LEGUME INOCULANT PRODUCTION
MANUAL
Joe C. Burton, Ph.D.
Inoculant Production Specialist
1984
NifTAL Center - MIRCEN
University of Hawaii
Department of Agronomy
and Soil Science
College of Tropical Agriculture
and Human Resources
1000 Holomua Road
Paia, Maui, Hawaii, 96779, USA
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Acknowledgements: This manual was edited from the original version by
Sally Ekdahl, Patty Nakao and Harold Keyser.
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Table of Contents
I. INTRODUCTION ...................................................................................................................... 1
II. RHIZOBIA ................................................................................................................................
A. Nature of Bacteria .........................................................................................................
B. Rhizobium Strains and Species ......................................................................................
C. Fast and Slow Growers .................................................................................................
D. Cultural Requirements ..................................................................................................
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III. RHIZOBIUM STRAIN SELECTION...................................................................................... 5
A. Strain Variation and Host Specificity ........................................................................... 5
B. Effectiveness Groupings of Leguminous Plants ........................................................... 5
C. Competitiveness ........................................................................................................... 10
D. Stock Culture Maintenance .......................................................................................... 10
IV. INOCULANTS ....................................................................................................................... 11
A. Desirable Qualities of a Legume Inoculant.................................................................. 11
B. Soil Inoculants .............................................................................................................. 11
V. MASS CULTURE OF RHIZOBIA ......................................................................................... 12
A. Media for Fermentors................................................................................................... 12
B. Preparation of Yeast Water .......................................................................................... 15
C. Culture Vessels ............................................................................................................. 15
D. Fermentor Requirements for Various Production Goals.............................................. 18
VI. CARRIER MATERIALS ....................................................................................................... 21
A. Sterilization .................................................................................................................. 23
B. Pure Culture Rhizobium Inoculants .............................................................................. 24
VII. MAKING INOCULANTS .................................................................................................... 26
A. Medium Preparation ..................................................................................................... 26
B. Monitoring Growth of Rhizobia................................................................................... 26
C. Multiple Strain Inoculants ............................................................................................ 27
D. Peat Inoculant In Bulk With Non-Sterile Carrier......................................................... 27
E. Soil or Granular Inoculant With Non-Sterile Carrier ................................................... 28
F. Pure Culture Rhizobium Inoculant ............................................................................... 28
VIII. PACKAGING ...................................................................................................................... 30
A. Evolution ...................................................................................................................... 30
B. Requirements ................................................................................................................ 30
C. Sizes ............................................................................................................................. 30
D. Label............................................................................................................................. 32
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IX. QUALITY CONTROL........................................................................................................... 32
A. Rhizobium Strain Selection .......................................................................................... 32
B. Monitoring Rhizobium Growth in Fermentors ............................................................. 33
C. Mixing Broth Culture and Carrier ................................................................................ 33
D. Lot Numbers ................................................................................................................ 33
E. Finished Inoculant ........................................................................................................ 34
F. Minimum Standards...................................................................................................... 34
X. INOCULATION METHODS .................................................................................................. 35
A. Sprinkle or Slurry Method ........................................................................................... 35
B. Lime and Phosphate Coatings ...................................................................................... 35
XI. REFERENCES AND SUGGESTED READING .................................................................. 38
XII. APPENDICES ....................................................................................................................... 42
APPENDIX A - Organic Medium Ingredients for Rhizobia ............................................ 43
APPENDIX B - Stains Useful in Studying Rhizobia ....................................................... 46
APPENDIX C - Bead Storage for Stock Cultures ............................................................ 48
APPENDIX D - Lyophilizing Rhizobium Cultures .......................................................... 50
APPENDIX E - Seeds of Leguminous Plants................................................................... 52
APPENDIX F - Sieves-Sizes and Designations ............................................................... 53
APPENDIX G - Enumerating Rhizobia in Broth, Inoculants and Soils ........................... 54
APPENDIX H - Plant Growth Systems ............................................................................ 66
APPENDIX I - To Produce Broth Cultures in Simple Glass FERMENTORS ................ 78
APPENDIX J - Conversion Factors: Length, Area, Mass, Volume, Temperature ........... 87
APPENDIX K - Forms Useful in Keeping Records in Legume Inoculant
Production and Quality Control ............................................................................ 89
APPENDIX L - Preservation of Nodulated Leguminous Roots for Exhibition
Purposes ................................................................................................................ 92
APPENDIX M - Recommended Rhizobium Strains ......................................................... 93
APPENDIX N - Differential Media for Rhizobia............................................................. 96
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I. INTRODUCTION
Leguminous plants have the remarkable ability to work with special bacteria (Rhizobium
spp.) in nodules on their roots and gather or fix atmospheric nitrogen. Through this
phenomenal process, inert nitrogen gas is taken from the inexhaustible supply in the air
and used by plants to build amino acids and proteins so essential to all life. This process
is called "Biological Nitrogen Fixation" (BNF). Since nitrogen fertilizer is the most
expensive input for food production, the BNF approach becomes a very attractive
alternative to expensive nitrogen chemical fertilizers.
Rhizobia are not universally present in soils and often those present gather little nitrogen.
In order to take advantage of this miraculous association of bacteria and leguminous
plants, it is often necessary to provide dependable legume inoculants to assure effective
nodulation of leguminous crops. The application of these bacteria to seed or soil is called
"inoculation".
This manual is concerned with methods of growing rhizobia, making effective legume
inoculants, and using them properly to maximize food, forage and fiber production. It
describes culture media; systems of screening and culturing Rhizobium spp.; selection
and processing of carrier materials; preparation, processing, and packaging of legume
inoculants; quality control; regulatory standards; and proper application of inoculants to
leguminous seeds.
Throughout this manual, the term rhizobia is used generically to apply to all root nodule
bacteria including species of Rhizobium, Bradyrhizobium, Sinorhizobium and
Azorhizobium.
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