ADDIS ABABA UNIVERSITY

SCHOOL OF GRADUATE STUDIES

Phenotypic and Symbiotic characterization of grass pea

(Lathyrus sativus) rhizobial isolates from some major
growing areas of South Wollo and West Shoa , Ethiopia

By
Musa Adal
A Thesis submitted to the school of graduate studies of Addis Ababa
University inpartial fulfillment of the requirements for the degree of
Master’s of science in Biotechnology, Industrial Biotechnology

July 2009
 ADDIS ABABA UNIVERSITY
SCHOOL OF GRADUATE STUDIES

Phenotypic and Symbiotic characterization of grass pea

(Lathyrus sativus) rhizobial isolates from some major
growing areas of South Wollo and West Shoa , Ethiopia.

By
Musa Adal

A Thesis submitted to the school of graduate studies of Addis Ababa

University inpartial fulfillment of the requirements for the degree of
Master’s of science in Biotechnology, Industrial Biotechnology

Approved by examining Board:

_________________________________ ______________________
_________________________________ ______________________
_________________________________ ______________________
_________________________________ ______________________
 Declaration
I, the under signed, declare that this thesis is my first original work. It has never been
done and submitted in any institution and sources of material used for thesis have been
dully acknowledged.

Name: Musa Adal

Place: Addis Ababa University
Signature: ___________________
Date: ___________________

This thesis has been submitted for examination with my approval as university advisor

Name: Fassil Assefa (PhD)

Signature: _________________
Date: _________________
ACKNOWLEDGEMENTS

I would like to express my gratitude and appreciation to my advisor Dr. Fasil Assefa for
his invaluable suggestions, critical comments, assistance and supervision starting from
problem identification up to finalization of my thesis.

I am highly indebted to the National Soil Research Centre, particularly to Dr. Asfaw
Hailemariam and the laboratory technicians of the Microbiology Department of the
centre for their greenhouse service and technical assistance.

I am very much pleased to express my indebtedness and genuine appreciation to AAU,

Department of Biology and the laboratory technicians: W/t Hirut Teshome and W/o
Tigist Mengesha for their full and free laboratory service and unreserved laboratory
assistance.

I would like to thank the Department of Biotechnology for permitting me to pursue my

study in the stream for paying my tuition fee and research financial assistance.

I am very much grateful to my brother, Hussien Adal for his invaluable support and
encouragement to upgrade my professional career. I am also grateful to my wife and
children for their encouragement.

I would also like to extend my appreciation to my friends: Tassew Siraj, Girmaye

Kenasa, Anteneh Aragaw, Getaneh Tesfaye, Kindu Nibret, Yirgalem Kumlachew, and
Shewakena Belayneh for their encouragement and assistance during my project work.

Last but not least I thank W/t Meseret Alemu for her typing this manuscript.

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TABLE OF CONTENTS
Page
Acknowledgement ..........................................................................................................................i
Table of contents........................................................................................................................... ii
List of tables ..................................................................................................................................v
List of figures ...............................................................................................................................vi
List of appendices ...................................................................................................................... vii
List of symbols and abbreviations........................................................................................... viii
Abstract ........................................................................................................................................x
1. Introduction ..............................................................................................................................1
2. Objective....................................................................................................................................4
2.1. General Objective................................................................................................................4
2. 2.Specific objectives ..............................................................................................................4
3. Review literature .....................................................................................................................5
3. 1. Legumes............................................................................................................................ 5
3. 2. Grass pea (Lathrys sativus) .............................................................................................. 6
3. 2. 1. Ecology of grass pea ................................................................................................6
3. 2. 2. Agronomic and economic importance of grass pea..................................................6
3. 3. Rhizobia............................................................................................................................7
3. 3. 1. Taxonomy of Rhizobia ............................................................................................ 7
3. 3. 2. Rhizobial diversity and characterization methods....................................................9
3. 4. Inoculation and their benefit .......................................................................................11
3. 5. Nitrogen fixation........................................................................................................... 11
3. 6. Biochemistry of biological nitrogen fixation (BBNF) ................................................ 14
3. 7. Ecological factors that affect nodulation and BNF.....................................................16
3. 7.1. Soil pH......................................................................................................................17
3. 7.2. Salt stress ...................................................................................................................19
3. 7.3. Temperature ............................................................................................................ 20
3. 7.4. Water stress...............................................................................................................21

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 3. 7. 5. Carbon and energy sources……………………………………………………...23
3. 7. 6. Soil nutrients…………………………………………………………………….23
4. Materials and methods………………………………………………………………….26
4. 1. Study sites…………………………………………………………………………….26
4. 2. Nodule collection and isolation of rhizobia ………………………………………….28
4. 2. 1. Nodule collection ………………………………………………………………..28
4. 2. 2. Isolation of rhizobia……………………………………………………………...28
4. 3. Purification of isolates ………………………………………………………………..31
4. 4. Preservation of isolates……………………………………………………………….31
4. 5. Designation of the isolates …………………………………………………………...31
4. 6. Seed samples …………………………………………………………………………31
4. 7. Authentication of the isolates and preliminary screening of their
symbiotic effectiveness on pouch experiment ………………………………………32
4.8. Relative effectiveness of the isolates………………………………………………….33
4.9. Characterization of the isolates………………………………………………………..34
4. 9. 1. Colony morphology……………………………………………………………..34
4. 9. 2. Acid – base production …………………………………………………………34
4. 9. 3. Determination of mean generation time (MGT) ……………………………….34
4. 10. Biochemical and physiological tests……………………………………………….35
4. 10. 1. pH tolerance …………………………………………………………………35
4. 10. 2. Salt tolerance…………………………………………………………………35
4. 10. 3. Temperature tolerance ……………………………………………………….36
4. 10. 4. Intrinsic antibiotic resistance (IAR) …………………………………………36
4. 10. 5. Carbohydrate utilization ……………………………………………………..36
4. 10. 6. Amino acid utilization………………………………………………………..37
4. 11. Soil analysis ………………………………………………………………………...38
4. 12. Screening of symbiotic effectiveness on potted field soil…………………………...38
4. 13. Plant total nitrogen content analysis………………………………………………...38
4. 14. Numerical analysis…………………………………………………………………..39
4. 15. Data analysis………………………………………………………………………...39

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5. Result………………………………………………………………………………………40
5. 1.Isolation and Authentication of rhizobia…………………………………………….40
5. 2. Characteristics of the isolates………………………………………………………..40
5. 2. 1. Morphological and cultural characteristics……………………………………..40
5. 2. 2. Mean generation time of the isolates …………………………………………..42
5.3. Physiological tests……………………………………………………………………43
5. 3. 1. pH tolerance…………………………………………………………………….43
5. 3. 2. Salt tolerance ……………………………………………………………………45
5. 3. 3. Temperature tolerance ………………………………………………………….47
5. 3. 4. Intrinsic antibiotic resistance……………………………………………………49
5. 3. 5. Utilization of carbon sources……………………………………………………51
5. 3. 6. Amino acid utilization…………………………………………………………..53
5. 4. Numerical analysis……………………………………………………………………55
5. 5. Soil analysis…………………………………………………………………………..56
5. 6. Symbiotic effectiveness test on pouch culture for preliminary screening …………...56
5. 7. Symbiotic effectiveness of selected isolates on potted soil…………………………..59
6. Discussion…………………………………………………………………………………62
7. Conclusion and recommendations………………………………………………………69
7.1. Conclusion…………………………………………………………………………….69
7.2. Recommendations……………………………………………………………………..70
8. References…………………………………………………………………………………71
9. Appendices ………………………………………………………………………………..83

iv
List of tables
Page
Table I: Rising number of species in the genera of rhizobia…………………………9
Table II: The Location map of sample collection…………………………………….29
Table III: N-free nutrient solution……………………………………………………..33
Table IV: Growth characterization of the isolates…………………………………….41
Table V: The tolerance of grass pea rhizobial isolates to different levels of pH…….44
Table VI: Tolerance of rhizobia nodulating grass pea at different level of salt
concentrations ………………………………………………………….46
Table VII: Table VII. Tolerance of grass pea rhizobia to different
levels of temperature……………………………………………………. 48
Table VIII. Antibiotic resistance of isolates after 5-7 days of incubation……………..50
Table IX. Grass pea rhizobial utilization to different
carbohydrate sources………………………………………………………52

Table X. Utilization of grass pea rhizobia on different nitrogen sources……………...54

Table XI. The most resistant and sensitive isolates to physiological characters ……...55
Table XII. Physical and chemical analysis of selected soils…………………………..56
Table XIII. Nodulation data of collected isolates of grass pea rhizobia………………57
Table XIV. Rate of effectiveness of isolates by sampling regions…………………….59
Table.XV. Nodulation data of selected isolates of grass pea rhizobia
on potted soil………………………………………………………………60
Table XVI. Shoot dry weight of selected isolates on both pouch and pot cultures…….61
Table XVII. Comparative anlysis of maximum tolerance and effectiveness of
different rhizobial isolates………………………………………………..61

v
List of figures Page

Fig. 1: The role of nitrogenous in nitrogen fixation……………………………………...15

Fig. 2: Location map of study sites……………………………………………………...27
Fig. 3: Dendrogram highlighting the phenotypic diversity of the isolates………………56

vi
List of Appendices Page

Appendix 1. Inoculated and control plants after 45 days of growth on pouch

culture in greenhouse……………………………………………………83
Appendix 2. Inoculated and control plants after 45 days of growth on
potted soil in greenhouse…………………………………………………85
Appendix 3. Authenticated and non-authenticated isolates that are not
Characterized…………………………………………………………….86

vii
List of symbols and Abbreviations

ATP Adenosine triphosphate

AAUGR Addis Ababa University Grass Pea rhizobia
BNF Biological nitrogen fixation
BTB Bromothymol blue
CR Congo red
E Effective
g/l gram per liter
ha hectare
HE highly effective
I ineffective
IAR intrinsic antibiotic resistance
ICARDA international central for agricultural research for in the dry areas
Kg kilogram
LE lowly effective
LM large mucoid
LW large watery
Mg milligram
MGT mean generation time
MM millimole
Min minute
MT metric tone
N nitrogen
N2 nitrogen molecule
PGA Peptone – Glucose – Agar

viii
pH hydrogen ion potential
PPM parts per million
rpm revolutions per minute
SW South Wollo
SDM Shoot dry matter
SDW Shoot dry weight
μg /ml microgram per milliliter
UPGMA Unpaired Group Method Analysis
V/V volume per volume
W/V weight per volume
WS West Shoa
YEMA Yeast Extract Mannitol Agar
YEMA-BTB Yeast Extract Mannitol Agar Bromothymol Blue
YEMA-CR Yeast Extract Mannitol Agar Congred

