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

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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 Signature Date Heluf Gebrekidan (PhD) ____________________ ___________________ Name of Co-Advisor Signature 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 Signature Date ________________________ ___________________ ____________________ Name of Internal Examiner Signature Date ________________________ ___________________ ____________________ Name of External Examiner Signature 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 candidate’s major department.

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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.

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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 Signature: ______________ Place: Haramaya University, Haramaya Date of Submission: ______________

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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.

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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.

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LIST OF ACRONYMS AND ABBREVIATIONS

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

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YEMA Yeast Extract Mannitol Agar YEMA-BTB Yeast Extract Mannitol Agar-0.25% Bromothymol Blue YEMA-CR Yeast Extract Mannitol Agar-0.25% Congo Red YEMB Yeast Extract Mannitol Broth

viii

TABLE OF CONTENTS

STATEMENT OF THE AUTHOR iii BIOGRAPHICAL SKETCH v ACKNOWLEDGMENTS vi LIST OF ACRONYMS AND ABBREVIATIONS vii LIST OF TABLES x LIST OF FIGURES xi LIST OF TABLES IN THE APPENDIX xii LIST OF FIGURES IN THE APPENDIX xiii ABSTRACT xiv 1. INTRODUCTION 1 2. LITERATURE REVIEW 5 2.1. General Description Legumes 5 2.2. Description of Groundnut 5 2.3. Symbiotic Rhizobium-Legume Nitrogen Fixation 6 2.4. Taxonomy and Host Specificity of Rhizobia 8 2.5. Significance of Biological Nitrogen Fixation 10 2.6. The Process of Nodulation and Fixation 12 2.6.1. The mechanism 12 2.6.2. Recognition between symbiotic partners 13 2.7. Characteristics of Nodules 13 2.7.1. Shape, size and number 13 2.7.2. Structure and function of nodule 14 2.8. Factors Affecting Symbiotic Nitrogen Fixation 15 2.8.1. Soil reaction (pH) 15 2.8.2. Mineral nutrient status 16 2.8.3. Photosynthesis 17 2.8.4. Legume management 17 2.8.5. Climate 18 2.9. Response of Groundnut to Inoculation 18 3. MATERIALS AND METHODS 21 3.1. Description of the Study Area, Site Selection and Sampling 21 3.1.1. Description of the study area 21 3.1.2. Site selection and sampling procedure 22 3.2. Isolation of Rhizobium Strains 22 3.3. Purification and Preservation of the Isolates 24 3.4. Characterization of Isolates 24 3.5. Physiological Tests 25 3.6. Enumeration of Rhizobia 25 3.7. Evaluation of Symbiotic Effectiveness of Groundnut Rhizobia 26 3.7.1. Effectiveness of isolates on sterilized sand 26 3.7.2. Evaluation of isolates in two unsterilized soil 28 3.8. Statistical Analysis 29

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TABLE OF CONTENTS (Continued)

4. RESULTS AND DISCUSSION 31 4.1. Presumptive Tests of Isolates 31 4.2. Morphology and Cultural Characteristics 31 4.3. Evaluation of Symbiotic Effectiveness on Sand Culture 33 4.3.1. Peg number per plant 33 4.3.2. Nodule number per plant 33 4.3.3. Nodule dry weight per plant 34 4.3.4. Shoot dry weight per plant 36 4.3.5. Symbiotic effectiveness 37 4.3.6.Correlation analysis for the selected parameter on sand culture 37 4.4. Physiological Characterization 37 4.4.1. Temperature tolerance 37 4.4.2. Salt tolerance 38 4.4.3. Tolerance to pH 40 4.5. Soil Properties and Enumeration of Rhizobia 40 4.6. Symbiotic Effectiveness of Selected Isolates on Fedis and Babile

Soils

43 4.6.1. Peg number per plant 43 4.6.2. Nodule number per plant 43 4.6.3. Nodulation ratings per plant 46 4.6.4. Nodule dry weight per plant 48 4.6.5. Shoot dry weight per plant 49 4.6.6. Total nitrogen percent and content per plant 51 4.6.7. Correlation of some selected parameters on Babile and Fedis

soils 52

4.6.8. Leaf and nodule color assessment 53 5. SUMMARY AND CONCLUSIONS 54 6. REFERENCES 57 7. APPENDICES 67 7.1. Appendix Tables 68 7.2. Appendix Figures 72

