EVALUATION OF SYMBIOTIC EFFECTIVENESS OF RHIZOBIA (Bradyrhizobium spp L.) WITH GROUNDNUT (Arachis hypogaea L.

) IN EASTERN HARERGHE ZONE OF OROMIYA REGIONAL STATE, ETHIOPIA

MSC THESIS

AYELE AKUMA

DECEMBER 2010
HARAMAYA UNIVERSITY

Evaluation of Symbiotic Effectiveness of Rhizobia (Bradyrhizobium spp L.) with Groundnut (Arachis hypogaea L.) in Eastern Harerghe Zone of Oromiya Reginal State, Ethiopia

A Thesis Submitted to the School of Natural Resource Management and Environmental Science, School of Graduate Studies HARAMAYA UNIVERSITY

In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE (SOIL SCIENCE)

By Ayele Akuma

December 2010 Haramaya University

SCHOOL OF GRADUATE STUDIES HARAMAYA UNIVERSITY

As thesis research advisor, I hereby certify that I have read and evaluated the Thesis entitled Evaluation of Symbiotic Effectiveness of Rhizobia (Bradyrhizobium spp L.) with Groundnut (Arachis hypogaea L.) in Eastern Harerghe Zone of Oromiya Reginal State, Ethiopia prepared under my guidance by Ayele Akuma, and recommend that it be submitted as fulfilling the thesis requirement.

L.M. Pant (PhD) Name of Major Advisor Heluf Gebrekidan (PhD) Name of Co-Advisor

____________________ Signature ____________________ Signature

___________________ Date ___________________ Date

As members of the Board of Examiners of the MSc Thesis Open Defense Examination, we certify that we have read and evaluated the Thesis prepared by Ayele Akuma and examined the candidate. We recommend that the Thesis be accepted as fulfilling the requirement for the degree of Master of Science in Agriculture (Soil Science).

________________________ Name of Chairman ________________________ Name of Internal Examiner ________________________ Name of External Examiner

___________________ Signature ___________________ Signature ___________________ Signature

____________________ Date ____________________ Date ____________________ Date

Final approval and acceptance of the Thesis is contingent upon the submission of the final copy to the Council of Graduate Studies (CGS) through the department graduate committee (DGC) of the candidates major department.

DEDICATION

I dedicate this thesis manuscript to my Father, Akuma Aga, and my wife, Manalush Worku, for nursing me with affection and love and for their dedicated partnership in the success of my life.

ii

STATEMENT OF THE AUTHOR

First, I declare that this thesis is my bonafide work and that all sources of materials used for the thesis have been duly acknowledged. This thesis has been submitted in partial fulfillment of the requirements for an MSc degree at the Haramaya University and is deposited at the University Library to be made available to borrowers under rules of the Library. I solemnly declare that this thesis is not submitted to any other institution anywhere for the award of any academic degree, diploma, or certificate. Brief quotations from this thesis are allowable without special permission provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of school of Natural Resource Management and Environment Science or the Dean of the School of Graduate Studies when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

Name: Ayele Akuma Place: Haramaya University, Haramaya Date of Submission: ______________

Signature: ______________

iii

BIOGRAPHICAL SKETCH
The author was born on November 22, 1982 at Dibatie town in Metekel Zone of Benishangul Gumuz National Regional State. He attended his primary education at Addis Alem, his junior secondary school education at Dibatie Junior Secondary School and his Senior Secondary School at Dibatie and Pawe Comprehensive Secondary School from 1990-2003. Following the completion of his high school education, he joined the then Alemaya University, now Haramaya University on September 2003 to pursue his tertiary education and graduated with a BSc degree in Agriculture (Crop Production and Protection) in July, 2006. Right after graduation, he was employed by the Haramaya University and served as graduate assistant until the year 2008, and then joined the School of Graduate Studies of the same University to study for his Master of Science degree in Agriculture (Soil Science) on October 2008.

iv

ACKNOWLEDGMENTS
The author is highly thankful to his instructors and advisors, the Late Prof. L.M. Pant and Prof. Heluf Geberkidan, for their helpful and endless supports, intellectual guidance, and critical suggestions, which were instrumental in planning and implementation of the research work and production of this thesis in its present form. The author also gratefully acknowledges Mr. Anteneh Argaw for his annotation, technical guidance during bacterial isolation work and facilitating other field works. The author extends his gratitude to the Haramaya University and the MOE for providing financial support for his study including research work and for facilitation of laboratory as well as field works. He whole heartedly, acknowledges staffs of the School of the Natural Resource Management and Environmental Science, for various kinds of supports rendered to him. His special thanks go to Rahel Berhanu and Ferezewed Feleke for their technical supports, the Head of Natural Resource Management Department, Dr. Lisanework Nigatu, and Head of the then Crop Production and Protection Department, Dr. Mashilla Dejene, for encouraging him during his study. The author extends his special thanks to his wife, Manalush Worku and to his child Kalkidan and heartfelt thanks to his parents for helping him during his study. His special attribute goes to his friends, and those not mentioned by name for their valuable encouragement and moral supports. The author would like to thank his colleagues for their valuable discussions and motivation from the beginning of proposal preparation to the final thesis write-up. Above all, the author is grateful to his almighty God who has given him the power to accomplish this piece of work.

LIST OF ACRONYMS AND ABBREVIATIONS

AvP BSc BNF C/N CEC CSA CV CRD CGS DGC d EC e e.g. FAO HUGR ICARDA ICRISAT m masl MOE MPN NDW NN NR OC OM OSSEP PGA PGA-BCP PN SDW SEM SE UK USA US TN VE v v/v w/v Available Phosphorus Bachelor of Science Biological Nitrogen Fixation Carbon to Nitrogen Ratio Cation Exchange Capacity Central Statistical Authority Coefficient of Variation Complete Randomized Design Council of Graduate Studies Department Graduate Committee Lowest Dilution Electrical Conductivity Electron Example Food and Agriculture Organization of the United Nation Haramaya University Groundnut Rhizobia International Center for Agricultural Research in the Dry Areas International Crops for Research Institute for the Semi-Arid Tropics Likely Number from Table for the Lower Dilution of the Series Meters Above Sea Level Ministry of Education Most Probable Number Nodule Dry Weight Nodule Number Nodule Ratings Organic Carbon Organic Matter Oromia State Socio Economic Profile Peptone Glucose Agar Peptone Glucose Agar-0.25% Bromocrosol Purple Peg Number Shoot Dry Weight Standard Error of Mean Symbiotic Effectiveness United Kingdom United States of America United State Total Nitrogen Very Effective Volume of Aliquot Applied to Plant Volume per Volume Weight per Volume

vi

YEMA YEMA-BTB YEMA-CR YEMB

Yeast Extract Mannitol Agar Yeast Extract Mannitol Agar-0.25% Bromothymol Blue Yeast Extract Mannitol Agar-0.25% Congo Red Yeast Extract Mannitol Broth

vii

TABLE OF CONTENTS

STATEMENT OF THE AUTHOR BIOGRAPHICAL SKETCH ACKNOWLEDGMENTS LIST OF ACRONYMS AND ABBREVIATIONS LIST OF TABLES LIST OF FIGURES LIST OF TABLES IN THE APPENDIX LIST OF FIGURES IN THE APPENDIX ABSTRACT 1. INTRODUCTION 2. LITERATURE REVIEW 2.1. General Description Legumes 2.2. Description of Groundnut 2.3. Symbiotic Rhizobium-Legume Nitrogen Fixation 2.4. Taxonomy and Host Specificity of Rhizobia 2.5. Significance of Biological Nitrogen Fixation 2.6. The Process of Nodulation and Fixation 2.6.1. The mechanism 2.6.2. Recognition between symbiotic partners 2.7. Characteristics of Nodules 2.7.1. Shape, size and number 2.7.2. Structure and function of nodule 2.8. Factors Affecting Symbiotic Nitrogen Fixation 2.8.1. Soil reaction (pH) 2.8.2. Mineral nutrient status 2.8.3. Photosynthesis 2.8.4. Legume management 2.8.5. Climate 2.9. Response of Groundnut to Inoculation 3. MATERIALS AND METHODS 3.1. Description of the Study Area, Site Selection and Sampling 3.1.1. Description of the study area 3.1.2. Site selection and sampling procedure 3.2. Isolation of Rhizobium Strains 3.3. Purification and Preservation of the Isolates 3.4. Characterization of Isolates 3.5. Physiological Tests 3.6. Enumeration of Rhizobia 3.7. Evaluation of Symbiotic Effectiveness of Groundnut Rhizobia 3.7.1. Effectiveness of isolates on sterilized sand 3.7.2. Evaluation of isolates in two unsterilized soil 3.8. Statistical Analysis

iii v vi vii x xi xii xiii xiv 1 5 5 5 6 8 10 12 12 13 13 13 14 15 15 16 17 17 18 18 21 21 21 22 22 24 24 25 25 26 26 28 29

viii

TABLE OF CONTENTS (Continued)

4. RESULTS AND DISCUSSION 4.1. Presumptive Tests of Isolates 4.2. Morphology and Cultural Characteristics 4.3. Evaluation of Symbiotic Effectiveness on Sand Culture 4.3.1. Peg number per plant 4.3.2. Nodule number per plant 4.3.3. Nodule dry weight per plant 4.3.4. Shoot dry weight per plant 4.3.5. Symbiotic effectiveness 4.3.6.Correlation analysis for the selected parameter on sand culture 4.4. Physiological Characterization 4.4.1. Temperature tolerance 4.4.2. Salt tolerance 4.4.3. Tolerance to pH 4.5. Soil Properties and Enumeration of Rhizobia 4.6. Symbiotic Effectiveness of Selected Isolates on Fedis and Babile Soils 4.6.1. Peg number per plant 4.6.2. Nodule number per plant 4.6.3. Nodulation ratings per plant 4.6.4. Nodule dry weight per plant 4.6.5. Shoot dry weight per plant 4.6.6. Total nitrogen percent and content per plant 4.6.7. Correlation of some selected parameters on Babile and Fedis soils 4.6.8. Leaf and nodule color assessment 5. SUMMARY AND CONCLUSIONS 6. REFERENCES 7. APPENDICES 7.1. Appendix Tables 7.2. Appendix Figures

31 31 31 33 33 33 34 36 37 37 37 37 38 40 40 43 43 43 46 48 49 51 52 53 54 57 67 68 72

ix

LIST OF TABLES
Table 1. 2. 3. 4. 5. 6. 7. 8. Description of soil sampling sites Chemical compositions of N-free nutrient solutions Colony characteristics and presumptive test of isolates after 5-7 days of incubation Nodulation and symbiotic effectiveness of isolates tested on groundnut in sand culture Physiological (temperature. salt and pH tolerance) characterization of the isolates Chemical and physical properties of Babile and Fedis soils Nodulation data of selected effective isolates of groundnut rhizobia on Fedis soil Nodulation data of selected effective isolates of groundnut rhizobia on Babile soil Page 23 27 32 35 39 42 45 47

LIST OF FIGURES
Figure 1. Location map of the study area Page 21

xi

LIST OF TABLES IN THE APPENDIX

Appendix Table Page

1. 2. 3. 4. 5. 6. 7. 8. 9.

Analysis of variance for nodulation parameters on sand experiment Scale of nodule and leaf color on sand culture just before harvest Nodulation and dry matter accumulation of groundnut on sand culture and correlation among parameters Nodulation of groundnut plant during MPN determination in Fedis and Babile soils Analysis of variance for the different parameters of Fedis soil culture Nodulation and dry matter accumulation of groundnut on Fedis soil and correlation among parameters Scale of leaf and nodule color on Fedis and Babile soil culture just before harvest Analysis of variance for the different parameters on Babile soil culture Correlation coefficient of selected parameters on Babile soil experiment

68 68 69 69 70 70 71 71 72

xii

LIST OF FIGURES IN THE APPENDIX

Appendix Figure Page

1. 2.

Stand of groundnut inoculated with different isolates on sand culture Performances of inoculated and uninoculated groundnut seedlings on sand culture in greenhouse condition

72 73

xiii

EVALUATION OF SYMBIOTIC EFFECTIVENESS OF RHIZOBIA (Bradyrhizobium sp L.) WITH GROUNDNUT (Arachis hypogaea L.) IN EASTERN HARERGHE ZONE OF OROMIYA REGIONAL STATE, ETHIOPIA By
Ayele Akuma (BSc), Haramaya University, Ethiopia Advisors: Prof. L.M. Pant (PhD), G.B. Pant University of Agric. & Technology, India Prof. Heluf Gebrekidan (PhD), University of Arizona, USA

ABSTRACT
A way of improving the success of inoculants can be to use native strains that are effective as well as competitive for nodulation as inoculants. Therefore, this study comprised isolation, characterization, selection and evaluation of potential indigenous rhizobial isolates for effective symbiosis with groundnut under sterilized sand and unsterilized different soils in greenhouse condition. Soil samples and nodules were collected from the major groundnut producing areas of Fedis, Babile and Gursum Districts of East Harerghe Zone. Rhizobia were isolated using plant Induction following the standard procedures. Experiments were arranged in CRD with three replications and two control units (positive and negative). Data on peg number, nodule number, nodulation ratings, nodule dry weight, shoot dry weight and total N-content were subjected to statistical analysis. The results of presumptive test revealed that all the isolates were gram negative, rod-shaped and milky color on YEMA-CR media without absorbed congo-red under dark condition. Similarly, no isolate grew on PGA-BCP media. All isolates turned YEMA-BTB medium into moderately deep blue color, and showed small dry and large mucoid, a buttery texture, raised and circular margin, with colony diameter of <1 to 2.5 mm on YEMA medium incubated at 28 2 oC. All isolates were authenticated as root nodule bacteria. Moreover, isolates were significantly (P < 0.05) superior to the negative control in terms of peg and nodule numbers and nodule and shoot dry weight plant-1. The SE result showed that 37.5 and 62.5% of the isolates were found to be highly effective and effective, respectively. Almost all of the selected isolates grew between 15 0 C and 40 0C and failed to grow at 4 and 10 0C. All isolates selected failed to grow at 1% NaCl, except HUGR18. The isolates also grew on a wide range of neutral to alkaline. The top ten SE scores (76-116%) were the isolates HUGR (3, 10, 11, 12, 13, 16, 18, 19, 22 and 24). Isolate HUGR22 had the highest symbiotic performed (more than 100%) on the Fedis soil and HUGR18 was the lower scorer from top ten on the sand culture. The inoculation results of the above ten isolates on both soils, having 2.6 x 102 and 3.3 x 103rhizobia g-1 of Fedis and Babile soil, respectively, but most of the parameter such as nodule number, nodule dry weight, shoot dry weight and N content significantly increased as compared to the negative control for most of the isolates for both soil. Furthermore, isolates HUGR (3, 12 and 22) performed well compared to other isolates in all experiments. The poor nodulation of groundnut on the Fedis soil indicates that the soil-related factors severely affected survival, nodulation and symbiotic nitrogen fixation of the indigenous rhizobia and the process can be rectified by inoculation of effective rhizobia.

xiv

1. INTRODUCTION
Groundnut (Arachis hypogaea L.) is an annual legume which is also known as peanut, earthnut, monkeynut and goobers. It is the 13 th most important food crop and 4th most important oilseed crop of the world. Groundnut seeds (kernels) contain 40-50% fat, 20-50% protein and 10-20% carbohydrate. Groundnut seeds are nutritional sources of vitamin E, niacin, falacin, calcium (Ca), phosphorus (P), magnesium (Mg), zinc (Zn), iron (Fe), riboflavin, thiamine and potassium (K). Groundnut kernels are consumed directly as raw, roasted or boiled kernels or oil extracted from the kernel is used as culinary oil. It is also used as animal feed (oil pressings, seeds, green material and straw) and industrial raw material (oil cakes and fertilizer). These multiple uses of groundnut plant make it an excellent cash crop for domestic markets as well as for foreign trade in several developing and developed countries (Chaudhary, 1986).

Cultivated groundnut originated from South America (Weiss, 2000). It is one of the most popular and universal crops cultivated in more than 100 countries in six continents (Nwokoto, 1996). It is grown in 25.2 million hectares (ha) with a total production of 35.9 million metric tons (FAO, 2006). Its cultivation is mostly confined to the tropical countries ranging from 40 N to 40 S latitudes. Major groundnut producing countries are China (40.1%), India (16.4%), Nigeria (8.2%), USA (5.9%) and Indonesia (4.1%).

The cultivated groundnut is one of the world's most important legume crops and is primarily grown in tropical and subtropical areas (Lemon et al., 2000). It is currently grown in Ethiopia with annual nationwide planted acreage coverage during the 2008/2009 of 41,761 ha with a production of 468,872 quintal. The average yield was estimated to be 11.23 quintal per ha (CSA, 2009). The crop is relatively new to Ethiopia. It was introduced from Eritrea to Hararghe in the early 1920s by the Italian explorers (Daniel, 2009). Major groundnut

producing areas in Ethiopia are Babile, Beles, Didessa, Gambella and Pawe. Gamu Gofa, Illubabor, Gojam, Wello and Wellega are identified as potential production areas (Daniel, 2009).

Average productivity of groundnut (seed in shell) in the world is about 13.1 quintals/ha. In USA, where most of the cultivation is commercial, an average yield of 26.3 quintals/ha is obtained and pod yields in excess of 4050 quintals/ha are not uncommon. Average productivity at the subsistence system in Africa and Asia, however, remains very low. In addition, a big gap exists between the realized yield and potential yield of groundnut at both subsistence and commercial systems. Several abiotic and biotic factors limit the realized yield of groundnut at the farm level. Genetic improvement and improved management practices can help bridge this gap (ICRISAT, 1995).

Groundnuts grow best on soils that are well drained, light textured and well supplied with Ca, K and P. The soil should be well aerated and contain moderate amounts of organic matter (OM). Heavier clay soils or those that tend to have surface crusting are unsuitable due to their high resistance to peg penetration and pod expansion (Frederich et al., 1991). Groundnut grows best in slightly acidic soils with a pH of 6.0 to 6.8 but a range of 5.5 to 8.0 is acceptable. Saline soils are not suitable since groundnut has a very low salt tolerance. In Ethiopia, groundnut is planted with the onset of Belg rain. At Babile, Fedis and Gursum; planting date depends on the onset of the rain but mid April is the right planting time. In some parts of western Ethiopia, it is also planted till mid June. Research findings indicate that mid May to mid June is the appropriate planting time for middle Awash (Daniel, 2009).

Legumes, because of their high protein content, require large amounts of nitrogen (N) to produce good yield. Groundnut being a legume is capable of obtaining its N requirements from both symbiotic fixations by root nodules and soil N. The source for the reduction of N (N-fixation) is gaseous N2, while soil N is absorbed mainly as nitrate (NO3) N. Groundnut genotypes need approximately 1 kg of assimilated N to produce around 36 kg biomass, in contrast to cereals such as sorghum, that can produce as much as 120 kg biomass kg -1 assimilated N (Nambiar, 1986). This large amount of N is supplied to the groundnut plant mainly by its root nodules. Available N in soils is at its highest level soon after fertilizer application, and it decreases thereafter, depending on such factors as plant uptake, leaching, mineralization, and nitrification. In contrast, symbiotic N2 fixation is a part of the plants

metabolism and, if well established, the nodules supply the plant with a regulated and continuous supply of N, depending on its growth stage.

The groundnut plant can obtain much of its N requirement through symbiotic N2 fixation when grown in association with effective and compatible Bradyrhizobium strains (van Rhijn and Vanderieyden, 1995). The multi-step symbiotic process between rhizobia and leguminous plants, under N limitation, leads to the development of N2-fixing nodules that are formed on the roots (Schultze and Kondorosi, 1998). These unique structures are agronomically significant, as they provide an alternative to the use of energy expensive ammonium fertilizer (Fisher and Long 1992; Sessitsch et al. 2002).

In agricultural systems, symbiotically fixed N can be an immediate source of N to the fixing species for dry matter and seed production and released from the fixing species to companion crops to supplement their N needs and useful as a green manure providing N to crops grown in rotations (Peoples et al., 1995). The inherent capacity for N2 fixation by the legume rhizobial symbiosis is a mainstay in cost-effective, ecologically sound approaches to sustainable agricultural practices. The expanded interest in ecology has drawn attention to the fact that biological N fixation (BNF) is ecologically benign and that its greater exploitation can reduce the use of fossil fuels and can be helpful in reforestation and in restoration of misused lands to productivity (Burris, 1994). Currently, the subject of BNF is of great practical importance because the use of nitrogenous fertilizers has resulted in unacceptable levels of water pollution (increasing concentrations of toxic nitrates in drinking water supplies) and the eutrophication of lakes and rivers (Al-Sherif, 1998). Further, while BNF may be tailored to the needs of the organism, fertilizer is usually applied in a few large doses, up to 50% of which may be leached (Burris, 1994). This not only wastes energy and money but also leads to serious pollution problems, particularly in water supplies.