ix
Abstract

Grass pea (Lathyrus sativus) is the most widely distributed and the third most important pulse
crop in Ethiopia. It grows in a wide range of altitudes and survives in scarce moist conditions and
fixes nitrogen with rhizobia and performs well in less fertility soils. However, the effectiveness in
nitrogen fixation varies depending upon the host varieties, type of strain and different
environmental characters. It is, therefore, necessary to evaluate the efficiency of different isolates
from different sampling areas. Thus, 68 grass pea rhizobial isolates (47 from South Wollo and 21
from West Shoa) were isolated and characterized on the basis of different characters. These
isolates were reinoculated on pouch culture using “Wasse” variety of grass pea to authenticate
nodulation and determine their symbiotic effectiveness. All but 5 isolates were authenticated as
root nodule bacteria with their capacity to infect and nodulate their parent host. Most of the
isolates displayed fast doubling time (1.2-4hrs) and large colonies (2-6.0 mm) and changed the
BTB-YEMA media to yellow which showed that they are fast growing rhizobia and then
categorized into R. leguminosarum bv. viceae. The preliminary screening of the isolates for
symbiotic effectiveness on pouch culture showed that isolates AAUGR (31, 37, 41 and 47) of
South Wollo and AAUGR (48, 49, 52, 59, 60, 61, 66 and 67) of West Shoa that comprised of
19% of the isolates were highly effective whereas, 43% were found to be effective. Three highly
effective isolates from South Wollo (AAUGR 31, AAUGR 37, and AAUGR 41) and four highly
effective isolates from West Shoa (AAUGR 48, AAUGR 59, AAUGR 61, and AAUGR 67) were
reinoculated into soil culture to evaluate their performance in the natural environment (soil). All
the selected isolates were found to accumulate 112-149 % of SDM that was higher than the
controls. Isolate AAUGR 37 (SW) accumulated the highest SDM of all the inoculated treatments
and the controls. All the selected isolates were found to perform better on pot culture than on
pouch culture. Almost all of the isolates were able to grow at extremely low and high pH, low and
high salt, low and high temperature, and utilized a wide range of carbohydrate and nitrogen
sources and displayed high resistance to several antibiotics indicating that the isolates can
compete in their natural environment. The result of the numerical analysis also showed the
existence of diversity among the tested isolates.
.

x
1. Introduction

Nitrogen is the fourth most abundant element in living organisms. It is part of the
proteins, nucleic acids, adenosine triphosphate and other nitrogenous compounds.
Although it makes up about 80% of the atmosphere mostly as dinitrogen (N2), it is one of
the most limiting factors that affect plant growth and crop production. It is often present
in the atmosphere in an inert form and the oxidized form in the soil (NO3) is available to
plants. It should be necessarily reduced into biologically useful ammonia (NH3, NH4+) or
nitrates (NO3). This process is accomplished through industrial synthesis and biological
nitrogen fixation (BNF).

BNF is the conversion of dinitrogen into ammonia (NH3) which is carried out only by
bacteria that are endowed with the enzyme, nitrogenase (Crews et al., 2004; Postgate,
1998). The N2- fixing microbes (many genera of bacteria, cyanobacteria, frankia) can
exist as free-living organisms or in association with other microbes, plants or animals. It
accounts to about 60% of the global nitrogen budget (Burdass, 2002) and is eco-friendly
and cost effective (Zahran, 1999).

The most important of endosymbiotic association is between the plants belonging to the
family Leguminasae and the root nodule bacteria, generally known as rhizobia. These
plants include pulses (food legumes), shrubs and trees that account to the third largest
group of flowering plants. The legumes are multi-purpose plants that serve as a source of
food, feed, medicine, firewood, timber and other products. Food legumes (pulses) provide
high quality protein food and feed, minerals and other nutrients. Consequently, 25% of
the world’s major crop production, more than one-third of human protein needs and two-
third of human livelihood is derived from pulses (Graham and Vance, 2003). The ability
of many legumes to establish symbiosis with nitrogen fixing bacteria makes them a
valuable source of soil nitrogen for other crops (Allen and Allen, 1981). Among the
legumes, the pulses have played a great role as a source of food and feed and an integral
part of the low-input agriculture. These plants fall into two principal groups based on

1
climatic conditions and geographical distribution over the world, namely cool season and
warm season legumes (Jayasundara et al.,1998).The cool season legumes consist of the
tribes viceae (Vicia, Lens, Lathyrus and Pisum), Cicereae (Cicer), and Genisteae
(Lupinus) whereas, the warm season lowland tropical food legumes include cow pea
(Vagina unguiculata), common bean ( Phaseolus vulgaris), soy bean (Glycine max),
ground nut (Arachis hypogea) and Pigeon pea (Cajanus cajan).

The genus Lathyrus is one of the cool season legumes belonging to the tribe viceae. It is
widely distributed in Eurasia, America, Africa, India and Asia where its primary center of
diversity is believed to be the Mediterranean region (Muehlbauer and Abebe, 1997). It is
known by many common names, including chickling, vetch, Indian vetch (UK and USA),
Khesari or Batura (Indian) and Guaya (Ethiopia) (Jennifer, 2003). However, the five
different species that are distributed in Ethiopia are Lathyrus sativus, L. pratensis, L.
sphaericus, L. apaca, L adoratus (Dawit, 1991). It is mostly grown in the highlands of
Ethiopia at altitudes ranging from 1700-2220m above sea level with a maximum
temperature of 15-20 OC in an operative range of 5-30 OC (Asmare et al., 2000).

The seed contains 18.2-34.6% protein, 0.6% fat, 58.2% carbohydrates (about 35% starch)
and it also contains 1.5% glucose, 6.8% Pentosans, 3.6% Phytin, 1.5% lignin, 6.69%
albumin, 1.5% prolamine 13.3% globulin, and 3.8% guttllin (Muehlbauer and Abebe,
1997). The same authors (Muehlbauer and Abebe, 1997) also reviewed that the essential
amino acids found in grass pea seeds are (in grams per 16 grams of nitrogen): arginine
7.85, histidine 2.51, leucine 6.57, isoleucine 2.34, tryptophan 0.40, and valine 4.68 (like
other cool season food legume, grass pea is deficient in methionine and tryptophan).

Grass pea (Lathyrus sativus) is the third most important pulse crop in Ethiopia after faba
bean and chickpea with 142,170 ha of production area and 104,744 tones of production
(Wuletaw and Endashaw 2001). It occupies 8.7% of the total area and 7.6% of the total
production of food legumes in Ethiopia. It performs well on heavy black soils which are
characterized by water logging. Grass pea production is mainly concentrated in the North

2
West Zone (58%), the central zone (16.3%), the North East (12%) and the Northern as
well as the Southeast (12.9%) regions of Ethiopia (Wuletaw and Endashaw, 2001).
However, the major grass pea producing administrative regions of Ethiopia are Shoa,
Gojam, Gonder, Tigray, Wollo and Arsi (Adugna and Shelemew, 2001).

In the highlands of Ethiopia, major soils suitable for cool season food legumes are
predominantly brown and chestnut vertisols (Adugna and Shelemew, 2001). Major soil
related constraints to cropping are poor fertility, soil erosion on the slopes, acidity, and
low phosphate availability (Angaw and Asnakew, 1994). Grass pea besides the
nutritional benefits, has an important role as a legume crop in crop rotations, reportedly
adding around 67 kgha-1of nitrogen to the soil from symbiosis with Rhizobium sp. in a
single season and conferring yield and protein benefits on the subsequent non-legume
crop (Jennifer, 2003).

The development of grass pea inoculants can improve the fertility status of the soil and
increase the crop yield. However, the development of such inoculants requires collection,
isolation, identification and phenotypic characterization of large number of rhizobial
isolates from different growing regions. Above all, testing the infectiveness and
effectiveness of the isolates is also required.

Some research works have been done on grass pea with regard to its epidemiology,
amount of neurotoxin, its production and its genetic resource conservation and utilization
in some Agricultural Research Centers of Ethiopia (Berhanu et al., 2000). However;
works on taxonomic status and symbiotic effectiveness of grass pea rhizobia is totally
absent. Thus, this particular study is initiated with the aim of investigating the phenotypic
diversity and symbolic effectiveness of rhizobia that nodulate grass pea from some major
growing areas of South Wollo and West shoa.

3
2. Objectives
2.1. General Objective

To isolate and characterize the diversity and symbiotic effectiveness of root nodule
bacterial isolates of grass pea (Lathyrus sativus) from major growing areas of South
Wollo and West Shoa.

2.2. Specific Objectives

• To determine the different phenotypic characteristics of the isolates

• To evaluate the symbiotic effectiveness of the isolates on pouch culture
• To evaluate the performance of selected isolates on potted soil.

4
3. Literature Review
3.1. Legumes

Legumes are a family of plants Leguminosiae (Fabaceae) that have the ability to react in
a symbiotic (mutually beneficial ) relationship with root nodule bacteria to convert
atmospheric nitrogen (N2) from the soil atmosphere to ammonia nitrogen (NH3) (Miller,
2008). Leguminosiae (Fabaceae) is composed of 18,000 species that are divided into
three sub-families, Mimosoideae, Caesalpinoiodeae and Papilionoideae that are ranging
from tiny annual herbaceous plants to a huge and perennial trees (Giller, 2001). They are
used by mankind as a source of food and forage to mankind and animals (Howieson et
al., 2000). According to De-Faria et al. (1989), about 23% of Caesalpinoiodeae, 90%
Mimosoideae and 97% of Papillionoideae are known for their ability to form nitrogen
(N2) fixing root nodules with rhizobia.

The majority of legumes play a paramount role for agricultural purposes. Thus, some of
the legumes that are frequently used in agriculture include Vicia faba, Pisum sativum,
Phaseolus vulgaris, Trifolium repens, Medicago sativa, Melilotus officinalis, Glycine
max and Lathyrus sativa (Alexander, 1997; Postgate, 1998). The symbiotic associations
of legumes with rhizobia contribute a lot for the global nitrogen cycle in different
ecosystems and also for ecological stability (Zahran, 1999). However; all legumes are not
necessarily nodulated by root nodule bacteria due to genetic and several environmental
factors. The association between Rhizobium and legume is a cheaper and usually more
effective agronomic practice for ensuring an adequate supply of nitrogen for legume
based crop and pasture production than application of chemical fertilizers.