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LIST OF TABLES Table Page

1. Description of soil sampling sites 23 2. Chemical compositions of N-free nutrient solutions 27 3. Colony characteristics and presumptive test of isolates after 5-7 days of incubation 32 4. Nodulation and symbiotic effectiveness of isolates tested on groundnut in sand

culture 35

5. Physiological (temperature. salt and pH tolerance) characterization of the isolates 39 6. Chemical and physical properties of Babile and Fedis soils 42 7. Nodulation data of selected effective isolates of groundnut rhizobia on Fedis soil 45 8. Nodulation data of selected effective isolates of groundnut rhizobia on Babile soil 47

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LIST OF FIGURES

Figure Page 1. Location map of the study area 21

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LIST OF TABLES IN THE APPENDIX Appendix Table Page

1. Analysis of variance for nodulation parameters on sand experiment 68 2. Scale of nodule and leaf color on sand culture just before harvest 68 3. Nodulation and dry matter accumulation of groundnut on sand culture and correlation

among parameters 69

4. Nodulation of groundnut plant during MPN determination in Fedis and Babile soils 69 5. Analysis of variance for the different parameters of Fedis soil culture 70 6. Nodulation and dry matter accumulation of groundnut on Fedis soil and correlation

among parameters 70

7. Scale of leaf and nodule color on Fedis and Babile soil culture just before harvest 71 8. Analysis of variance for the different parameters on Babile soil culture 71 9. Correlation coefficient of selected parameters on Babile soil experiment 72

xiii

LIST OF FIGURES IN THE APPENDIX

Appendix Figure Page

1. Stand of groundnut inoculated with different isolates on sand culture 72 2. Performances of inoculated and uninoculated groundnut seedlings on sand culture in

greenhouse condition

73

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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 0C 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.

1. INTRODUCTION

Groundnut (Arachis hypogaea L.) is an annual legume which is also known as peanut,

earthnut, monkeynut and goobers. It is the 13th 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 40º

N to 40º 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).

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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 40–50 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 plant’s

3

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

4

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.

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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.

6

Over 90% of the world groundnut crop is produced in developing countries and roughly two-

thirds 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 fix 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 fix 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

7

been identified as diazotrophs or organisms that can fix 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

8

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 O2

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

9

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 world’s 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 earth’s 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:

Nitrogenase N2 + 8H+ + 8e - ------------------------------------------2NH3 + H2

(Fe, Mo-protein)

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 0C (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 m-

1, 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

* 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

Location Designation of isolate

Name of site

District

Altitude

(masl) Latitude (N)

Longitude (E)

Cropping systems

EC

(dsm-1)

pH

HUGR09 Audal 1 Gursum 1801 90 17’ 24” 420 26’ 19” LLR 0.031 7.81

HUGR10 Audal 2 Gursum 1840 90 18’ 24” 420 26’ 03” FLR 0.029 7.91 HUGR13 Haro Bate 1 Gursum 1659 90 16’ 15” 420 23’ 52” LLR 0.060 7.64

HUGR14 Haro Bate 2 Gursum 1631 90 15’ 41” 420 25’ 09” LLR 0.039 8.01 HUGR11 Oda Oromia 1 Gursum 1779 90 18’ 40” 420 28’ 08” LLR 0.025 6.62 HUGR12 Oda Oromia 2 Gursum 1809 90 19’ 11” 420 27’ 02” LLR 0.029 6.58

HUGR15 Kasa Oromia 1 Gursum 1706 90 14’ 60” 420 27’ 18” LLR 0.055 7.65 HUGR16 Kasa Oromia 2 Gursum 1725 90 16’ 10” 420 26’ 51” LLR 0.028 8.04

HUGR01 Shek Hussien 1 Babille 1603 90 10’ 48” 420 21’ 56” LLR 0.045 7.59 HUGR19 Shek Hussien 2 Babille 1601 90 10’ 46” 420 21’ 52” LCR 0.048 7.57

