAR CELLS FROM PERIPHERAL BLOOD CONCLUSIONS TRANSCRIPTOME ANALYSIS RNA PREPARATION NORTHERN ANALYSIS PRINCIPLE PROCEDURE APPLICATIONS IN SITU HYBRIDIZATION DOT BLOT AND SLOT BLOT REVERSE TRANSCRIPTASE–POLYMERASE CHAIN REACTION APPLICATION OF RT-PCR DNA MICROARRAY RNAI TECHNOLOGY: A TOOL FOR FUNCTIONAL VALIDATION OF NOVEL GENES MACHINERY INVOLVED IN RNAI INDUCER DICER RNA-DEPENDENT RNA POLYMERASE RNA-INDUCED SILENCING COMPLEX MIRNA AND SIRNA RNAI AS A TOOL OF FUNCTIONAL GENOMICS PRODUCTION OF DSRNA CONSTITUTIVE AND INDUCIBLE RNAI ANTISENSE RNA AND RNAI POTENTIAL AREAS OF APPLICATION MOLECULAR MATCHMAKING: TECHNIQUES FOR BIOMOLECULAR INTERACTIONS TOOLS FOR THE STUDY OF PROTEIN–PROTEIN INTERACTIONS THE TWO-HYBRID SYSTEM THE SPLIT-UBIQUITIN SYSTEM REVERSE TWO-HYBRID SYSTEM SOS RECRUITMENT SYSTEM (CYTO TRAP YEAST TWO-HYBRID SYSTEM) YEAST ONE-HYBRID SYSTEM DOUBLE INTERACTION SCREEN YEAST THREE-HYBRID OR TRI-HYBRID SYSTEM REAGENTS, MATERIALS, AND EQUIPMENT NOTES AND POINTS TO WATCH 10 ENVIRONMENTAL PROTEOMICS: EXTRACTION AND IDENTIFICATION OF PROTEIN IN SOIL SAMPLE PREPARATIONS PROTOCOLS FOR PROTEIN EXTRACTION FROM SOIL 10 3 1 EXTRACTION OF EXTRACELLULAR PROTEIN 10 3 2 EXTRACTION OF WHOLE-CELL PROTEIN PROTEIN LOADING PROTEIN EXPRESSION ANALYSES 10 5 1 SDS-PAGE 10 5 2 TWO-DIMENSION SDS-PAGE ANALYSIS 10 6 GEL STAINING 10 6 1 COOMASSIE BRILLIANT BLUE STAINING PROTOCOL (FOR MINI GELS) 10 6 2 SILVER STAINING PROTOCOL IMAGE ANALYSIS SPOT CUT PROTEIN DIGESTION 10 10 MASS SPECTROMETRY ANALYSIS 10 11 SPECTRAL ANALYSIS 10 12 N-TERMINAL AMINO ACID SEQUENCING 10 13 CONCLUSIONS 11 DGGE AND RISA PROTOCOLS FOR MICROBIAL COMMUNITY ANALYSIS IN SOIL SOIL DNA EXTRACTION 11 2 1 EQUIPMENT 11 2 2 CHEMICALS 11 2 3 DNA EXTRACTION PROTOCOL POLYMERASE CHAIN REACTION PROTOCOL FOR DGGE 11 3 1 FIRST ROUND PCR 11 3 2 GC CLAMP 16S PCR (SECOND ROUND PCR) DGGE TECHNIQUES 11 4 1 EQUIPMENT 11 4 2 CHEMICALS 11 4 3 ASSEMBLING THE GEL CHAMBER 11 4 4 CASTING THE GEL 11 4 5 LOADING OF THE SAMPLES 11 4 6 STAINING AND IMAGING OF THE GELS RIBOSOMAL INTERGENIC SPACER ANALYSIS 11 5 1 EQUIPMENT 11 5 2 CHEMICALS 11 5 3 PCR PROTOCOL 11 5 4 GEL PREPARATION AND LOADING 11 5 5 GEL RUNNING 11 5 6 STAINING AND IMAGING OF THE GELS 12 SOIL MICROBIAL COMMUNITY STRUCTUREAND FUNCTION ASSESSED BY FAME, PLFA AND DGGE – ADVANTAGES AND LIMITATIONS MICROBIAL COMMUNITY STRUCTURE BASED ON FATTY ACID PATTERNS 12 2 1 FAME EXTRACTION AND DATA ANALYSIS 12 2 2 PLFA ANALYSIS 12 2 3 ADVANTAGES AND LIMITATIONS