A way of improving the success of inoculants can be to use native strains that are effective as well as competitive for nodulation as inoculants. As step to increase groundnut yield as well as to improve soil N status in Ethiopia, use of a very effective indigenous rhizobial strain as an inoculants is vital. At present, we lack adequate information on the diversity, symbiotic

effectiveness, as well as the competitiveness for nodule occupancy of this native population of rhizobia (Vlassak and Vandurleyden, 1997).

Most farmers of East Hararghe are engaged in cultivation of groundnut as monocropping. Groundnut is grown by most of the farmers as cash and food crop of interest. The yield is extremely low due to low soil fertility, smallholder farming and limited access to external inputs. One of the most important factors of soil fertility is N deficiency of most Ethiopian soils (Desta and Angaw, 1986). Groundnut and other legume crops were not usually introduced as intercrops between others except at Fedis area because growers were not well aware of the benefits of using legumes in crop production system. In Ethiopia, the work on the nodulation status and N fixation potential of legumes is very scarce and has concentrated on highland pulses such as peas, beans, chickpeas, and lentils (Fassil, 1993). So far, works have not been reported on symbiotic effectiveness concerning groundnut of Ethiopia. Even if agricultural professionals and extension workers of the area were aware of the fact that groundnut and other legumes form symbiosis for N fixation, there had not been any attempt to isolate, select and evaluate potentially effective rhizobia for groundnut and popularization of inoculation practices.

Havlin et al. (2003) stated that a response to compatible native or inoculated rhizobia depends on soil physical, chemical, biological and past management practices. Therefore, this study was proposed to select and evaluate potential indigenous rhizobial isolates for effective symbiosis with groundnut in different soil types under greenhouse conditions.

2. LITERATURE REVIEW
2.1. General Description of Legumes
Legumes are dicotyledonous plants categorized into the third largest family of flowering plants, the family leguminosae. They are found in various behaviors of herbs, shrubs and trees. The family leguminosae is estimated to contain 18,000-19,000 species in about 750 different genera (Allen and Allen, 1981) and divided into three subfamily; the Papilionoideae (pea-like flowers), the Mimossoideae (compound inflorescences with reduced petals) and the Caesalpinioideae (flowers usually with five petals apparently radially symmetrical) (Polhill and Raven, 1981), of which 500 genera and approximately 10,000 species belong to the subfamily Papilionoideae. All legumes do not bear nodules on their root system and it is known that certain tree forms do not possess them at all. Hardly, 16% have so far been examined for nodulation of which 95% of Mimosoideae, 26% of Ceasalpinioideae and 90% of Papilionoideae (Subba Rao, 1999).

The Mimosoideae and Caesalpinioideae are almost completely restricted to the tropics, whereas Papilionoideae contains the majority of the most important legumes (Sprent and Raven, 1992). The majority of the latter contains herbaceous plants that include the genera Arachis, Lotus and Vicia (Rendle, 1979). Most of the genera in this subfamily are nodulated. The seeds, rich in starch and proteins, are a good source of food, as in the various beans, peas, vetches, lentils and others (Rendle, 1979).

2.2. Description of Groundnut

The groundnut or peanut (Arachis hypogaea) is a tetraploid species within a predominantly diploid South American genus which has yet to be fully described (Smartt, 1990, 1994). Three main types of groundnut are recognized: the erect, bunch Spanish with compound inflorescences; species fastigiata var. vulgaris, Valencia with simple inflorescences; species fastigiata var. fastigiata types and the more spreading Virginia type; species hypogaea (Smartt, 1994). The Virginia type can be further subdivided into Virginia Bunch; var. hypogaea and Virginia Runner; var. hirsuta.

Over 90% of the world groundnut crop is produced in developing countries and roughly twothirds of this is used for oil making and it is the second most important source of vegetable oil after soybean (Freeman et al., 1999). It is also important as a subsistence food crop throughout the tropics and although groundnut is principally a warm-temperature crop, varieties exist that are adapted to altitudes of 1500 masl. Groundnut is generally nodulated by Bradyrhizobium strains (Urtz and Elkan, 1996) and has an unusual mechanism of infection. Groundnut is an annual herbaceous plant that grows to a maximum height of 60 cm. It is characterized by bearing of fruits that develop and mature underground. Fertilization of the ovary results in the development of an elongated stalk (peg) which grows downwards and carries the ovary into the soil to a depth of 2-7 cm. Pegs can attain a length of 15-30 cm. Once penetration of the soil surface has occurred, fruit enlargement proceeds at the peg tip with eventual formation of the groundnut pod. Pods can contain 1-5 seeds (Frederich et al., 1991).

The groundnut, grown mainly for human consumption, has several uses as whole seeds or processed to make groundnut butter, oil and other products. The seed contains 25- 30% protein (average of 25% digestible protein) and 42-52% oil (Freeman et al., 1991). One kilogram of groundnuts is high in food energy and provides approximately the same energy value as two kilograms of beef, 1.5 kg of cheddar cheese, nine liters of milk or 36 medium size eggs (Frederich et al., 1991). Groundnut kernels are widely consumed as snack food and can be processed in a variety of ways (e.g. groundnut butter, roasted, fried and salted).

2.3. Symbiotic Rhizobium-Legume Nitrogen Fixation

Organisms that can x N, i.e., convert the stable N gas (N2) in the atmosphere into a biologically useful form; all belong to a biological group known as prokaryotes. All organisms which reduce N2 to ammonia do so with the aid of an enzyme complex, nitrogenase. The nitrogenase enzymes are irreversibly inactivated by oxygen, and the process of N fixation uses a large amount of energy (Postgate, 1982). A wide range of organisms have the ability to x N. However, only a very small proportion of species are able to do so; about 87 species in 2 genera of archaea, 38 genera of bacteria, and 20 genera of cyanobacteria have

been identied as diazotrophs or organisms that can x N (Sprent and Sprent, 1990). This wide variety of diazotrophs ensures that most ecological niches will contain one or two representatives and that lost N can be replenished. Root-nodule-based N fixing associations include Rhizobium-legume and Actinorhizobium symbiosis. These interactions between diazotrophic bacteria and plants are major biological contributors of fixed N in soil-based ecosystems. The predominance of this fixed N source results from the agricultural exploitation of Rhizobium-legume symbioses and the advantage conferred on these symbiotic diazotrophs by the ready availability of plant produced photosynthate. That is, N fixation is energy-intensive process and those heterotrophic organisms living in the legume or Actinorhizobium nodule have more energy available to devote to N fixation than do diazotrophs living non-associated or loosely linked with plants (Tate III, 2000).

Bacteria belonging to the genus rhizobia live freely in soil and in the root region of leguminous and non-leguminous plants. However, they can enter in to symbiosis only with leguminous plants, by infecting their roots and forming nodules on them. In legume-root nodule symbiosis, the legume is the larger partner while the rhizobia are the smaller partner, often referred to as the microsymbiont (Subba Rao, 1999).

Due to the extensive cultivation of legumes, the greatest documented contribution of fixed N in to land-based systems results from the infection of legume root by species of the bacterial genera Rhizobium and Bradyrhizobium. A tremendous potential for contribution of fixed N to soil ecosystems exists among the legumes. Estimates are that the Rhizobium symbiosis with the somewhat greater than 100 agriculturally important legumes contributes nearly half the annual quantity of biologically fixed N entering the soil ecosystems (Tate III, 2000).

As indicated by Hardarson (1993), legume species and their microsymbioants do have different effectiveness. The study shows faba bean (Vicia faba), lupin (Lupinus spp.) and pigeon pea (Cajanus cajan) are found to be very efficient; soyabean (Glycin max), groundnut (Arachis hypogae) and cowpea (Vigna unguculata) to be average; and common bean (Phaseolus vulgaris) and pea (Pisum sativum) are poor in fixing atmospheric N. Hardarson (1993) stated that there are great differences among the grain legume species, with groundnut

being intermediate, common bean rather poor and others very effective. However, forage or pasture legumes are usually more efficient in N fixation and derive high percentage of their N from the atmosphere. Hence, it will be possible to enhance the efficiency of the genotypically poor ones but more difficult for those plants that derive a high proportion of total N from the atmosphere.

2.4. Taxonomy and Host Specificity of Rhizobia

Rhizobia are genetically diverse and physiologically heterogeneous group of bacteria that were originally classified together with their nodulating members of leguminosae (Somasegaran and Hoben, 1994). Morphologically, they are medium-sized, rod-shaped cells, 0.5-0.9 mm in width and 1.2-3.0 mm in length. They are gram-negative, motile by a single polar filagellum or six peritrichous flagella. Rhizobia are predominantly aerobic chemoorganotrophs and are relatively easy to culture. They grow well in the presence of O 2 and utilize relatively simple carbohydrates and amino compounds. Optimal growth of most strains occurs at a temperature range of 25-30 0C and pH of 6.0-7.0 (Somasegaran and Hoben, 1994).

Until the early 1980s, all symbiotic N fixing bacteria from leguminous plants were classified in the single genus Rhizobium. Six species were identified in to R.

leguminosarum, R. meleloti, R. trifolii, R. phaseoli, R. Lupine and R. japonicum based on their cross-inoculation groups with pea, alfalfa, clover, bean, lotus, and soybean, respectively. Taxonomy based on the concept of cross-inoculation groups failed because of the many exceptions to this rule. It was also widely recognized that rhizobial classification should adjust to general bacterial taxonomy and include a panel of genomic, phenotypic and phylogentic features instead of the sole nodulation properties (Zakhia and de Lajudie, 2001).

Differences in rates of growth allowed early separation of rhizobia into two basic groups, fast growers and slow growers. Fast growers have generation times of less than 6 hours and generally forms visible colonies (2-4 mm in diameter) on agar media within 2-5 days; whereas slow growers have generation times exceeding 6 hours and give detectable

growth after more than 5 days under standardized conditions. Most of the slow growing rhizobia are produced alkali reaction (blue color) while fast growers produce acid reaction (yellow color) (Jordan, 1984).

According to the current classification rhizobia belong to the alpha subdivision of protobacteria, that were first classified into two genera, the genus Rhizobium including the fast growing strains and the new genus Bradyrhizobium, created for the slow growing ones (Jordan, 1984). Since, isolation of rhizobia from an increasing number of plant species around the world and their characterization by modern polyphasic taxonomy has necessitated the description of additional new genera and species. A total of 6 genera; Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium, Allorhizobium and 28 species have been recognized (Zakhia and de Lajudie, 2001). Associated with legume host, rhizobia is indispensable in biological nitrogen fixation. Although fast-growing rhizobia have been discovered, slow-growing bradyrhizobia are predominant population in soybean and peanut rhizobia. Strains of bradyrhizobia have miscellaneous host specificity and outstanding ecological adaptability because they not only inhabit in soil and rhizosphere but also can inhabit aquatic ecosystems and nodulating Aeschynomene species (Willems et al., 2000). They can nodulate legumes, nonlegume Parasponia andersonii as the nitrogen fixation endosymbionts Han et al. (2005) or even in rice as the endophytic bacteria (Chaintreuil et al., 2000).

Until present, six Bradyrhizobia species have been identified (Vinuesa et al., 2005). Among them, Bradyrhizobium japonicum, Bradyrhizobium elkanii, and Bradyrhizobium liaoningense have been originally isolated from soybean. The reported rhizobia nodulating on peanut are all slow-growing bradyrhizobia and described as Bradyrhizobium spp. However, some peanut bradyrhizobia have been identified, others are still uncharacterized (Yang et al., 2005).

10

2.5. Significance of Biological Nitrogen Fixation

Nitrogen is an essential element for plant growth and reproduction. Lack of mineral N in the soil often limits plant growth (Trevaskis et al., 2002). The atmosphere contains about 1015 tons of N2 gas, and the nitrogen cycle involves the transformation of 3 x 109 tons of N2 per year on global bases (Posgate, 1982). However, N2 fixation is not exclusively biological, lightning probably accounts for about 10% of the worlds supply of fixed nitrogen (Sprent and Sprent, 1990). FAO (1990) reported that world production of fixed N in the

form of chemical fertilizers accounts for about 25% of the earths newly fixed N and biological processes accounts for about 60%. The need of plants for N fertilizer is relatively immense. For successful production of crops, the soil should be fertile enough to meet the demands of these crops. For fulfillment of this need, the contribution of biological N fixation (BNF) should not be over looked, because N fixation by microorganism is the major source of soil N contributing to as much as 60% of total N content (Subba Rao, 2001).

Subba Rao (2001) stated that organic form of N is the major components of living organisms and it is the most abundant elements in the atmosphere. However, it is not available to most organisms because of their inabilities to use it in the elemental form. He further reported that only a few groups of bacteria have the ability to combine it with other elements and use it directly and all other organisms depend on combined forms of N, mostly produced by these bacteria.

Experiments in various countries showed that introduction of legumes which form associations with some bacteria in the cropping systems could significantly maximize yields of the legume crops. Subba Rao (2001) reported that experiment at Rothamsted Experiment Station in UK, extending over a century showed that in a wheat field which was regularly weeded, production was only 2 tons per ha, while in a field left without weeding, it was 4 tons, indicating a gain of N by the wheat crop from the weeds which are mainly legumes.

Due to the inevitable losses of fixed N from all soils, some external involvement is demanded to enhance the fixation process in the soil. It can be stated that a portion of the foundation for

11

sustained productivity of soil-based systems is the insurance of renewal of the fixed N pool through existence of a functional N-fixing microbial population in situ. Fortunately, for the development of high productive, sustainable terrestrial systems fixed N inputs do balance losses (Robert and Claudia, 2000). Biological N fixation ensures an environmentally friendly N supply system for plant growth. Robert and Claudia (2000) declared that although intensive agricultural systems are usually sustained through liberal use of industrially fixed N, economic and environmental pressure dictate reduction fertilizer use and maximization of in situ BNF.

The legume BNF is responsible for the better performance of succeeding crops in the field. Van Slyke (2001) reported that the practical experience of hundreds of years led farmers to believe that leguminous crops (clovers, alfalfa, beans, peas, etc) possess some peculiar power to make succeeding crops give better yields. The importance of BNF is now not limited to plant nutrition; but also reduce environmental problems, e.g., nitrate and nitrite pollution, volatilization of ammonia from the surface application of urea, eutrophication of streams and lakes (Srivastava and Singh, 1999).

Biological N fixation is, after photosynthesis, the second most important biological process on the earth (Graham, 1992). He reported that N2 fixation accounts for 65% of the N currently required for agriculture and in Brazil alone it is equivalent to applying some 2.5 million Mg (mega gram) of N fertilizer per year, a saving of 1.8 billion US Dollars per year.

Therefore, adoption of systems of cropping that involve legume crops will be a key to sustaining or improving agricultural production at a time when population pressures threatens food self-sufficiency in countries like Ethiopia. The economic importance of symbiosis is realized when it is said that the extent of the growth of legumes is a major factor in the attainment and maintenance of a high level of agriculture (Brockwell, 1995). Numerous Rhizobium spp exist each requiring a specific legume plant. For instance, bacteria that live symbiotically with groundnut will not fix N2 with alfalfa (Havlin et al., 2003).

12

2.6. The Process of Nodulation and Fixation

2.6.1. The mechanism
Symbiotic Rhizobium fixes N2 in nodules present on the root of legumes. Nodulation in legumes results from molecular signaling between host and rhizobia (Giller, 2001). Nodule formation in legume is a genetic process. Graham (1992) indicated that both common and host specific nodulation genes have been identified, with the majority of them only expressed in the presence of an appropriate host.

The bacterial groups that form symbiotic relationship with groundnut are Bradyrhizobium spp. regardless of the organisms involved; the key to BNF is the enzyme nitrogenase, which catalyses the reduction of N2 gas to ammonia (Burton, 1976). Exposure of nitrogenase enzyme to free oxygen may destroy it. So, it is up to the organisms to protect it. When N fixation takes place in root nodules, one means of protecting the enzyme from free O2 is the formation leghemoglobin (Burton, 1976).

The symbiotic bacteria begin by infecting root hairs, causing an inquiry inward through several cells. When these bacteria come in contact with the roots of the legume, some of them enter the single-celled root hairs. A rapid increase in the growth rate and in the number of bacteria then takes place because of the abundance of easily accessible food. These bacteria moves from an infection thread toward the base of the root hairs that eventually penetrates the cortex of root. This infection injures the legume plant and, in response to this stimulus, numerous plant cells in the meristematic tissue are produced in the immediate vicinity of the infection, eventually forming the nodule. The nodules then are essentially nothing more than masses of root tissue in which the bacteria live (Millar and Turk, 2001). The process of converting the N2 in to nutrient N (ammonia) is a nitrogenase enzyme catalyzed and takes place in the nodules. Sharma (2002) reported that N fixation in root appears immediately after nodule formation.

13

Smith and Hamel (1999) reported that epidermal cells with immature or as yet unformed root hairs are the usual sites for bacterial penetration. They further indicated that prior to attachment, communication between the two symbiotic partners, groundnut and

Bradyrhizobium sp bacteria is required and a certain minimum period of contact is needed. Infected root hairs are always shorter than mature intact root hairs, due to marked curling up on infection and at the point of infection; the root hairs wall forms a depression that probe deeply, forming an infection thread lined by a continuation of the root hair cell wall and membrane (Smith and Hamel, 1999). According to Burton (1976) and Simpson and Burris (1984), the mechanism is as follows: N2 + 8H+ + 8e - ------------------------------------------2NH3 + H2
(Fe, Mo-protein) Nitrogenase

2.6.2. Recognition between symbiotic partners

The molecular mechanisms for recognition between bradyrhizobia and groundnut can be considered as a form of interorganismal cell-to-cell communication (Smith and Hamel, 1999). The apparent exchange of signals involves the secretion of phenolic compounds (flavonoids, flavones and isoflavones) by groundnut plant. These signal compounds are often excreted by the portion of the root with emerging root hairs, a region that is highly susceptible to infection by rhizobia. These compounds activate the expression of nod genes in rhizobia, stimulating production of the bacterial nod factor. This nod factor has been identified as lipo-oligo sacchride, able to induce many of the early events in nodule development, including deformation and curling of plant root hairs, the initiation of cortical cell divisions, and induction of root nodule meristems. Tate III (2000) also observed that there is an impact of some root exudates components and expression of nodulating genes by the rhizobial cell.

2.7. Characteristics of Nodules

2.7.1. Shape, size and number The presence of nodules on legume roots does not necessarily indicate N2 fixation by active rhizobia (Havlin et al., 2003). Mature effective alfalfa nodules tend to be large, elongated (2

14

to 4 by 4 to 8 mm), often clustered on the primary roots, and have pink to red centers. The red color is attributed to the occurrence of leghemoglobin, which indicates rhizobia are fixing N2 (Havlin et al., 2003). Nodules that are small (< 2 mm in diameter), usually numerous, and scattered over the entire root system are mostly said to be ineffective nodules. In some cases, root nodules may be very large (> 8 mm in diameter), few in number, and have white or pale green centers.

The individual nodule may vary greatly in size and shape on different kinds of legumes. For example, the cultivated annual legumes generally have large sphere like nodules, while those on the biennial and perennial legumes tend to be smaller, elongated and in clusters (Hoa et al., 2002). As reported by Tate III (2000), the roots were initially susceptible to infection 3 to 4 days following germination and the number of nodules formed was proportional to the bacterial density up to an optimal concentration.

The size and shape of nodules formed on roots of different plants are different. For instance on red clover, they sometimes are as large as a pea and more or less ball-shaped; on cow pea and soybean they are much larger, and on velvet-bean may even reach the size of a baseball; on vetches they vary irregular in both size and shape (van Slyke, 2001).

2.7.2. Structure and function of nodule

The outer most layer of the nodule constitutes the bacteriod zone, which is enclosed by several layers of cortical cells. The rate of N fixation of nodule is directly proportional to the volume of the effective nodules (Purohit, 2001). Some nodules contain poorly developed tissue, which is associated with morphological abnormalities. Effective nodules have a red colored pigment called leghemoglobin, which is similar to hemoglobin of blood, is found in nodules between bacteriods and membrane envelops, enclosing them. The amounts of leghemoglobin in nodules have the direct relationship between amounts of atmospheric N fixed by legumes (Purohit, 2001). Leghemoglobin regulates the oxygen supply to bacteriods in the nodule to the level sufficient enough for nodule respiration without deactivating the nitrogenase enzyme.