It is estimated that Legume-rhizobia association fix 180 billion tones of atmospheric

nitrogen (N2) per year into ammonia (NH3) (Freiberger et al., 1997) which is equivalent
to the expenditure of nitrogen fertilizer to about US 6.8 million dollar (Herridge and
Rose, 2000). Generally, the inclusion of grain legumes in cropping sequences increases
soil nitrate, grain yield, total nitrogen accumulation, food and feed (Badaruddin and

5
Meyer, 1994). Latta and carter (1998) also reviewed that their incorporation into the
cropping system also reduces soil erosion losses, improve structure of the soil and
nitrogen use efficiency of a subsequent cereal crop with inorganic fertilizer applied.

3.2. Grass pea

3.2.1. Ecology of Grass pea

The crop is widely cultivated in central, south and east Europe, the Mediterranean and
Africa and India. The mean fluctuations during the growing season ranges from 10-30 0C
with annual rainfall ranging from 600-1200mm and the crop is tolerant to extremely dry
conditions in drought prone areas as in Ethiopia and also tolerant to excessive floods as in
Bangladesh (Muehlbauer and Abebe, 1997). Asmare,et al.( 2000) have also found that
grass pea is grown in highlands of Ethiopia at altitude ranging from 1700-2220m with a
maximum temperature of 15-20 0C in an operative range of 5-300C. It is a hardy crop
suited to dry climates, producing good seed crops on poor soils. It is commonly cultivated
on heavy clay soils. Black deep retentive soils are considered best for grass pea and it is
sensitive to acidity and requires lime on acid soils (Muehlbauer and Abebe, 1997).

The growing conditions for grass pea have also been described at
http://www.Hydra.Org.uk.(2001) as follows: i) annual rainfall: grass pea requires
300mm-1300mm.It is useful for very wet and very dry areas. ii) temperature: it grows at
4.5 0C to 27.50C and does well in colder areas and iii) soil type: it tolerates almost all
types of soil from pH 4.5 to 8.3.It does best on clay soils.

3.2.2. Agronomic and economic importance of Grass pea

Grass pea seeds may be white, yellow-green, yellow, brown, gray or black. White seed is
the most popular for human consumption. Common foods prepared from grass pea in
Ethiopia includes roasted whole seeds (kolo), boiled whole seeds (nifro), pancake (kitta),

6
leavened bread (injera) and the traditional sauce (shiro-wot). Grass pea performs well
under adverse agricultural conditions, and its many cultivars possess different attributes
including the ability to resist both drought and flooding, high climatic adaptability and
the ability to grow in cool climates and at high altitudes (Jennifer, 2003).Grass pea
cultures also have the ability to adapt to saline, alkaline, clay or otherwise poor soils, and
are hardy and easy to cultivate (Sharma and Pandey, 2001).

One of the major drawbacks of grass pea is the fact that the seeds contain a major anti
nutritional compound B-N-oxyalylamino-lalanine (BOAA) (also known as B-N-oxalyl-L-

α, ß-diaminopropionic acid or ODAP (Jennifer, 2003). The neurotoxin causes a drastic

and irreversible paralytic disease known as “Lathyrism or neuro Lathyrism” upon

prolonged or excessive consumption of grass pea and the paralysis is manifested on leg
muscle, muscular rigidity and weakness (Jennifer, 2003).

3. 3. Rhizobia

Rhizobia are classically defined as symbiotic bacteria capable of eliciting and

invading root and stem tissue forming nodules on leguminous plants where they
undertake symbiotic nitrogen fixation (Sahgal and Johri, 2003). They are gram
negative rods, aerobic, motile and non-spore forming with one polar or sub-polar
flagellum or 2-6 peritichous flagella and that are pleomorphic under adverse growth
conditions (Jordan, 1984; Postgate, 1998). Rhizobia usually accumulate granules of
poly-β-hydroxyl butyrate when carbon is in excess and possess respiratory type of
metabolism with oxygen as the terminal electron acceptor (Jordan, 1984).

3. 3. 1. Taxonomy of rhizobia

The taxonomy of root nodule bacteria has changed considerably over the last 20
years. Beijerinck (1888) had for the first time isolated a bacterium from root nodule

7
of legume and named it Bacillus radicicola. This was subsequently renamed
Rhizobium (Frank, 1889). The earliest classification of rhibzobia was based on
specificity of symbiotic plant range of bacterial species (Sahgal and Johri, 2003). Fred
et al. (1932) recognized six species in the genus Rhizobium viz. R. japonicum
(Lathyrus, Lens, Pisum and Vicia), R. lupini (Lupinus), R. Meliloti (Melilotus,
Medicago, Trigonella) R. phaseoli (Phaseolus) and R. Trfolii (Trifolium) based on
their host range and certain morphological and physiological properties .

Based on their growth rate bacteria were grouped as fast growers and slow growers,
but were still placed in the genus Rhizobium till Jordan (1984) coined the new genus
Bradyrhizobium for isolates from Glycine max. Norris (1965) observed that fast
growers and slow growers differed in their symbiotic affinity. Accordingly, alkali
producing slow growers was isolated with the tropical legumes whereas; fast growers
from temperate legumes were acid producing (Sahgal and Johri, 2003).

Since the early nineties, sequence comparison of 16s r RNA genes and genetic finger
printing methods based on PCR have been used extensively for characterizing
rhizobia (Sahgal and Johri, 2003). These authors described that the use of sequencing
of full 16s rRNA gene and different PCR tools are reliable to ascertain the phylogeny
of rhizobial isolates. As Willems (2006) reviewed that the introduction of more
genetic characteristics (DNA-DNA and DNA-rRNA hybridizaions, rRNA catalogues,
rDNA sequencing) revealed more diversity among the rihizobia. This leads to a
gradual increase in number of genera (Table I).

8
Table I. Rising number of species in the genera of the rhizobia.
Genus Original Number of species
Publication Before 81-85 86-90 91-95 90- 01-
1980 00 06
Rhizobium Frank,(1889) 4 5 5 10 10 16
Brandy rhizobium Jordan,( 1982) 1 1 3 3 7
Sinorhizobium, Chen et al.,(1988) 2 5 8 11
Azorhizobium Dreeffus et al.,( 1 1 1 2
1988)
Merorhizobium Jarvis et al., 7 11
(1997)
Allorhizobium Delajudie et al.,( 1 1
1998)
Total 4 6 9 19 30 48
Source: Willems (2006)

3. 3. 2. Rizobial diversity and characterization methods

The diversity of rhizobia in a particular soil may be influenced by the method used to
isolate. Several arrays of techniques are used for detecting and describing rhizobial
diversity. These are host range, comparative growth in culture, intrinsic antibiotic
resistance, carbohydrate utilization, amino acid utilization and tolerance in pH,
temperature and salt. These are among the most common methods that are considered
as phenotypic characters and are used primarily to study rhizobial diversity (Ahmad
et al., 1984; Maatallaah et al., 2002). The size, shape, color and texture of colonies
and the ability to alter the pH of the media are generally stable characteristics useful
in defining strains of isolates. Colonies could be discrete, round varying from flat to
domed and even conical on agar surface. Moreover; they may be white, opaque, and
milky and watery translucent (Ahmad et al., 1984).

9
Rhizobial colonies after incubation on YEMA-CR indicator remain white opaque or
occasionally pink except some contaminants such as Rhizobum melliloti (Lupwayi
and Haque, 1994). No or little growth of rhizobial isolates occur when streaked on
Peptone Glucose Agar medium and incubated at 28 ± 20C for 3-5 days (Somasegaren
and Hoben, 1994).

The fast growers form visible colony with in 2-3 days and their generation time is less
than four hours. The fast growers are acid producers and the slow growers are base
producers. This identification can be done by growing them on YEMA media in the
presence of Bromthymol blue (BTB) indicator at pH 6.8 (Jordan, 1984; Lupwayi and
Haque, 1994). Tolerance to acid or alkali, temperature sensitivity, resistance to
antibiotics and heavy metals, tolerance to salinity, utilization of different carbon
sources, utilization of different nitrogen sources and symbiotic characteristics were
used by many researchers to determine a wide physiological diversity among tested
isolates (Ahmad et al., 1984 and Maatallah et al., 2002). These phenotypic methods
provide a valuable insight into rhizobial population structure and strain diversity.
Thus, they are best regarded as preliminary screening methods for relative screening
methods.

Modern molecular tools which are more preferred to phenotypic methods can also be
used to evaluate the specificity of the stains (Thiess et al., 2001). Some of these
molecular tools employed for rhizobial diversity identification includes: sequence
comparison of 16S rRNA genes and genetic finger printing (Sahgal and Johri, 2003),
plasmid profiling (Broughton et al, 1987), restriction fragment length polymorphism
(RFLP) (Odee et al., 2002), polymerase chain reaction based techniques (PCR)
(Richardson et al., 1998), PCR-RFLP of 16S rRNA genes (Sahgal and Johri, 2003).

10
3. 4. Inoculation and their benefit

Soil contains millions of rhizobia but they may not be specific to the particular
legume grown and in an active stage to induce infection and initiate N2 fixation. This
brings the necessity of inoculation of particular rhizobium inoculants into their
specific legume partner. Lewis (1967) defined inoculation of legume as the
introduction of legume bacteria into the soil or plant seedling to enable plants to fix or
change in to usable form of atmospheric nitrogen. Mulongoy (2004) has also
described inoculation as the introduction of rhizobia into the soil to ensure proper
nodulation and nitrogen fixation, if specific and effective rhizobia are absent in the
soil or if they are present in low numbers. The benefits of inoculation described by
Miller (2008) include: i) increased yield -yields may increase 10-100% depending on
specific soil conditions; soils with average fertility may have yield increase from 15-
25%, ii) improved protein content, and iii) increase soil nitrogen for future crops –
nitrogen fixation will vary depending on plant species, suitable bacterium population,
soil nitrogen content, soil fertility level, soil pH, moisture, and temperature.

3.5. Nitrogen fixation

Nitrogen is an essential plant nutrient which is most commonly deficient in soils,

contributing to reduced agricultural fields throughout the world (Montanez, 2000).
The element nitrogen is an essential part of many of the chemical compounds, such as
proteins, nucleic acids and other metabolites, which are the basis of all life forms. The
atmospheric nitrogen in order to be incorporated in to amino acids and subsequently
become an integral part of plant tissue, it has to be transformed in to ammonia (NH3)
through nitrogen fixation (Postgate, 1998). The different ways of N-transformation
and fixation are (Smil, 2000):
1. The natural formation of NO from N2 and O2 due to lightening.
2. Combustion of fossil fuels: through automobile engines and thermal power plants
that release different oxides of nitrogen.