OF FATTY ACID PATTERNS DENATURING GRADIENT GEL ELECTROPHORESIS 12 3 1 DNA EXTRACTION FROM SOIL 12 3 2 POLYMERASE CHAIN REACTION 12 3 3 DGGE PROCEDURES 13 MEASUREMENT OF MICROBIAL BIOMASS AND ACTIVITY IN SOIL PROTOCOLS FOR MICROBIAL BIOMASS DETERMINATION 13 2 1 CHLOROFORM FUMIGATION–EXTRACTION METHOD FOR MICROBIAL BIOMASS C AND N 13 2 2 HEXANOL EXTRACTION METHOD FOR MICROBIAL P PROTOCOL FOR TOTAL MICROBIAL ACTIVITY DETERMINATION 13 3 1 EQUIPMENT 13 3 2 REAGENTS 13 3 3 PROTOCOL FOR EXTRACTION PROTOCOL FOR SOIL DEHYDROGENASE ENZYME ANALYSIS 13 4 1 EQUIPMENT 13 4 2 REAGENTS 13 4 3 PROTOCOL FOR EXTRACTION 14 IMMUNO-TECHNOLOGY FOR THE LOCALIZATION OF ACID PHOSPHATASE USING NATIVE GEL BANDS IN PIRIFORMOSPORA INDICA AND OTHER SOIL MICROORGANISMS 14 1 1 TAXONOMIC STATUS 14 1 2 PHOSPHATASES IMMUNOTECHNOLOGY FOR THE DETECTION AND LOCALIZATION OF ACID PHOSPHATASE IN P INDICA 14 2 1 EXTRACTION OF PROTEIN AND ENZYME ASSAY 14 2 2 PURIFICATION OF PROTEIN BY COLUMN CHROMATOGRAPHY 14 2 3 PURIFICATION OF PROTEIN BY ION EXCHANGE CHROMATOGRAPHY 14 2 4 NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS 14 2 5 DETECTION OF ENZYME IN NATIVE PAGE 14 2 6 ISOLATION OF ACID PHOSPHATASE FOR RAISING ANTIBODY 14 2 7 PRODUCTION OF ANTIBODIES USING ACID PHOSPHATASE IN NATIVE GEL 14 2 8 ANTISERUM PREPARATION 14 2 9 PURIFICATION OF IMMUNOGLOBULIN FROM SERUM 14 2 10 WESTERN BLOT 14 2 11 IMMUNO-FLUORESCENCE 14 2 12 LOCALIZATION OF ACPASE BY IMMUNOGOLD TECHNIQUE TROUBLESHOOTING 15 USE OF SHORT OLIGONUCLEOTIDE PRIMERS IN RANDOM AMPLIFIED POLYMORPHIC DNA TECHNIQUES FOR SPECIES IDENTIFICATION POLYMORPHISM BETWEEN PIRIFORMOSPORA INDICA AND SEBACINA VERMIFERA, MEMBERS OF THE ORDER SEBACINALES GENERAL PROTOCOL FOR RAPD TECHNIQUE TO SHOW POLYMORPHISM 15 3 1 EXPERIMENTAL PROCEDURES 16 CO-CULTIVATION WITH SEBACINALES SEBACINACEAE – NOVEL FUNGI HOST SPECTRUM FUNCTIONS OF THE SEBACINACEAE ECO-FUNCTIONAL IDENTITY AXENIC CO-CULTIVATION OF SEBACINACEAE 16 6 1 PROCEDURE 16 6 2 PROTOCOL MEDIA COMPOSITIONS SEED SURFACE STERILIZATION AND GERMINATION 16 8 1 PROTOCOL FOR SEED SURFACE STERILIZATION 16 8 2 INOCULUM PLACEMENT IN THE POTS 16 8 3 RESULTS COMPARATIVE STUDY ON PLANT GROWTH WITH TREATED ENDOSYMBIONTS 16 10 IN VIVO CO-CULTIVATION OF SEBACINALES 16 11 CONCLUSIONS 17 QUANTITATIVE HISTOCHEMISTRY: A FORGOTTEN TOOL WITH NEW APPLICATIONS SAMPLE PREPARATION AND HANDLING MICROPHOTOMETRY BIOCHEMICAL ANALYSIS: REAL TIME MICROASSAYS SPATIAL RESOLUTION OF BASIC STEPS OF FUNGAL TREHALOSE