Handbook for Rhizobia

I. General Microbiology of Rhizobia.- 1. Collecting Nodules and Isolating Rhizobia.- 2. Observing the Infection Process.- 3. Cultural Properties, Cell Morphology, and Nutritional Requirements of Rhizobia.- 4. Demonstrating Genetic Diversity in Rhizobia Using Patterns of Carbohydrate Utilization and Intrinsic Antibiotic Resistance.- 5. Quantifying the Growth of Rhizobia.- 6. Counting Rhizobia by a Plant Infection Method.- 7. Counting Serologically Specific Rhizobia in Soil and Peat Inoculants Using Membrane Filters and Immunofluorescence.- Additional References and Recommended Reading.- II. Identification of Rhizobia.- 8. Developing Antisera.- 9. Somatic Agglutination Reactions with Pure Cultures of Rhizobia.- 10. Agglutinating Antigens from Root Nodules.- 11. Performing Rhizobial Antigen-Antibody Reactions by Gel Immunodiffusion.- 12. Determining Strain Occupancy in Soybean Nodules by Gel Immunodiffusion.- 13. Producing and Applying Fluorescent Antibodies.- 14. Identifying Rhizobia by the Indirect Enzyme-Linked Immunosorbent Assay.- 15. Identifying Rhizobia by Immunoblot.- 16. Isolating Spontaneous Antibiotic-Resistant Mutants of Rhizobia.- 17. Analyzing Nodule Occupancy Using Antibiotic-Resistant Markers.- 18. Distinguishing between Strains of Rhizobia by Rhizobiophage Susceptibility.- Additional References and Recommended Reading.- III. Evaluating Symbiotic Potential of Rhizobia.- 19. Testing For Genetic Compatibility between Rhizobia and Legumes.- 20. Screening Rhizobia for Nitrogen-Fixation Potential.- 21. Screening Effective Strains of Rhizobia in Potted Field Soil.- 22. Verifying the Nitrogen-Fixing Potential of Glasshouse-Selected Soybean Rhizobia in the Field Environment.- 23. Evaluating the Symbiotic Potential of Indigenous Rhizobial Populations of Soils Using the Whole-Soil Inocula Technique.- 24. Investigating the Importance of Optimal Soil Fertility in the Response of a Legume to Inoculation with Rhizobia.- Additional References and Recommended Reading.- IV. Inoculant Technology.- 25. Producing Broth Cultures in Simple Glass Fermentors.- 26. Producing Inoculum in a Steel Fermentor.- 27. Preparing a Range of Carrier Materials and Producing Inoculants.- 28. Preparing Inoculants Using Diluted Cultures of Rhizobia and Presterilized Peat.- 29. Testing the Survival of Rhizobia on Inoculated Seeds.- Additional References and Recommended Reading.- V. Genetic Techniques for Rhizobia.- 30. Analyzing Plasmid Profiles of Rhizobium spp. by a Modified Eckhardt Vertical Gel Electrophoresis Procedure.- 31. Isolating and Purifying Genomic DNA of Rhizobia Using a Large-Scale Method.- 32. Isolating and Purifying Genomic DNA of Rhizobia Using a Rapid Small-Scale Method.- 33. Digesting Genomic DNA of Rhizobia with Restriction Endonucleases.- 34. Separating Restriction Fragments of Genomic DNA by Horizontal Agarose Gel Electrophoresis.- 35. Transferring Electrophoretically Separated DNA from Agarose Gels to a Membrane by Southern Blotting.- 36. Preparing a DNA Probe for Detecting the nif Genes on Symbiotic Plasmids of Rhizobium spp.- 37. Incorporating a Nonradioactive Label into a DNA Probe by Nick Translation.- 38. Using a Nonradioactively Labeled nifKDH Gene Probe to Locate Complementary Sequences of Rhizobial DNA Immobilized on Membranes.- Additional References and Recommended Reading.- VI. Appendices.- 1. Characteristics of the Subfamilies of Legumes.- 2. The Nodule Preservation Vial.- 3. Bacterial Growth Media and Plant Nutrient Solutions.- 4. Reagents and Buffers.- 5. Molecular Biology Reagents and Buffers.- 6. McFarland Nephelometer Barium-Sulfate Standards.- 7. Preparing Seedling-Agar Slants and NifTAL-Tubes for Culturing Small-Seeded Legumes.- 8. Building a Rack for Growth Pouches.- 9. Recommendations of Legumes and Growth Systems for Authentication.- 10. Seed Surface Sterilization and Germination.- 11. Preparing Leonard Jars.- 12. Injecting and Bleeding Rabbits.- 13. The Indirect Fluorescent Antibody Technique.- 14. Additional Information on the Plant Infection Count.- 15. The Acetylene Reduction Assay for Measuring Nitrogenase Activity.- 16. Methods for Determining Lime Requirements of Acid Soils.- 17. Analysis of Variance for a Rhizobial Strain Selection Experiment.- 19. Replicators and Microtiter Plates.- 20. Seed Inoculation Procedures.- 21. Determining Field Capacity of Field Soil.- 22. The Simple Transfer Chamber.- 23. Freeze Drying Cultures of Rhizobia.- 24. Source of Rhizobia.- 25. Absorption of Antisera.- Supplemental Reading List.