15

2.8. Factors Affecting Symbiotic Nitrogen Fixation

The nodulation and subsequent N2 fixation processes will not be undertaken unless they are favored or promoted by a conducive environment. So, there are situations where a stress is created in legume plants which affect the mutual activity. The most important of these factors are soil pH, mineral nutrient status, photosynthetic activity, climate and legume management.

2.8.1. Soil reaction (pH)

Legume and their rhizobia exhibit varied responses to acidity. Some rhizobial species can tolerate acidity better than others, and tolerance may vary among strains within species (Brockwell et al., 1995). The optimum pH for rhizobial growth is considered to be between 6.0 and 7.0 (Jordan, 1984) and relatively few rhizobia grow well at pH less than 5.0. The fast growing strains of rhizobia have generally been considered less tolerant to acid pH than have slowly growing strains of Bradyrhizobium (Graham et al., 1994). Although the basis for differences in pH tolerance among strains of Rhizobium and Bradyrhizobium is not clear (Correa and Barneix, 1997), differences in lipopolysacchardes composition, proton exclusion and extrusion accumulation of cellular polyamines and synthesis of acid shock proteins (Zarhan, 1999) and composition and structure of outer membrane (Graham et al., 1994) have been implicated with pH tolerance of endosymbioants. Vlassak and Vanderleyden (1997) reported that nodulation of legumes is reduced in acid soil, mainly because of sensitivity of early nodulation events, such as attachment, root hair curling and initiation of infection thread formation. In addition, low pH can affect the production and excretion of nodulation factors in some strains of rhizobia. Lapinskas et al. (2005) showed that soil acidity was a decisive factor in spread and symbiotic efficiency of rhizobia.

In general, low soil pH is often associated with increased aluminum (Al) and manganese (Mn) toxicity and Ca, P and molybdenum (Mo) deficiencies (Hungria and Vargas, 2000). These stresses affect the growth of rhizobia, the host legume and symbiosis. The effect on symbiosis is evident from the fact that nodulated legumes are more sensitive to Al and Mn toxicity than plants receiving mineral N (Hungaria and Vargas, 2000).

16

Soil acidity is a significant problem facing agricultural production in many areas of the world and limits legume productivity (Bordeleau and Prevost, 1994; Correa and Barnex, 1997). Most leguminous plants require a neutral or slightly acidic soil for growth, especially when they depended on symbiotic N2 fixation (Bordeleau and Prevost, 1994). Liming has been considered the most efficient practice in overcoming soil acidity, with some of the benefits to legume crops not only due to increased soil pH, but also to increased availability of Ca to plant, bacteria and the symbiosis (Hungria and Vargas, 2000).

2.8.2. Mineral nutrient status

Nodulation and N2-fixation by many legumes are limited by deficiencies in soil nutrients such as N, P, and micronutrients (Sanginga et al., 1995). Sakala (1984) found that inoculation in combination with a starter dose of N increased yield of common bean by 73%. Although mineral N in the soil affects the process of nodulation, it may be promoted by relatively low levels of available nitrate or ammonia. However, higher concentrations of nitrogen always depress nodulation (Eaglesham, 1989). Application of N for Phaseolus vulgaris L. was found to suppress nodulation but resulted in yield increase on a vertisol at Alemaya (Mitiku, 1990). Danso et al. (1990) found that soybean N2 fixation is inhibited at higher N levels (83 mg of N kg-1 of soil) which subsequently reduced production. The inhibitory effect of nitrate on N2 fixation has been attributed to a direct competition between nitrate reductase and nitrogenase for reducing power or to the hypothesis that nitrite as intermediate of nitrate reductase inhibits the function of nitrogenase or leghaemoglobin (Straub et al., 1997). Gates and Miller (1979) observed that nodulation in soya bean is affected by unbalanced nutritional conditions of N, P and sulfur (S).

N fixing organisms have a relatively high requirement for Mo, Fe, P and S, because these nutrients are either part of the Nitrogenase molecule or needed for its synthesis and use (Burton, 1976). It is known also that accumulation of ammonia in the soil will inhibit N fixation. High levels of available N, whether from the soil or added in fertilizers, tend to

17

depress biological N fixation (Burton, 1976). The amounts of N2 fixed by soil bacteria and actinomycetes vary enormously and are usually less in soils that have high N levels or have had N fertilizers added (Miller and Donahue, 1997). Formation of nodules can be reduced with applied fertilizer N even as low as 30 kg N/ha in the soil. Soil nutrients, both major and minor, not only contribute to the growth of the plants but also to N fixation. N fixation begins when plants are in quadrifoliate stage (25-30 days after sowing).

2.8.3. Photosynthesis A high rate of photosynthate production is strongly related to increased N2 fixation by rhizobia (Havlin et al., 2003). In spite of the high carbon cost of N2 fixation, recent investigations (Maury et al., 1993) suggested that plant photosynthesis could be adjusted to the photosynthate requirements of the nodules. The process of N fixation requires a considerable energy input. This energy is provided by the plant which obtains it form photosynthesis. Soil acidity, low available P and removal of the biologically active top soil through erosion limit the potential of N fixation (Giller et al., 1998). More commonly, a combination of several soils physical and chemical conditions stress soil microbial populations sufficiently to cause them to operate at sub-optimal levels.

2.8.4. Legume management Management practices one follows in legume crop decide the quantity of N fixed. Havlin et al. (2003) stated that any management practice that results in reduced legume stands or yield will reduce the quantity of N fixed by legumes. They further stated that these factors include water and nutrient stress, excessive weed and insect pressure and improper harvest management. Recommended doses of insecticides and fungicides do not unduly harm the nodulation. However, care should be taken that soil insecticides should not come in contact with rhizobial inoculums. A seed-treating fungicide affects the rhizobial population considerably. Therefore, one should preferably inoculate groundnut through other methods like liquid inoculation, seed pelleting or rhizobia mixed in compost. Higher plant population,

18

as a result of competition of moisture, nutrients, light and space, affect the process of nodulation and N fixation.

2.8.5. Climate
Temperature and light are among the climatic factors affecting nodulation. Effects of day temperature on nodulation of soybean have been studied. One of the bacterial strains was most effective at 33 0C on soybean, while others showed no difference in effectiveness at 21
0

C (Subba Rao, 1986). It was found on a certain study that photoperiods influenced the

formation, size and number of nodules on the root system. Nodulation and N fixation show rapid decline under drought conditions. Prolonged desiccation leads to nodule loss with partial inability to further form nodules. Proper soil moisture should therefore be maintained by supportive, irrigation, wherever possible. Groundnut is grown during the winter/summer in certain parts of the country. Survival of rhizobia under waterlogged conditions is reduced which leads to poor N fixation. Hence groundnut cultivated after the rice needs rhizobial inoculation. Rhizobial populations can be reduced in hot, dry soils particularly at planting or may not be available to shallow-planted seed (Piha and Munnus, 1987). Cool soil temperatures also slow down the movement of bacteria into the roots. Moisture is needed for rhizobia to survive. Prolonged drought, combined with high temperatures, can reduce bacteria levels. Flooding and the depletion of oxygen in the root zone will also kill the bacteria (Giller, 2001). Other strains of bacteria and soil organisms competing for moisture and nutrients may reduce the population of rhizobia (Eaglesham and Ayanaba, 1984). Any practice or a condition that puts stress on the plant can reduce the nutrients available to the bacteria thereby reducing formation of nodules.

2.9. Response of Groundnut to Inoculation

In some crop particular combinations of strains and cultivar have been shown to be especially efficient at fixing N (Buttery et al., 1997). Nodulation and yield of groundnut is influenced by the effectiveness of rhizobial strains introduced in to the soil. Ravuri and Hume (1993) said that a linear and positive effect on the increasing of N2 fixation efficiencies. Early

19

investigation of the effective of rhizobial strains, major component of rhizobia populations on the groundnut plant revealed that genotypic variability exists in the response (chlorosis and early shoot growth) to nodulation (Erdman et al., 1957).

The response of a legume to inoculation with rhizobia depends on involving the bacterium, the plant and the environment in which the symbiosis is established. Phosphorous has been demonstrated to be one of the most limiting factors to N fixation in legume grown in tropical soils in Africa (Kenya, 1977). The population of the nodules occupied by inoculated rhizobial strains dependant on the population of the indigenous soil organisms.

Groundnut is nodulated by the rhizobia that also nodulate many species of tropical leguminous plants, and are classified as the cowpea miscellany (Allen and Allen, 1981). These rhizobia have recently been classified as Bradyrhizobium (Jordan, 1984), and most cultivated soils of the tropics appear to have relatively large populations (> 102 g-1 dry soil) of them. Groundnut nodules are formed at the junctions of root axils where lateral roots emerge (Nambiar et al., 1983). During the early stages of seedling growth rhizobia colonize the rhizosphere, enter the junction of root axils, penetrate into deeper cell layers of the root, and infect a cell. Soon after intracellular infection, the bacteria multiply rapidly. Further development of the nodule occurs by repeated division of the infected host cells (Chandler, 1978). However, rhizobia differ in their ability to Fix N2 and the presence of nodules on the roots of a groundnut plant does not necessarily mean that sufficient N2 is being fixed to maximize its growth (Nambiar et al., 1982). It may therefore be necessary to introduce superior strains of Bradyrhizobium, to ensure adequate N2 fixation for maximum growth and yield of the host plant.

Rhizobium or Bradyrhizobium inoculation is a cheaper and usually more effective way of ensuring an adequate N supply to legumes than the application of fertilizer N. The development of an inoculant industry in many countries has largely been motivated by the desire to introduce legume species to new areas, mainly in temperate zones where more specific rhizobia are required (Burton, 1982). Rhizobium or Bradyrhizobium inoculation of newly introduced crops has resulted in dramatic yield increases in several countries (Burton,

20

1976). In USA, 80% of the total inoculants are for soybeans and alfalfa that are introduced crop species (Burton, 1982). However, results of inoculation trials on many other legume crops have been neither consistent nor encouraging (Subba Rao, 1976; Lopes, 1977; Graham, 1981; Hegde, 1982; Hadad et al., 1982). Reviewing the prospects for inoculating groundnut, Lopes (1977) observed that "since advantages from seed inoculant peanuts are not clearly established, the practice of inoculating this legume is not usual".

21

3. MATERIALS AND METHODS

3.1. Description of the Study Area, Site Selection and Sampling
3.1.1. Description of the study area
The study involved survey and soil sampling, (field work), greenhouse studies and laboratory characterization. The field survey was conducted to identification of representative sampling sites and the subsequent collection of soil samples covered the major groundnut growing areas (Fedis, Babile and Gursum Districts) of East Harerghe Zone, Oromia Regional State (Figure 1). The Zone falls under Weinadega and Kola traditional agro-climatic zones and the altitude, range from 500 to 2950 masl whereas the altitudes where soil samples were collected range from 1601-1899 masl, and the geographical location of the study area is between 090 02 52 N and 420 06 03 E to 090 19 11 N and 420 27 02 E latitude and longitude, respectively, (Table 1). Based on the three years meteorological data of the Babile, the area has mean annual rainfall between 500-875 mm with much variation among years and with mean annual maximum and minimum daily temperatures of 28.27 and 14.18 0C, respectively.

Figure 1. Location map of study area

22

The major soil types in the area were Leptosols/ Lithosols, Regosols, Cambisols, Luvisols and Arenosols with clay loam, loam, sandy loam to loamy sand textural class. The soil reaction and electrical conductivity of the study area range between 6.4 to 8.04 and 0.025 to 0.08 ds m1

, respectively. Sorghum, maize, groundnut and haricot bean are the major crops grown in the

areas. The major cropping systems of the areas were monocropping of groundnut (Gursum and Babile), legume-legume rotation, and legume cereal rotation and intercropping with cereals (Fedis). Unreliability of rainfall, low adoption of modern agricultural inputs and deterioration of soil fertility are the major problems of the area (OSSEP-East Harerghe Zone, 2009).

3.1.2. Site selection and sampling procedure

Soil samples were collected from the major groundnut producing areas of Fedis, Babile and Gursum Districts of East Harerghe Zone (Table 1). These Districts are the potential major groundnut growing areas in the Zone with high potential for increasing yield level. In each of these districts, four peasant associations and two farms per peasant association were selected based on past and present management and status of the groundnut production as presented in Table 1. The variations in these elements were served as a basic tool to select representative peasant associations. Farms with previous history of groundnut inoculation were excluded from sampling. Three healthy plants were uprooted but only one was considered for nodule collection per farm. Twenty four isolates were recovered from groundnut nodules at late flowering and early pod-setting stage of the plants during august 2009.

3.2. Isolation of Rhizobium Strains

Rhizobial strains were isolated from the soil samples using plant induction method (Vincent, 1970). Each representative soil sample was thoroughly mixed and sieved using 2 mm sieve. The soil from each sample was filled into 4 kg capacity plastic pots, which had been surface sterilized by swabbing with 70% alcohol. Undamaged and selected seeds of groundnut were surface sterilized briefly with 95% ethanol for 10 seconds and 0.2% acidified mercuric chloride solutions for 3 minutes (Vincent, 1970). The seeds were rinsed with sterile water and

23

five seeds placed carefully in to each pot and the germinated seedlings were reduced to three per pot. The pots were watered twice a week at full field capacity, and arranged in a complete random design (CRD) to allow plant growth in a glasshouse with 12/12 hours (hrs) light/dark cycle. Table 1. Description of soil sampling sites
Location Designation of isolate HUGR09 HUGR10 HUGR13 HUGR14 HUGR11 HUGR12 HUGR15 HUGR16 HUGR01 HUGR19 HUGR05 HUGR06 HUGR03 HUGR04 HUGR07 HUGR08 HUGR21 HUGR22 HUGR23 HUGR24 HUGR02 HUGR20 HUGR17 HUGR18 Name of site Audal 1 Audal 2 Haro Bate 1 Haro Bate 2 Oda Oromia 1 Oda Oromia 2 Kasa Oromia 1 Kasa Oromia 2 Shek Hussien 1 Shek Hussien 2 Kito 1 kito 2 Shek Abdi 1 Shek Abdi 2 Iffa 1 Iffa 2 Hussien 1 Hussien 2 Tuka kanesa 1 Tuka Kanesa 2 Umer Kulle 1 Umer Kulle 2 Ido Basso 1 Ido Basso 2 District Gursum Gursum Gursum Gursum Gursum Gursum Gursum Gursum Babille Babille Babille Babille Babille Babille Babille Babille Fedis Fedis Fedis Fedis Fedis Fedis Fedis Fedis Altitude (masl) 1801 1840 1659 1631 1779 1809 1706 1725 1603 1601 1694 1710 1619 1644 1670 1643 1705 1681 1724 1710 1783 1804 1841 1899 Latitude (N) 90 17 24 9 18 24 90 16 15 9 15 41 9 18 40 90 19 11 9 14 60 9 16 10 9 10 48 90 10 46 9 15 51 9 15 55 9 11 40 90 12 22 9 1418 9 14 54 9 05 24 90 07 02 9 03 39 9 02 52 9 09 36 90 11 24 9 12 48 9 13 56
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Longitude (E) 420 26 19 42 26 03 420 23 52 42 25 09 42 28 08 420 27 02 42 27 18 42 26 51 42 21 56 420 21 52 42 17 50 42 18 02 42 21 40 420 21 44 42 18 32 42 19 19 42 04 57 420 04 50 42 06 02 42 06 03 42 04 18 420 04 30 42 04 52 42 05 44

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Cropping systems LLR FLR LLR LLR LLR LLR LLR LLR LLR LCR LLR LCR LLR LLR LCR LCR IC IC IC IC IC IC IC IC

EC (dsm-1) 0.031 0.029 0.060 0.039 0.025 0.029 0.055 0.028 0.045 0.048 0.034 0.039 0.065 0.047 0.031 0.038 0.080 0.079 0.097 0.049 0.044 0.042 0.055 0.077

pH 7.81 7.91 7.64 8.01 6.62 6.58 7.65 8.04 7.59 7.57 6.90 7.50 7.45 6.96 6.45 6.15 7.94 8.00 8.02 7.93 7.33 7.46 7.94 8.00

* LCR = Legume-cereal rotation, LLR = legume rotation, IC = intercropping with cereals, FLR=Fallow-legume rotation

After 60 days of planting, plants were uprooted and intact, pink, multi lobed and large nodules were separated from the taproot with a portion of the root attached to the nodule for ease of handling and kept in a vial. Nodules were then transported through vial containing silica gel covered with a cotton plug to prevent contact of nodules and desiccant. The nodules were

24

thoroughly washed with distilled water so as to remove gross surface contamination. After an overnight soaking in distilled water, the nodules were again immediately immersed in 70% ethanol and 0.1 % acidified HgCl2 for 3 minutes and 1minute respectively. A loopful of the suspension was streaked out on yeast extract mannitol agar (YEMA) medium containing 0.25% (w/v) congo red (Subba Rao, 1999). The plates were inverted and incubated at 28 2
O

C for 5-7 days.

3.3. Purification and Preservation of the Isolates

Following the procedure mention on Jordan (1984) a single isolated colony was picked with sterile inoculating loop and transferred in to 10 ml of sterilized YEM broth (YEM without agar) in test tubes, vortex dispersed and placed on rotary shaker at room temperature for more than 48 hrs. A loopful of culture suspensions was streaked on sterile YEMA plates and incubated. The purity of cultures was checked by repeatedly streaking the bacteria on YEMA medium (Jordan, 1984). A single well-isolated colony was transferred to YEMA slant containing 0.3% (w/v) CaCO3. When sufficient growth was observed, the slants were stored at 4 0C (Vincent, 1970).

3.4. Characterization of Isolates

Using standard microbiological techniques (Schiner et al., 1996), all the isolates were characterized for some cultural and morphological parameters like gram reaction, colony morphology, and acid/base production. In doing so, a loop-full of test isolates from YEM broth culture was inoculated in to YEMA plate. After 5-7 days the strains were characterized by colony shape, texture and color. The ability of the isolates to produce either acid or base and release to the medium was detected through color changes on YEMA containing 0.25% (w/v) Bromothymol Blue medium as described by (Jordan, 1984). Similarly, the isolates were tested for presumptive purity using gram staining (Subba Rao, 1999). The isolates were given a designation HUGR (Haramaya University Groundnut Rhizobia) with different numbers representing each isolate.

25

3.5. Physiological Tests

In this study, the bacterial isolates were tested for their reactions to temperature, salt and pH of growing medium. Before incubation, isolates were grown on YEMB to 109 cells/ml. When test plates were used, inoculation was performed with 3.0 l of these cultures. The results were scored after 5 days of incubation at 28 + 2 oC unless stated otherwise (Somasegaran and Hoben, 1994). All tests were carried out in triplicate

Accordingly, to verify the tolerance to low and/or high temperatures, a loop of each bacterium was streaked on triplicate plates containing YEMA medium and allowed to grow at 35 and 40
o

C as indicated by Hungria et al. (2000). Moreover, growth of isolates was also detected at 4,

10, 15, 20 and 45 oC on YEMA medium (Jordan, 1984).

The tolerance of isolates to salt was determined on YEMA medium plates containing different concentration of salt [0.1, 0.2, 0.5, 1.0, and 2.0% sodium chloride (NaCl)] as described by Bernal and Graham (2001). Similarly, the tolerance of the isolates to extreme pH was tested on YEMA medium set at 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 8.5 and 9.0, pH values as indicated by Bernal and Graham (2001).

3.6. Enumeration of Rhizobia

The numbers of indigenous rhizobia present in the Fedis and Babille soils, which could nodulate Arachis hypogaea, were estimated by the most-probable-number (MPN), plant infection technique as indicated by Somasegaran and Hoben (1994). Seeds were surface sterilized with 95% ethanol and in 3% (v/v) solution of sodium hypochlorite. The seeds were successively rinsed in sterilized distilled water, allowed to germinate, and stored at 25+ 2 oC on sterilized Petri-dish plates containing filter paper. One healthy well-grown seedling with similar size and radicle length were transferred into growth pots aseptically.

26

One groundnut variety (Roba) and two soils from Fedis and Babile with two combinations, Roba with Babille; Roba with Fedis were tested for estimation of numbers of indigenous rhizobia. For each combination, tenfold soil dilutions (10 -1-10-8) and one control pot following each group of inoculated pots were used with four replicate pots. After three weeks, nodulation was assessed and the number of rhizobia was calculated based on the following formula: X= m x d Where v m = Likely number from the most probable number table for the lower dilution of the series, d = Lowest dilution (first unit used in the tabulation), v = Volume of aliquot applied to plant, and, X = The most probable number per gram of inoculants.