METABOLISM IN SYMBIOSIS 18 ION CYCLOTRON RESONANCE FOURIER TRANSFORM MASS SPECTROMETRY FOR NON-TARGETED METABOLOMICS OF MOLECULAR INTERACTIONS IN THE RHIZOSPHERE THE CHEMICAL BIOLOGY APPROACH COMPLEMENTARY ANALYTICAL APPROACHES 18 3 1 TARGETED ANALYSIS 18 3 2 METABOLITE PROFILING 18 3 3 NON-TARGETED ANALYSIS RESOLVING STRUCTURAL INFORMATION FROM MOLECULAR COMPLEXITY WITH ICR-FT/MS 18 4 1 TOP-DOWN APPROACH: FROM ICR-FT/MS-PROFILING ANALYSIS TO STRUCTURAL HYPOTHESIS 18 4 2 COMPLEMENTARY ANALYTICAL TOOLS 18 4 3 BOTTOM-UP APPROACH: FROM HYPOTHESIS-DRIVEN EXPERIMENTS UPWARDS TO ICR-FT/MS 19 APPLICATION OF TERMINAL-RESTRICTION FRAGMENT LENGTH POLYMORPHISM FOR MOLECULARANALYSIS OF SOIL BACTERIAL COMMUNITIES A GENERAL PROTOCOL FOR TAXONOMIC T-RFLP PROFILING OF SOIL BACTERIAL COMMUNITIES 19 2 1 MATERIALS 19 2 2 EXPERIMENTAL PROCEDURE 19 2 3 TROUBLESHOOTING STANDARDIZATION OF T-RFLP PROFILES OTHER APPLICATIONS OF T-RFLP TO SOIL BACTERIAL COMMUNITIES 20 MOLECULAR SYMBIOTIC ANALYSIS BETWEEN ARABIOPSIS THALIANA AND PIRIFORMOSPORA INDICA BENEFICIAL INTERACTION BETWEEN PLANTS AND FUNGI: PIRIFORMOSPORA INDICA AND ARABIDOPSIS THALIANA AS A MODEL SYSTEM CO-CULTIVATION OF P INDICA AND ARABIDOPSIS UNDER STANDARDIZED GROWTH CONDITIONS MAP-BASED CLONING OF A MUTATED GENE RAPID DNA EXTRACTION CONFIRMATION OF A MUTATED PHENOTYPE OF AN EMS MUTANT BY THE ANALYSIS OF AN INDEPENDENT T-DNA INSERTION LINE DIFFERNTIAL DISPLAY TO IDENTIFY GENES WHICH ARE REGULATED IN RESPONSE TO P INDICA ACTIVATION TAGGED LINES IDENTIFICATION OF BIOCHEMICAL PATHWAYS IN A THALIANA WHICH ARE REGULATED BY P INDICA 21 BIOPHYSICAL PHENOMICS REVEALS FUNCTIONAL BUILDING BLOCKS OF PLANTS SYSTEMS BIOLOGY: A CASE STUDY FOR THE EVALUATION OF THE IMPACT OF MYCORRHIZATION WITH PIRIFORMOSPORA INDICA BIOPHYSICAL PHENOMICS OF THE FAST FLUORESCENCE RISE O-J-I-P 21 2 1 THE ENERGY CASCADE IN THE PHOTOSYNTHETIC APPARATUS 21 2 2 MICROSTATES – FUNCTIONAL BUILDING BLOCKS OF PHOTOSYNTHESIS 21 2 3 MEASURING FLUORESCENCE TRANSIENTS WITH PEA, HANDY-PEA AND FIM- FLUORIMETERS 21 2 4 HOW FLUORESCENCE KINETICS PROVIDE AN INSIGHT TO THE MICROSTATES – FUNCTIONAL BLOCKS OF PSII CASE STUDY 21 3 1 MYCORRHIZATION AND THE ADVANTAGES OF PIRIFORMOSPORA INDICA, AN EMERGING GROWTH BOOSTER 21 3 2 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 21 3 3 CORRELATION OF PHYSIOLOGICAL WITH BIOPHYSICAL PARAMETERS 22 ANALYSIS OF THE PLANT PROTECTIVE POTENTIAL OF THE ROOT ENDOPHYTIC FUNGUS PIRIFORMOSPORA INDICA IN CEREALS