METHODS IN LEGUME-RHIZOBIUM TECHNOLOGY by P. Somasegaran and H. J. Hoben University of Hawaii NifTAL* Project and MIRCEN* Department of Agronomy and Soil Science Hawaii Institute of Tropical Agriculture and Hunan Resources College of Tropical Agriculture and Human. Resources May, 1985 This document was prepared under United States Agency for International Development (USAID) contract No. DAN-0613-C-00-2064-00 * NifTAL and MIRCEN are acronyms for Nitrogen fixation in Tropical Agricultural Legumes and Microbiological Resources Center, respectively FOREWORD There is no doubt that in the near future the emerging biotechnology based on genetic engineering and somatic cell fusion will contribute significantly to solving agricultural problems. Presently, however, so much of the available technology, i.e., inoculum technology, is not being fully enough utilized in agriculture. It would be prudent to devote major efforts to their adoption. Serious obstacles to adoption of modern technologies, especially in developing countries, is the shortage of trained personnel. It is, therefore, essential for all development support projects to include a training component. This book is the culmination of several years of experience in training of scientists and technicians from developing countries. The six-week Training Course, for which this book is intended, was developed at NifTAL and, in the early years, taught there. Subsequently, the course was taken to the field and offered at host institutions in Africa, Asia and Latin America. Somasegaran and Hoben have done a commendable job of drawing from their experience with these courses. They have compiled an “All You Ever Wanted To Know About …” style book that is not only valuable to developing country scientists, but also useful for technicians and graduate students starting work with the legume/Rhizobium symbiosis. B. Ben Bohlool Professor, University of Hawaii Director, NifTAL project May, 1985 INTRODUCTION The symbiosis between the root-nodule bacteria of the genus Rhizobium and legumes results in the fixation of atmospheric nitrogen in root-nodules. This symbiotic relationship is of special significance to legume husbandry as seed inoculation with effective strains of Rhizobium can meet the nitrogen requirements of the legume to achieve increased yields. Obviously, such a phenomenon is of world-wide interest because it implies lesser dependence on expensive petroleum based nitrogen fertilizers for legumes. In all regions of the world where food consumption exceeds production or where nitrogenous fertilizer has to be imported, leguminous crops have a special relevance. Self- sufficiency for nitrogen supply and the high protein and calorific values of food, forage and feed legumes make them increasingly attractive. Greater use of legumes can have a significant beneficial impact in tropical countries where population increase and food production are most out of balance, and where the purchasing power for imported fertilizers is least adequate. The University of Hawaii NifTAL Project was funded by the United States Agency for International Development to promote greater use of symbiotic nitrogen fixation through Legume-Rhizobium Technology. An essential component in NifTAL’s overall objective was specialized training in Legume-Rhizobium Technology. This strategy would provide the means of transferring the techniques in Legume- Rhizobium Technology for the implementation of viable research and development programs in nitrogen fixation in tropical countries. Training was initiated in 1976 when Professor J.M. Vincent prepared the first course outline for NifTAL. Since then, the authors have conducted similar training courses in Hawaii, Kenya, Malaysia, Mexico, Thailand and India. The valuable experiences gained in these intensive six-week training courses led the authors to identify and develop the key research and development activities essential for Legume-Rhizobium Technology. PREFACE It was in 1970 that Professor J.M. Vincent published his excellent book entitled “A Manual for Practical Study of Root-Nodule Bacteria.” Unfortunately this book is out of print at the present time. The motivation for this book of methods grew out of the ever-increasing role of the Legume-Rhizobium symbiosis in agricultural production in tropical countries where the benefits of this unique symbiosis can only be realized through correct practices in Legume-Rhizobium Technology. This book is designed for the practicing technologist to provide competent technical support to research and development activities relevant to Legume-Rhizobium Technology. Teachers and students will also find this volume useful in addressing the applied aspects of the Legume-Rhizobium symbiosis especially when exercises are