3.7. Evaluation of Symbiotic Effectiveness Groundnut Rhizobia

The symbiotic effectiveness (SE) of rhizobial isolates were tested in pot experiments under greenhouse using sterilized sand and two different unsterilized soil samples collected from Babile and Fedis from sites which are favorable for groundnut growing. The methods followed for the evaluation of SE in the two different media are briefly described in the following subsections.

3.7.1. Effectiveness of isolates on sterilized sand

Fine graded river sand was well washed in tap water and immersed in 98% sulfuric acid for two days. It was washed in several changes of tap and distilled water to get rid of the last traces of the acid and autoclaved for 1.5 hrs before filled into surface sterilized plastic pots at 4 kg sand per pot (Subba Rao, 1988). Groundnut variety (Roba)that locally recommended for the study areas was selected and suspended in 3% H2O2 for 10 minutes to free from superficial microorganisms and washed several times with distilled and steriled water (Subba Rao, 1988). The pot surfaces were sterilized with 95% ethanol as well.

27

Six pre-germinated groundnut seeds were soaked in 0.75 (w/v) distilled water and incubated in 25 0C for 3 days and then transplanted into each plastic pot. After three weeks of growth, three plants were maintained in each pot. A three milliliter of active culture of each isolate grown in YEMB was poured on to seedlings. The experimental setup was replicated three times and laid out in CRD. There were two (negative and positive) control/standard treatments. The negative control was that lack both sources of N while the positive control was supplied with 70 mg N kg-1 soil fertilizer which was added as 0.05% (w/v) solution week 1

. The plants were irrigated to field capacity with distilled water. All pots were fertilized with

N-free nutrient solution, comprising the chemical compounds shown in Table 2.

Table 2. Chemical compositions of N-free nutrient solutions Solution 1 Chemical g l-1 CaCl2.2H2O 294.1 Solution 2 Chemical g l-1 KH2PO4 136.1 Solution 3 Chemical g l-1 FeC6H5O7.3H2O 6.700 MgSO4.7H2O 123.300 K2SO4 87.000 MnSO4.H2O 0.338 H3BO3 0.247 Solution 4 Chemical ZnSO4.7H2O CuSO4.5H2O CoSO4.7H2O Na2MoO2.2H2O

g l-1 0.288 0.100 0.056 0.048

Source: Somasegaran and Hoben (1994)

After Sixty days of planting, plants in all the replications were carefully uprooted to expose the whole root system. The adhering soils were removed by washing the roots with water over a sieve. The important parameters such as leaf color, nodule color, peg number, nodule number, nodule dry weight, shoot dry weight and relative symbiotic effectiveness were used to confirm the candidate isolate for evaluation of symbiotic effectiveness of isolates under sterilized sand condition.

Leaf color of plants in each pot was given subjective relative greenness scale. Four representative nodules were taken from the same sample and dissected with blade to observe their color in the center. The color score was made in 1-4 scale as: 1 = white, 2 = pink, 3 = slightly dark red and 4 = deep dark red (Tekalign and Asgelel, 1994). Total numbers of nodules in all the uprooted plants of each replication (3 plants) were counted after carefully

28

detaching them from the roots. Fresh nodules obtained after number of nodules were pooled together including the dissected nodules for color determination and number of nodules was averaged to per plant and their dry weight was measured by drying at 70 0C to constant weight. The nodule dry weight was reported as mg plant-1. All the plants under the same replication of each treatment were placed in a paper bag and oven-dried at 70 oC to constant weight. The average dry weight of the plants was taken as dry weight per plant and reported as g plant-1. Finally, the percent of relative symbiotic effectiveness (RSE) of the isolates was computed to select effective isolates as:

R SE (%)

Shoot dry weight of plants inoculated with test strain 100 % Shoot dry weight of plants supplied with nitrogen

where the RSE (%) values were rated as : >80% = highly effective, 50-80% = effective, 35-50 = lowly effective, and <35% = ineffective (Lalande et al., 1990).

3.7.2. Evaluation of isolates in two unsterilized soil

Composite soil samples for the pot experiment were collected from Fedis farmer field and Babile Research site. Surface (0-30 cm) depth the Composite soil samples were uniformly mixed, by removing any debris and filled 4 kg soil into plastic pots while the composite soil samples were subjected to the determination of selected physicochemical properties after airdried, ground and sieved through 2 mm sieve size. The particle size distribution (Bouyoucus Hydrometer Method), organic carbon (wet oxidation/dichromate digestion), total N (modified micro-Kjeldahl procedure), available P (Olsen extraction method), soil reaction or pH (1:2.5 soil to water ratio suspension), electric conductivity (1:2.5 soil to water ratio extract) and cation exchange capacity (ammonium acetate extract) were made following the standard analytical procedures of the respective parameter compiled by Sahlemedhin and Taye (2000).

Evaluation of symbiotic effectiveness of ten most relatively effective isolates was tested on two unsterilized soils. The powdered carrier base inoculants were prepared through mixing of 5-7 days old isolate suspension (108-109 cells) and sterile charcoal powder in 1:1 ratio. Healthy and surface sterilized seeds of groundnut were sown after being uniformly coated

29

with powdered inoculants of 10% sugar solution in cool and clean water at a rate of 7 g inoculants kg-1 of seed. Inoculated seeds were immediately planted into irrigated pots. These pots were arranged in CRD with two control treatments: the negative control, which was without N and inoculants and the positive control, which was fertilized with 70 mg N kg -1 soil in greenhouse. The purpose of N fertilization was to measure the biomass yield potential of groundnut in the experiment. The greenhouse condition had 103 and 322 0C mean minimum and maximum temperatures, respectively, with an average of 11-12 sunlight hours during the study period.

After 60 days, data on peg number, nodule number, nodulation rating, leaf and nodule color, nodule dry weight and shoot dry weight were collected. Furthermore, N content of plant tissue were determined after oven-drying at 70 0C to a constant weight using the micro-Kjeldahl method (Sahlemedhin and Taye, 2000), and the N content in the plant tissue (g per plant) was determined by: multiplying the percent N by the shoot dry weight in grams divided by hundred.

Total number of pegs in all the uprooted plants of each replication (3 plants) was counted after washing carefully and then was averaged to per plant. Nodulation rating was determined after counting number of plants out of total uprooted plants from a treatment with tap-root nodulation (a), number of plants with nodules close to tap root (b), number of plants with scattered nodules (c) and number of plants without nodules (d) where 10, 5, 1 and 0 points were assigned, respectively. It was calculated as described by Nif Tal Project, 1976). Nodulation rating = (a x 10) + (b x 5) + (c x 1) + (d x 0) Total number of plants

3.8. Statistical Analysis

Data on peg number, nodulation rating, nodule number, nodule dry weight, shoot dry weight, percent N and N content were subjected to one-way analysis of variance (ANOVA) following procedures appropriate for the design using statistical analysis software (SAS) Version VIII

30

(Gomez and Gomez, 1984). A least significant difference (LSD) test was used for separating the significant mean at P < 0.05. Pearsons correlation coefficient (r) was determined to confirm direction and magnitude of the association between the studied parameters.

31

4. RESULTS AND DISCUSSION

4.1. Presumptive Tests of Isolates
All of the tested isolates were gram negative and rod shaped bacteria with little or no absorption of Congo red grown on YEMA-CR medium. No isolate was grown on peptone glucose Agar supplemented with 25 mg l-1 bromocresol purple medium (Table 3). A total of 24 isolates from groundnut were characterized for alkaline reaction on YEMA-BTB. Acid and alkaline production on YMA-BTB medium has been used as a tool to indicate the general character of rhizobia. Slow growing rhizobia produce alkaline while fast growing rhizobia produce acid (Jordan, 1984). Accordingly, all of the tested isolates were alkaline producers. However, Kennedy and Greenwood (1982) have shown that slow growth and alkali production are not mutually exclusive characteristics. Moreover, Wolde-meskel et al. (2004) have also reported the presence of fast growing alkaline producing and slow growing acid producing strains that were isolated from native woody legumes in southern Ethiopia. Generally, in this study, all of groundnut nodulating isolates from East Hararghe Zone were found to be slow growers. Besides, the stimulation of nod (nodulation) genes by flavonoids/isoflavonoids secretion of legumes is by no means completely specific as exudates from incompatible legume species can often activate the nod gene of a given rhizobial strain to some degree (Giller, 2001).

4.2. Morphology and Cultural Characteristics

The diversity of the isolates was confirmed using colony diameter and colony morphology (Table 3). The slow growing strains attain < 1 mm colony size within 5-7 days of incubation. Accordingly, 62.5% of the isolates had less or equal to 1 mm and 37.5% isolates had 1.5 to 2.5 mm colony size. According to Jordan (1984), fast-growing root nodule bacteria (genus Rhizobium) form visible colonies (2-4 mm diameter) on YEMA within 3-5 days and Bradyrhizobium (slow-growers) need 5-7 days to form 1 mm diameter under the same conditions. The color of all of the isolates recovered from root nodules of groundnut in different locations showed milky colony and a buttery colony texture (Table 3).

32

Table 3. Colony characteristics and presumptive test of isolates after 5-7 days of incubation
Isolate Colony size (mm) <1.0 1.5-2.5 <1.0 <1.0 <1.0 1.5-2.5 1.5-2.5 <1.0 <1.0 <1.0 1.5-2.5 <1.0 1.5-2.5 <1.0 1.5-2.5 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 1.5-2.5 1.5-2.5 1.5-2.5 YEMA-BTB reaction Blue M. Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue M. Blue Blue Blue YEMA-CR PGABCP NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG Colony texture on YEMA medium Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Buttery Colony Type SD LM SD SD SD LM LM SD SD SD LM SD LM SD LM SD SD SD SD SD SD LM LM LM

HUGR1 HUGR2 HUGR3 HUGR4 HUGR5 HUGR6 HUGR7 HUGR8 HUGR9 HUGR10 HUGR11 HUGR12 HUGR13 HUGR14 HUGR15 HUGR16 HUGR17 HUGR18 HUGR19 HUGR20 HUGR21 HUGR22 HUGR23 HUGR24

Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky Milky

YEMA- CR = Yeast Extract Mannitol Agar Congo red; YEMA-BTB = Yeast Extract Mannitol Agar with 25 mgl-1 Bromothymol Blue, PGA = Peptone glucose agar supplemented with 25 mgl-1 Bromocresol purple, SD=Small dry, LM = Large mucoid, M = Moderate, YEMA =Yeast extract mannitol agar, NG = No growth.

Sixty two point five percent of the isolates displayed small dry colony and 37.5% characterized by large mucoid colony morphology. During purification, all strains exhibited longer time to grow on YEMA medium.

Therefore, the strains isolated from groundnut root nodule in the present study showed the characteristics of slow growing Bradyrhizobium according to the classification of the family Rhizobiaceae and the isolates fall into the Bradyrhizobium (Cowpea miscellany) (Jordan, 1984). Although large or small colony and mucoid or dry colony are the basis of

33

differentiating into fast growing Rhizobium and slow-growing Bradyrhizobium, 37.5% of the isolates attained relatively larger diameter (1.5-2.5 mm) and displayed relatively large mocuid colony growth. Furthermore, two isolates showed moderate blue colony growth on YEMABTB containing medium (Table 3). The appearances of such variations have also been reported by several workers (Dakora and Vincent, 1984; Hernandez and Foscht, 1984).

4.3. Evaluation of Symbiotic Effectiveness on Sand Culture

The rhizobial isolates were tested in pot experiment using sterilized sand culture for their symbiotic effectiveness on groundnut under greenhouse condition. All the tested isolates formed nodules and obtained a 100% infection of bradyrhizobial isolates upon reinoculation on groundnut. As strains of bradyrhizobia tend to be more effective on the host from which they were trapped than other species (Giller, 2001). The results confirmed that all strains considered in this study were authenticated to be true bradyrhizobia that infected their host as described by Subba Rao (1988) and Giller (2001).

4.3.1. Peg number per plant

According to the present finding, peg number significantly differed among (P < 0.05) the isolates and both positive and negative controls (Table 4). Accordingly, inoculation with all the isolates significantly increased peg number over the negative control except for a few isolates. The highest peg number was obsebed with the inoculation of HUGR22 and positive control. Whereas there were no significance differences among isolate HUGR8, HUGR9 and negative control. The highest peg numbers were observed on isolate HUGR22 (9) and followed by HUGR20 (6) but the lowest peg numbers were observed on the isolate HUGR1 and HUGR15 (0). The average peg number recorded in the present finding was 2.54 (Appendix Table 1).

4.3.2. Nodule number per plant

Inoculation significantly increased nodule number per plant (P < 0.05) as compared to the negative and positive controls (Table 4 and Appendix Table 1). The maximum nodule number

34 recorded per plant (plant -1) was 131 for isolate HUGR22 followed by HUGR3 (121), whereas the minimum number of nodules recorded was 39 from isolate HUGR9 followed by HUGR14 (43). The average nodule number produced by groundnut plants in this study (70.37 nodules plant-1) was more than those obtained by Numbier et al. (1983) which was 93 and 48 nodules per plant in groundnut. Dashti et al. (1998) reported that inoculation of soya bean with Bradyrhizobium strains increased nodule numbers, plant weight and seed yields under greenhouse and field conditions. Similarly, Wange (1989) recorded 19 and 37 nodule numbers plant-1 as the minimum and maximum nodule numbers, respectively, in experiment conducted on groundnut on soil culture which was less than what was obtained in the present work. The result on nodule number was also higher than the highest average nodule numbers plant-1 (37) recorded by Hoque (1993) on groundnut in Bangladesh.

On the other hand, inoculation with isolates HUGR 9 and HUGR14 exhibited light yellow color of leaf and small white color of nodules on lateral root compared to other isolates (Appendix Table 2). The formation of white nodules could be associated with weak vigor and poor yield that may be related to poorly developed bacteroid as described by Subba Rao (1988). Even if it produced relatively yellow leaf as well as small white nodule color on lateral root, the strain was displayed symbiotic effectiveness (Table 4). This implies that the presence of nodules on plant tap root does fix N which benefits the host but, still it does not necessarily mean sufficient N is being fixed for maximum benefit to the host plant. This situation might indicate that the isolate is ineffective in N fixation where an indigenous rhizobial is present in the field condition where competition is high (Wange, 1989).

4.3.3. Nodule dry weight per plant

In line with the nodule number per plant, there was a significant difference (p < 0.05) in nodule dry weight among the isolates and the controls (Appendix Table 1 and Table 4). The maximum (155.33 mg plant -1) and minimum (13.33 mg plant -1) were displayed by HUGR22 and HUGR9, respectively (Table 4). The average nodule dry weight recorded in this study was 52.06 mg plant-1. Dashti et al. (1998) has reported that inoculation of soya bean with Bradyrhizobium strains increased nodule weight 29-83% over control under greenhouse and

35

field conditions. The difference in nodule dry weight might be attributed to difference in efficiency of nodules in fixing N and the host species since isolates from different agroecologies were tested on different host varieties as it was explained by Zhang et al. (1996).

Table 4. Nodulation and symbiotic effectiveness of isolates tested on groundnut in sand culture
SE (%) Rate 64 E HUGR1 0.33+0.58k 48.67+1.53r 23.0+1.00pq 0.83+0.01qr 73 E HUGR2 1.67+0.58hgji 77.0+1.00j 46.0+0.00kj 0.94+0.01kj 100 VE HUGR3 3.67+0.58c 121.33+1.15b 119.33+8.96b 1.29+0.01b 74 E HUGR4 2.0+1.00hgfi 78.0+0.00ji 47.67+0.58kj 0.96+0.01ji 71 E HUGR5 1.67+0.58hgji 74.0+1.73k 44.33+0.58kjl 0.92+0.01k 75 E HUGR6 1.67+0.58hgji 78.0+0.00ji 49.67+2.08ji 0.97+.01hi 70 E HUGR7 2.67+0.58dgfe 69.67+2.08L 42.33 +1.53kl 0.90+0.00L 65 E HUGR8 0.67+0.58jk 52.67+2.08q 26.33+1.151po 0.84+0.00pq 60 E HUGR9 0.67+0.58jk 39.33+1.15t 13.33+2.311r 0.78 +0.01s 81 VE HUGR10 2.0+1.00hgfi 81.33+1.15h 59.33+2.08hg 1.04 +0.01g 92 VE HUGR11 3.33+0.58dce 89.0+1.00f 79.67+4.93e 1.19+0.01e 89 VE HUGR12 1.33+0.58hjki 84.0+1.73g 63.0+1.73g 1.15+0.00f 98 VE HUGR13 2.67+0.58dgfe 118.33+1.26c 100.0+7.00c 1.26+0.01b 64 E HUGR14 1.0+1.00jki 43.0+1.00s 19.0+2.65qr 0.82+.01r 67 E HUGR15 0.33+0.58k 55.0+1.00p 29.0+10npo 0.86+0.01pno 97 VE HUGR16 1.33+0.58hjki 95.67+2.08d 92.67+3.21d 1.25+0.01c 67 E HUGR17 1.33+0.58hjki 60.67+1.16n 35.33+0.58mn 0.87+0.01mn 76 E HUGR18 1.33+0.58hjki 79.33+0.58hi 55.33 +2.08hi 0.98+0.01h 95 VE HUGR19 3.0+1.00dfe 91.33+0.58e 86.0+2.00e 1.23+0.02d 66 E HUGR20 6.0+0.58 b 53.0+1.00qp 27.33+0.58po 0.85+0.01pqo 67 E HUGR21 4.33+0.58c 58.0+1.00O 32.0+1.00mno 0.87+0.01no 116 VE HUGR22 8.67+0.58a 131.33+1.53a 155.33+14.47a 1.49+0.01a 69 E HUGR23 2.67+0.58dgfe 64.33+0.58m 38.0+1.00ml 0.89+0.01ml 91 VE HUGR24 2.33+0.58hgfe 87.33+1.00f 69.67+1.15f 1.17+0.01fe 48 N0.67+0.58jk 0 0 0.62+0.012t 100 N+ 8.67+0.58a 0 0 1.29+0.01b - LSD(0.05) 1.11 2.02 6.55 0.023 SEM 0.46 1.51 16.00 0.0002 - figures within a column followed by same letter(s) are not significantly different at P 0.05. N- = Uninoculated and unfertilized control; N+ = N fertilized control; LSD = Least significant difference; SEM = Standard error of mean; NDW = Nodule dry weight; SDW = Shoot dry weight; NN = Nodule number; PN = peg number; SE = Symbiotic effecetiveness; VE = Very effective
PN(plant-1) NN(plant-1) NDW(mg plant-1) SDW(g plant-1)

36

4.3.4. Shoot dry weight per plant

In this experiment, pronounced physical (stand) differences were also apparently observed between the controls and inoculated treatments (Appendix Figure 1). The maximum mean shoot dry weight was 1.49 g plant -1 for HUGR22 while the minimum mean was 0.62 g plant -1 for the negative control (Table 4 and Appendix Table 1). Moreover, isolate HUGR22 accumulated significantly (p < 0.05) higher shoot dry matter than even the positive control. This isolate had a shoot dry weight of 140 and 16% over the negative and positive controls, respectively. In general, inoculation sustained significantly (p < 0.05) different shoot dry weight of groundnut seedlings as compared to the negative and positive controls and among the isolates (Appendix Table 1 and Table 4). The finding of this study was in agreement with that of Nambiar and Dart (1980) who reported that the inoculation of bradyrhizobia increased shoot dry weight of groundnut grown on the soil culture.

Inoculation of faba bean with Rhizobium significantly increased shoot dry weight, and number and dry weight of nodules under field conditions (Ahmed and Elsheikh, 1998). Similarly, Abere (2010) observed a significant increase in shoot dry weight of faba bean inoculated with isolates collected from East and West Hararghe highlands compared to the positive control. Single Rhizobium inoculation of Phaseolus vulgaris showed significant difference for the nodule dry weight, shoot dry weight and root dry weight (Mohamed et al., 2009). Somasegaran and Hoben (1994) and Peoples et al. (2002) explained that shoot dry matter is a good indicator of relative isolate effectiveness and there exists often positive correlation between the N fixing capacity of legumes and their shoot dry matter accumulation. Similarly, all of the isolate collected from Fedis, Babile and Gursum Districts of East Hararghe Zone and evaluated in this study were better as compared to the negative (uninoculated) control in terms of biomass accumulation in groundnut plant tissue.

In accordance the results obtained from the present study, various researchers have reported findings where inoculation increased shoot dry weights of many other legume crops (Zhang et al., 1996; Dashti et al., 1998).