PLANT RESPONSES AND RESISTANCE TO PATHOGENS 22 2 1 LOCAL REACTIONS 22 2 2 SYSTEMIC REACTIONS AND RESISTANCE IN CEREALS 22 2 3 BENEFICIAL MICROBIAL ENDOPHYTES PROTECTING CEREALS FROM PATHOGENS INTERACTION OF P INDICA WITH CEREALS 22 3 1 P INDICA COLONIZES ROOT CORTICAL CELLS IN BARLEY 22 3 2 P INDICA ENHANCES BIOMASS AND YIELD IN BARLEY APPROACHES TO STUDY THE MECHANISM OF P INDICA-INDUCED PATHOGEN RESISTANCE 22 4 1 P INDICA INDUCES DISEASE RESISTANCE AGAINST ROOT PATHOGENS 22 4 2 P INDICA INDUCES SYSTEMIC DISEASE RESISTANCE 22 4 3 ASSESSMENT OF THE ANTIOXIDANT CAPACITY OF P INDICA- INFESTED ROOTS 22 4 4 GENE EXPRESSION INDUCED BY P INDICA IN BARLEY LEAVES 23 MEMBERS OF SEBACINALES CONFER RESISTANCE AGAINST HEAVY METAL STRESS IN PLANTS SCIENTIFIC BACKGROUND DIFFERENTIAL DISPLAY TO UNDERSTAND CD2+ RESISTANCE MEDIATED BY ENDOPHYTIC FUNGI STUDIES ON PROTEIN LEVEL 23 4 1 TWO-DIMENSIONAL GEL ELECTROPHORESIS, PREPARATION OF PROTEINS 23 4 2 MASS SPECTROMETRY, PREPARATION OF SAMPLES BY TRYPTIC DIGESTION 24 SCREENING OF PLANT GROWTH-PROMOTING RHIZOBACTERIA CANDIDATURE FOR BEING A RHIZOBACTERIA SCREENING METHODS 24 3 1 CRITERIA FOR SCREENING 24 3 2 SELECTION OF SCREENING METHODS 24 3 3 CLASSIC METHODS 24 3 4 MODERN METHODS 24 3 5 MOLECULAR METHODS METAGENOMICS TRACKING OF GEMS 25 RESEARCH METHODS IN ARBUSCULAR MYCORRHIZAL FUNGI ASSESSMENT OF AM FUNGAL PROPAGULES IN SOIL 25 2 1 SOIL SAMPLING 25 2 2 SPORE EXTRACTION 25 2 3 QUANTIFICATION OF SPORE NUMBERS 25 2 4 INFECTIVITY ASSAYS 25 2 5 IDENTIFICATION OF AM FUNGI 25 2 6 USE OF FATTY ACIDS FOR IDENTIFICATION OF AM FUNGI QUANTIFICATION OF AM FUNGAL ROOT COLONIZATION IN ROOT 25 3 1 CLEARING AND STAINING ROOTS 25 3 2 MODIFICATIONS OF STAINING PROCEDURE 25 3 3 MEASUREMENT OF ROOT COLONIZATION BY AM FUNGI EXTRACTION AND QUANTIFICATION OF EXTRA-RADICAL MYCELIUM OF AM FUNGI IN SOILS ASSESSMENT OF GROWTH RESPONSE OF EFFECTIVE ISOLATES INOCULUM PRODUCTION OF AM FUNGI 25 6 1 ON-FARM PRODUCTION OF AM FUNGI 25 6 2 TRADITIONAL CULTURE METHODS 25 6 3 AM FUNGAL CULTURE USING AEROPONIC AND HYDROPONIC CULTURE 25 6 4 MONOAXENIC CULTURE OF AM FUNGI 25 6 5 STORAGE OF AM FUNGAL INOCULUM 26 FIELD TRIALS OF BIOINOCULANTS EFFECT OF MYCORRHIZAL INFECTION ON NUTRIENT UPTAKE EFFECT OF SOIL FUMIGATION AND MYCORRHIZAL INOCULATION ON PLANT GROWTH UNDER FIELD CONDITIONS EFFECT OF MYCORRHIZAL INOCULATION ON PLANT GROWTH AND NUTRIENT UPTAKE UNDER NON-STERILE FIELD CONDITIONS SOIL AND CROP MANAGEMENT SYSTEM INOCULATION TECHNIQUES