11
3. Industrial nitrogen fixation: in the Haber-Bosch process, N2 is converted together
with hydrogen gas (H2) into ammonia (NH3) fertilizer.
4. Biological nitrogen fixation: bacteria are able to fix and assimilate it as organic
nitrogen. Symbiotic nitrogen fixing bacteria such as rhizobium live in association
with leguminous plants whereas, free-living nitrogen fixing bacteria such as
azotobacter fix nitrogen without any association with plants.

Biological nitrogen fixation (BNF) is the process whereby atmospheric nitrogen

(N=N) is reduced consequently in to ammonia and other reactive and usable forms in
the presence of nitrogenase which is a biological catalyst found naturally only in
prokaryotes. BNF is exclusively carried out by prokaryotes. The ability to reduce and
siphon out an appreciable amount of nitrogen from the atmospheric reservoir and
enrich the soil is confined to bacteria and archea (Tilak et al., 2005). These include a)
symbiotic nitrogen fixing (N-fixing) forms, viz. rhizobium the obligate symbionts in
leguminous plants and Frankia in non-leguminous plants and b) Non- symbiotic (free-
living, associative or endophytic) N2 – fixing forms such as Cyanobacteria,
Azosprillum, Azotobacter, Acetobacter, Diazotrophicus, Azoarcus, etc.

BNF takes place in all ecological environments and conditions such as soils, fresh
and salt water and sediments, on or within roots, stems, leaves of different type of
leguminous plants (Postage, 1998; Zahran, 1999). Biological nitrogen fixation (BNF)
is becoming more important for not only reducing energy costs but also in seeking
more sustainable agricultural production and agricultural systems. Nitrogen is
continually depleted by such processes as microbial denifrification, soil erosion,
leaching, chemical volatilization and perhaps most important, removal of nitrogen-
containing crop residues from the land. Since its deficiency is a problem in most soils
of the tropics and subtropics (Hungria and Vargas, 2000), it’s adequate supply, proper
management and utilization efficiency are crucial to ensure improved agriculture in
sustainable manner (Graham and Vance, 2003).

12
The atmosphere comprises of approximately 80% molecular nitrogen (N2) that is
about 4 X 1015 tones in the form of ditinrogen molecules, but only about 2 X 106
tones is found in compound form (Hubbell and Kidder, 2002). This molecular
nitrogen is inert and unavailable for plants. It has to be transformed into utilizable
form such as NH4 through the process known as nitrogen fixation (Smith and
Douglas, 1987; Postgate, 1998). The nitrogen reserve of agricultural soils must
therefore be replenished periodically in order to maintain an adequate (non-growth
limiting) level for crop production. This replacement of soil nitrogen is generally
accomplished by the addition of chemically fixed nitrogen in the form of biological
nitrogen fixation (BNF) systems (Hubble and Kidder, 2003).

The application of nitrogen fertilizer has increased approximately 10 fold to 90

million Mt between 1950 and 1995 with significant energy consumption for N-
fertilizer synthesis and application (Frink et al., 1999 and FAO, 1994). However, in
developing countries, several constraints such as the high cost of nitrogen fertilizer,
the energy requirements for production and the suboptimal transportation capacities
made the chemical fertilizers neither available nor affordable in food production on
small farms (Bohlool et al., 1992; and Vance, 1997).

BNF is estimated to contribute 180 X 106 mt /year globally, of which 80% comes
from symbiotic association and the rest 20% from free-living or associative systems
(Tilak et al., 2005). This is by far greater than that of the industrially fixed nitrogen
supply which is 65 X 106 tons per year. Roughly, half of the 23 million mt of nitrogen
consumed as human food sources (grains and live stocks) come from BNF by
prokaryotes (Frink et al., 1999). Out of this, rhizobia in root nodules are estimated to
carry out about 80% of the world’s BNF (Burris and Roberts, 1993).

Symbiotic nitrogen fixation (SNF) by rhizobium with legume crops fixes about 90 X
109 Kg of N2 annually (Vance, 1998). In agricultural systems, symbiotically fixed
nitrogen can: (i) be an immediate source of N to the fixing species for dry matter and

13
seed production, (ii) be released from the species to comparison crops to complement
their N needs and (iii) be useful as a green manure providing N to crops grown in
relations (Peoples et al., 1995). SNF is dependent on several factors including the
host plant genotype, the Rhizobium strain and the interaction of these symbionts with
the pedoclimatic factors and the environmental conditions (Bordeleau and Prevost,
1994). Both the legume plant and the bacteria derive mutual benefit form the
association. The bacterium’s enzyme system supplies a constant source of reduced
nitrogen to the host plant and the plant furnishes nutrients and energy for the activities
of the bacterium (Burdass, 2002).

3. 6. The biochemistry of biological nitrogen fixation (BBNF)

BNF, the process of reducing the triple bond of atmospheric nitrogen (N=N) in to
ammonia and other usable forms take place in the presence of an oxygen sensitive
enzyme, the nitrogenase. Nitrogenase is a complex – enzyme containing two oxygen
sensitive metalloprotein components that are active independently but work
synergetically (Dilworth and Glenn, 1991; Leigh, 2002).

Dinitrogenase (Component I), the larger heterotetramer component of nitrogenase

with two different metalloclusters has two α- protein subunits and two β-protein
subunits, 24 molecules of iron, two molecules of molybdenum and an iron
molybdenum cofactor (FeMoCo) (Leigh, 2002). Component I, the dinitrogenase
reductase, catalyses the actual conversion of N2 to ammonia. Similarly, component II
is composed of two protein subunits (different from that of component I) and a large
number of iron molecules or iron-protein which plays a role in donating electrons to
component I (Giller and Wilson, 1991; Dean and Jacobson, 1992; Maschner, 1995;
Peters et al., 1995; Haward and Rees, 1996).

Each individual α, β dimmer component containing Fe-Mo-Co and a P-cluster is

considered as a functional unit of nitrogen fixation and the Fe-protein is a

14
 homodimmer component containing two MgATP binding sites and a single (4Fe-45)
cluster (Peters et al., 1995). Dinitrogenase, a 240-KDa heterotertramer, binds N2 and
holds it while it is being reduced whereas; Dinitrogenase reductase a 64-KDa
homodimer provides dinitrogenase with high energy electrons. Following binding, an
electron is transferred from the Fe-protein to the Mg Fe-protein with concomitant
hydrolysis of both bound ATP molecules to ADP; ultimately, the two components of
nitrogenase dissociate (Giller, 2001). Thus, nitrogenase catalyses the conversion of
N2 to NH3 which is represented as:

+ ++ + +
N2 + 16 ATP + 8e + 8 H Mg 2NH 4 +H 2 + 16 ADP + 16 pi

Fig. 1. The role of nitrogenase enzyme in nitrogen fixation

For the reaction to be catalyzed, nitrogenase requires anaerobic environment and a source
of large amount of energy. BNF is energy intensive, efficient but requires much lower
energy than Haber process (Zahran et al., 1992). In symbiotic association, nitrogenase
activity is protected form oxygen damage in two ways:one with strong and variable
physical barrier to oxygen diffusion into nodule cortex and the second component of
oxygen protection is with the presence of leg-hemoglobin within the infected cells. The

15
role of leg-hemoglobin in the nodule is to facilitate oxygen supply for respiration, so that
high oxygen tension support bacteroidal oxidative phosophorylation while keeping
oxygen sequestered away from nitrogenous enzyme (Shaw, 1983).

3. 7. Ecological factor that affect BNF

BNF is a key to sustainable agricultural systems in tropical soils which are frequently
deficient in nitrogen. This is achieved where there is establishment of effective
symbiosis which requires the following factors (Bordeleau and Prevost, 1994).
A. Colonization and survival in soil by rhizobia as saprophytes in competition
with other endogenous microbes which is also associated with soil infertility
and extremes of temperature,
B. Rapid colonization of the rhizosphere prior to root infection and genetic
compatibility between host and root nodule bacteria to establish an effective
nodule, and
C. A favorable environment to allow maximum fixation.

In addition to the competitiveness of the rhizobia in forming nodule and the effectiveness
of the rhizobium-host plant to fix N2, a series of edaphic, chemical and biophysical
factors affects N2 fixation (Montanez, 2000). In the Rhizobium-legume symbiosis, N2
fixation is strongly related to the physiological state of the host plant (Zahran, 1999). All
environmental limitations that adversely affect plant growth and vigor also influence the
symbiosis (Giller, 2001). Plants are highly dependent on physical and chemical
conditions of the soil to give their natural potential for growth and nitrogen fixing activity
(Negales et al., 2002; Zahran, 1999). As a result, an efficient rhizobial strain is not
expected to express its full capacity for nitrogen fixation, if limiting factors imposed on
the vigor of the host legume (Negales et al., 2002; Zahran, 1999).

Environmental factors can influence the ratio between rhizobia in the soil and
rhizospehere microorganisms, and can also influence other steps in the nodulation

16
process such as attachment, infection and nodule formation (Al-Falih, 2002). There are
several environmental stresses (factors) that influence the legumes and the symbiotic
partners, (Rhizobium) and the symbiosis which are soil acidity, drought, temperature,
salinity, water stress, unfavorable soil pH, nutrient deficiency, insufficient or excess soil
moisture, energy and carbon sources (Zahran, 1999).

3. 7.1. Soil PH

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 Barneix,
1997). Most leguminous plants require a neutral or slightly acidic soil for growth,
especially when they depend on symbiotic nitrogen fixation (Bordeleau and Prevost,
1994).

Acid soil conditions pose problems for the plant, the bacteria and the symbiosis (Giller
and Wilson, 1991). Legume and their rhizobia exhibit varied response to acidity. Some
rhizobial species can tolerate acidity better than others, and tolerance may vary among
strains within a species (Vargas and Graham, 1998). The optimum pH for rhizobial
growth is considered to be between 6.0 and 7.0 (Jordan, 1984) and a relatively few
rhizobia grow well at pH less than 5.0 (Graham et al., 1994). Soil acidity constrains
symbiotic N2 fixation in tropical and temperature soils, limiting Rhzobium survival and
persistence in soils and reducing nodulation (Graham et al., 1982).