37

4.3.5. Symbiotic effectiveness

The relative effectiveness expressed as percentage of shoot dry mass of the inoculated over the positive control, showed in Table 4 that 37.5 and 62.5% of the isolates were found to be highly effective (80-116%) and effective (60-76%), respectively. The highest scores of 80116% effectiveness of symbiotic nitrogen fixation were displayed by HUGR 22, HUGR 3, HUGR 13, HUGR 16, HUGR 19, HUGR 11, HUGR 24, HUGR 12 and HUGR 10 with shoot dry mass greater than 1.04 g plant-1. The remaining 15 isolates were found to be effective which were observed to be slightly higher than the 0.62 g plant-1 obtained in the uninoculated and non N fertilized negative control plant.

4.3.6. Correlation analysis for the selected parameter on sand culture

The correlation analysis revealed a highly significant association existed (p < 0.01) between the nodule dry weight and shoot dry weight (r = 0.82), nodule dry weight and nodule number (r = 0.93) and significant (p < 0.05) nodule number and shoot dry weight (r = 0.68), peg number and shoot dry weight (r = 0.55), peg number and nodule dry weight (r = 0.30). (Appendix Table 3) Wange (1989) reported some yield parameter agree with this study.

4.4. Physiological Characterization

4.4.1. Temperature tolerance
All of the selected rhizobial isolates were able to grow between the ranges of 15 to 40 0C without any variation among isolates except for isolates HUGR3, HUGR10, HUGR11, HUGR16 and HUGR24 which produced large mucus at 40 0C as shown in Table 5. Similarly, previous studies reported that maximum survival for bradyrhizobia ranged from 33.7 to 48.7
0

C (Munvar and Wollum, 1982). Nevertheless, in studies by La Favre and Eaglesham (1986),

12% of the bradyrhizobial strains tested could not survive temperatures of 37 0C on agar. Furthermore, the 42 strains tested by Munevar and Wollum (1981), 20% could not survive at 38 0C in broth culture. However, for most rhizobia, the optimum temperature range for growth

38 in culture is 28 to 37 0C Somasegaran and Hoben (1994) and many other (Graham, 1992) were unable to grow at 37 0C. On the other hand none of the isolates showed growth at temperatures of 4 and 10 0C in the present finding (Table 5). In contrary to the present study, Amanuel (2008) reported that 55 and 70% of mung bean and groundnut isolates, respectively, were able to grow at 5 0C. This may be due to genetic and climatic variability or other growth limiting factor. In the present study temperature of 28 0C was used as a control temperature while temperatures 4, 10, 15, 20, 35 and 40 0C were randomly chosen. As the present result shows, all the tested isolates tolerated 40 0C (Table 5).

4.4.2. Salt tolerance

No variations were observed among the isolates to NaCl tolerance (Table 5). All of the tested isolates were able to grow up to 0.5% NaCl while HUGR18 isolate weakly continued to grow up to 1% NaCl. Graham and Parker (1964) report that out of Rhizobium leguminosarum strains tested, none could tolerate NaCl concentrations of 2-3%. According to a study that assess salinity tolerance level on bradyrhizobial isolates from lupine in YEMB, the number of rhizobial cells showed an inverse relationship with respect to increasing NaCl concentration, but with strain to strain variation (Raza, 2001). None of the groundnut isolates tested in this study tolerated more than 1% of NaCl. In contrarily to this study, 44% of groundnut isolates from Gamo Gofa and 21% of from Awassa tolerated up to 7% NaCl, while it was 33 and 9% for mung bean isolates from Awassa and Gamo Gofa, respectively (Amanuel, 2008). This indicates that no specific pattern of resistance was observed location wise.

39

Table 5. Physiological (temperature. salt and pH tolerance) characterization of the isolates Treatment Temp (0C) 4 10 15 20 30 35 40 Salt (%) 0.1 0.2 0.5 1.0 2.0 pH 4.5 5.0 5.5 6.0 6.5 8.0 8.5 9.0 + + + + + + + + + + + + + + + + + + + + HUGR 03 + + + + + + + + HUGR 10 + + + + + + + + HUGR 11 + + + + + + + + HUGR HUGR HUGR 12 13 16 Temperature tolerance* + + + + + + + + + + + + + + + Salt tolerance + + + + + + pH tolerance + + + + + + + + + + + + + HUGR 18 + + + + + + + + + + + + + + HUGR 19 + + + + + + + + + + + + + HUGR 22 + + + + + + + + + + + + + HUGR 24 + + + + + + + + + + + + +

*Temp = Temperature; + = denotes presence of rhizobial growth; - = denotes absence of rhizobial growth

40

Generally, the salt tolerance of different strains of rhizobia and bradyrhizobia is not related to their ecological origin (Elshikh-Elsiddig, 1998). Similarly, Hosney et al. (2002) have concluded that there was no correlation between salt tolerance of a given isolate and its origin.

4.4.3. Tolerance to pH

All of the isolates had shown growth on YEMA medium from pH 6 up to pH 9 (Table 5). On the other hand, the isolates failed to tolerate pH levels between 4.5 up to 5.5. Similarly, Cooper (1982) found that none of 20 strains of Bradyrhizobium (Lotus) developed at pH 4.6. In present study, all isolates of groundnut were able to tolerate 8-9 pH range, which similar to the study results reported by Amanuel (2008) who found that all isolates of mung bean and groundnut from Awassa were able to grow on pH values from 8.5-10. The isolates of the present study were collected from soils with a relatively higher pH (6.4-8.0) (Table 1) this attributed to all isolates to tolerate high pH, that is a reflection of adaptation of indigenous rhizobia to local soil conditions.

Generally, we can conclude that most of the rhizobial isolates in this study are tolerant to high pH. Zhang et al. (2006) reported that most of the rhizobial strains isolated from root nodules of cowpea (Vigna unguiculata) and mung bean (Vigna radiata) grown in different regions of China, could grow from pH 5.0 to pH 11.0. Several authors have also reported tolerance to high pH for rhizobial strains isolated from different legumes. Nour et al. (1994) found pH tolerance of rhizobial strains isolated from chickpea (Cicer arietinum) up to 10.0. Rhizobial strains isolated from Sesbania formasa, Acacia farnesiana and Dulbergia sissoo were well adapted to grow at pH 12.0 (Surange et al., 1997). However, Jordan (1984) reported that the slow-growing strains are more tolerant to low pH than the fast-growing strains.

4.5. Soil Properties and Enumeration of Rhizobia

The physical and chemical properties of soil samples from the experimental sites were presented in Table 6. The soil at Fedis had high total N content (0.17%) while the Babile soil was low (0.07%) According to Desta and Anagw (1986). This showed the additional N

41

requirement of inputs to Babile soil. The available P of the experimental soil in Fedis (5.42 mg kg-1) was bellow critical level whereas the Babile soil (9.81 mg kg-1) was above the critical level. Tekalign and Haque (1991) established the critical Olsen extractable available soil P content for crops in some Ethiopian soils estimated to be 8.5 mg kg-1. Generally, it can be concluded that the available P contents of the experimental soils may not at optimum to enhance nodulation and nitrogen fixation of groundnut especially in Fedis soil (Tsvetkova and Georgiev, 2003). The pH values indicate that the soils have neutral (6.6) and slightly alkaline (7.4) soil reaction for Babile and Fedis soil, respectively. The electrical conductivity of the soils at Fedis and Babile were (0.49 ds m-1 and 0.06 ds m-1), respectively. With regard to soil texture, Fedis and Babile soils are sandy loam and loamy sand, respectively (Table 6).

The plant infection tests were undertaken to evaluate the most probable number (MPN) density of indigenous population of rhizobia at Fedis and Babile soils on Roba variety of groundnut. The indigenous rhizobial population in Fedis and Babile soils capable of forming nodules on groundnut were estimated 2.6 x 102 and 3.3 x 103 cells g-1 soil, respectively. The Babile soil harbor more number of rhizobial population as compared to the Fedis soil might be due to the frequent sole cropping history of the trapping host attributed to in regardless of sandy loam soil texture, more OM and higher CEC in Fedis soil that helps the rhizobial cells to stabilize. The populations of rhizobia (in the surface soil) capable of nodulating groundnut observed in the present study also fall within the range reported by other researchers. for instance Nambiar (1985) revealed that the resident groundnut nodulating rhizobia population was found to be greater than 102 rhizobia g-1 of soil. Hadada and Loynachan (1985) also reported an indigenous rhizobia population of 3 x 102 to 2.4 106 g-1 of soil from groundnut growing areas of Sudan.

On the other hand, Boonkerd and Wadisirisuk (1993) in Thailand reported that the numbers of groundnut nodulating rhizobia in most of the fallow fields and some of the non cultivated shrub or forest locations were about 1.6 103 cells g-1 of soil. Generally, both Fedis and Babile soils contain a reasonable number of rhizobial populations residing in the soil.

42

Table 6. Chemical and physical properties of the soils for the 0-30 cm depth Parameter pH EC (ds m-1) Total nitrogen (%) Carbon to nitrogen ratio Available phosphorus (mg kg -1) CEC (cmol(+) kg-1) Organic matter (%) Sand (%) Silt (%) Clay (%) Textural class Fedis soil 7.4 0.49 0.17 6.59 5.42 22.8 1.93 56.0 33.8 10.2 Sandy loam Babile soil 6.6 0.06 0.07 13.00 9.81 9.80 1.57 74.0 17.5 8.5 Loamy sand

According to Thies et al. (1991), the present results were above the recommended values for inoculation (20-50 cell g-1) for a likely yield response and hence there seems to be no need of inoculation for the crop under study. However, it should be noted that the MPN result only tells us the infectiveness and therefore, information regarding the effectiveness, the competitiveness of the resident strains as well as soil fertility status and N demand of the host plant should be well studied before deciding up on inoculation. Moreover, Singleton and Tavares (1986) stated that both size and effectiveness of indigenous rhizobial populations are primary factors that determine the incidence and magnitude of response of legumes to inoculation. Nevertheless, Hamdi (1982) and Beck and Duc (1991) reported a significant response to inoculation in spite of the presence of 1.8 104 indigenous population of rhizobia g-1 of soil.

Slattery and Pearce (2002) also claimed that the presence of background rhizobia was not always in itself, to ensure optimal N fixation in the host legume as the effectiveness of isolates to fix N within naturalized populations might vary considerably. Reports from various parts of the world confirmed that low soil N status and overall crop failure insurance strategy together with its relative low cost often make inoculation an ordinary practice for crop and pasture production. Keeping in view the above results and explanations, symbiotic efficiency test of

43

isolates of groundnut plant was conducted to select well adapting and high N fixing isolates in the soil environment.

4.6. Symbiotic Effectiveness of Selected Isolates on Fedis and Babile Soils

4.6.1. Peg number per plant
In the present study, peg numbers on both the Babile and Fedis soils showed significant differences among the treatments (Appendix Tables 5 and 8 and Tables 7 and 8). Inoculation of the selected isolates increased peg number in groundnut inconsistently in both Babile and Fedis soils. However, there was no evidence which supported inoculation can influence peg formation. As indicated in Tables 7 and 8, there were significant variations (p < 0.05) among some inoculant isolates and the negative control on both soils. The three different experiments (sand culture, Fedis soil and Babile soil) conducted in greenhouse; the peg formation was strongly correlated with the superior inoculants and the positive control. However, an influence of Rhizobium inoculants on peg formation was strongly rejected by Wynne et a1. (1983). They added that the data they obtained suggest that while the presence of rhizobia soil inoculum influences nodule formation on groundnut plants, there was no indication that this will assist the plant in the production of pegs and pods. Generally, responses of different plant genotypes and rhizobial strains have been found to be specific to peg formation.

4.6.2. Nodule number per plant

On Babile soil both inoculated isolates and the negative control displayed significantly (p < 0.05) higher nodule numbers than the positive control. Inoculation brought 8-57 and 8-35% increased nodule numbers over the positive and negative controls, respectively, except with isolate HUGR11 for the negative control (Table 8). Wynne et al. (1980) reported that nodulation, plant weight, nitrogenase activity and N content of groundnut greenhouse grown condition varied significantly. Similarly, soil in this particular study, inoculated isolates displayed (16.67-72 and 4-68%) increased in nodule number over both positive and negative controls, respectively, on the Fedis.

44

However, the average nodule number on the Fedis soil was relatively lower than that of the Babile soil (Tables 7 and 8). This showed that the host plant might be an alternative option for the N requirements which inhibits the Rhizobium infection process and also reduced N2 fixation. These were probably resulted from impairment of the recognition mechanisms by NO3, while the latter was probably due to diversion of photosynthates toward assimilation of nitrates. Nitrates may allow the plant to conserve its energy, since in overall terms more energy is required to fix N2 than to utilize NO3. The poor nodulation was probably attributed to the deficiency of available phosphorus in the soil as well as population of the indigenous rhizobial in the soil. The soil and MPN rhizobial enumeration results showed that Fedis soil had relatively more soil N, soil organic matter cation exchange capacity and low population of indigenous rhizobial strains. This result was similar to the report of Abdel-ghaffar (1988) who indicated reduction of nodule number and suppression of N fixation when fertilized with 180 kg N ha-1. Absence of nodules was also reported by Singleton and Tavares (1986) on different legumes fertilized with 600 mg N kg-1 of soil regardless of the presence of rhizobia in Hawaiian soils. In contrast to the above result, the conservative estimate of Singleton and Tavares (1986) showed that inoculation did not enhance nodule number when the number of invasive native rhizobia were greater than or equal to 1 x 102 g-1 of soil, whereas inoculation always increased nodule numbers when resident rhizobia were below 6 x 100 g-1 of soil.

On both Fedis and Babile soils, the poor nodulation of groundnut plants in the positive control was primarily accounted to the reserve of mineral N to nodule formation and functioning on root colonization of N fixing legumes despite its dependence on several factors. Musa, (1982), Abdel-ghaffar (1988) and Giller (2001) reported that the presence of large amounts of mineral N, beyond 48 kg N ha-1 depressed nodulation entirely due to exhaustion of carbonaceous material within the plant N. In general, the absence of good nodulation for the N treated control was attributed to the detrimental effect of the applied N combined partially to the soil N on nodulation (Table 6).

45 Table 7. Nodulation data of selected effective isolates of groundnut rhizobia on Fedis soil

PN (plant-1) HUGR3 5+0.00b HUGR 10 4+0.00cd HUGR 11 4+0.00cd HUGR 12 4.67+0.58cb HUGR 13 3+0.00de HUGR 16 2.67+0.58e HUGR 18 4.0+0.00dcd HUGR 19 2.67+1.15e HUGR 22 5.0+0.00b HUGR 24 4.0+0.00cd N+ 7.67+0.58a N3.33+0.58de LSD(0.05) 0.79 SEM 0.22

NN (plant-1) 35.67+8.08b 27+4.00dc 27.67+1.53c 39.67+8.50b 21.67+2.89dce 18.67+1.15def 22.33+1.15dce 18+2.65ef 54+1.00a 25+1.00dc 15+1.00f 17.33+1.53ef 6.51 14..94

NR NDW SDW -1 -1 (plant ) (mg plant ) (g plant-1) 10.0+0.00a 116.67+11.55b 1.94+0.02b 6.67+2.89b 93.33+15.28c 1.85+0.07c 5+0.00b 96.67+5.77c 1.87+0.04c 10.0+0.00a 120+17.32b 1.97+0.04b 5+0.00b 71.67+2.89de 1.64+0.01ef 2.33+2.31c 60+10.00ef 1.59+0.02f 5+0.00b 71.67+2.89de 1.7+0.03d 2.33+2.65c 56.67+11.55e 1.6+0.06f 10.0+0.00a 170+10.00a 2.23+0.04a 5.0+0.00b 83.33+5.77dc 1.83+0.02c 1.0+0.00c 28.33+10.41f 2.28+0.02a 5.0+0.00b 65+5e 1.69+0.03ed 2.12 16.97 0.06 1.58 101.39 0.0013

N % 2.2+0.00c 2.03+0.06d 2.07+0.06d 2.27+0.06c 1.7+0.06f 1.67+0.10f 1.87+0.06e 1.73+0.06f 2.5+0.01b 2.1+0.02d 3.5+0.10a 1.88+0.01e 0.11 0.005

TN (g plant-1) 0.04+0.00d 0.04+0.00d 0.04+0.00d 0.05+0.001c 0.03+0.00e 0.03+0.001e 0.03+0.00e 0.03+0.00e 0.06+0.01b 0.04+0.001d 0.08+0.00a 0.03+0.00e 0.004 0.0000056

Key: Levels not connected by same letter are significantly different (P<0.05). N- = Uninoculated and unfertilized control; N+ = N fertilized control; LSD = Least significant difference; SEM = Standard error of mean;PN = Peg number; NN = Noduotal number; NDW = Nodule dry weight; NR = Nodulation ratings; SDW = shoot dry weight; N% = percentage of nitrogen; TN = total nitrogen content

46

On the other hand, there was a significant difference (p < 0.05) between the inoculated and the negative control on the Babile soil. This is may be because of the inoculants strain was relatively more competent than that of the indigenous rhizobia present in the soil (Graham, 1981; Buttery et al., 1987).

On Fedis soil, isolate HUGR22 was the top scoring that displayed significantly (p < 0.05) higher nodule number as compared to others (Table 7). The maximum nodule number difference between the remaining top scoring (HUGR3 and HUGR12), medium scoring (HUGR10, HUGR11 and HUGR24) and the least scoring (HUGR19, HUGR16, HUGR13 and HUGR18) isolates were about 18, 21 and 22 nodules, respectively, which were quite smaller values compared to the difference of 39 nodules obtained in this study from the respective sand culture. On the Babile soil, isolate HUGR19 was relatively the top scoring displaying significantly (p < 0.05) higher nodule number as compared to other isolates. All the remaining isolates enhanced nodule number next to HUGR19 except HUGR11 which produced lower nodule number even less than the negative control. However, high number of nodule always does not mean, high N fixation in legumes. Similarly, Wynne et al. (1980) reported that the greater number of nodules did not result in greater top dry weights.

4.6.3. Nodulation ratings per plant

Nodulation ratings showed a significant difference at (p < 0.05) among some inoculated isolates and the controls on both soils (Tables 7 and 8). In this study, nodulation rating does not mean number of nodules but it refers to the presence or absence of nodule on the root (taproot and lateral). In the present findings, inoculation significantly increased the nodulation ratings over the controls on both soils. The minimum and maximum nodulation scored were 2 and 10 on Fedis soil and 1 and 10 on Babile soil, respectively. The average value of the nodulation on both Fedis and Babile soils were 5.27 and 7.86, respectively. Wynne et al. (1980) reported that inoculation of rhizobial strains significantly (p < 0.05) influenced nodulation rating in groundnut grown in greenhouse.

47

Table 8. Nodulation data of selected effective isolates of groundnut rhizobia on Babile soil

HUGR 3 HUGR 10 HUGR 11 HUGR 12 HUGR 13 HUGR 16 HUGR 18 HUGR 19 HUGR 22 HUGR 24 N+ NLSD(0.05) SEM

PN (plant-1) 5.0+0.00b 5.0+0.00b 3.33+ 0.58ed 3.0+0.00e 3.0+0.00e 2.67+0.58e 4.67+0.27bc 6.0+0.27a 4.67+0.27bc 1.33+0.27f 4.0+0.00cd 1.33+0.58f 0.84 0.25

NN (plant-1) 41.67+ 1.53e 55.33+ 0.58 c 27.67+2.08g 51.00+2.31d 58.67+1.16b 51.00+1.00d 56.67+1.05bc 62.33+1.05a 42.33+1.05e 42.33+1.05e 25.33+0.58g 38.00+2f 3.05 3.28

NR (plant-1) 8.33+ 2.8 9ba 10.0+0.00a 5.0+0.00c 10.0+0.00a 10.0+0.00a 8.33+2.89ba 10.0+0.82a 10.0+0.82a 10.0+0.82a 5.0+0.82c 1.0+0.00d 6.67+2.87bc 2.43 2.08

NDW (mg plant-1) 98.33+ 7.23 a 92.00+2.00ba 83.00+6.92c 79.33+2.08c 70.77+3.66d 62.00+2.65e 95.33+4.73a 99.67+8.39a 84.67+8.50bc 47.33+2.52f 34.67+0.58g 54.33+0.58fe 8.44 25.09

SDW (g plant-1) 2.1+0.00bc 1.97+0.06dc 1.80+0.10de 1.80+0.00de 1.73+0.06fe 1.73+0.06fe 1.90+0.10de 2.33+0.25a 1.87+0.06de 1.57+0.12f 2.23+0.15ba 1.57+0.06f 0.18 0.011

N % 1.89+0.01dc 1.59+0.01h 1.84+0.02dce 1.74+0.06fg 1.79+0.01fe 2.1+0.12b 1.91+0.01c 1.93+0.03c 1.80+0.01dfe 1.05+0.13i 2.33+0.06a 1.66+0.02hg 0.09 0.003

TN (g plant-1) 0.040+0.00cb 0.030+0.00e 0.033+0.01ed 0.030+0.00e 0.030+0.00e 0.037+0.01cd 0.037+0.01cd 0.043+0.01b 0.030+0.00e 0.020+0.00f 0.053+0.01a 0.030+0.00e 0.006 0.000014

Key: Levels not connected by same letter are significantly different (P < 0.05). N- = Uninoculated and unfertilized control; N+ = N fertilized control; LSD = Least significant difference; SEM = Standard error of mean; PN = Peg number; NN = Noduotal number; NDW = Nodule dry weight; NR = Nodulation ratings; SDW = shoot dry weight; N% = percentage of nitrogen; TN = total nitrogen content

48

The greater number of nodules did not result in greater top dry weights. The nodules where mostly located on the main root, had red interiors, and were associated with large top dry weights.