HUGR05 Kito 1 Babille 1694 90 15’ 51” 420 17’ 50” LLR 0.034 6.90 HUGR06 kito 2 Babille 1710 90 15’ 55” 420 18’ 02” LCR 0.039 7.50

HUGR03 Shek Abdi 1 Babille 1619 90 11’ 40” 420 21’ 40” LLR 0.065 7.45 HUGR04 Shek Abdi 2 Babille 1644 90 12’ 22” 420 21’ 44” LLR 0.047 6.96

HUGR07 Iffa 1 Babille 1670 90 14’18” 420 18’ 32” LCR 0.031 6.45 HUGR08 Iffa 2 Babille 1643 90 14’ 54” 420 19’ 19” LCR 0.038 6.15

HUGR21 Hussien 1 Fedis 1705 90 05’ 24” 420 04’ 57” IC 0.080 7.94 HUGR22 Hussien 2 Fedis 1681 90 07’ 02” 420 04’ 50” IC 0.079 8.00

HUGR23 Tuka kanesa 1 Fedis 1724 90 03’ 39” 420 06’ 02” IC 0.097 8.02 HUGR24 Tuka Kanesa 2 Fedis 1710 90 02’ 52” 420 06’ 03” IC 0.049 7.93

HUGR02 Umer Kulle 1 Fedis 1783 90 09’ 36” 420 04’ 18” IC 0.044 7.33 HUGR20 Umer Kulle 2 Fedis 1804 90 11’ 24” 420 04’ 30” IC 0.042 7.46

HUGR17 Ido Basso 1 Fedis 1841 90 12’ 48” 420 04’ 52” IC 0.055 7.94 HUGR18 Ido Basso 2 Fedis 1899 90 13’ 56” 420 05’ 44” IC 0.077 8.00

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

OC 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 µl 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 oC 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

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

Solution 1 Solution 2 Solution 3 Solution 4 Chemical g l-1 Chemical g l-1 Chemical g l-1 Chemical g l-1 CaCl2.2H2O 294.1 KH2PO4 136.1 FeC6H5O7.3H2O 6.700 ZnSO4.7H2O 0.288 MgSO4.7H2O 123.300 CuSO4.5H2O 0.100 K2SO4 87.000 CoSO4.7H2O 0.056

MnSO4.H2O H3BO3

0.338 0.247

Na2MoO2.2H2O 0.048

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 100nitrogenwithpliedsupplantsofweightdryShoot

straintestwithinoculatedplantsofweightdryShoot(%) SE %

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 air-

dried, 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 10±3 and 32±2 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. Pearson’s 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)

YEMA-BTB reaction

YEMA-CR PGA- BCP

Colony texture on YEMA medium

Colony Type

HUGR1 <1.0 Blue Milky NG Buttery SD HUGR2 1.5-2.5 M. Blue Milky NG Buttery LM HUGR3 <1.0 Blue Milky NG Buttery SD HUGR4 <1.0 Blue Milky NG Buttery SD HUGR5 <1.0 Blue Milky NG Buttery SD HUGR6 1.5-2.5 Blue Milky NG Buttery LM HUGR7 1.5-2.5 Blue Milky NG Buttery LM HUGR8 <1.0 Blue Milky NG Buttery SD HUGR9 <1.0 Blue Milky NG Buttery SD

HUGR10 <1.0 Blue Milky NG Buttery SD HUGR11 1.5-2.5 Blue Milky NG Buttery LM HUGR12 <1.0 Blue Milky NG Buttery SD HUGR13 1.5-2.5 Blue Milky NG Buttery LM HUGR14 <1.0 Blue Milky NG Buttery SD HUGR15 1.5-2.5 Blue Milky NG Buttery LM HUGR16 <1.0 Blue Milky NG Buttery SD HUGR17 <1.0 Blue Milky NG Buttery SD HUGR18 <1.0 Blue Milky NG Buttery SD HUGR19 <1.0 Blue Milky NG Buttery SD HUGR20 <1.0 Blue Milky NG Buttery SD HUGR21 <1.0 Blue Milky NG Buttery SD HUGR22 1.5-2.5 M. Blue Milky NG Buttery LM HUGR23 1.5-2.5 Blue Milky NG Buttery LM HUGR24 1.5-2.5 Blue Milky NG Buttery LM

