Investigation of the symbiotic associations of Acacia ligulata Benth. and Acacia tetragonophylla F.Muell: the potential for use in the rehabilitation of excavated sites at Shark Bay Salt Pty. Ltd. by Yvette Joan Hill This thesis is presented for the degree of Doctor of Philosophy of Murdoch University 2015 i DECLARATION I hereby declare that, unless otherwise stated, the work presented in this Thesis is my own and has not been submitted for a degree at any other institution. Yvette Joan Hill ii For Jason and Sophia, you are the smile in my heart and sunshine in my soul. iii TABLE OF CONTENTS Publications arising from the thesis............................................................................x Acknowledgements...................................................................................................xi Abbreviations..........................................................................................................xiii Summary..................................................................................................................xv CHAPTER 1: Literature Review.......................................................................................1 1.1 Challenges for rehabilitation in Shark Bay Salt, Western Australia.........................2 1.2 Disturbance and rehabilitation of Useless Loop......................................................4 1.3 Climate classification of Australian drylands...........................................................5 1.4 Dryland disturbance ecology...................................................................................6 1.4.1 Impacts of climate change for Australian drylands........................................10 1.4.1.1 Rainfall and temperature: trends and predictions...................................10 1.4.1.2 Vegetation................................................................................................12 1.4.1.3 Soil microbial communities......................................................................14 1.4.2 Australian dryland soils: edaphic characteristics and mineral nutrition.........15 1.4.3 Provenance of flora species............................................................................18 1.5 Leguminosae..........................................................................................................20 1.6 The genus Acacia sens. lat.....................................................................................21 1.6.1 The subgenera Acacia s.s................................................................................23 1.6.1.1 Acacia ligulata Benth................................................................................26 1.6.1.2 Acacia tetragonophylla F. Muell...............................................................27 1.7 Root nodule bacteria.............................................................................................27 1.7.1 Australian RNB...............................................................................................29 1.8 Legume-RNB symbioses.........................................................................................30 1.8.1 Bacterial infection..........................................................................................31 1.8.1.1 Signalling pathways..................................................................................31 1.8.1.2 Process of infection..................................................................................32 1.8.2 Nodule organogenesis....................................................................................33 1.8.2.1 Nodule development................................................................................34 1.8.2.2 Nitrogen fixation.......................................................................................35 1.9 Dryland environmental constraints on N2 fixation................................................36 1.9.1 Temperature and water availability...............................................................36 iv 1.9.2 Soil pH and salinity..........................................................................................38 1.10 RNB inoculants for dryland rehabilitation...........................................................39 1.11 Aims of thesis.......................................................................................................41 CHAPTER 2: Disturbance effects on soil characteristics and RNB..............................44 2.1 Introduction...........................................................................................................45 2.2 Materials and Methods..........................................................................................46 2.2.1 Site assessment of abiotic conditions.............................................................46 2.2.1.1 Site selection............................................................................................46 2.2.1.2 Soil chemical analysis...............................................................................49 2.2.1.2.1 Soil sampling....................................................................................49 2.2.1.2.2 Analysis............................................................................................49 2.2.1.3 Temperature and Rainfall........................................................................49 2.2.2 Vegetation survey...........................................................................................50 2.2.2.1 Flora survey and vegetation profile.........................................................50 2.2.2.2 A. ligulata Benth. and A. tetragonophylla F.Muell. survey......................50 2.2.3 Most probable number of RNB.......................................................................50 2.2.3.1 Soil sampling............................................................................................50 2.2.3.2 Seed and growth jar preparation.............................................................51 2.2.3.3 Infection test and RNB enumeration.......................................................51 2.3 Results....................................................................................................................53 2.3.1 Chemical characteristics of soils.....................................................................53 2.3.2 Rainfall and Temperature...............................................................................53 2.3.2.1 Rainfall.....................................................................................................53 2.3.2.2 Temperature............................................................................................56 2.3.3 Vegetation survey...........................................................................................57 2.3.3.1 Flora survey and vegetation profile.........................................................57 2.3.3.2 A. ligulata Benth. and A. tetragonophylla F.Muell. survey......................58 2.3.4 Most probable number of RNB in soil............................................................64 2.4 Discussion..............................................................................................................66 Chapter 3: RNB from Shark Bay Salt lease area soils: isolation and effectiveness…..73 3.1 Introduction...........................................................................................................74 3.2 Materials and Methods..........................................................................................75 v 3.2.1 Isolation and symbiotic effectiveness of A. ligulata Benth. RNB………………...75 3.2.1.1 .Collection of root-nodules from A. ligulata Benth..................................75 3.2.1.2 Isolation of RNB........................................................................................75 3.2.1.3 Authentication of RNB isolated from A. ligulata Benth...........................76 3.2.1.4 RPO1-PCR fingerprinting of root nodule occupants.................................77 3.2.1.5 Statistical analysis.....................................................................................78 3.2.2 Trapping RNB from Shark Bay Salt lease area soils........................................78 3.2.2.1 Selection of trap species..........................................................................78 3.2.2.2 Collection and preparation of soil............................................................78 3.2.2.3 Glasshouse trapping of RNB.....................................................................78 3.2.3 Authentication and N2 fixation effectiveness of A.ligulata and A. tetragonophylla F.Muell. RNB isolates and WattleGrow™.............. 81 3.2.3.1 Experimental design.................................................................................81 3.2.3.2 A. ligulata Benth. RNB isolates.................................................................81 3.2.3.3 A. tetragonophylla F.Muell. RNB isolates.................................................82 3.2.3.4 Nitrogen content of A. ligulata Benth. and A. tetragonophylla F.Muell.82 3.3 Results...................................................................................................................82 3.3.1 Isolation, authentication and symbiotic effectiveness of A. ligulata Benth. RNB………………..………………….…….…………………….……………….…………..……….…..82 3.3.2 RNB trapped from Shark Bay Salt lease area soils.........................................84 3.3.3 Authentication and N2 fixation effectiveness of A.ligulata Benth. and