4.6.4. Nodule dry weight per plant

On Fedis soil, inoculations of all the isolates were showed significantly (p < 0.05) higher nodule dry weight than the positive and the negative controls except HUGR19 and HUGR16 (Table 7). Similarly, on Babile soil except isolate HUGR24, all the isolates produced significantly (p < 0.05) higher nodule dry weight than the negative and the positive controls as indicated on Table 8. The negative control produced higher nodule dry weight than the isolates HUGR19 and HUGR16 on Fedis soil, whereas isolate HUGR24 on Babile soil. Moreover, the minimum and maximum nodule dry weight records were 56.67 11.55 and 170 11.55 mg plant-1 on the Fedis soil, respectively, whereas on the Babile soil the minimum and maximum nodule dry weight were 47.33 + 2.52 and 99 7.23 mg plant-1, respectively. These two extreme values were displayed by the isolates HUGR19 and HUGR22 on the Fedis soil and the isolates HUGR24 and HUGR19 on the Babile soil, respectively.

On the other hand, HUGR19 showed an irrespective property being superior on the Babile soil but inferior on Fedis soil. This may be because of the strain lack of adaptation to the Fedis soil characteristics which may inhibit the effectiveness of the isolate. However, this strain was superior on the Babile soil. This may be due to the strain was isolated from soil ecology of the Babile soil that it was easily adapted soil properties which have relatively higher population of indigenous rhizobia. Inoculation of groundnut with bradyrhizobial isolates in northeast Thailand increased yields and N uptake, even in soils that contain native bradyrhizobia. It is apparent that strains of Bradyrhizobium differ in their effectiveness in different soils and are also affected by the host cultivar (Arunchalum et al., 1984). High inoculation rates were also reported to result in increased recovery of inoculant rhizobia in nodules (Bromfield and Ayanaba., 1980). In the present findings, the recovery of the inoculant strains of groundnut in

49 the presence of > 103 indigenous rhizobia g-1 soil indicated that inoculant rhizobia were highly competitive in the infection nodulation process.

4.6.5. Shoot dry weight per plant

According to in Tables 7 and 8, all inoculated isolates increased shoot dry weight significantly (p < 0.05) over the negative control except the isolates HUGR16 (1.59 g plant-1), HUGR19 (1.6 g plant-1) and HUGR13 (1.64 g plant-1) on the Fedis soil. Similarly, on the Babile soil all inoculated isolates increased shoot dry weight significantly over the negative control except isolate HUGR24 (1.56 g plant-1). The previous research (Reid and Cox, 1973; Walker et al.,
1976) reported inconsistent response of groundnut to inoculation that agreed with the present findings. The average shoot dry matters accumulated by groundnut with the tested isolates

were 1.85 and 1.88 g plant-1 on the Fedis soil and Babile soil respectively. The positive control gave the highest shoot dry weight of all the inoculated isolates except HUGR22 whereas the negative control produced higher shoot dry weight over the inoculated isolate HUGR16, HUGR19 and HUGR13 but insignificant difference with isolates HUGR18 on Fedis soil. On the other hand, Isolate HUGR22 was superior over all inoculated isolate as well as negative control. The remaining six isolates had better performance next to the isolate HUGR22. However, isolate HUGR16 and HUGR19 perform below the negative control on the Fedis soil.

On Babile soil, isolate HUGR19 produce significantly higher shoot dry matter over all the inoculated isolates and the negative control. Similar with the Fedis Soil, the positive control significantly higher shoot dry weight than the other inoculated isolates and the negative control except isolate HUGR19. Isolate HUGR24 was not significantly (p < 0.05) different from the negative control.

The greenhouse study designed to select strains of useful Rhizobium for groundnut and to estimate potential variation in the N fixing ability of these strains in symbiosis with diverse groundnut germplasm, both the host genotype and the strain treatments influenced the effectiveness of the symbiotic association (Wynne, et al., 1980). Some of the strains were able

50

to successfully compete for infection sites and were more effective than the naturally occurring strains (Tables 7 and 8). Earlier studies elsewhere showed that rhizobial inoculant has a favorable effect on legumes like groundnut (Joshi et al., 1989). Fifty nine percent of the observed variation in inoculation response was substantiated to the population density of the indigenous rhizobia (Thies et al., 1991). Generally, the Fedis soil has low population 2.6 x 102 rhizobia g-1 soil and poor nodulation observed is compared to the Babile soil where 3.1 x 103 rhizobia g-1 soil response better nodulation this may be related to the low soil N in Babile soil and low soil P in the Fedis soil (Ghosh and poi, 1998). The presence of low soil P and population of indigenous rhizobia in Fedis soil and low nitrogen content and relatively ineffective indigenous rhizobia in Babile soil inoculation of the selected isolates with phosphorous solublizer bacteria to the Fedis soil and inoculation of the superior isolates in Babile soil to support the hosts N requirements in the present situation may be feasible.

The isolate HUGR22, HUGR3 and HUGR12 well performed in all the three different experiments in terms of shoot dry weights. Moreover, these isolates demonstrated quite comparable observations with the positive control. Generally, shoot dry matter was increased (36-50%) for the ten isolates inoculated on the Babile soil as compared to their respective sand cultures whereas (33-51%) on the Fedis soil. However, the increment in shoot dry matter was common for nitrogen fixing legumes. Similarly, results were reported by (Aynabeba et al., 2001; Zerihun, 2006 and Getaneh, 2008). This increment may be attributed to the

available N of the soil, additional nodulation by the background indigenous isolates of the tested soils, Fe, K and P assimilation, siderophores, plant growth promoting rhizomicrobes and organic and inorganic plant phosphorus mobilization (Alikhani et al., 2006) and enhancement of nutrient uptake of legumes (Zafar-ul-Hye et al., 2007).

The first criterion for a Rhizobium strain to be used in legume inoculant was that it must be highly effective in N fixation (Matos and Schroder, 1989). Generally, in the present studies all strains tested provided N to the plant, as shown shoot dry weight and tissue N content (Tables 7 and 8). Furthermore, there were significant differences (p < 0.05) among rhizobial strains for various parameters such as nodule dry weight, shoot dry weight, N concentration, and N contents. For example, significant differences among the rhizobial strains were observed

51

under growth room, greenhouse, and field conditions for lentil (Bremer et al., 1990), pigeon pea (Matos and Schroder, 1989), clover (Ferreira and Marques, 1992) and chickpea (Chandra and Pareek, 1985; Somasegaran et al., 1988; Icgen et al., 2002).

4.6.6. Total nitrogen percent and content per plant

With respect to percent N, the results in Tables 7 and 8 revealed that the positive control on both soils were significantly higher in N than plants inoculated with all isolates. Isolate HUGR22 also showed significantly (p < 0.05) higher percent of N than plants inoculated with all other isolates and the negative control on the Fedis soil. Similarly, plants inoculated with isolates HUGR16 and isolate HUGR19 had higher percent of N than the plants inoculated with the other isolates on Babile soil. The isolates HUGR 13 and HUGR16 on Fedis soil and HUGR24 and HUGR10 on Babile soil had accumulated significantly lower percent of N (Tables 7 and 8). Despite the absence of a significant difference (p < 0.05) for some strains, the average percent of N value of inoculated isolates in this study was 1.80 and 2.13% for Babile and Fedis soil that was 6% and 7% over their respective negative control, respectively. The determination of N content (g plant -1) (the product of shoot dry weight and percent N) showed that the positive control was significantly (p < 0.05) higher than most of the inoculated isolates and the negative control on both soils (Tables 7 and 8). Besides, isolates HUGR19 and HUGR3 were significantly (p < 0.05) higher in tissue N content than the rest of the isolates on the Babile soil. The highest (0.043 0.01 g plant-1) and the lowest (0.02 g plant-1) tissue N content were displayed by HUGR19 and HUGR24, respectively. Similarly, on the Fedis soil, the positive control showed significantly (p < 0.05) higher than the inoculated isolates and the negative control (Table 7). On the other hand, isolate HUGR22 was superior than the other inoculated isolates followed by the isolate this HUGR12 on the Fedis soil. Some inoculated plants had N content equivalent to or below the negative control which implies that the N fixing capacity of those inoculated isolates were not effective in the respective soil.

52

4.6.7. Correlation of selected parameters on Babile and Fedis soils

The peg number was positively and highly significantly correlated with shoot dry weight and tissue N content on the Fedis soil (Appendix Table 6). Moreover, nodule number was correlated significantly with nodulation ratings and nodule dry weight (r = 0.85** and 0.97**) respectively, and nodulation ratings positively and significantly correlated with nodule dry weight. Shoot dry weight was correlated with content of N fixed (0.90**) and percent of N (r = 0.92**) respectively. Tissue N content was correlated with percent N positively (r = 0.98**) in Appendix Table 6 on Fedis soil.

Similarly, on Babile soil, peg number was correlated significantly (p < 0.05) with nodule dry weight and shoot dry weight. Nodulation ratings significantly correlated with nodule number. Shoot dry weight was positively correlated significantly with percent N (p < 0.01) and nodule dry weight (p < 0.05). Percent N correlated with shoot dry weight and total N content (p < 0.05) in Appendix Table 9 with highly significant manner (p < 0.01). Wynne et al. (1980) reported that nodulation, plant weight, nitrogenase activity and N content of groundnut were significantly correlated with each other. This indicates, as suggested by Date (1976), that screening of rhizobial strains in the greenhouse may be effective as a preliminary evaluation of rhizobial strain performance in the field. Similarly, significant correlation for other legumes were also reported by Beck and Duc (1991) and (Shah et al., 1994).

The above result confirmed the presence of strong and positive associations among some parameters. This may imply that N fixation is a function of photosynthate availability and/or translocation, or of interactions between fixed and soil N (Beck and Duc, 1991). The strong association between N content and shoot dry weight in this study substantiate the declaration of Atici et al. (2005). This help to use N content in legume plants as one of the best parameters to measure symbiotic N fixation under field as well as in greenhouse experimental conditions.

53

4.6.8. Leaf and nodule color assessment

The qualitative measurement carried out in the present studies was the relative redness or whiteness and greenness of the interior nodule color and the leaf color, respectively, in the inoculated plants (Appendix Table 7). According to the scale used, most of the plants produced pink and some slightly dark red nodules except the positive control which had few white color nodules. Indicating that ineffective nodules to fix N in both Babile and Fedis soils. On the other hand, all plants including the controls had light green and some dark green leaves. All these visual observations nearly explain the presence of active nodules and positive nitrogen fixation (Bromfield and Roughley, 1980; Mutch and Young, 2004).

54

5. SUMMARY AND CONCLUSIONS

Biological N-fixation is believed to contribute sufficient N to legumes and thus enhance N and grain yields if supplied with a microsymbiont that can be evaluated and selected for its high effectiveness. Therefore, this study comprised isolation, characterization, selection and evaluation of potential indigenous rhizobial isolates for effective symbiosis with groundnut under sterilized sand and unsterilized different soils under greenhouse condition.

The presumptive test results confirmed that all the isolates were gram negative and rod shaped without absorbing up congo red in dark growth condition. Similarly, no isolate growth was observed on PGA media. All isolates turned the BTB-YEMA medium into moderately deep blue color. As to the cultural characterization, 66% of the isolates had small dry and 33% mucoid, all isolates had a buttery texture and milky color, raised and circular margin of colonies on YEMA medium with a diameter of <1.0 to 2.5 mm when incubated at 28 2 0C.

The result in sand culture showed that all the 24 isolates produced nodules. On the basis of host-plant specificity, all of the rhizobial isolates in this particular study were authenticated as groundnut nodulating Bradyrhizobium spp. Furthermore, inoculation of these isolates significantly improved (p < 0.05) peg number, nodule number, nodule dry weight and shoot dry weight plant-1 as compared to the negative control. The result also displayed a positive and significant (p < 0.01) association of shoot dry weight with nodule dry weight plant -1 and nodule number with nodule dry weight plant-1. Moreover, 62.5% of the tested isolates were found to be symbiotically effective and 37.5% highly effective with groundnut. Isolate HUGR22 had the highest symbiotic effectiveness (116%) which was higher than even the positive control. In addition, HUGR22, HUGR3, HUGR13, HUGR16, HUGR19, HUGR11, HUGR24, HUGR12 and HUGR10 isolates scored between 81 and 116% SE while HUGR18 scored about 76% and considered as the 10th top performing isolate.

Some physiological tests (temperature, salt and pH tolerance) for those of the ten best isolates were carried out on YEMA medium and all isolates have capability to grow on high pH (6-9),

55

low content of NaCl salt 0.1-0.5% (except HUGR18 which can grow on 1%) and high temperature (15-40 0C). However, all isolates, failed to grow on low pH (4.5-5.5), temperature (4 -10 0C) and high NaCl salt (1-2%). Similarly, Symbiotic effectiveness test for the ten best isolates were also carried out in soil having 2.6 x 102 and 3.3 x 103 rhizobia g-1 of soil on the sites of Fedis and Babile. Inoculation of the isolates in groundnut significantly increased peg number, nodule number, nodulation ratings, nodule dry weight, shoot dry weight and N content in this particular study. All isolates had significantly higher (p < 0.05) nodule number, nodulation ratings and nodule dry weight on both soils than the positive control with some exception. On the other hand, the positive control had significantly higher (p < 0.05) shoot dry weight and N content over all isolates on the both Babile and Fedis soils apart from HUGR22 on Fedis and HUGR19 on the Babile soil where no significant different was observed. All isolates had significantly higher (p < 0.05) peg number and shoot dry weight than the negative control except HUGR16, HUGR19 and HUGR13 on the Fedis soil and HUGR24 on the Babile soil. All isolates were significantly (P < 0.05) produced higher nodule number than the negative control on the Fedis soil except HUGR16, HUGR19 and HUGR13. On the other hand, the negative control was significantly higher nodule number than HUGR24, HUGR10 and HUGR11 isolates on Babile soil. All isolates were significantly increased nodule dry weight over the negative control on both soils except HUGR24 on the Babile soil and HUGR19 and HUGR16 on the Fedis soil. Despite of the absence of a significant difference (p < 0.05) for some strain, the average N percentage of groundnut inoculated with the isolates in Babile and Fedis soils this study were 1.80 and 2.13% that increased 6 and 7% over their respective negative control, respectively. The highest (0.043 g plant-1) and the lowest (0.020 g plant-1) N contents were displayed by HUGR19 and HUGR24, respectively, on Babile soil. Whereas on Fedis soil, isolates HUGR3, HUGR10, HUGR11 and HUGR24 were not significantly different (p < 0.05). Moreover, isolate HUGR3, HUGR16, HUGR18, HUGR19 were not statistically significantly different from the negative control. On the other hand, isolate HUGR22 followed by isolate HUGR12 was superior to the other inoculated isolate on Fedis soil.

Generally, Isolate HUGR22 on the Fedis and HUGR19 on the Babile soils were significantly superior in all parameters to other isolates. The poor nodulation of the positive control on both

56

soils was associated with the inhibition effect of mineral N. In relation to correlation of selected parameters, peg number was positively associatived with shoot dry weight and total N content (r = 0.88 and 0.90, respectively); nodule number with nodulation rating and nodule dry weight (r = 0.85 and 0.97); nodulation ratings with nodule dry weight(r = 0.87); shoot dry weight with N % and N content (r = 0.90, 0.92) finally N% with total N content (r = 0.98) on Fedis soil whereas on Babile soil, total N was positively associated with shoot dry weight and N% (r =0.08 and 0.083, respectively) in significant at (p < 0.01).

East Hararghe Zone is one of the important groundnut production areas, where more effective strains of Bradyrhizobium spp can be isolated from other cross inoculation group of warm season legumes of other parts of Ethiopia. Considering these observations, it remains to be a follow-up research on screening using molecular techniques such as REP/PCR or RFLP/PCR, sequencing of 16S rDNA genes and DNA/DNA hybridization to study the competitiveness of the strains in terms of nodule occupancy. Further investigation should be carried out to evaluate their effectiveness under different environmental conditions both in the greenhouse and field trials. Generally, similar studies should be encouraged to obtain local isolates that can form more effective symbiosis under a wide range of environmental conditions across the country.

57

6. REFERENCES
Abdel-ghaffar, A.S., 1988. Effect of edaphic factors on biological N fixation in Vicia faba under Egyptian soil condition. pp. 303-316. In: D.P. Beck and L.A. Materon (eds.). Proceedings of a Workshop on Biological Nitrogen Fixation on Mediterranean-type Agriculture. Aleppo, Syria, 14-17 April 1986, ICARDA Abere Mnalku, 2010. Isolation and selection of Rhizobium leguminosarum bv. Viciae strains for effective symbiotic association with faba bean (Vicia faba L.) in Eastern and Western Hararghe Highlands, Ethiopia. MSc Thesis, Haramaya University. Ahmed, A.E. and E.A.E. Elsheikh, 1998. Effects of biological and chemical fertilizers on growth and symbiotic properties of faba bean (Vicia faba L.) under salt stress. U.K. Agric. Sci. 6: 150-166 Alikhani, H.A., N. Saleh-Rastin and H. Antoun, 2006. Phosphate solubilization activity of rhizobia native to Iranian soils. Plant and Soil, 287: 35-41. Allen, O.N. and E.K. Allen, 1981. Leguminosae. Wisconsin, USA: University of Wisconsin Press. Al-Sherif, E.M., 1998. Ecological studies on the flora of some aquatic systems in Beni-Suef district. M.Sc. Thesis. Cairo University (Beni-Suef Branch), Beni-Suef, Egypt. Amanuel Asrat, 2008. Exploration and characterization of mung bean (vigna radiata L.) and groundnut (Arachis hypogaea L.) Nodulating indigenous rhizobia in Awassa and Gofa soils.Ethiopia. MSc Thesis, Awassa University. Arunchalum, V., G.D.M. Pungle, M. Dutta, P.T.C. Nambiar and P.J. Dart, 1984. Efficiency of nitrogenase activity and nodule mass in predicitng the relative performance of genotypes assessed by a number of characters in groundnut (Arachis hypogaea).Expl. Agric. 20:303-309. Atici, O., H. Ogutcu and O.F. Algur, 2005. Effect of putrescence on inducing symbiosis in chickpea and vetch inoculated with commercial or indigenous strains of Rhizobium. Symbiosis, 38: 163-174. Ayanaba, A., Asanuma, S. and Munns D.N., 1983. An agar plate method for rapid screening of Rhizobium for tolerance to acid-aluminium stress. Soil Science Society of America Journal, 47: 256-268. Ayneabeba Adamu, Fassil Assefa, Asfaw Hailemariam and Endashaw Bekele, 2001. Studies of Rhizobium inoculation and fertilizer treatment on growth and production of faba bean (Vicia faba) in some yield-depleted and yield sustained regions of Semien Shewa. SINAT: Ethiop. J. Sci. 24(2): 197-211. Beck, D. and G. Duc, 1991. Improving N2-fixation in faba bean: Rhizobium inoculation and N nutrition. Options Mediterraneans, 10: 97-103. Bernal, G. and P. H. Graham, 2001. Diversity in the rhizobia associated with Phaseolus vulgaris L. in Ecuadore, and comparisons with Mexican bean rhizobia. Can. J. Microbiol. 47: 526-534. Bordeleau, L.M. and D. Prevost, 1994. Nodulation and nitrogen fixation in extreme environments. Plant Soil, 161: 115124 Bouhmouch, I., Brhaoda, F., Filali-Maltouf, A., Aurag, J., 2001. Selection of osmotolerant and effctive strains of Rhizobiaceae for inoculation of common bean (Phaseolus vulgaris) in Moroccan saline soils. Agronomie, 21:2072-2077.