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 YEMA-

BTB 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 agro-

ecologies 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

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) SE (%) Rate HUGR1 0.33+0.58k 48.67+1.53r 23.0+1.00pq 0.83+0.01qr 64 E HUGR2 1.67+0.58hgji 77.0+1.00j 46.0+0.00kj 0.94+0.01kj 73 E HUGR3 3.67+0.58c 121.33+1.15b 119.33+8.96b 1.29+0.01b 100 VE HUGR4 2.0+1.00hgfi 78.0+0.00ji 47.67+0.58kj 0.96+0.01ji 74 E HUGR5 1.67+0.58hgji 74.0+1.73k 44.33+0.58kjl 0.92+0.01k 71 E HUGR6 1.67+0.58hgji 78.0+0.00ji 49.67+2.08ji 0.97+.01hi 75 E HUGR7 2.67+0.58dgfe 69.67+2.08L 42.33 +1.53kl 0.90+0.00L 70 E HUGR8 0.67+0.58jk 52.67+2.08q 26.33+1.151po 0.84+0.00pq 65 E HUGR9 0.67+0.58jk 39.33+1.15t 13.33+2.311r 0.78 +0.01s 60 E HUGR10 2.0+1.00hgfi 81.33+1.15h 59.33+2.08hg 1.04 +0.01g 81 VE HUGR11 3.33+0.58dce 89.0+1.00f 79.67+4.93e 1.19+0.01e 92 VE HUGR12 1.33+0.58hjki 84.0+1.73g 63.0+1.73g 1.15+0.00f 89 VE HUGR13 2.67+0.58dgfe 118.33+1.26c 100.0+7.00c 1.26+0.01b 98 VE HUGR14 1.0+1.00jki 43.0+1.00s 19.0+2.65qr 0.82+.01r 64 E HUGR15 0.33+0.58k 55.0+1.00p 29.0+10npo 0.86+0.01pno 67 E HUGR16 1.33+0.58hjki 95.67+2.08d 92.67+3.21d 1.25+0.01c 97 VE HUGR17 1.33+0.58hjki 60.67+1.16n 35.33+0.58mn 0.87+0.01mn 67 E HUGR18 1.33+0.58hjki 79.33+0.58hi 55.33 +2.08hi 0.98+0.01h 76 E HUGR19 3.0+1.00dfe 91.33+0.58e 86.0+2.00e 1.23+0.02d 95 VE HUGR20 6.0+0.58 b 53.0+1.00qp 27.33+0.58po 0.85+0.01pqo 66 E HUGR21 4.33+0.58c 58.0+1.00O 32.0+1.00mno 0.87+0.01no 67 E HUGR22 8.67+0.58a 131.33+1.53a 155.33+14.47a 1.49+0.01a 116 VE HUGR23 2.67+0.58dgfe 64.33+0.58m 38.0+1.00ml 0.89+0.01ml 69 E HUGR24 2.33+0.58hgfe 87.33+1.00f 69.67+1.15f 1.17+0.01fe 91 VE N- 0.67+0.58jk 0 0 0.62+0.012t 48 - N+ 8.67+0.58a 0 0 1.29+0.01b 100 - LSD(0.05) 1.11 2.02 6.55 0.023 - - SEM 0.46 1.51 16.00 0.0002 - -

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 80-

116% 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 0C (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

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

Treatment HUGR 03

HUGR 10

HUGR 11

HUGR 12

HUGR 13

HUGR 16

HUGR18

HUGR 19

HUGR 22

HUGR 24

Temp (0C) Temperature tolerance* 4 - - - - - - - - - - 10 - - - - - - - - - - 15 + + + + + + + + + + 20 + + + + + + + + + + 30 + + + + + + + + + + 35 + + + + + + + + + + 40 + + + + + + + + + +