Acidity affects early steps in the infection process including the exchange of molecular
signals between symbiotic partners and attachment to the roots (Hungria and Vargas,
2000). Release of nod-gene inducers by soybean and common bean roots is less at pH 4.5
than at pH 5.8 (Hungria and Stancey, 1997), with some nodulation genes including nod
A, switched off as the pH falls (Richardson et al., 1988). Low pH can affect the
production and exeretion of nodulation factors (Mcklay, 1993). Low pH is often
associated with increase in Al and Mn toxicity and reduced Ca supply that affect the

17
growth of rhizobia (Campo, 19995), of the host legume and symbiosis (Kim et al., 1985).
The number of nodules, the nitrogenase activity, the nodule ultra-structure and the fresh
and dry weights of nodules are affected to a greater extent at a low medium pH (< 4.5)
(Vassileva et al., 1997).

Tolerance to high alkalinity has also been observed for rhizobial strain isolates with
tolerance up to pH 9.5 for rhizobiaceae (Jordan, 1984). Nour et al. (1994) found the pH
tolerance of rhizobial strains isolated from chickpea (Cicer arietinum) to be 10.0 and
Shenbagarathi (1993) reported that rhizobial strain SBS- R100 isolated form Sesbania
procumbens is capable of growing at pH 11. Rhizobia appear to be more tolerant to
alkalinity than do their legume plants (Zahran, 1999). Rao and Sharama (1995) reported
that while germination of pigeon pea was decreased at pH values of > 8.8, growth of
rhizobia was unaffected up to pH 11.5. Rhizobial strains isolated form Seseania formasa,
Acacia farnesiana and Dulbergia sissoo were well adapted to grow on pH 12.0 (Surange
et al., 1997).

Rhizobia with a higher tolerance to acidity have been identified (Graham et al., 1982).
Aarons and Graham (1991) reported high cytoplasmic potassium and glutamate levels in
acid-stressed cells of Rhizobium leguminosarum are linked with low pH tolerance.
Furthermore, differences in LPS composition, proton exclusion and extrusion (Chen et
al.; 1993), accumulation of cellular polyamines (Fujihara and Yoneyama, 1993), and
synthesis of acid shock proteins (Hickey and Hirshfield, 1990) have been associated with
the growth of cells at acid pH.

The composition and structure of outer membrane could also be a factor in pH tolerance
(Graham et al., 1994). At least two loci of either megaplasmid or chromosomal location
for pH genes are necessary for the growth of rhizobia at low PH (Chen et al., 1993). The
expressions of one membrane proteins of 49.5 KDa and three soluble proteins of 66.0,
85.0, and 44.o KDa increased when the cells of acid-tolerant strains were grown at pH
4.0 (correa and Barneix, 1997). Soil acidity can be corrected by different practical

18
measures. Two strategies that have been adopted to solve the problem of soil acidity are
liming the acidic soil to ameliorate the effects of acidic conditions and selecting and
isolating tolerant strains of rhizobia and plant cultivars to increase plant production on
acidic soils (Graham, 1992; Bordeleau and prevost, 1994; Zahran, 1999).

3. 7.2. Salt stress

Salinity is a serious threat to agriculture in arid and semiarid regions (Zahran, 1999).
Nearly 40% of the world’s land surface can be categorized as having potential salinity
problems (Cordovilla et al., 1994). Increasing salt concentrations may have a detrimental
effect on soil microbial populations as a result of direct toxicity as well as through
osmotic stress (Tate, 1995). Salt tolerance in plants is a complex phenomena that
involves morphological and developmental changes as well as physiological and
biochemical processes. Salinity decreases plant growth and yield, depending up on the
plant species, salinity levels and ionic composition of the salts (Delgado et al., 1994).

The response of legumes to salinity causes depends on such factors as climatic

conditions, soil properties and the stage of growth and crop cultures (cordovilla et al.,
1995). Zahran (1999) and (Cordovilla et al., 1995) reported variability in salt tolerance
among legumes such as Phaseolus vulgaris, Vicia faba, Glycine max, alfalfa, clover and
soybean. Legumes are generally more sensitive to osmotic stress than their
microsymbionts are (Zahran, 1999). In contrast to their host legumes, some rhizobia can
survive in the presence of extremely high levels of salt both in culture and in soil
(Bordeleau and Prevost, 2002). According to Bordeleau and Prevost (2002) salt tolerant
legumes include alfalfa and narrow- leafed lupins (L. angustifolius) that can grow in
salinities equivalent to seawater.

Salt stress result in growth depression which can be attributed to the accumulation of
toxic ions such as sodium and chlorine in plant tissue, where they can disturb enzyme
activities (Bordeleau and Prevost, 2002). Increase in salt concentration affect microbial

19
population, plant growth and yield as the result of inhibition of rhizobia-legume
symbiosis, nodule initiation and nodule formation (Zahran, 1999). In addition, constraints
of salt stress causes nutrient imbalance such as potassium, iron, boron and calcium when
nodulated pisum sativum were analyzed (El-hamdaoui et al., 2003). The reduction on
nitrogen fixing activity by salt stress is usually the result of reduction in respiration of
nodules and in cytosolic protein production particularly leg-hemoglobin by nodules.

Rhizobial cells subjected to high salt stress change their morphology (Zahran, 1999). The
cell ultrastructure is severely affected and the cell envelop is distorted. These alterations
affect the hosts and the symbiotic interactions that lead to changes in lipopolysaccharides
(LPS) and surface antigens; which are the major determinants for adaptation and
symbiosis (Zahran, 1999). These structures affect the capacity of rhizobium to infect and
form effective nodules and hence the amount of nitrogen fixed per unit weight of nodules
with salt stress (El-Shinnawi et al., 1989; Miller and Wood, 1996).

Rhizobial cells have various mechanisms for adapting high salt-stress conditions.
Rhizobial adaptations to salt-stress are attributed to the accumulation of intracellular low
molecular weight organic solvents (Osmolytes) such as glutamate, proline, glycine-
betaine, glycine amino acids, ectoine, amide, polyoles called osmotically active solutes
(Zahran, 1999). The accumulations of osmolytes (Osmotic adaptations) are thought to
counteract the dehydration effect of low water activity in the medium but not to interfere
with macromolecleular structure or function. They are involved in osmoregulation and
prevention of cytoplasmic dehydration in saline environments (Bostford and Lewis,
1990; Miller and Wood, 1996). The disaccharide trehalose plays a role in osmoregulation
when rizobia are growing under salt or osmotic stress (El-Sheikh and Wood, 1990).
Trehalose accumulates to higher levels in cells of R. leguminosarum and peanut rhizobia
under the increasing osmotic pressure of hyper salinity (Gittoni and Bueno, 1996).

20
3. 7.3. Temperature

Rhizobial species as other organisms do have their own range of optimum temperature
for survival and growth. However; this optimum temperature differs from species to
species. For most rhizobia, the optimum temperature range for growth in culture is 28 to
310C (Graham, 1992). Rhizobia isolated from nodules of Acacia senegal and Proscopis
chilensis, growing in hot dry regions of Sudan, has had high maximum growth
temperatures (44.20c) (Zahran et al., 1999; Zhang et al., 1991). Hangar and Vargas
(2000) have reviewed that the upper limits for rhizobial growth range between 32 and
470c although tolerance varies among species and strains. The upper limits for N2 fixation
in tropical legumes vary between 27 and 400c (Gibson, 1971). It is also reported that
Strange et al. (1997) has isolated highly temperature (500c) tolerant strains of rhizobium
nodulating trees from tropical soils.

High temperature affects the growth and survival of rhizobia (Zahran, 1999) and
nodulation by influencing adherence, penetration of bacteria to form infection-thread
(Vincent, 1980; Hungria and Vargas, 2000). It also influences nitrogen fixation by
decreasing leg-hemoglobin synthesis, nitrogenase activity and allocation of fixed nitrate
in nodules (Hungaria et al., 1989; Bordeleau and Prevost, 1994). Furthermore, high
temperature accelerates nodule senescence (Hungria and Franco, 1993). The remedy is
administration of inoculum in deeper soils and application of surface mulch to reduce soil
temperature (Roughley, 1980). Likewise, it was also found that low temperature decrease
nodulation and nitrogen fixation (Waughman, 1997).

The over all detrimental effects that come as a result of high temperature are due to
physiological and genetic modifications in bacteria, including plasmid deletion and
genomic rearrangement. Although, heat shock proteins have been found in rhizobium
(Aarons and Graham, 1991), they cannot be produced during salt and osmotic stress
(Zharan et al., 1999), which indicates that it is a specific response to heat stress.

21
3. 7.4. Water Stress

The survival and activity of micro-organisms may depend on their distribution among
microhabitats and changes in soil moisture. Low water activity affects the legume
cultivar, the micorsymbiont and the symbiosis. One of the immediate responses of
rhizobia to water stress (low water potential) concerns the morphological changes. The
modification of rhizobial cells by water stress will eventually lead to a reduction in
infection and nodulation of legumes (Zahran, 1999). Water stress limits rhizobial
survival, growth and their population structure in soil (Zahran, 1999; Hangria and
Vargas, 2000).

Chemo tactic movement of rhizobia is influenced if there is no enough film of moisture

in the medium (Bordeleau ad Prevost, 1994, Zahran, 1999) and they respond to low water
activity by morphological changes (Zahran, 1999). This alteration together with low
mobility results in low rhizosphere colonization and low infection of root hairs
(Roughley, 1980). Furthermore, reduction in soil moisture affects formation and
longevity of nodules and leghemoglobin synthesis (Hangria and Vargas, 2000). In
general, nodule initiation, growth and activity are very sensitive to water (Zahran, 1999).
Moreover, severe stress may lead to irreversible cessation of N2 fixation (Vincent, 1980).

Water stress reduces both N2 fixation and respiration of nodules by influencing legumes
to produce short and stubby roots which are not suitable for infection by rhizobia. The
effect of water stress on vegetative growth is more detrimental to nodulation and nitrogen
fixation than during reproduction stage (Zahran, 1999). Sensitivity to moisture varies for
a variety of rhizobial strains and different legume cultivars (Bordeleau and Prevost, 1994;
Zahran, 1999). Water logging in the rhizospere exerts anaerobic condition which results
in the build up of carbondioxide and ethylene reduction which thus restrict nodulation
and nitrogen fixation (Bordeleau and Prevost, 2000).