58

Boonkerd, N.P. and Wadisirisuk, 1993. Population size and nitrogen-fixing activity of native peanut rhizobia in soils of Thailand. Biology and Fertility of Soils, 15(4): 275-278. Bremer, E., C. Van Kessel, L. Nelson, R.J. Rennie and D.A. Rennie, 1990. Selection of Rhizobium leguminosarum strains for lentil (Lens culinaris) under field conditions. Plant and Soil, 121: 47-56. Brockwell, J., P.J. Bottomley, and J.E. Thies. 1995. Manipulation of rhizobia micro flora for improving legume productivity and soil fertility: a critical assessment. Plant Soil, 174: 143180. Bromfield, E.S.P., and A. Ayanaba, 1980. The efficiency of soybean inoculation on acid soil in tropical Africa. Plant Soil, 54: 95-106. Bromfield E.S.P. and R.J. Roughley, 1980. Characterization of rhizobia isolated from nodules on locally adapted Glycine max grown in Nigeria. Annals of Applied Biology, 95:185190. Burris, R.H., 1994. Biological nitrogen fixation past and future, p. 111. In N.A. Hegazi, M. Fayez, and M. Monib (ed.), Nitrogen fixation with non legumes. The American University in Cairo Press, Cairo, Egypt. Burton J.C., 1976. Pragmatic aspects of the Rhizobium leguminous plant association. Pp 429 - 446. In Newton, W. E. and Nyman, C. J. (Eds.) Proc. 1st Inter. Symp. On N fixation, Vol. 2, Wash. State Univ. Press. Burton, J.C., 1982. Modern concepts in legume inoculation. Pp 105-114 in Biological nitrogen fixation technology for tropical agriculture: papers presented at a Workshop, 9-13 Mar, 1981. Cali, Colombia: (Graham, P.H. and Harris, S.C., eds) : Cali, Colombia: Centro Internacional de Agricultura Tropical. Buttery, B.R., S.J. Park and P. Van Berkum, 1997. Effects of common bean cultivar and Rhizobium strain on plant growth, seed yield and N content. Canadian Journal of Plant Science,77: 1-4. Chaintreuil, C., Giraudm, E., Prin, Y., Lorquin, J., B.A.M. Gillis, de Lajudie, P., B. Dreyfus, 2000. Photosynthetic bradyrhizobia are natural endophytes of the African wild rice Oryza breviligulata. Appl Environ Microbiol. 66: 5437 5447. Chandler, M.R., 1978. Some observations on infections of Arachis hypogaea L. by Rhizobium. Journal of Experimental Botany, 29: 749-755. Chaudhary, G.A., 1986. Groundnut; a usefull crop for Barani areas and Sandy lands. Pp 100 103. proc. Nti.seminar on Oilseed Research and development in Pakistan A perspective Organized by Pak. Agric. Res. Council in Islamabad on May 6-9, 1985. Chandra, R. and R.P. Pareek, 1985. Role of host genotype in effectiveness and competitiveness of chickpea (Cicer arietinum L.) Rhizobium. Tropical Agriculture, 62: 90 - 94. Cooper, J.E., 1982. Acidic production, acid tolerance and growth rate of lotus rhizobia in laboratory media. Soil Biology and Biochemistry, 14: 127 - 131. Correa, O. S. and A. J. Barneix, 1997. Cellular mechanisms of pH tolerance in Rhizobium loti. World J. Microbiol. Biotechnol. 13: 153 157 CSA (Central Statistics Authority), 2009. Agricultural sample survey 2009/2010 (2001E.C.). Conducted from September to December 2009. Vol. 1. Report on Area and Production of Crops (for private peasant holdings, meher season). Addis Ababa, Ethiopia. Date, R.A. 1976. Principles of Rhizol>irrni strain selection, pp. 137-150. In P. S. Nutman (ed.) Symbiotic Nitrogen Fixation in Plants. Cambridge Univ. Press, Cambridge.

59

Daniel Endale, 2009. Groundnut research Pp 1-3. In: Presentation for workshop, Werer Research Center, Ethiopia. Unpublished. Danso, S.K.A., Zapata, F. and K.O.Awonaike, 1990. Effect of post emergence, supplemental inoculation on nodulation and symbiotic performance of soybean (Glycin max L) Merrill) at three levels of soil nitrogen. Appl. Environ. Microbiol. 56: 1793 - 1798. Dakora, F.D. and Vincent, J.M., 1984. Fast growing bacteria from nodules of cowpea (Vigna ungucuilata L. Walp.) J. Appl. Bacteriol. 56: 327 - 330. Dashti, N., F. Zhang, R. Hynes and D.L. Smith, 1998. Plant growth promoting rhizobacteria accelerate nodulation and increase nitrogen fixation activity by field grown soybean [Glycine max L. Merr.] under short season conditions. Plant and Soil, 200: 205 213. Desta Beyene and Angaw Tsige, 1986. The response of pulse crops to N and P fertilizers. In: Soil Science Research. Proceedings of the First Soil Science Research Review Workshop, Addis Ababa, Ethiopia, 11 - 14 Feb. Eaglesham, A.R.J. and A. Ayanaba, 1984. Tropical stress ecology of rhizobia, root nodulation and legume fixation, p. 1 35. In N. S. Subba Rao (ed.), Current developments in biological nitrogen fixation. Edward Arnold Publishers, London, United Kingdom. Eaglesham, A.R.G., 1989. Nitrate inhibition of root nodule symbiosis in doubly rooted soybean plants. Crop sci. 29: 115 - 119. Elsheikh-Elsiddig, A.E., 1998. Effects of salt on rhizobia and bradyrhizobia: a review. Annals of. Appllied Biology, 132: 507 524. Erdman, L.W., H.W. Johnson and F. Clark, 1957. Varietals Response of Soya bean to a Bacterial induced chlorosis. Argon. J. 49: 267-271. Fassil Assefa, 1993. Nodulation and Fixation by Rhizobium and Bradyrhizobium spp of some indigenous legumes of Ethiopia. PhD dissertation. FAO (Food and Agriculture Organization), 1990. Fertilizer yearbook, vol. 39. FAO, Rome, Italy. FAO (Food and Agriculture Organization), 2006. FAO Production Yearbook, Vol. 60, Rome, Italy. Ferreira, E.M. and J.F. Marques, 1992. Selection of Portuguese Rhizobium leguminosarum bv. trifolii strains for production of legume inoculants. Plant and Soil, 147: 151 - 158. Fisher, R.F. and S. R. Long, 1992. "Rhizobium Signal Exchange." Nature, 357: ' 655-660. Frederich, W.S., S.B. Wall, A. Rehman and M.S. Nawaz, 1991. Groundnut production in Pakistan. BARD Project, PARC, Islamabad. Freeman, H.A., Nigam, S.N., Kelley, T.G., Ntare, B.R., Subrahmanyam, P. and Boughton, D., 1999. The World Groundnut Economy: Facts, Trends and Outlook. ICRISAT, Hyderabad, Andhra Pradesh, India. Gates, C.T. and Muller, W.J., 1979. Nodule and plant development in the soyabean, Glycine max (L.) Merr.: Growth response to nitrogen, Phosphorus and Sulfure. Aust. J. Bot. 27: 203 -215. Getaneghe Tesfaye, 2008. Symbiotic and Phenotypic Diversity of Rhizobium Isolates Nodulating (Vicia faba) from Western Shewa and Western Hararghe, Ethiopia. MSc Thesis, Addis Ababa University. Pp. 23-52. Ghosh, G. and Poi, S.C., 1998. Response of Rhizobium, phosphorus soluilizing bacteria and micorrhizal organisms on some legume crops. Env. Ecol. 16 (3), 607 - 610.

60

Giller, K.E., Witter, E., McGrath, S.P., 1998. Toxicity of heavy metals to microorganism and microbial processes in agricultural soils: a review. Soil Biol. Biochem. 30, 1389 1414. Giller, K.E., 2001. Nitrogen fixation in tropical cropping system 2ed. CAB International Wageninge, Netherlands. Gomez, K.A. and A.A. Gomez, 1984. Statistical Procedure for Agricultural Research. 2nd Ed. John Wiley and sons Inc., New York. Graham, P.H. and Parker, C. A., 1964. Diagnostic features in the characterization of the root nodule bacteria of legumes. Plant and Soil, 28: 383 396. Graham, P.H., 1981. Some problems on nodulation and symbiotic nitrogen fixation in Phaseolous vulgaris L. a review. Field Crops Research 4:92-112. Graham, P.H., 1992. Stress tolerance in Rhizobium and Bradyrhizobium, and nodulation under adverse soil conditions. Canadian Journal of Microbiology, 38: 475 484. Graham, P.H., K. Draeger, M. L. Ferrey, M. J. Conroy, B. E. Hammer, E.Martinez, S. R. Naarons and C. Quinto, 1994. Acid pH tolerance in strains of Rhizobium and Bradyrhizobium and initial studies on the basis for acid tolerance of Rhizobium tropici UMR1899. Can. J. Microbiol. 40: 198 207. Hadad M.A. and Loynachan, T.E., 1985. Abundance and characterization of cowpea Miscellany Rhizobium from Sudanese soils. Department of Agronomy, Iowa state university, Ames, IA 50011, U.S.A. Hadad, M.A., Loynachan, T.E., and Musa, M.M., 1982. Inoculation trials on groundnut (Arachis hypogaea) in Sudan. pp 249 - 256 in Biological nitrogen fixation Technology for tropical agriculture: papers presented at a Workshop, 9-13 Mar, 1981, Cali, Colombia (Graham, P.H. and Harris, S.C. eds) Cali, Colombia: Centro Internacional de Agricultura Tropical. Hamdi, Y.A., 1982. Symbiotic Nitrogen fixation in faba beans. pp. 127-138. In: G. Hawtin and C. Webb (eds.). Proceedings of the faba bean Conference. Cairo, Egypt, 7-11 March 1981. ICARDA. Hardarson, G., 1993. Methods for enhancing symbiotic N fixation. pp. 1 - 15. In: F.A. Bliss and G. Hardarson (eds.). Enhancement of Biological N Fixation of Common Bean in Latin America, vol. 52. Kluwer Academic. Havlin, J.L., J.D. Beaton, S.L. Tisdale and W.L. Nelson, 2003. Soil Fertility and Fertilizers. 6th ed. An Introduction to Nutrient Management. Pearson Education, Inc., Singapore. 437p. Hegde, S.V., 1982. Field responses to Rhizobium inoculation in Arachis hypogaea, Vigna spp. and Dolichos spp. in India. Pp 257 - 264 in Biological nitrogen fixation technology for tropical agriculture: papers presented at a Workshop, 9-13 Mar, 1981. Cali, Colombia (Graham, P.H., and Harris, S.C., eds.). Cali, Colombia: Centro Internacional de Agricultura Tropical. Hernandez, B. S. and Foscht, D.D., 1984. Invalidity of the concept of slow growth and alkali production in cowpea rhizobia. Appl. Environ. Microbiol. 48: 206 - 210. Hoa, N.T.L., T.Y. Thao, P. Lieu and D. Herridge, 2002. N2 Fixation of Groundnut in the Eastern Region of South Vietnam. In: D. Herridge (ed.), Inoculants and Nitrogen Fixation of Legumes in ACIAR Proceedings Vietnam. 109p. Hosney, I., H. K. Abd-El-Maksoud and F.H. Abd-EI-Zaher, 2002. Ecological studies on soil indigenous rhizobia in Egypt. Egyptian Journal of Microbiology, 37(2): 173 - 183.

61

Hoque, M.S., 1993. Bradyrhizobium technology: a promising substitute for chemical Nitrogen fertilizer in Bangladesh agriculture. Department of Soil Science, Bangladesh Agricultural University, Mymensigh, Bangladesh. Kluwer Academic Publishers, printed in the Netherlands. Plant and soil, 155/156: 337-340. Hungaria, M. and Vargas, M.A.T., 2000. Environmental factor affecting N2 fixation in grain legumes in tropics, with an emphasis on Brazil. Elsevier Science, B.V. 65: 151 - 164. Hungria, M., Vargas, M. T., Campo, R. J., Chueire, L. O. and Andrade, D. S., 2000. The Brazilian experience with the soybean (Glycine max) and common bean (Phaseolus vulgaris) symbioses. In: Nitrogen Fixation: From Molecules to Crop Productivity, 515p, (Pedrosa, F. O., Hungria, M., Yates, G. and Newton, W. E. eds.). Kluwer Academic Publishers, Dordrecht, the Netherlands. Icgen, B., G. Ozcengiz and N.G. Alaeddinolu, 2002. Evaluation of symbiotic effectiveness of various Rhizobium cicer strains. Research in Microbiology, 153: 369 - 372. ICRISAT (International Crops Research Institute for the Semi-Arid Tropics), 1995. Annual Report, 1995. Patancheru, A.P. 502 324, India: ICRISAT. (Bulletin no. 30) Jordan, D.C., 1984. Rhizobiumceae. In: Krieg, N. and J.G. Holt (eds.), Bergeys manual of systematic bacteriology, Williams and Wilkins, Baltimore 1: 235-256. Joshi, P.K., Kulkarni, J.H. and Reddy, P.S., 1989. IGR6 and IGR40: two effective Bradyrhizobium strains for rabi / summer groundnut production. Junagadh , Gujarat 362001, India : National Research Centre for Groundnut, Indian Council of Agricultural Research.12p. Kennedy, L.D. and R.M. Greenwood, 1982. 6-Phosphogluconate and glucose-6-phosphate dehydrogenase activities, growth rate, and acid production as taxonomic criteria for Rhizobium. N.Z. J. Sci. 25: 361 - 366. Kenya, S.O., 1977. Nodulation and N Fixation in Legumes in East Africa. In: A. Ayanaba and P.J. Dart (Eds). Biological N Fixation in Farming Systems of the Tropics. pp 233-244. John Wiley and Sons, Chichester. Kulkarni, S., Surange, S. and Nautiyal, S.C., 2000. Crossing the limits of Rhizobium existence in extreme conditions. Curr. Microbiol. 41: 402 409. Lalande, R., Bigwaneza, P.C. and Antoun, H., 1990. Symbiotic effectiveness of strains of Rhizobium leguminosarum biovar Phaseoli isolated from soils of Rwanda. Plant Soil, 121: 41-46. Lapinskas, E., Ambrazaitiene, D. and Piaulokaite-motuziene, L., 2005. Estimation of soil microbiological properties in relation to soil acidity and fertilization. Latvian J. Agro. 8: 39-43 Lemon, R., T. Lee, M. Black, W.L. Grichar, T. Baughman, P. Dotray, C. Trostle, M. McFarland, P. Baumann, C. Cmmbley, J.S. Russell, and G. Norman, 2000. "Texas Peanut Production Guide." Texas Agricultural Extension Service Texas A & M University: 1 - 60. Lopes, E.S. 1977. Ecology of legume-Rhizobium symbiosis. Pp 173-190 in Limitations and potentials for biological nitrogen fixation in the tropics (Dobereiner, J., Burris, R.H. and Hollaender, A., eds.). New York, USA: Plenum Press. Matos, I. and C. Schroder, 1989. Strain selection for pigeon pea Rhizobium under greenhouse conditions. Plant Soil, 116, 19-22. Maury, P., S. Suc, M. Berger and C. Planchon, 1993. Response of Photochemical Processes of photosynthesis to dinitrogen fixation in Soybean. Plant Physiol. 101: 493-497.

62

Nif Tal, 1979. Legume inoculation trials procedures. Nif Tal Project, University of Hawaii, USA. Millar, C.E. and L.M. Turk, 2001. Fundamental of Soil Science. Biotech Books. New Delhi. 420p. Miller, R.W. and R.L. Donahue, 1997. Soils in Our Environment. 7th Ed. Prentice-Hall of India, New Delhi. Mitiku Haile, 1990. Preliminary studies of biological nitrogen fixation by haricot bean on two soil types in Hararghe, Ethiopia. In: Proceedings of the Second Regional Workshop on Bean Research in Eastern Africa, Nairobi, Kenya. Mohamed Ahmed, T.H., E.A.E. Elsheikh and A.A. Mahadi, 2009. The in vitro compatibility of me Rhizobium and Bradyrhizobium strains with fungicides. Afr. Crop Sci. Conf. Proc., B: 1171-1178. Munevar, F., and A.G. Wollum II, 1981. "Growth of Rhizobium japonicum strains at temperatures above 27C." Appl Envir. Microbiol. 42: 272 - 276. Munevar, F., and A.G. Wollum, 1982. Response of soybean plants to high root temperature as affected by plant cultivar and Rhizobium strain. Agron. J. 74:138142. Musa, M.M, 1982. Symbiotic N fixation in faba beans in Sudan. Pp 139-142. Proceedings of the faba bean Conference held in Cairo, Egypt, March 7-11, 1981. ICARDA. Mutch, L.A. and P.W. Young, 2004. Diversity and specificity of Rhizobium leguminosarum bv. viciae on wild and cultivated legumes. Nambiar, P.T.C, and Dart, P.J., 1980. Studies on nitrogen fixation by groundnut at ICRISAT. Pp 110 - 124 in Proceedings of the International Workshop on Groundnuts, 13-17 Oct 1980, ICRISAT Center, India. Patancheru, A.P. 502 324, India: International Crops Research Institute for the Semi-Arid Tropics. (CPE 012) Nambiar P.T.C, Dart, P.J., Srinivasa Rao, B., and Rao, V.R., 1983. Nodulation in the hypocotyl region of groundnut (Arachis hypogaea L.). Experimental Agriculture 18: 203-207. (JA 178) Nambiar, P.T.C, Dart, P.J., Srinivasa Rao, B. and Ravishankar, H.N., 1982. Response of groundnut (Arachis hypogaea) to inoculation. Pp 241-248 in Biological nitrogen fixation technology for tropical agriculture: papers presented at a Workshop, 9-13 Mar 1981, Cali, Columbia (Graham, P.H., and Harris, S.C., eds.). Cali, Colombia:Centro Internacional de Agricultura Tropical. (CP 58) Nambiar, P.T.C., 1985. Response of groundnut (Arachis hypogaea L.) to Rhizobium inoculation in the field : problems and prospects. MIRCEN Journal of Applied Biology and Biotechnology, 1:293-309. (JA 475) Nambiar, P.T.C., 1986. The use of enzyme-linked immunosorbent assay for quality assessment of bacterial inoculants. Page 113 in Cereal nitrogen fixation: proceedings of the Working Group Meeting, 9-12 Oct 1984, ICRISAT Center, India. Patancheru, A.P. 502 324, India: International Crops Research Institute for the Semi arid Tropics. (Abstract.) (CPE 037). Nwokoto, E., 1996. Peanut (Arachis hypogaea L.). In: Food and Fee from Legumes and Oil seeds. E. Nwokoto and J. Smartt, Eds. Pp. 49-63. New York: Chapman and Hall.ODA (Overseas. Development Administration). 1984. Annual report no.13.Response of groundnut to the distribution of rainfall or irrigation. Nottingham, UK: University of Nottingham.

63

OSSEP-East Hararghe Zone, 2009. The Oromia government socio economic profile of East Hararghe Zone. Unpublished. Peoples, M. and E. Craswel.1992. Biological nitrogen fixation: Investments, expectation and actual contribution to agriculture. Plant soil, 141:13-39. Peoples, M.B., D.F. Herridge, and J.K. Ladha. 1995. Biological nitrogen fixation: an efficient source of nitrogen for sustainable agricultural production. Plant Soil, 74: 3 28. Piha, M. I., and D. N. Munnus. 1987. Sensitivity of the common bean (Phaseolus vulgaris L.) symbiosis to high soil temperature. Plant Soil, 98: 183194. Polhill, R.M. and Raven, P.H., 1981. Advances in Legumes Systematics. Part I. Royal Botanic Gardens, Kew, UK. Postgate, J.R., 1982. The fundamentals of nitrogen fixation. Cambridge University Press, Cambridge, United Kingdom. Purohit, S.S., 2001. Biotechnology. Fundamentals and Applications. 3rd Ed. Annis Offset Printers, New Delhi. Rai, R., 1992. Effect of acidity factors on aspects of symbiotic N2 fixation of Lens vulinaris in acid soils. J. Gen. Appl. Microbiol. 38: 391406. Ravuri, V. and D.J. Hume, 1993. Soya bean Stover Nitrogen Affected by Dinitrogen fixation and Cultivar. Agron. J. 85: 328-333. Raza, S., B. Jrnsgaerd, H. Abou-Taleb and J.L.Christiansen, 2001. Tolerance of Bradyrhizobium sp. (Lupini ) strains to salinity, pH, CaCO3 and antibiotics. Applied Microbiology, 32: 379-383. Reid, P.H. and Cox, F.R., 1973. Soil properties, mineral nutrition and fertilization practices. Pp 271-297 in Peanuts: culture and uses (Wilson, C.T., ed.). Stillwater, Oklahoma, USA: American Peanut Research and Education Association. Rendle, A.B., 1979. The Classification of Flowering Plants. Vol. II, Dicotyledons. Vikas Publishing House, p.v.t. Ltd., India, Pp.348-370. Robert M. and Claudia P., 2000. Techniques for the Quantification of Plant-Associated Biological Nitrogen Fixation. Department of Crop Science, University of Florida. Sahelemedhin Sertsu and Taye Bekele (2000). Procedures for Soil and Plant analysis. National Soil Research Center, Ethiopian Agricultural Research Organization, Addis Ababa,Ethiopia. Pp 70-76. Sakala, M. K., 1984. Field beans (Phaseolus vulgaris) response to inoculation in Zambia. In: Proceeding of the First Conference of the African Association for biological Nitrogen Fixation. Nairobi, Kenya. Sanginga, N., Vanlauwe, B. and Danso, S. K. A., 1995. Management of biological N2 fixation in alley cropping systems: Estimation and contribution to N balance. Plant soil, 174: 119-141. Schiner, F., R. Olhiner, E. Kandeler and R. Margesin, 1996. Methods in soil biology. Springer- Verlag, Berlin Heidelberg. Schultze, M. and A. Kondorosi, 1998. "Regulation of symbiotic root nodule development." WM. Rev. Genet. 32: 33 - 57. Sessitsch, A., J.G. Howieson, X. Perret, H. Antoun, and E. Martinez-Romero, 2002. "Advances in Rhizobium Research." Critical Reviews in Plant Sciences, 21: 323 - 378. Sharma, A.K., 2002. A Handbook of Organic Farming. Central Arid Zone Research Institute. Alberta. 81: 248 249.