Salt (%) Salt tolerance

0.1 + + + + + + + + + + 0.2 + + + + + + + + + + 0.5 + + + + + + + + + + 1.0 - - - - - - + - - - 2.0 - - - - - - - - - -

pH pH tolerance 4.5 - - - - - - - - - - 5.0 - - - - - - - - - - 5.5 - - - - - - - - - - 6.0 + + + + + + + + + + 6.5 + + + + + + + + + + 8.0 + + + + + + + + + + 8.5 + + + + + + + + + + 9.0 + + + + + + + + + +

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 Fedis soil Babile soil pH 7.4 6.6 EC (ds m-1) 0.49 0.06 Total nitrogen (%) 0.17 0.07 Carbon to nitrogen ratio 6.59 13.00 Available phosphorus (mg kg-1) 5.42 9.81 CEC (cmol(+) kg-1) 22.8 9.80 Organic matter (%) 1.93 1.57 Sand (%) 56.0 74.0 Silt (%) 33.8 17.5 Clay (%) 10.2 8.5 Textural class Sandy loam 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

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

PN (plant-1)

NN (plant-1)

NR (plant-1)

NDW (mg plant-1)

SDW (g plant-1)

N %

TN (g plant-1)

HUGR3 5+0.00b 35.67+8.08b 10.0+0.00a 116.67+11.55b 1.94+0.02b 2.2+0.00c 0.04+0.00d HUGR 10 4+0.00cd 27+4.00dc 6.67+2.89b 93.33+15.28c 1.85+0.07c 2.03+0.06d 0.04+0.00d HUGR 11 4+0.00cd 27.67+1.53c 5+0.00b 96.67+5.77c 1.87+0.04c 2.07+0.06d 0.04+0.00d HUGR 12 4.67+0.58cb 39.67+8.50b 10.0+0.00a 120+17.32b 1.97+0.04b 2.27+0.06c 0.05+0.001c HUGR 13 3+0.00de 21.67+2.89dce 5+0.00b 71.67+2.89de 1.64+0.01ef 1.7+0.06f 0.03+0.00e HUGR 16 2.67+0.58e 18.67+1.15def 2.33+2.31c 60+10.00ef 1.59+0.02f 1.67+0.10f 0.03+0.001e HUGR 18 4.0+0.00dcd 22.33+1.15dce 5+0.00b 71.67+2.89de 1.7+0.03d 1.87+0.06e 0.03+0.00e HUGR 19 2.67+1.15e 18+2.65ef 2.33+2.65c 56.67+11.55e 1.6+0.06f 1.73+0.06f 0.03+0.00e HUGR 22 5.0+0.00b 54+1.00a 10.0+0.00a 170+10.00a 2.23+0.04a 2.5+0.01b 0.06+0.01b HUGR 24 4.0+0.00cd 25+1.00dc 5.0+0.00b 83.33+5.77dc 1.83+0.02c 2.1+0.02d 0.04+0.001d N+ 7.67+0.58a 15+1.00f 1.0+0.00c 28.33+10.41f 2.28+0.02a 3.5+0.10a 0.08+0.00a N- 3.33+0.58de 17.33+1.53ef 5.0+0.00b 65+5e 1.69+0.03ed 1.88+0.01e 0.03+0.00e LSD(0.05) 0.79 6.51 2.12 16.97 0.06 0.11 0.004 SEM 0.22 14..94 1.58 101.39 0.0013 0.005 0.0000056

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

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

PN (plant-1)

NN (plant-1)

NR (plant-1)

NDW (mg plant-1)

SDW (g plant-1)

N %

TN (g plant-1)