22
Drought tolerant N2 fixing legumes can be selected although the majority of legumes are
sensitive to drought stress. Several mechanisms have been suggested to explain the
various physiological responses of several legumes to water stress (Zahran, 1999). The
legume with a high tolerance to water stress usually exhibit osmotic adjustment; this
adjustment is partly accounted for by changing cell turgore and by accumulation of some
osmotically active solutes (Ford, 1984). The accumulation of specific organic solutes
(osmotica) such as proline, free amino acids and low molecular weight solutes (pinitol)
and potassium is a characteristics response of plants subjected to prolonged severe water
stress (Hungria and Vargas, 2000; Zahran, 1999).

3.7.5. Carbon and energy sources

Energy is required for the reaction of N2 fixation from the host plant. The rhizobia use
photon assimilated carbon via the carbon cycle and generate energy as ATP. The
functioning of nodules depends directly on photosynthesis (Kouchi et al., 1986). As
much as 25% of a legume’s net photosynthate may be allocated below ground to
support N2 fixation (Atkins, 1984). Some of this carbon may be used directly to
maintain nodule function while some is returned to the host plant in the form of
carbon skeletons of the exported nitrogenous solutes (Crews, 2004). If the bacteroids
face deprivation of their energy source (photosynthate), the proper functioning of the
nodules becomes impaired.

3.7.6. Soil Nutrients

Soil salinity and acidity are usually accompanied by mineral toxicity (Specific ion
toxicity), nutrient deficiency, and nutrient disorder (Zahran, 1999). In addition to their
importance in the growth and survival of free-living rhizobia, some essential nutrients
have been shown to have specific roles in the development and functioning of N-
fixing symbioses. A group of essential nutrients Ca, P, Fe, and Mo are required at
specific stages for the development of legume symbiosis and the symbiotic N fixation

23
to the extent that their deficiencies limit the productivity of host legumes in some
agricultural systems (Graham and Vance, 2000).

Phosphorous is one of several elements which affects N2 fixation and along with N, it
is a principal yield limiting nutrient in many regions of the world (Pereira and Bliss,
1989). Phosphorous appears essential for both nodulation and nitrogen fixation
(Pereira and Bliss, 1989). Nodules are strong links for phosphorous (P) and range in P
content from 0.72 to 1.2% (Hart, 1989); consequently, N2 fixation dependent plants
will require more of this element than those supplied with combined nitrogen
(Cassman, 1981). Nodulation, N2 fixation and specific nodule activity are directly
related to the P supply (Singleton et al., 1983). Phosphorous is required for signal
transduction, membrane biosynthesis, nodule development and function (Israel, 1987)
and nitrogenase activity (Al- Niemi et al., 1997). Adaptive mechanisms of legumes
are different to acquire P under phosphate limiting conditions in soil ranging from
acidification of the rhizosphere, exudation of acid phosphase, changes in root
architecture, enhance P transport and to mycorhizal symbiosis (Johnson et al., 1996).

Calcium deficiency has specific effects on the legume symbiosis during the pre-
infection and nodule development stages (O. Hara et al., 1988). Calcium has been
suggested to have role in maintaining cell wall rigidity specifically, in
lipopolysaccharides (LPS) structure (Ballen et al., 1998) and expression of outer
membrane proteins. Since the cell surface component of rhizobia are calcium
dependent, attachment of rhizobia to root hair, appearance of nodule and nodulation
can be affected by calcium deficiency (Caetano-Anollen et al., 1989; Graham, 1992).

Molybdenum has a major role in symbiotic N fixation as a fundamental component

for nitrogenase. Mo deficiency affects nodule development and nodule functioning by
reducing bacteroid replication and delaying or preventing the onset of nitrogenase
activity (Jongruaysup et al., 1997).

24
Iron is required for legume nodulation, possibly for the proliferation of the infecting
rhizobia in the host tissue (Tang et al., 1992). Lupines are sensitive to Fe deficiency
at an early stage of nodule initiation (Tang et al., 1992) whereas; peanut seem to be
sensitive to Fe stresses at latter stages in nodule development (O’Hara et al., 1988).
The source of iron from seed reserves or supplied from soil, during nodule
development may be an important factor in the efficiency of transfer of Fe from host
tissues to the infecting rhizobia (O’Hara et al., 1993).

Regulation of dinitrogen fixation in response to available fixed nitrogen is mediated

by the host legume rather than the bacterial symbiont. The soil nitrate will also inhibit
or regulate the nitrogen fixation in three ways; low concentration of nitrate (1-2mm)
actually promote nodulation by ensuring early rapid growth of the plant and
development of a healthy root system that is able to nodulate, the intermediate
concentration of nitrate partially inhibit nodulation and the effect may be manifested
in the developing nodules being smaller, such that the nodule mass per plant is
reduced while the total number of nodules remain almost unaltered and if the
concentration of nitrate is excess, the effect will be actual inhibition of fixation and
inactive nodules because nitrate could be restricted to the cytoplasm of nodule cells
and the nitrogenase activity decreased due to an increase in the resistance of oxygen
diffusion barrier (Arrese-Ignor et al., 1997).

25
4. Materials and methods
4. 1. Study sites

The sampling area covers 47 sampling sites from Kutaber, Legehida, Wereilu, and
Jamma woredas (South Wollo) and 21 sampling sites from Gindeberet, Jeldu, and Dendi
woredas (West Shoa). The nodule sample collection was done from these areas where
grass pea has been growing for a long time with no earlier history (legend) of inoculation
with rhizobia. The root nodules were collected at the end of October and at the beginning
of November, 2008 during their flowering stage. The study sites are indicated in Figure 2
and Table 2.

26
 3000 00 4000 00 5000 00 6000 00

1300000
1300000
Y
#

Kutab er

So uth W ollo

1200000
1200000
W ere Ilu
Legehid a

Jam a

1100000
1100000

Leg end Abuna

Ginde be re t
Sampling W ered a

Jeldu

1000000
1000000

W est Sho a
N Dendi

0 50 100 km

3000 00 4000 00 5000 00 6000 00

Fig.2. Location map of the study areas

27
4. 2. Nodule collection and isolation of rhizobia.

4. 2. 1. Nodule collection

After the study sites were selected, nodule samples were collected from the farmer’s field
randomly. The nodules were immediately kept in sealed vials containing a desiccant
(Silica gel) covered with 1cm of cotton wool (Somasegaren and Hoben, 1994) till
isolation of the rhizobia was undertaken.

4. 2. 2. Isolation of Rhizobia

Dehydrated or desiccated root nodules were immersed in sterile distilled water for
overnight in labeled sets of peri-dishes (Vincent, 1970). The imbibed nodules were
surface sterilized according to Somasegaren and Hoben (1994). They were first subjected
to 70% ethanol for 10 seconds and then to 5 % (v/v) solution of sodium hypochlorite
(NaoCl) for 4 minutes. The surface sterilized nodules were then rinsed in five changes of
sterile distilled water to completely rinse the sterilizing chemicals.

The surface sterilized nodules were transferred into sterile Petri-dishes and crushed with
alcohol flamed sterile glass rod in a drop of normal saline solution (0.85%NaCl) inside a
laminar air flow hood. A loopful of suspensions (crushed nodule saps) were streaked
across the surface of Yeast Extract Mannitol Agar (YEMA) plates containing 25 ppm of
congored (YEMA-CR) and incubated at 28 ± 20C fro 3-5 days.

The YEMA-CR medium comprises of the following components (g / l):

Mannitol ---------------------------------10g
K2 HP04 -----------------------------------------------------0.5g
Mg So4. 7H2O----------------------------0.2g
NaCl ---------------------------------------0.1g
Yeast extracts ------------------------------1g

28
 Agar ---------------------------------------15g
Congored ----------------------------------10ml
Distilled water ----------------------------1000ml
They were autoclaved for 1210C for 15 minutes.
Source: Somasegaren and Hoben (1994).

Table 2. The study area of nodule collection

Isolates Zone Woreda Kebele Soil
PH
AAUGR1 South Wollo Kutaber Chistiane 6.3
AAUGR2 South Wollo Kutaber Gono 6.1
AAUGR3 South Wollo Kutaber Jamlako 6.5
AAUGR4 South Wollo Kutaber Gono 6.4
AAUGR5 South Wollo Kutaber Ruga 6.6
AAUGR6 South Wollo Kutaber Dihamatebia 6.8

AAUGR7 South Wollo Kutaber Boru 6.7

AAUGR8 South Wollo Kutaber Kemmeda 6.5
AAUGR9 South Wollo Legehida Teftef 6.6
AAUGR10 South Wollo Legehida Aley 6.2
AAUGR11 South Wollo Legehida Sirgut 6.1
AAUGR12 South Wollo Legehida Adalo 6.4
AAUGR13 South Wollo Legehida Mendel 6.3
AAUGR14 South Wollo Legehida Godo 6.7
AAUGR15 South Wollo Legehida Nifas Amba 6.8
AAUGR16 South Wollo Legehida Guramba 6.9
AAUGR17 South Wollo Legehida Tatity 7.1
AAUGR18 South Wollo Legehida Ablko 6.5
AAUGR19 South Wollo Legehida Shefait 6.2
AAUGR20 South Wollo Legehida Zerkamy 7.2
AAUGR21 South Wollo Legehida Liguama 6.2
AAUGR22 South Wollo Legehida Shikif 6.7
AAUGR23 South Wollo Woreilu Amayo 6.5
AAUGR24 South Wollo Woreilu Abewold 6.6
AAUGR25 South Wollo Woreilu Gurachalie 6.9
AAUGR26 South Wollo Woreilu Tulumojo 6.8
AAUGR27 South Wollo Woreilu Gerbo 6.5
AAUGR28 South Wollo Woreilu Mechela 6.8
AAUGR29 South Wollo Woreilu Dereba 6.7