64

Simpson, F.B. and Burris, R.H., 1984. A nitrogen pressure of 50 atmospheres does not prevent evolution of hydrogen by nitrogenase. Singleton, P.W. and J.W. Tavares, 1986. Inoculation response of legumes in relation to the number and effectiveness of indigenous Rhizobium populations. Applied Environmental Microbiology 51:1013-1018. Slattery, J. and D. Pearce, 2002. The impact of background rhizobial population on inoculation response. Pp 33 - 44. In: D. Herridge (ed.). Proceedings of Inoculants and N Fixation of Legumes in Vietnam. ACIAR. Smartt, J., 1990. Grain Legumes. Evolution and Genetic Resources. Cambridge University Press,Cambridge, UK. Smartt, J., 1994. The Groundnut. Chapman and Hall, London. Smith, D.L. and C. Hamel, 1999. Crop Yield Physiology and Processes. Springer-Verlag Berlin. Heidelberg, New York University. 586p. Somasegaran, P., H.J. Hoben and V. Gurgun. 1988. Effects of inoculation rate, rhizobial strain competition, and nitrogen fixation in chickpea. Agronomy Journal, 80: 68 - 73. Somasegaran, R.C. Abaidool and F. Kumaga, 1989. Host-Bradyrhizobium relationships and nitrogen-fixation in the Bambarra groundnut [Voandzeia subterrane (L.)Thouars nom. cons.] NifTAL Project, University of Hawaii, 1000 Holomua Avenue, Paia, Hawaii 96779, USA Somasegaran, P. and Hoben. H. J., 1994. Handbook for Rhizobium. Methods in Legume Rhizobium technology. Pp. 1-380, Springer-Verlang. New York. Sprent, J.I. and P. Sprent, 1990. Nitrogen fixing organisms. Pure and applied aspects. Chapman and Hall, London, United Kingdom. Sprent, J. I. and Raven, J. A., 1992. Evolution of nitrogen fixing symbioses. In:Biological Nitrogen Fixation, pp 461-496, (Stacey, G.S., Burris, R.H. and Evans, H.J., eds). Chapmen and Hall, New York. Srivastava, H.S. and R.P. Singh, 1999. Nitrogen Nutrition and Plants Growth. RhizobiumLegume Association Oxford & IBH publishing Co. Pvt. Ltd, New Delhi. 687p. Straub, P.F., Shearer, G., Reynolds, P.H.S., Sawyer, S. A. and Kohl, D., 1997. Effect of disabling bacteroid proline catabolism on the response of soybean to repeated drought stress. Subba Rao, N.S., 1976. Field response of legumes in India to inoculation and fertilizer applications. Pp 255-268 in Symbiotic nitrogen fixation in plants (Nutman, P.S., ed.). Cambridge, UK: Cambridge University Press. Subba Rao, N.S., 1986. Soil Microorganisms and plant growth. 2nd Ed. Indian Agricultural Research Institute. Oxford & IBH Publishing Co. Pvt. Ltd, New Delhi. 543p. Subba Rao, N.S., 1988. Biofertilizers in Agriculture. Oxford & IBH Publishing Co. Pvt. Ltd, New Delhi, Bombey, Calcutta. Pp 16 - 65. Subba Rao, N.S, 1999. Soil Microbiology. Fourth Edition of Soil Microorganisms and Plant Growth. Pp 166-228. Oxford & IBH Publishing Co. Pvt. Ltd, New Delhi, Calcutta. Subba Rao, N.S., 2001. Introduction to Microbiology. Metabolism of Bacteria. Microorganisms in the Environment. Prentice-Hall of India, New Delhi, 425p. Surange, S., A.G. Sollum, N. Kumar and S. Nautiyal. 1997. Characterizations of Rhizobium from root nodules of leguminous tress growing in alkaline soils. Canadian Journal of. Microbiology, 43: 891-894.

65 Tate III, R.L., 2000. Soil Microbiology.2 nd Ed. N Fixation: The Gate Way to Soil Nitrogen Cycling. John Wiley and Sons, Inc., Toronto. 647p. Tekalign Mamo and Asegelil Dibabe, 1994. Soil Microbiology research. In: Cool- Season Food Legumes of Ethiopia, pp. 293-311, (Asfaw Telaye, Geletu Bejiga, Saxena, M. C. and Solh, M. B., eds) Proceedings of the First National Cool-season Food legumes review Conference, 16-20 December 1993, Addis Ababa, Ethiopia. ICARDA/ Institute of Agricultural Research, ICARDA, Syria. VII 440P. Tekalign Mamo and I. Hague, 1991. Phosphorus status of some Ethiopian soils. Plant and soil, 102: 261-266. Thies, J.E., P.W. Singleton and B.B. Bohlool. 1991. Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes. Applied Environmental Microbiology, 57: 19 - 28. Trevaskis, B., Colebatch, G., Desbrosses, G., Wandrey, M., Wienkoop, S., Saalbach, G. and Udvardi, M., 2002. Differentiation of plants cells during symbiotic nitrogen fixation. Comp. Funct. Genom. 3: 151-157. Tsvetkova, G.E., G.I. Georgiev, 2003. Effects of phosphorous nutrition on the nodulation, nitrogen fixation and nutrient use efficiency of Bradyrhizobium japonicum soybean (Glycine max L. Merr.) symbiosis. Bulg. Journal of Plant Physiology, special issue, 331-335. Urtz, B. and Elkan, G., 1996. Genetic diversity among Bradyrhizobium isolates that Effectively nodulate peanut (Arachis hypogaea). Canadian Journal of Microbiology, 42, 11211130. Van Rhijn, P.and, J. Vanderieyden, 1995. "The Rhizobium-Plant Symbiosis." Microbiological Reviews, 59: 124-142. Van Slyke, L.L., 2001. Fertilizers and Crop Production. Relation of Microorganisms to Plant Food. Updesh Purohit for Agro bios (India), Jodhpur. 523p. Vincent, J.M., 1970. A manual for the practical study of root-nodule bacteria. IBP Handbook no. 15. Oxford, UK: Blackwell Scientific Publications. Vinuesa, P., Leon-Barrios, M., Silva, C., Willems, A., Jarabo-Lorenzo, A., Perez-Galdona, R., Werner, D., Martinez-Romero, E., 2005. Bradyrhizobium canariense sp. nov., an acidtolerant endosymbiont that nodulates endemic genistoid legumes (Papilionoideae: Genisteae) from the Canary Islands, along with Bradyrhizobiu japonicum bv. genistearum, Bradyrhizobium genospecies alpha and Bradyrhizobium genospecies beta. Int J Syst Evol Microbiol. 55: 569575. Vlassak, K.M., and J. Vandurleyden, 1997. Factors influencing nodule occupancy by inoculant rhizobia. Crit. Rev. Plant Sci. 16: 163229. Walker, M.E. , Minton, N.A. and Dowler, C.C., 1976. Effects of herbicides, a nematicide and Rhizobium inoculant on yield, chemical composition and nodulation of Star peanuts (Arachis hypogaea L.). Peanut Science, 3: 49 - 51. Wange, S. S., 1989. Response of groundnut (Arachis hypogaea L.) to inoculation with Rhizobium strains isolated from wild arboreal legumes. J. Appl.Microbiol. Biotechnol. 5: 135141. Weiss, E.A., 2000. Oil Seed Crops. 2nd ed. Blackwell Science Ltd., Oxford, London, Berlin Carlton, Paris.

66

Willems, A., Doignon-Bourcier, F., Coopman, R., Hoste, B., de Lajudie, P., Gillis, M., 2000. AFLP fingerprint analysis of Bradyrhizobium strains isolated from Faidherbia albida and Aeschynomene species. Syst Appl Microbiol. 23: 137147. Wolde-meskel, E., Z. Terefework., K. Lindstrom and A. Frostegard, 2004. Metabolic and genomic diversity of rhizobia isolated from field standing native and exotic Woody legumes in Southern Ethiopia. Systematic and applied Microbiology, 27: 603 - 611. Wynne, J.C., Elkan, G.H. and Schneeweis, T.J., 1980. Increasing nitrogen fixation of the groundnut by strain and host selection. Pp 95-109 in Proceedings of the International Workshop on Groundnuts, 13-17 Oct 1980, ICRISAT Center, India. Patancheru, A.P. 502 324, India: International Crops Research Institute for the Semi arid Tropics. Wynne, J.C., Elkan, G.H., Isleib, T.G., and Schneeweis, T.J., 1983. Effect of host plant, Rhizobium strain and host x strain interaction on symbiotic variability in peanut. Peanut Science, 10:110-114. Yang J.K, Xie FL, Zou J, Zhou Q, Zhou J.C., 2005. Polyphasic characteristics of bradyrhizobia isolated from nodules of peanut (Arachis hypogaea) in China. Soil Biol Biochem. 37: 1411. Zafar-ul-Hye, M., Z.A. Zahir, S.M. Shahzad, U. Irshad and M. Arshad, 2007. Isolation and screening of rhizobia for improving growth and nodulation of lentil (Lens culinaris Medic) seedlings under axenic conditions. Soil & Environ., 26: 81-91. Zahran, H.H., 1999. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol. Mol. Biol. Reviews 63: 968 989. Zakhia, F. and de Lajudie, P., 2001. Taxonomy of rhizobia. Agronomi,. 21: 569 - 576. Zerhari, A., Aurag, J., Khbaya, B., Kharchat, D. and Filali-Maltouf, A., 2000. Phenotypic characteristics of rhizobia isolate nodulating Acacia species in the arid and Saharan regions of Morocco. Lett. Appl. Micrbiol. 30: 351-357. Zerihun Belay (2006). Symbiotic and phenotypic diversity of Rhizobium leguminosarum var Isolates (Vicia faba) from Northern Gondar. MSc Thesis, Addis Ababa University, Ethiopia. Zhang, F., N. Dashti,H.Hynes, and D.L. Smith, 1996. Plant growth promoting rhizobacteria and soybean (Glycine max (L.) Merr) nodulation and nitrogen fixation at suboptimal root zone temperatures. Annals of Botany, 77: 453459. Zhang W., Yuan, T., Y.J. Yang, and J. Zhou, 2006. Studies on genetic diversity and phylogeny of slow-growing rhizobia isolated from Vigna radiata at main ecotypes of China. Wei Sheng Wu Xue Bao. 46(6): 869-74. .

67

7. APPENDICES

68

7.1. Appendix Tables

Appendix Table 1. Analysis of variance for nodulation parameters on sand experiment Treatment MS 14.855 2893.420 3996.510 0.129 Error MS 0.462 1.513 16.00 0.0002 CV (%) 26.76 1.75 7.68 1.39 LSD (0.05) 1.113 2.015 6.554 0.023 Mean 2.54 70.37 52.06 1.01

Parameter PN (plant-1) NN (plant-1) NDW (mg plant-1) SDW (g plant-1)

DF 25 25 25 25

F-value 32.19* 1912.60* 249.78* 659.11*

DF 52 52 52 52

* PN = Peg number per plant; NN = Nodule number; NDW = Nodule dry weight; SDW = Shoot dry weight; ns = Nonsignificant; * = Significant at P 0.05; CV = Coefficient of variation; DF = Degree of freedom; MS = Mean square; LSD = Least significant different

Appendix Table 2. Scale of nodule and leaf color on sand culture just before harvest
Treatments on sand culture*
HUGR6 HUGR7 HUGR8 HUGR9 HUGR10 HUGR11 HUGR12 HUGR17 HUGR13 HUGR14 HUGR15 HUGR16 HUGR18 HUGR19 HUGR20 HUGR21 HUGR22 HUGR23 HUGR24 HUGR1 HUGR2 HUGR3 HUGR4 HUGR5 N+ N-

2 2

3 3

2 2

3 2 2 3 1 3 2 2 2 2 2 2 1 3 2 2

2 3

1 1

3 3

3 3

2 3

3 3

2 3

3 2

2 2

3 3

2 2

2 3

1 -

3 -

NC

*NC = Nodule inside color; 1= White, 2= Pink, 3= slightly dark red; LC = Leaf color; 1= Yellow; 2 = Light green; 3 = Dark green; HUGR = Haramaya University groundnut rhizobia; N- = uninoculated and N+ = N fertilized plant

69

Appendix Table 3. Nodulation and dry matter accumulation of groundnut on sand culture and correlation among parameters Parameter PN NDW PN SDW NN NDW NDW SDW NN SDW Mean 2.54 52.06 2.54 1.01 70.37 52.06 52.06 1.01 70.37 1.01 Std Dev 2.27 36.17 2.27 0.21 30.67 36.17 36.17 0.21 30.67 0.21 Minimum 0.0 0.0 0.0 0.6 0.0 0.0 0.0 0.6 0.0 0.6 Maximum 9.0 172 9.0 1.5 133 172 172 1.5 133 1.5 r - value 0.30* 0.55* 0.93** 0.82** 0.68*

*PN = Peg number per plant; NN = Nodule number; NDW = Nodule dry weight; SDW = Shoot dry weight; ** = Significance at P 0.01 and P < 0.05

Appendix Table 4. Nodulation of groundnut plant during MPN determination in Fedis and Babile soils
Dilution level Nodulation under the respective replication (Fedis soil) I II III IV + + + + + + + Nodulation units* Nodulation under the respective replication (Babile soil) I II III IV + + + + + + + + + + + + Nodulation units*

10-1 4 4 -2 10 2 4 10-3 1 3 -4 10 0 1 -5 10 0 0 10-6 0 0 -7 10 0 0 -8 10 0 0 Total 7 12 +: Presence of nodulation; - : Absence of nodulation; *: Total nodulated replications in a dilution level

70

Appendix Table 5. Analysis of variance for the different parameters of Fedis soil culture Treatment MS F-Value 5.61 25.23* 383.67 25.67* 33.57 21.20* 4102.02 40.46* 0.16 123.24* 0.73 164.63* 0.00066 118.18* Error DF MS 24 0.22 24 14.94 24 1.58 24 101.39 24 0.013 24 0.005 24 0.0000056 CV (%) 11.31 14.41 23.84 11.69 1.93 3.14 5.47 LSD (0.05) 0.79 6.51 2.12 16.97 0.06 0.11 0.004

Parameter PN plant-1 NN plant-1 NR plant-1 NDW (mg plant-1) SDW (g plant-1) N (%) TN (g plant-1)

DF 11 11 11 11 11 11 11

Mean 4.17 26.83 5.28 86.11 1.85 2.13 0,04

*PN = Peg number per plant; NN = Nodule number; NR = Nodulation ratings; NDW = Nodule dry weight; SDW = Shoot dry weight; TN = total nitrogen content; * = Significant at P 0.05; CV = Coefficient of variation; DF = Degree of freedom; MS = Mean square; LSD = Least significant different

Appendix Table 6. Nodulation and dry matter accumulation of groundnut on Fedis soil and Correlation among parameters

Parameter PN SDW PN N% PN TN NN NR NN NDW NR NDW N% SDW TN SDW N% TN

Mean 4.17 1.85 4.17 2.13 4.17 0.04 26.83 5.28 26.83 86.11 5.28 86.11 2.13 1.85 0.04 1.85 2.13 0.04

Std Dev 1.38 0.22 1.38 0.48 1.38 0.02 11.44 3.41 11.44 36,86 3.41 36,86 0.48 0.22 0.02 0.22 0.48 0.02

Minimum 2.00 1.57 2.00 1.60 2.00 0.03 14.00 1.00 14.00 20.00 1.00 20.00 1.60 1.57 0.03 1.57 1.60 0.03

Maximum 8.00 2.30 8.00 3.60 8.00 0.08 55.00 10.00 55.00 180.00 10.00 180.00 3.60 2.30 0.08 2.30 3.60 0.08

r-value 0.88** 0.82** 0.90** 0.85** 0.97** 0.87** 0.90** 0.92** 0.98**

*PN = Peg number per plant; NN = Nodule number; NR = Nodulation ratings; NDW = Nodule dry weight; SDW = Shoot dry weight; TN = total nitrogen content;** = Significance at P 0.01

71

Appendix Table 7. Scale of leaf and nodule color on Fedis and Babile soil culture just before harvest
Treatments of Fedis soil
HUGR10 HUGR12 HUGR13 HUGR16 HUGR18 HUGR19 HUGR22 HUGR24 HUGR11 HUGR3 N+ N-

Colors LC NC

3 3 2 3 2 3 3 2 2 3 3 2 2 2 2 2 2 2 2 1 Treatments of Babile soil LC 3 2 3 3 2 3 2 3 3 2 2 3 NC 2 3 2 2 2 2 2 3 2 2 2 1 *LC = Nodule inside color; 1 = Green, 2 = Pink, 3= slightly dark red; LC = Leaf color; 1= Yellow; 2 = light green; 3 = Dark green

3 2

2 3

Appendix Table 8. Analysis of variance for the different parameters of Babile soil culture Parameters Treatment Error CV (%) DF MS F-Value DF MS -1 PN plant 11 6.55 26.18* 24 0.25 13.64 NN plant-1 11 421.66 128.64* 24 3.28 3.92 -1 NR plant 11 25.30 12.14* 24 2.08 18.36 NDW (mg plant-1) 11 1373.90 54.77* 24 25.09 6.67 SDW (g plant-1) 11 0.17 15.30* 24 0.011 5.67 N (%) 11 0.29 100.11* 24 0.0029 2.98 TN (g plant-1) 11 0.00021 15.42* 24 0.000014 10.82 LSD (0.05) 0.84 3.05 2.43 8.44 0.18 0.09 0.006

Mean 3.67 46.17 7.86 75.12 1.88 1.81 0.034

*PN = Peg number per plant; NN = Nodule number; NR = Nodulation ratings; NDW = Nodule dry weight; SDW = Shoot dry weight; TN = Total nitrogen content; * = Significant at P 0.05; CV = Coefficient of variation; DF = Degree of freedom; MS = Mean square; LSD = Least significant different .

72

Appendix Table 9. Correlation coefficient of selected parameters in Babile soil experiment

Parameter PN NDW PN SDW NDW SDW NN NR SDW N% TN SDW N% TN

Mean 3.67 75.12 3.67 1.883 75.12 1.883 46.17 7.86 1.883 1.803 0.034 1.883 1.803 0.034

Std Dev 1.49 21.19 1.49 0.250 21.19 0.250 11.61 3.06 0.250 0.322 0.009 0.250 0.322 0.009

Minimum 1.00 34.00 1.00 1.50 34.00 1.50 25.00 1.00 1.50 0.97 0.02 1.50 0.97 0.02

Maximum 6.00 105.00 6.00 2.60 105.00 2.60 64.00 10.00 2.60 2.40 0.06 2.60 2.40 0.06

r value 0.68* 0.79* 0.51* 0.79* 0.56* 0.80** 0.83**

*PN = Peg number per plant; NN = Nodule number; NR = Nodulation ratings; NDW = Nodule dry weight; SDW = Shot dry weight; TN = total nitrogen content;** = Significance at P 0.01 and P < 0.05

7.2. Appendix Figures

Appendix Figure 1. Stand of groundnut inoculated with different isolates on sand culture

73

Appendix Figure 2. Performances of inoculated and uninoculated groundnut seedlings on sand culture in greenhouse condition