HUGR 3 5.0+0.00b 41.67+ 1.53e 8.33+ 2.8 9ba 98.33+ 7.23 a 2.1+0.00bc 1.89+0.01dc 0.040+0.00cb HUGR 10 5.0+0.00b 55.33+ 0.58 c 10.0+0.00a 92.00+2.00ba 1.97+0.06dc 1.59+0.01h 0.030+0.00e HUGR 11 3.33+ 0.58ed 27.67+2.08g 5.0+0.00c 83.00+6.92c 1.80+0.10de 1.84+0.02dce 0.033+0.01ed HUGR 12 3.0+0.00e 51.00+2.31d 10.0+0.00a 79.33+2.08c 1.80+0.00de 1.74+0.06fg 0.030+0.00e HUGR 13 3.0+0.00e 58.67+1.16b 10.0+0.00a 70.77+3.66d 1.73+0.06fe 1.79+0.01fe 0.030+0.00e HUGR 16 2.67+0.58e 51.00+1.00d 8.33+2.89ba 62.00+2.65e 1.73+0.06fe 2.1+0.12b 0.037+0.01cd HUGR 18 4.67+0.27bc 56.67+1.05bc 10.0+0.82a 95.33+4.73a 1.90+0.10de 1.91+0.01c 0.037+0.01cd HUGR 19 6.0+0.27a 62.33+1.05a 10.0+0.82a 99.67+8.39a 2.33+0.25a 1.93+0.03c 0.043+0.01b HUGR 22 4.67+0.27bc 42.33+1.05e 10.0+0.82a 84.67+8.50bc 1.87+0.06de 1.80+0.01dfe 0.030+0.00e HUGR 24 1.33+0.27f 42.33+1.05e 5.0+0.82c 47.33+2.52f 1.57+0.12f 1.05+0.13i 0.020+0.00f N+ 4.0+0.00cd 25.33+0.58g 1.0+0.00d 34.67+0.58g 2.23+0.15ba 2.33+0.06a 0.053+0.01a N- 1.33+0.58f 38.00+2f 6.67+2.87bc 54.33+0.58fe 1.57+0.06f 1.66+0.02hg 0.030+0.00e LSD(0.05) 0.84 3.05 2.43 8.44 0.18 0.09 0.006 SEM 0.25 3.28 2.08 25.09 0.011 0.003 0.000014

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 host’s 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

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7. APPENDICES

68

7.1. Appendix Tables

Appendix Table 1. Analysis of variance for nodulation parameters on sand experiment

* PN = Peg number per plant; NN = Nodule number; NDW = Nodule dry weight; SDW = Shoot dry weight; ns = Non- significant; * = 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

*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

Treatment Error Parameter DF MS F-value DF MS

CV (%)

LSD (0.05)

Mean

PN (plant-1) 25 14.855 32.19* 52 0.462 26.76 1.113 2.54 NN (plant-1) 25 2893.420 1912.60* 52 1.513 1.75 2.015 70.37 NDW (mg plant-1) 25 3996.510 249.78* 52 16.00 7.68 6.554 52.06 SDW (g plant-1) 25 0.129 659.11* 52 0.0002 1.39 0.023 1.01

Treatments on sand culture*

HU

GR

1

HU

GR

2

HU

GR

3

HU

GR

4 H

UG

R5

HU

GR

6 H

UG

R7

HU

GR

8 H

UG

R9

HU

GR

10

HU

GR

11

HU

GR

12

HU

GR

13

HU

GR

14

HU

GR

15

HU

GR

16

HU

GR

17

HU

GR

18

HU

GR

19

HU

GR

20

HU

GR

21

HU

GR

22

HU

GR

23

HU

GR

24

N-

N+

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

NC 2 2 3 2 2 2 2 2 1 3 2 2 3 1 3 3 3 3 3 2 2 3 2 3 - -

69

Appendix Table 3. Nodulation and dry matter accumulation of groundnut on sand culture and correlation among parameters

*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

Nodulation under the respective replication (Fedis soil)

Nodulation under the respective replication (Babile soil)

Dilution level

I II III IV

Nodulation units*

I II III IV

Nodulation units*

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

Parameter Mean Std Dev Minimum Maximum r - value PN 2.54 2.27 0.0 9.0 NDW 52.06 36.17 0.0 172

0.30*

PN 2.54 2.27 0.0 9.0 SDW 1.01 0.21 0.6 1.5

0.55*

NN 70.37 30.67 0.0 133 NDW 52.06 36.17 0.0 172

0.93**

NDW 52.06 36.17 0.0 172 SDW 1.01 0.21 0.6 1.5

0.82**

NN 70.37 30.67 0.0 133 SDW 1.01 0.21 0.6 1.5

0.68*

70

Appendix Table 5. Analysis of variance for the different parameters of Fedis soil culture