29
AAUGR31 South Wollo Woreilu Geshober 6.6
AAUGR32 South Wollo Woreilu Kabie 6.5
AAUGR33 South Wollo Woreilu Gebrelu 6.9
AAUGR34 South Wollo Woreilu Korkie 7.1
AAUGR35 South Wollo Woreilu Dolu 6.9
AAUGR36 South Wollo Woreilu Tulu 6.7
AAUGR37 South Wollo Woreilu Chifa 6.8
AAUGR38 South Wollo Jamma Soramicha 7.0
AAUGR39 South Wollo Jamma Ketari 7.1
AAUGR40 South Wollo Jamma Faji 6.8
AAUGR41 South Wollo Jamma Kubi 7.1
AAUGR42 South Wollo Jamma Jarso 7.2
AAUGR43 South Wollo Jamma Boren 7.1
AAUGR44 South Wollo Jamma Yedo 7.0
AAUGR45 South Wollo Jamma Amuma 7.2
AAUGR46 South Wollo Jamma Shilafaf 7.1
AAUGR47 South Wollo Jamma Aejerti 6.9
AAUGR48 West shoa A/Gindeberet Berbabo 6.8
AAUGR49 West shoa A/Gindeberet Mendida 6.6
AAUGR50 West shoa A/Gindeberet Korkie 6.9
AAUGR51 West shoa A/Gindeberet Kersa 6.7
AAUGR52 West shoa A/Gindeberet Gitre 6.5
AAUGR53 West shoa A/Gindeberet Bekekelate 6.6
AAUGR54 West shoa A/Gindeberet Erjajo 6.7
AAUGR55 West shoa Jeldu Gojo 7.2
AAUGR56 West shoa Jeldu Tulubultuma 6.7
AAUGR57 West shoa Jeldu Sunko 6.8
AAUGR58 West shoa Jeldu Felo 6.7
AAUGR59 West shoa Jeldu Chilankie 6.8
AAUGR60 West shoa Dendi Golelo Bolo 6.7
AAUGR61 West shoa Dendi Awash Bolto 6.8
AAUGR62 West shoa Dendi Denbi 6.7
AAUGR63 West shoa Dendi Ubdo legabatu 6.8
AAUGR64 West shoa Dendi Jemiem legabaut 6.9
AAUGR65 West shoa Dendi Ababor 6.7
AAUGR66 West shoa Dendi Awaro kolo 6.8
AAUGR67 West shoa Dendi Tulu meda 6.9
AAUGR68 West shoa Dendi Chonkie 6.9

30
4. 3. Purification of isolates

Single and seemingly different colonies were picked from plates with sterile inoculating
loop and transferred into 6ml of sterilized YEM broth in test tubes. The test tubes were
vortexed and placed on rotary shaker for 48 hrs at room temperature. After two days, a
loop-full of culture suspensions from each test tube was taken and streaked on sterile
YEMA plates and incubated at 28 ± 20C for 3-5 days. The purity and uniformity of the
colony type was exhaustively tested through repeated re-streaking.

4. 4. Preservation of isolates

A single well isolated colony which was obtained after repeated purification was picked
and streaked to YEMA slant containing 0.3% (w / v) CaCO3 in a culture test tube and
incubated at 28 ± 20C for 3 days. After sufficient growth was observed, the culture slants
were then transferred to and preserved inside refrigerator adjusted at 40C (Vincent, 1970).

4. 5. Designation of the isolates

All the isolates were designated as AAUGR (Addis Ababa University Grass pea
Rhizobia) with different numbers representing each strain.

4. 6. Seed samples

The seeds of variety “Wasse” with gray seeds were provided by Debrezeit Agricultural
Research centre

31
4. 7. Authentication of the isolates and preliminary screening of their
symbiotic effectiveness on pouch experiment

To identify the definitive test of all the rhizobial isolates, they were then screened for
infectivity and effectivity using pouch. The pouches were washed, surfaced sterilized and
autoclaved as indicated in Somasegaren and Hoben (1994).

The grass pea variety called “Wasse” was used for the greenhouse experiment. Gray
undamaged seeds of uniform size were selected and surface sterilized and were rinsed in
five changes of sterile water and placed to 0.75 (w/v) water agar plates. They were
incubated at 28 ± 20C for 3 days. After three days, two germinated seeds were transferred
into each of pouch for germination.

After 3 days, each seedling was inoculated with 1ml of 72 hr YEMA grown culture. The
experiment was statistically laid out with three replications using a complete random
design in a greenhouse with a 12hr. photoperiod and an average of 280C and 150C day
and night temperature. Each block contained three pouches for a negative control devoid
of both the chemical N-fertilizer (KNO3) and the inoculum whereas, the other three
contained positive control treated with 70mg/l nitrogen( 0.05 KNO3 (w/v) )solution every
week. All the pouches were fertilized with quarter strength of Broughton and Dilworth N-
Free medium per week as described in Somasegaren and Hoben (1994) Table 3.

32
Table 3. N-Free nutrient solution (Brouthton and Dilworth, 1970)

Stock solution Chemical G / liter

1 Ca Cl2.2H2O 294.1
2 K H2 PO4 136.1
3 Fe C6 H5O7. 3H2O 6.7
Mg SO4. 7H2O 123.3
K2 SO4 87.0
Mn SO4. H2O 0.33
4 H3BO3 0.247
ZnSO4. 7H2O 0.288
CU SO4. 5H2O 0.100
CoSO4. 7H2O 0.056
Na2MoO2. 2H2O 0.048

Source: Adapted form Somasegaren and Hoben (1994)

4. 8. Relative effectiveness of the isolates

After sixty days of cultivation, the plants were uprooted to record nodule number, nodule
dry weight and shoot dry weight. The effectiveness of the isolates in accumulating plant
shoot dry matter was calculated as described in Somasegaren and Hoben (1984) and
Molungoy (2004):
%SE= Inoculated plant DM X 100
N – Fertilized plant DM
Where, DM = dry matter, N= nitrogen, SE= symbiotic effectiveness

The rate of nitrogen fixing effectiveness is evaluated as: Highly effective > 80%,
Effective 50-80%, Lowly effective 35-49% and Infective <35%.

33
4. 9. Characterization of isolates
4.9.1. Colony morphology

The morphological characteristics of the isolates were determined according to Lupwayi

and Haque (1994). A loopful of rhizobial isolates from 48 hrs old broth culture was
inoculated by streak plating onto YEMA and incubated at 28 ± 20C for 3-5days. After
5days, colony diameter and morphology, colony texture were recorded as large mucoid
(LM), large watery(LW), Elastic and buttery respectively as indicated in Martinez-
Romero et al., (1991).

4. 9.2. Acid-base production

To determine the ability of the rhizobial isolates to produce acid or alkaline in the
medium, YEMA containing bromthymol blue (BTB) (0.025 w/v) was used. A loopful of
the isolates from a 48 hrs old culture broth was streaked on the YEMA –BTB medium
and incubated for 3-7 days so as to record the color changes (Jordan, 1984).

4. 9.3. Determination of Growth rate

Each isolate was inoculated into a 10ml YEM broth (YEMB), vortex-dispersed and
-1
shaken on orbital shaker at 120 rev. min for 48 hrs at room temperature. Then, half ml
of each broth culture (cell suspensions) was inoculated into 50ml sterilized YEM broth in
125ml Erlenmeyer flask and kept on orbital shaker at a rev. of 120min-1. Turbidity was
measured by taking an optical density (OD540nm) reading of the YEM broth cultures
just at the time of inoculation (0hr) and every 6hrs interval by using spectrophotometry
(Jenway, 6405 UV/ VIS spectrophotometer) after calibrating it to zero with sterile
uninoculated YEM broth as a blank. Isolates were immediately taken, serially diluted (10-
1
– 10-10 with sterile distilled water). 0.1 ml sample from each solution was dispensed on

34
to sterilized YEMA plates and spread by using alcohol flamed hockey-like glass rod
spreader to determine the colony forming units (CFU) (Somasegaren and Hoben, 1994).
Mean generation (doubling) time was calculated from the logarithmic phase (White,
1995).

4. 10. Biochemical and physiological tests

For each biochemical and physiological test, an inoculation of a loopful of 48hrs old
broth culture was streaked on YEM medium. The inoculated YEMA plates were
incubated at 28 ± 20C for 3-5 days unless stated other wise (Somacegaren and Hoben,
1994). For each experiment, three replicates and controls were used per each test as
indicated in (Maatallah et al., 2002). Ultimately, the growth of each rhizobial isolate was
determined as (+) for slight growth, (++) for abundant growth and (-) for no growth.

4. 10.1. pH tolerance

The capacity of each rhizobial isolate to grow on acidic and alkaline media was
determined by inoculating each isolate on YEMA adjusted at a pH of 4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11.0, and 12.0 as described by Bernal and
Graham (2001).

4. 10.2. Salt tolerance

The ability of the isolates to grow at different level of salt concentrations was determined
by inoculating each isolate on the YEMA media containing 0.5%, 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 11%, 12% and 13% of NaCl as indicated in Bernal and Graham
(2001).

35
4. 10. 3. Temperature tolerance

The growth of each isolate at different incubation temperatures was evaluated by

inoculating each isolate on YEMA plates. The inoculated plates were incubated at a
temperature of 40C, 100C, 150C, 200C, 250C, 300C, 350C, 400C, 450C and 500C as
indicated in (Lupwayi and Haque, 1994).

4. 10.4. Intrinsic antibiotic resistance

The resistance of the isolates to intrinsic antibiotics was evaluated by streaking each
isolate on YEMA containing freshly prepared filter sterilized antibiotics using 0.22μm
sized membrane filters. The stock solution of each antibiotic was first prepared as
described in Lupwayi and Hagqe (1994). The antibiotics were Streptomycin,
Erythromycin, Ampicillin, Chloroamphinicol, and Nalidixic acid. Each antibiotic was
used at a concentration of 2.5 µg/ml, 5 µg/ml, 10 µg/ml. Streptomycin and erythromycin
were dissolved in ethanol whereas, the other three were dissolved in sterile water. The
stock solution of each antibiotic was prepared by dissolving 2 g of it in 100ml of water.
The required concentrations were then added to the media aseptically with single pipette
for each antibiotic. If the required concentration is 10 µgml,-1 0.05ml stock was added to
100ml YEMA.

4.10.5. Carbohydrate utilization

The carbohydrate requirement of the rhizobial isolates was evaluated on 16 carbon

sources: fructose, mannitol, sucrose, lactose and glucose that are heat resistant
carbohydrates whereas, sorbitol, arabinose, galactose, xylose, maltose, mannose,
cellulobiose, cellulose, starch, Na-citrate and Trehalose that are of heat liable carbon
sources (Somasegaren and Hoben, 1994).

36
One gram (1g) carbohydrate from each heat stable and heat liable was dissolved in 10ml
of distilled water and was kept in refrigerator. Basal media (carbohydrate free media
containing the following ingredients (g/l) was also prepared :
• K2 HPO4----------------------------1 g
• K H2PO4----------------------------1 g
• Fe Cl3.6H2O------------------------0.01 g
• MgSO4.7H2O----------------------0.2 g
• CaCl2--------------------------------0.1 g
• (NH4)2SO4--------------------------1 g
• Agar ---------------------------------15 g

The heat stable carbohydrate sources were autoclaved together with the basal media. The
heat liable carbon sources were first filter sterilized using sterile 0.22µm disposable
membrane filters. They were then added to the autoclaved and cooled basal media. 48 hrs
old rhizobial broth cultures were inoculated on to the basal media containing the different
carbon sources.