supported by well prepared lectures. There are four sections to this book and they related the sequence of activities which should be followed in Rhizobium research. Each exercise is structured to include all the steps required to accomplish the particular experiment. Certain activities in the exercise require a knowledge or a source of information and this is given in the Appendix. At the end of each section are recommended journal articles and textbooks for more background on principles or greater detail on methods. In putting together this book, we have indicated to the user by cross- reference that the various sections support each other. For example, the serological techniques in Section C are not meant only for strain identification in nodules but also for checking strain contamination in inoculant production and quality control. It is hoped that this volume will serve as an instrument of self-instruction since the skills can be acquired by careful practice of techniques. Satisfactory completion of the exercises should impart to the user a good working knowledge and competence in Legume-Rhizobium Technology. The exercises in this volume were tested successfully in all the NifTAL training courses and intern training programs and we hope that this volume will be useful in organizing similar courses by other institutions. The authors would appreciate comments and suggestions for effecting further revisions to improve this volume. Padmanabhan Somasegaran Heinz J. Hoben May 1985 ACKNOWLEDGEMENTS The authors are indebted to several colleagues who are specialists and who contributed constructively in the review of preliminary versions of this volume. We gratefully acknowledge the support of professor J.M. Vincent who reviewed and supplied us with detailed notes and comments for each exercise. We are indebted to him as the outline of this volume is based on a course outline originally conceived by him while he was a consultant to the NifTAL Project. The authors express their sincere thanks to Dr. J.C. Burton for his encouragement and support. His review, comments, and suggestions especially in Section D were invaluable. We wish to express our gratitude to Dr. J. Halliday for his continued interest, encouragement and support in the preparations of this volume. The authors are indebted to the invaluable review support given by Drs. P.H. Graham, D.H. Hubbell, B.B. Bohlool, P.W. Singleton, J.W. King, R.W. Weaver, J.A. Thompson and L.A. Materon. The authors wish to express their sincere gratitude to Dr. Velitin Gurgun of the University of Ankara (Turkey) who did an excellent review on the final version of this volume. We appreciate all participants of the NifTAL Training courses, intern trainees, and visiting scientists whose comments and suggestions were invaluable in making this work more comprehensive. In the final analysis, the production of this volume was the result of teamwork and support given to us by several NifTAL Project staff members. We express our sincere appreciation to all these helpful people. They are Ms. Princess Ferguson who handled all logistics pertaining to publication besides editing; Ms. Judith Dozier for the painstaking checking, proofreading and editing of the typed manuscripts; Mr. Keith Avery who patiently handled much of the graphics and artwork; and Ms. Karean Zukeran and Ms. Mary Rohner for the administrative and secretarial support. CONTENTS Introduction. 1 Exercise 1. TO COLLECT NODULES AND ISOLATE RHIZOBIUM. 7 a. Recognizing legumes and identifying them in the field b. Recovering nodules in the field c. Preserving nodules d. Examining nodules and bacteroids e. Isolating Rhizobium from nodule f. Performing the presumptive test g. Authenticating the isolates as Rhizobium h. Preserving Rhizobium cultures Requirements Exercise 2. TO OBSERVE THE INFECTION PROCESS. 30 a. Culturing strains of rhizobia in YM broth b. Germinating seeds c. Preparing a Fahraeus-slide d. Inoculating the seedlings e. Observing the root-hairs under the microscope f. Comparing root hair deformations Requirements Exercise 3. TO STUDY CULTURAL PROPERTIES, CELL MORPHOLOGICAL CHARACTERISTICS AND SOME NUTRITIONAL REQUIREMENTS OF RHIZOBIUM. 40 a. Preliminary subculturing of different bacterial cultures b. Comparing cell morphology and gram stain reactions of Rhizobium with those of other microorganisms c. Determining gram stain reactions of various bacteria d. Characterizing growth of rhizobia using a range of media e. Observing growth reactions on modified media Requirements Exercise 4. TO QUANTIFY THE GROWTH OF RHIZOBIUM. 53 a. Preliminary culturing of fast and slow- growing rhizobia b. Determining the total count with a Petroff- Hausser chamber c. Using the Petroff-Hausser counting chamber d. Estimating cell concentration