*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

*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

Treatment Error Parameter DF MS F-Value DF MS

CV (%)

LSD (0.05)

Mean

PN plant-1 11 5.61 25.23* 24 0.22 11.31 0.79 4.17 NN plant-1 11 383.67 25.67* 24 14.94 14.41 6.51 26.83 NR plant-1 11 33.57 21.20* 24 1.58 23.84 2.12 5.28 NDW (mg plant-1) 11 4102.02 40.46* 24 101.39 11.69 16.97 86.11 SDW (g plant-1) 11 0.16 123.24* 24 0.013 1.93 0.06 1.85 N (%) 11 0.73 164.63* 24 0.005 3.14 0.11 2.13 TN (g plant-1) 11 0.00066 118.18* 24 0.0000056 5.47 0.004 0,04

Parameter Mean Std Dev Minimum Maximum r-value 4.17 1.38 2.00 8.00 PN

SDW 1.85 0.22 1.57 2.30

0.88** PN 4.17 1.38 2.00 8.00 N% 2.13 0.48 1.60 3.60

0.82**

PN 4.17 1.38 2.00 8.00 TN 0.04 0.02 0.03 0.08

0.90**

NN 26.83 11.44 14.00 55.00 NR 5.28 3.41 1.00 10.00

0.85**

NN 26.83 11.44 14.00 55.00 NDW 86.11 36,86 20.00 180.00

0.97**

NR 5.28 3.41 1.00 10.00 NDW 86.11 36,86 20.00 180.00

0.87**

N% 2.13 0.48 1.60 3.60 SDW 1.85 0.22 1.57 2.30

0.90**

TN 0.04 0.02 0.03 0.08 SDW 1.85 0.22 1.57 2.30

0.92**

N% 2.13 0.48 1.60 3.60 TN 0.04 0.02 0.03 0.08

0.98**

71

Appendix Table 7. Scale of leaf and nodule color on Fedis and Babile soil culture just before harvest

*LC = Nodule inside color; 1 = Green, 2 = Pink, 3= slightly dark red; LC = Leaf color; 1= Yellow; 2 = light green; 3 = Dark green Appendix Table 8. Analysis of variance for the different parameters of Babile soil culture

*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 .

Treatments of Fedis soil

Colors H

UG

R3

HU

GR

10

HU

GR

11

HU

GR

12

HU

GR

13

HU

GR

16

HU

GR

18

HU

GR

19

HU

GR

22

HU

GR

24

N-

N+

LC 3 2 3 3 2 3 2 3 3 2 2 3 NC 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

Treatment Error Parameters DF MS F-Value DF MS

CV (%)

LSD (0.05)

Mean

PN plant-1 11 6.55 26.18* 24 0.25 13.64 0.84 3.67 NN plant-1 11 421.66 128.64* 24 3.28 3.92 3.05 46.17 NR plant-1 11 25.30 12.14* 24 2.08 18.36 2.43 7.86 NDW (mg plant-1) 11 1373.90 54.77* 24 25.09 6.67 8.44 75.12 SDW (g plant-1) 11 0.17 15.30* 24 0.011 5.67 0.18 1.88 N (%) 11 0.29 100.11* 24 0.0029 2.98 0.09 1.81 TN (g plant-1) 11 0.00021 15.42* 24 0.000014 10.82 0.006 0.034

72

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

*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

Parameter Mean Std Dev Minimum Maximum r – value PN 3.67 1.49 1.00 6.00 NDW 75.12 21.19 34.00 105.00

0.68*

PN 3.67 1.49 1.00 6.00 SDW 1.883 0.250 1.50 2.60

0.79*

NDW 75.12 21.19 34.00 105.00 SDW 1.883 0.250 1.50 2.60

0.51*

NN 46.17 11.61 25.00 64.00 NR 7.86 3.06 1.00 10.00

0.79*

SDW 1.883 0.250 1.50 2.60 N% 1.803 0.322 0.97 2.40

0.56*

TN 0.034 0.009 0.02 0.06 SDW 1.883 0.250 1.50 2.60

0.80**

N% 1.803 0.322 0.97 2.40 TN 0.034 0.009 0.02 0.06

0.83**

73

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