Working with rhizobia J.G. Howieson and M.J. Dilworth (Eds.) Working with rhizobia J.G. Howieson and M.J. Dilworth (Eds.) Centre for Rhizobium Studies Murdoch University 2016 The Australian Centre for International Agricultural Research (ACIAR) was established in June 1982 by an Act of the Australian Parliament. ACIAR operates as part of Australia’s international development cooperation program, with a mission to achieve more productive and sustainable agricultural systems, for the benefit of developing countries and Australia. It commissions collaborative research between Australian and developing- country researchers in areas where Australia has special research competence. It also administers Australia’s contribution to the International Agricultural Research Centres. Where trade names are used this constitutes neither endorsement of nor discrimination against any product by ACIAR. ACIAR MONOGRAPH SERIES This series contains the results of original research supported by ACIAR, or material deemed relevant to ACIAR’s research and development objectives. The series is distributed internationally, with an emphasis on developing countries. © Australian Centre for International Agricultural Research (ACIAR) 2016 This work is copyright. Apart from any use as permitted under the Copyright Act 1968, no part may be reproduced by any process without prior written permission from ACIAR, GPO Box 1571, Canberra ACT 2601, Australia, [email protected]. Howieson J.G. and Dilworth M.J. (Eds.). 2016. Working with rhizobia. Australian Centre for International Agricultural Research: Canberra. ACIAR Monograph No. 173 ACIAR Monographs – ISSN 1031-8194 (print), ISSN 1447-090X (online) ISBN 978 1 925436 17 4 (print) ISBN 978 1 925436 18 1 (PDF) Editing by Kate Langford Text design by Peter Nolan, Canberra Cover design by CRS and Joke Hollants Printing by Bytes ’n Colours, Canberra Foreword The legume symbiosis with rhizobia (root nodule bacteria) has been acknowl- edged as fundamental to sustainable agriculture because this intimate relationship between soil bacteria and flowering plants can alleviate the need to provide manu- factured nitrogen for farming systems. Prior to the invention of the Haber–Bosch process, which led to the manufacture of fertiliser nitrogen early last century, rhizobial nitrogen fixation was the dominant source of nitrogen in agriculture. We now understand that much of the change to our climate has resulted from the burning of fossil fuels, which is essential for generating the high temperature and pressure for the Haber–Bosch process. Any anthropomorphic activity that can limit consumption of fossil fuels must therefore be embraced. This has brought a renewed focus to the science of biological nitrogen fixation because the reduct- ant and metabolic energy needed for the key enzyme nitrogenase to function in rhizobia are instead derived from solar radiation. The Australian Centre for International Agricultural Research (ACIAR) has a fundamental commitment to the development of sustainable agricultural prac- tices. Further, many of the ACIAR aid projects are directed at landscapes that are infertile. Nitrogen infertility is a global production constraint for farmers who cannot afford manufactured fertiliser, and thus adoption of legumes inoculated with appropriate rhizobia in their farming systems can lead to greatly increased food production. This manual provides scientists and technicians with modern guidelines for en- suring that the legume symbiosis with rhizobia is optimised for nitrogen fixation in their environments. It builds upon similar manuals produced over the last hun- dred years and updates our knowledge of this fundamental biological process and our ability to use it in agriculture. Nick Austin Chief Executive Officer, ACIAR 3 Contents Foreword ............................................................................................................3 Preface ................................................................................................................9 About this manual ....................................................................................................9 About the authors and compilation of the manual ..............................................9 Correct citation for the manual ..............................................................................9 Acknowledgements ................................................................................................10 Authors ....................................................................................................................10 1 The legume-rhizobia symbiosis and assessing the need to inoculate .......15 1.1 Legumes in agriculture, society and the environment ...........................15 1.2 Rhizobia and nodules .................................................................................16 1.3 The legumes and their nodule characteristics .........................................17 1.4 Assessing the need to inoculate .................................................................20 1.5 Selecting inoculant quality strains ............................................................24 1.6 References ....................................................................................................24 2 Collecting nodules for isolation of rhizobia ..............................................25 2.1 Introduction .................................................................................................25 2.2 Collecting new strains of rhizobia ............................................................26 2.3 Respecting the international biodiversity convention ............................27 2.4 Collecting nodules ......................................................................................27 2.5 Many nodules from one plant or a few nodules from many plants? ....31 2.6 Storage of nodules .......................................................................................31 2.7 Trapping rhizobia from soil or ruptured nodules ...................................34 2.8 Specific host in situ trapping .....................................................................34 2.9 Collection of passport data ........................................................................35 2.10 Collection of seed (for authentication) ....................................................36 2.11 References ....................................................................................................37 3 Isolation and growth of rhizobia ................................................................39 3.1 Introduction .................................................................................................39 3.2 Preparing solid growth media ...................................................................39 3.3 Preparing the nodules .................................................................................40 3.4 Isolating bacteria from nodules .................................................................40 3.5 Recognising nodule bacteria growing on solid media ...........................42 3.6 Some useful diagnostic features of species of nodule bacteria ..............46 5 3.7 Routine culture media for rhizobia ...........................................................47 3.8 Antibiotics and indicators ..........................................................................54 3.9 Avoiding contamination .............................................................................57 3.10 Authentication of isolates and Koch’s postulates.....................................58 3.11 References ....................................................................................................60 4 Preservation of rhizobia .............................................................................61 4.1 The need to preserve cultures ....................................................................61 4.2 Options for preservation ............................................................................61 4.3 Longer-term preservation ..........................................................................63 4.4 Quality control .............................................................................................68 4.5 Cryopreservation .........................................................................................69 4.6 Developing a parent/working-lot system .................................................70 4.7 References ....................................................................................................71 5 Authentication of rhizobia and assessment of the legume symbiosis in controlled plant growth systems ............................................................73 5.1 Introduction .................................................................................................73 5.2 Host-range, cross nodulation and effectiveness ......................................74 5.3 Plant growth systems with which to assess nodulation and effectiveness .................................................................................................74 5.4 Screening for N2 fixation ............................................................................78 5.5 Substrates for effectiveness experiments ..................................................84 5.6 Assessment of N2-fixing potential in non-sterile soil cores ...................87 5.7 Nutrient media for legume cultivation in glasshouse experiments ......89 5.8 General procedures for