4.3.2.2. DNA Sequence Analysis and Design of the Universal Primers and Probe
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lima2809585515 _ ^ _ REFERENCE ONLY UNIVERSITY OF LONDON THESIS Degree Year 1 (5 1 5 1 Name ofAuthor k j \ $ P / COPYRIGHT t \ This is a thesis accepted for a Higher Degree of the University of. ItLondon is an unpublished typescript and the copyright is held by the author. All persons consulting this thesis must read and abide by the Copyright Declaration below. COPYRIGHT DECLARATION I recognise that the copyright of the above-described thesis rests with the author and that no quotation from it or information derived from it may be published without the prior written consent of the author. LOANS Theses may not be lent to individuals, but the Senate House Library may lend a copy to approved libraries within the United Kingdom, for consultation solely on the premises of those libraries. Application should be made to: Inter-Library Loans, Senate House Library, Senate House, Malet Street, London WC1E 7HU. 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HOW DO RHIZOSPHERE BACTERIA INTERACT WITH THEIR ENVIRONMENT AT THE MICROHABITAT SCALE? Paul Gareth Dennis Presented for the degree of Doctor of Philosophy in the Faculty of Science, University of London May 2007 Dept, o f Geography, University College London, Gower Street, London WC1E6BT, UK Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK The Natural History Museum, Cromwell Road, London SW7 5BD, UK The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author 1 UMI Number: U592006 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U592006 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Declaration of authenticity I, Paul Gareth Dennis, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 ABSTRACT The rhizosphere supports greater bacterial densities than root-free soil. Rhizosphere bacteria (RB) can affect plant health and nutrition; however, attempts to manipulate introduced and/or indigenous communities to benefit plants are unreliable. Current evidence indicates that habitat factors strongly influence bacterial communities. In the rhizosphere many processes give rise to a high degree of habitat heterogeneity; therefore, to understand how RB interact with their environment their ecology should be studied at the micro-spatial-scale. The objectives of this research were to develop a method for sampling RB at the microhabitat-scale, and to investigate techniques that can link these samples with key factors, such as substrate availability and pH. A novel method enabling non-destructive, micro-scale sampling of bacteria was developed. Its efficiency for removing bacteria from the root surface was similar to that of existing methods but offered greater accuracy in estimating RB densities. The novel method revealed that RB density was inversely proportional to distance from the apex of Brassica napus roots and that the composition of RB communities was highly variable at the micro-scale. Imaging of 14C-labelled root exudates revealed that RB density was not reflective of exudate availability but attempts to link RB with available C were unsuccessful. A key outcome of this work was the development of a strategy to combine micro sampling with microelectrode measurements. Microelectrodes revealed that pH at the root surface was highly variable at the micro-scale which, combined with similar observations for RB density/diversity, highlights the appropriateness of this scale for linking RB communities with their environment. This thesis considers the link between RB community structure, habitat and function and provides a detailed description of micro-sample analyses as well as a set of methods that will enable for the first time, the interactions between any surface-associated bacteria and their environment to be investigated at a microhabitat-scale. 3 TABLE OF CONTENTS ABSTRACT....................................................................................................................... 3 TABLE OF CONTENTS................................................................................................... 4 LIST OF FIGURES......................................................................................................... 14 LIST OF TABLES........................................................................................................... 23 ACKNOWLEDGEMENTS............................................................................................. 25 CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW................................ 26 1.1. Introduction ........................................................................................................... 26 1.2. Bacterial biogeography .........................................................................................29 1.2.1. What is biogeography? ...................................................................................29 1.2.2. Do bacteria exhibit similar biogeographical trends to macroorganisms? 30 1.2.3. The influence of habitat on bacterial biogeography ......................................32 1.2.4. Bacterial function and activity ...................................................................... 33 1.3. The soil environment ............................................................................................34 1.3.1. Pore spaces and water dynamics ................................................................... 35 1.3.2. Connectivity between microhabitats ............................................................. 41 1.3.3. Physicochemical conditions within microhabitats ........................................ 43 1.3.4. Summary........................................................................................................46 1.4. The rhizosphere environment ................................................................................46 1.4.1. Definition ....................................................................................................... 46 1.4.2. Root structure and anatomy ...........................................................................47 1.4.2.1. The root cap ............................................................................................. 48 1.4.2.2. Meristematic zone ...................................................................................52 1.4.2.3. Cell elongation zone ................................................................................52 1.4.2.4. Root hair zone ......................................................................................... 53 1.4.2.5. Mature zone ............................................................................................. 54 1.4.3. Influence of root processes on environmental conditions in the rhizosphere .................................................................................................................................. 55 1.4.3.1. Root growth and water uptake ................................................................55 1.4.3.2. Nutrient uptake ........................................................................................ 57 1.4.3.2.1. General uptake trends .....................................................................57 4 1.4.3.2.2. The influence of rhizosphere microorganisms on nutrient uptake 61 1.4.3.3. Rhizodeposition ......................................................................................62 1.4.3.3.1. Terms and definitions .......................................................................62 1.4.3.3.2. How large are different pools of rhizodeposits? ............................. 64 1.4.3.3.3. What compounds are usually found in rhizodeposits and what do they do? ............................................................................................................ 67 1.4.3.3.4. Range of influence of various rhizodeposits ................................... 70 1.4.3.3.5. Influence of rhizodeposition on bacterial distribution .................... 71 1.4.3.4.