Impact of farm management on the diversity of desulfonation genes and 16S rRNA genes in wheat rhizospheres

i-ii Acknowledgements iii Abbreviations & Acronyms iv-v Chapter 1 General introduction 1-65 1.1. Fertiliser: Scarcity of resources and global challenges 2-3 1.2. Functions of the rhizosphere 4-8 1.3. Hormonal effects of plant growth promoting rhizobacteria (PGPR) 9-12 1.4. Microbial nutrient cycling 12-13 1.5. Plant requirement for nitrogen 13-19 1.5.1. Terrestrial N-cycle 13-15 1.5.2. Biological nitrogen fixation (BNF) 16-19 1.6. Requirement for phosphorus 20-24 1.6. 1. Soil-phosphorus cycle 20 1.6. 2. Mechanisms of plant P solubilisation 21 1.6. 3. P solubilisation through rhizosphere microorganisms 21 1.6. 3.1. Mycorrhizal P solubilisation 22 1.6. 3.2. Mycorrhizal-bacterial interaction 23 1.6. 3.3. Rhizobacterial P solubilisation 23-24 1.7. Sulfur 25-42 1.7. 1. Soil sulfur pools 25-28 1.7. 2. Sulfur in bacteria 29 1.7. 2.1. Sulfur metabolism in bacteria 29-30 1.7. 2.2. Organosulfur metabolism under sulfur limitation: SSI proteins 30-31 1.7. 2.3. Sulfate ester utilisation 31-32 1.7. 2.4. Sulfonate utilisation 1.7. 2.5 Importance of the asfA and ssuD genes for soil sulfur cycling 33-35 36 1.7. 3. S in plants 37-39 1.7. 4. Impact of sulfonate and sulfate-ester mobilising microbial communities on plant nutrition 40-42 1.8. Agricultural measures and their impact on microbial nutrient solubilisation 43-51 1.8. 1. Influences of tillage on microbial communities 44-45 1.8. 2. Impact of pesticides on soil microbial communities 45-47 1.8. 3. Response of soil microbial communities to fertiliser 47-50 1.8. 4. Plant species and plant genotype 50 1.8. 5. Crop rotations 50 1.8. 6. Inoculation treatments 51-55 1.9. Microbial community analysis 56-64 1.9. 1. Culture based approaches 56 1.9. 2. PCR based approaches 57 1.9. 2.1. Denaturing gradient gel electrophoresis (DGGE) 58-59 1.9. 2.2. Terminal restriction fragment length polymorphism (T-RFLP) 57-58 1.9. 2.3. Multiplex PCR, multiplex T-RFLP 60-61 1.9. 2.4. Pyrosequencing and metagenomics 61-64 1.10 Aims of this project 65 Chapter 2 Materials and methods 66-93 2.1. Chemicals 67 2.2. Soil properties used for field and pot trials and sampling procedure 68-71 2.2.1. Soil preparation and fertilization 69-70 2.2.2 Mesocosm construction and root harvest 70-71 2.3. Measurement of plant growth 72-74 2.3.1. Plant varieties 72 2.3.2. Measurement of plant dry weight 73 2.3.3 Measurement of root length and root diameter 74 2.4. Bacterial growth 75-79 2.4. 1. Bacterial strains 75 2.4. 2. Bacterial growth and growth conditions 76-77 2.4. 3. Bacterial growth curves 78 2.4. 4. Whole cell arylsulfatase measurement 79 2.5. Plant inoculation with bacteria, recovery and enumeration 79-80 2.5.1. Inoculation suspension 79 2.5.2. Recovery and enumeration of rhizosphere bacteria 80 2.6. PCR-based methods for determining bacterial diversity of the 16S rRNA gene and the diversity of ssuD and atsA genes 81-89 2.6. 1. DNA extraction from rhizosphere soils 81-82 2.6. 2. PCR amplifications 83 2.6. 2.1. PCR primers 83 2.6. 2.2. PCR reactions 84 2.6. 2.3. PCR conditions 85 2.6. 3. DNA ligation and transformation 86 2.6. 4. Conditions for denaturing gradient gel electrophoresis 87 2.6. 5. Terminal restriction fragment length polymorphism (T-RFLP) 88 2.6. 5.1. Primer set for 16S rRNA gene T-RFLP PCR 88 2.6. 5.2. Primer set for ssuD gene T-RFLP PCR 88 2.6. 5.3. Primer set for atsA T-RFLP PCR 89 2.7. Primer design 90-91 2.8. Statistical analysis 91-93 Chapter 3 Degenerate primer design for the ssuD gene (alkanesulfonate monooxygenase) and development of T-RFLP protocol for analysis of ssuD gene diversity 94-132 3.1. General requirements on a degenerate primer set 95-97 3.2. Sequence retrieval and alignment for degenerate primers targeting the ssuD gene 97-99 3.3. Design of degenerate primers for the ssuD gene encoding alkanesulfonate monooxygenase 100-103 3.3.1. Assessing the diversity of the ssuD gene in soils 101 3.3.2. Coverage of the active sites 103 3.4. Primer pair ssuD_209F & ssuD_1001R (used for T-RFLP) 104-115 3.4. 1. Sequences used for the primer design and binding sites of the forward and reverse primer 104-107 3.4. 2. Evaluation of primer specificity for ssuD_209F & ssuD_1001R 108-113 3.4. 3 Development of T-RFLP restriction enzyme digest and fluorescent primer labelling 114-115 3.5. Primer pair ssuD_26F & ssuD_601R for alkanesulfonate monooxygenase with a product size of ~560 base pairs 116-121 3.5.1. Sequences for the primer design and primer binding sites 116-120 3.5.2. Evaluation of primer specificity 120-121 3.6. Other primer pairs targeting specific phylogenetic classes of ssuD representatives 122-125 3.6. 1. Degenerate primer pair ssuD_125F & ssuD_827R 122-123 3.6. 2. Degenerate primer pair ssuD_34F/1ssuD_636R 124-125 3.7. Degenerate Primers targeting atsA diversity 126-129 3.8. Discussion 130-132 Chapter 4 Sulfonate mobilising abilities of the PGPR Azospirillum brasilense and Herbaspirillum seropedicae and effects of inoculation on plant growth 133-161 4.