Preface by Dr. Sueo Machi, the FNCA Coordinator of Japan

Acknowledgements

Members of the FNCA Biofertilizer Project Group

Contributing Authors

I. Introduction

II. General Methods to Evaluate Microbial Activity

1. Isotopic Methods

                1.1. N2-fixing Activity

                1.1.1. Introduction

                1.1.2. Total N accumulation

                1.1.3. N difference method

                1.1.4. ¹⁵N dilution method

                1.1.5. ¹⁵N natural abundance method

                1.1.6. ¹⁵N2 fixation activity

                1.1.7. Appendix: Direct Analysis of 15N Abundance of 

                           Kjeldahl Digested Solution by Emission Spectrometry

1.2. P-solubilizing Activity

2. Conventional Methods

2.1. N-fixing Activity

                2.1.1. Acetylene reduction assay

                2.1.2. H2 production assay

                2.1.3. Relative ureide method

                2.2. P-solubilizing Activity

III. Carriers for Biofertilizers

1. Carrier Materials

                1.1. Introduction

                1.2. Carrier material

                1.3. Sterilization

                1.4. Appendix: Enhanced Growth and Nodule occupancy of Red Kidney

        Bean  and Soybean Inoculated with Soil Aggregate-Based Inoculant

2. Carrier Sterilization using γ-irradiation

             2.1. Introduction

             2.2. The properties of ionizing radiation

             2.3. The effects of radiation on microorganisms

             2.4. The necessity of radiation sterilization

             2.5. The practical example for carrier sterilization             

             2.6. Appendix: γ–ray irradiation facilities and electron-beam

             irradiation facilities potentially available for carrier sterilization in Asia

IV. Inoculant for Biofertilizers

             1. Rhizobium Inoculant

1.1. Introduction

1.2. Isolation of Rhizobium Strains

1.2.1. Collection and preservation of root nodules in field trips

1.2.2. Isolation of bradyrhizobia from dried root nodules

1.2.3. Isolation of rhizobia from fresh nodules

1.3. Rhizobial Inoculant Production

1.3.2. Production of sterile carrier-based inoculant

1.3.1. Production of broth culture

1.3.3. Production of non-sterile carrier-based inoculant

1.3.4. Production of liquid inoculant

1.4. Introduction to the Rhizobium Supplier and Field Experiments in Japan

                1.4.1. Production and Sales of Rhizobium Biofertilizers

                1.4.2. Facility of biofertilizer production in TFAC

                1.4.3. Outline of Rhizobium inoculant production in Tokachi

           Federation  of Agricultural Cooperation

1.5. Inoculant Application

1.6. Field Experiments Related to New Inoculation and Fertilization Methods

 for Soybean in Japan

2. Non-Symbiotic Nitrogen Fixers

                2.1. Introduction

                2.1.1. Azospirillum

                2.1.2. Other Associative Nitrogen Fixing Bacteria

2.2. Isolation of Microbial Strains

                2.2.1. Isolation of endophytic bacteria from grass/rice roots

2.2.2. Isolation of Associative Nitrogen Fixing Bacteria from rice roots

2.2.3. Evaluation of N2 Fixation of Associative Nitrogen Fixing Bacteria

2.3. Inoculant Production

 2.3.1. Associative Nitrogen Fixer

                2.3.2. Mass Inocula Production

2.4. Application of biofertilizer from Associative Nitrogen Fixing Bacteria

                 2.4.1. Benefits of Biofertilizer

 2.4.2. Application of Biofertilizer

2.4.3. Procedures for Growing Corn using Biofertilizer Inoculated Seeds in Indonesia

2.5. Tips on Buying and Storage of Biofertilizers

 2.6. Cautions and Limitations of Biofertilizer

3. Mycorrhiza

3.1. Introduction

3.2. Benefits of Mycorrhizal

 3.3. Isolation of Arbuscular Mycorrhizal Fungi

3.3.1. Taxonomy of AM fungi

3.3.2. Observation of arbuscular mycorrhizal fungi in roots

3.3.3. Isolation of spores from soils and their observation for identification

3.3.4. Morphological observation of spores for identification

                3.3.5. Culturing AM fungi

3.3.6. DNA extraction from spores

3.4. Inoculant Production

3.4.1 Preparation of trap culture inoculum of arbuscular mycorrhizal fungal spores

 3.4.2 Inoculation of AMF

 3.4.3 Problems and potential for AMF inoculum production and utilization

 3.5. Inoculant Application

3.6. Preservation and Precautions

4. Phosphate Solubilizers

 4.1. Isolation of Microbial Strains

4.1.1. Mechanisms of phosphate solubilization

4.1.2. Isolation of mineral phosphate solubilizer

4.2. Inoculant Production

4.2.1. Cultivation

4.2.2. Collection

4.2.3. Formulation of inoculants using carrier materials

4.3. Inoculant Application

                 4.3.1. Inoculation method of phosphate solubilizer

4.3.2. Improvement of phosphate solubilizers

4.3.3. Case study of improvement of soil condition for phosphate solubilizers 

                on the fifty-years long-term experiment in rice paddy soils in Korea

V. Quality Control of Biofertilizers

 1. General Concept of Quality Control

 1.1. Definition of

1.2. Microbial Functions Newly Recommended as Biofertilizer

1.3. Properties of Microbial Products

1.4. Quality Management

2. Procedures for Quality Control of Biofertilizer

2.1. Rhizobium

2.1.1. Mother culture test

 2.1.2. Broth test

2.1.3. Peat test