The potential for bradyrhizobia and phosphate solubilising microorganisms to improve soybean (Glycine max (L.) Merr.) production in acid soils in Ethiopia Daniel Muleta Fana Thesis submitted for the degree of Doctor Philosophy School of Agriculture, Food & Wine The University of Adelaide March 2018 Table of contents List of Figures ..................................................................................................................... vi List of Tables ....................................................................................................................vii Acronyms and abbreviations .............................................................................................. viii Abstract ..................................................................................................................... xi Declaration .................................................................................................................... xv Acknowledgements ............................................................................................................. xvi Chapter 1 Introduction ................................................................................................. 17 1.1. Aims ...................................................................................................................... 19 1.2. Thesis structure ...................................................................................................... 19 Chapter 2 Literature Review ........................................................................................ 22 2.1. Biological N fixation .............................................................................................. 22 2.2. Industrial inputs and sustainability.......................................................................... 24 2.3. Microbial Inoculants in sustainable agriculture ....................................................... 25 2.3.1 Microbial inoculants / Biofertilizers definition ................................................ 25 2.3.2 Microbial inoculants versus inorganic inputs ................................................... 26 2.3.3 Microbial inoculant types and function ............................................................ 27 2.4. Biological N fixing organisms ................................................................................ 27 2.5. Rhizobia ................................................................................................................. 28 2.5.1 Techniques to study rhizobial diversity............................................................ 28 i 2.5.2 Rhizobia taxonomy ......................................................................................... 32 2.5.3 Nodulation ...................................................................................................... 32 2.6. Factors affecting response to rhizobial inoculants ................................................... 34 2.6.1 Inoculant delivery method ............................................................................... 34 2.6.2 Inoculant quality ............................................................................................. 35 2.6.3 Competition for nodulation ............................................................................. 35 2.6.4 Environmental factors affecting inoculation response ...................................... 36 2.7. Biological N fixation in soybeans ........................................................................... 37 2.7.1 Soybean use and world production .................................................................. 37 2.7.2 Soybean biological N fixation ......................................................................... 37 2.7.3 Soybean nodulating rhizobia ........................................................................... 38 2.7.4 Soybean production and N fixation in Ethiopia................................................ 39 2.7.5 Assessment of resident soil population size and effectiveness .......................... 40 2.7.6 Screening of acid tolerant rhizobia .................................................................. 41 2.8. Phosphate solubilising microorganisms .................................................................. 42 2.9. Summary ................................................................................................................ 44 Chapter 3 The potential for rhizobial inoculation to increase soybean grain yields on acid soils in Ethiopia ............................................................................................................ 45 3.1. Manuscript summary .............................................................................................. 45 3.1.1 Context ........................................................................................................... 45 3.1.2 Research objective .......................................................................................... 46 ii 3.1.3 Methods .......................................................................................................... 46 3.1.4 Findings .......................................................................................................... 46 3.1.5 Implications .................................................................................................... 46 3.2. Statement of Authorship ......................................................................................... 47 3.3. Article .................................................................................................................... 48 Chapter 4 Evaluation of the diversity among acid tolerant soybean rhizobia from key agricultural regions of Ethiopia............................................................................................. 60 4.1. Introduction ............................................................................................................ 60 4.2. Material and methods ............................................................................................. 62 4.2.1 Rhizobia isolates ............................................................................................. 62 4.2.2 PCR amplification ........................................................................................... 62 4.2.3 PCR confirmation ........................................................................................... 63 4.2.4 DNA purification and sequencing.................................................................... 63 4.2.5 Phylogenetic analysis ...................................................................................... 63 4.2.6 Symbiotic effectiveness of acid tolerant strains ............................................... 64 4.3. Results ................................................................................................................... 64 4.3.1 PCR amplification ........................................................................................... 64 4.3.2 Phylogeny of test strains based on the 16S-23S rRNA ITS sequence ............... 64 4.3.3 Phylogeny and symbiotic effectiveness of test strains ...................................... 65 4.3.4 Phylogenetic comparison of test strains with previously identified strains. ...... 66 4.4. Discussion .............................................................................................................. 66 iii Chapter 5 Isolation and evaluation of phosphate-solubilising bacteria associated with soybean .................................................................................................................... 75 5.1. Introduction ............................................................................................................ 75 5.2. Materials and methods ............................................................................................ 76 5.2.1 Initial in-vitro phosphate dissolution ............................................................... 76 5.2.2 Determination of phosphate dissolution efficiency in liquid cultures ............... 77 5.2.3 Characterization of phosphate solubilising bacteria using inter transcribed region (ITS) sequence of 16S-23S rRNA. ..................................................................... 77 5.2.4 Field Experiment on phosphate solubilisation .................................................. 78 5.2.5 Experimental design ........................................................................................ 78 5.2.6 Data collection ................................................................................................ 79 5.2.7 Statistical analysis ........................................................................................... 79 5.3. Results ................................................................................................................... 79 5.3.1 Initial phosphate dissolution screening ............................................................ 79 5.3.2 Phosphate dissolution in liquid culture ............................................................ 80 5.3.3 ITS sequences of the phosphate solubilising bacteria ....................................... 81 5.3.4 Field experiments ............................................................................................ 81 5.4. Discussion .............................................................................................................. 81 Chapter 6 Assessment of symbiotic effectiveness and population