4. 10. 6. Amino acid utilization

Different types of amino acids which include L-alanine, L-arginine, L-asparagine, L-

glutamate, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-tryptophan and L-
tyrosine were used to determine the ability of the isolates to utilize amino acids. These
amino acids were added at a concentration of 0.5 g / l to a similar basal media used for
carbohydrate sources that lack ammonium sulfate and supplemented with 1g/ l of
mannitol. The membrane filter sterilized amino acids were added to the autoclaved and
cooled (approximately 550C) basal media as indicated in Amargar et al., (1997). 48hrs old
rhizobial suspensions were inoculated in to these basal media.

37
4. 11. Soil analysis

Soil sample for pot experiment was brought from Awash Bolto from where the nodules
were collected. Then, the soil was analyzed to determine its pH, total nitrogen, organic
carbon following the manual produced by Sahlemedhin Sertsu and Taye Bekele, (2002).

4. 12. Screening for symbiotic effectiveness of potted field soil

The effectiveness of seven (7) selected isolates (3 isolates from South Wollo and 4
isolates from West Shoa) was determined through pot experiment in a greenhouse. The
soil was properly mixed, sieved and air–dried. 3 Kg of this soil was distributed to plastic
pots. Grass pea “Wasse variety” was surface sterilized as before and rinsed in five
changes of distilled sterilized water. Five ungerminated seeds were sown in each pot and
later thinned down to three after germination for one week. After a week, each seedling
was inoculated with 1 ml of each isolate grown for 72 hrs YEM broth. The experiment
was set up in replicates under a greenhouse temperature of 19 ± 10C mean minimum and
30+/-10C mean maxmum. The pots were arranged in complete random design with each
block consisting of negative control (without N and uninoculated) and positive control
(uninoculated but with N). The nitrogen fertilizer (KNO3) was given at a concentration of
0.5 g / l per week until the plants were harvested. All the pots were watered every two
days.

4. 13. Plant total nitrogen content analysis

The nitrogen content of the plant samples was evaluated using the modified Kjeldhal
method (Bremer, 1965). The plant samples were grounded. At first 0.3 g of the ground
sample was measured in 100 ml digestion tube for the analysis. A 0.5 g of mixture (10 g
K2 SO4, 2 g CuSO4. 5H2O and 0.2 g Selenium) was added to the ground sample to be
used as a catalyst. Sulfuric and salicylic acid mixtures at an amount of 7 ml were mixed
to the ground sample and permitted to react for 30 minutes. 0.5 g of NO2S2O3.5 H2O was

38
also added and shaken to react for five minutes. A blank consisting of 0.5 g salt mixture
and 7 ml of sulfuric-salicylic mixture was also prepared. The digestion of the ground
sample was undertaken at a temperature of 3000C for 2hrs until the content turned to
colorless. Then after, the sample was distilled by adding 75 ml of 40% NaoH and its
nitrogen was collected with a flask containing 20 ml boric acid until the volume reached
110 ml. ultimately, the distillates become titrated using 0.1 N H2SO4 and the reading of
the burette was recorded and the percentage nitrogen was calculated by:

Nitrogen (%) = (V1 – V2) X N X 0.014 X 100

S

Where, V1 = ml of titrant used for the sample

V2 = ml of titrant used for the blank
N = Normality for the acid
S = weight of the plant material

4. 14. Numerical analysis

The isolates’s phenotypic variability was analyzed with a computer cluster analysis using
similarity coefficient. A denodrogram was constructed by the Unweighed Pair Group
Method with the Average (UPGMA) clustering method using NTSYS-Pc version 2.1.
The analysis was carried out on the basis of the parameters done during the physiological
tests (Tolerance of Temperature, pH, salt, antibiotic resistance and utilization of amino
acids and carbohydrates).

4. 15. Data analysis

Comparison between the treatments was done by one way analysis of variance (ANOVA)
(Turkey’s HSD test) using statistical program.

39
5. Result
5.1. Isolation and Authentication of rhizobia

A total of 68 Grass pea rhizobial bacteria were isolated from South Wollo (47
isolates) and West Shoa (21 isolates). In order to determine the capacity of all isolates
to form nodules, they were re-inoculated on the host plant on sterile pouch. As a
result, 63 out of 68 isolates formed nodules and were authenticated as root nodule
bacteria by forming nodules. The remaining five isolates that failed to nodulate and
authenticate were AAUGR (1, 5, 7, and 18) from South Wollo and AAUGR 65 from
West Shoa .

5. 2. Characteristics of the isolates

5. 2. 1. Morphological and cultural characteristics

The different isolates showed variations in colony texture and diameter (Table 4).
Accordingly, 22% of the isolates showed large watery colonies (LW) with
extracellular exopolysaccharide production whereas, 78% of the isolates displayed
large mucoid (LM) texture on YEMA media. The colony diameter of all the isolates
was between 2.0mm and 6.0mm. The largest colony diameter of 6.0 was displayed by
isolates AAUGR (35, 41and 47) from South Wollo and AAUGR (49 and 55) from
West Shoa whereas, the smallest diameter of 2.0mm was exhibited by isolates
AAUGR (21, 22, 29, 31, 34, and 37) from South Wollo and AAUGR(48, 54 & 66)
from West Shoa. The colony texture was distributed evenly among the sampling sites.
The number of isolates that showed large water (LW) was 5 and 3 from South Wollo
and West Shoa, respectively. Whereas, those that displayed large mucoid (LM) were
14 isolates from each sampling region.

40
Table 4 – Growth characterization of the isolates
Isolates Collection Colony (mm) Colony Mean generation
sites diameter morphology time (MGT)
AAUGR-4 SW 2.5 LM 1.34
AAUGR-9 SW 3.0 LM 1.36
AAUGR-19 SW 4.0 LM 2.03
AUGR-21 SW 2.0 LW 1.24
AAUGR-22 SW 2.0 LW 1.34
AAUGR-29 SW 2.0 LM 2.34
AAUGR-31 SW 2.0 LW 1.99
AAUGR-32 SW 5.0 LM 2.39
AAUGR-34 SW 2.0 LM 3.98
AAUGR-35 SW 6.0 LW 3.74
AAUGR-37 SW 2.0 LM 3.97
AAUGR-38 SW 3.0 LM 3.89
AAUGR-39 SW 4.0 LM 3.31
AAUGR-40 SW 5.0 LM 2.13
AAUGR-41 SW 6.0 LM 2.21
AAUGR-42 SW 4.5 LM 1.96
AAUGR-43 SW 3.0 LM 5.82
AAUGR-46 SW 4.0 LW 4.71
AAUGR-47 SW 6.0 LM 3.69
AAUGR-48 WS 2.0 LM 3.99
AAUGR-49 WS 6.0 LM 3.48
AAUGR-52 WS 5.5 LM 2.31
AAUGR-53 WS 4.0 LM 3.99
AUGR-54 WS 2.0 LM 3.85
AUGR-55 WS 6.0 LM 3.85
AUGR-56 WS 4.0 LM 3.82
AAUGR-57 WS 4.0 LM 3.41
AAUGR-58 WS 4.5 LM 3.11
AAUGR-59 WS 4.5 LM 2.33
AAUGR-60 WS 5.0 LM 2.57
AAUGR-61 WS 4.0 LM 3.97
AAUGR-63 WS 3.0 LW 2.99
AAUGR-64 WS 3.0 LW 6.12
AAUGR-66 WS 2.0 LW 3.91
AAUGR-67 WS 4.5 LM 3.49
AAUGR-68 WS 5.0 LM 3.41

WS= West Shoa, SW= South Wollo, LM= Large mucoid, LW= Large watery

41
5. 2. 2. Growth on YEMA-BTB and Mean generation time of the
isolates

All the isolates changed the YEMA-BTB medium into yellow colour (data not
shown). With regard to their growth rate, the isolates displayed different doubling
times that ranges from 1.24 – 6.12 hrs (Table 4). Six isolates (17% of the isolates)
that were all from South Wollo showed the fastest growth with doubling times less
than 2 hrs. These isolates were AAUGR 4, AAUGR 9, AAUGR21, AAUGR 22,
AAUGR 31 and AAUGR 42 with doubling time 1.34, 1.36, 1.24, 1.34, 1.99 and 1.96,
respectively. The doubling time of 75% of the isolates (27 isolates) was between 2.0 -
4.0 hrs. The remaining 8% (three) of the isolates exhibited slow growth with doubling
time of 4.71 – 6.12 hrs. Isolates AAUGR 43 and AAUGR 46 of South Wollo and
isolate AAUGR 64 of West Shoa showed slow growth with doubling time of 5.82,
4.71 and 6.12 hrs, respectively.

5. 3. Eco-physiological characters

The isolates showed diversity in physiological tolerance to pH, salt, temperature, IAR,
and utilization of carbohydrate and amino acid as substrates.

5. 3. 1. pH tolerance

All the isolates (100%) were found to tolerate pH 5.0 to pH 12 with abundant growth at
pH 6-10 (Table 5). Likewise, all but five isolates from South Wollo and four isolates
from West Shoa managed to grow on all the tested pH indicating that there are variations
amongst the isolates based on the site (soil) of isolation. Isolates AAUGR (31, 37, and
41) from South Wollo and isolates AAUGR (48,49, 52, 58, 59, 61, and 67) from West
Shoa showed abundant growth on all the tested pH values whereas, the isolates that were
found to be sensitive at pH 4 and pH 4.5 were isolates AAUGR (34, 35, 38, 43, 46, 54,
57, 64, and 66). In general, 75% of the isolates managed to grow on all pH media.

42
Table 5 Tolerance of rhizobial isolates to different pH
Isolates Site 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 11 12
AUGR-4 SW + + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-9 SW + + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-19 SW + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-21 SW + + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-22 SW + + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-29 SW + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-31 SW ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-32 SW + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-34 SW _ _ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-35 SW _ _ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-37 SW ++ ++ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-38 SW _ _ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-39 SW ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-40 SW ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-41 SW ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-42 SW + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-43 SW - - + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-46 SW - - + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-47 SW + + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-48 WS ++ ++ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-49 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-52 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-53 WS + + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-54 WS _ _ + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-55 WS + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-56 WS + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-57 WS _ _ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-58 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-59 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-60 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-61 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-63 WS + + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-64 WS _ _ + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-66 WS - - + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +
AUGR-67 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AUGR-68 WS + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
TOTAL 27 27 36 36 36 36 36 36 36 36 36 36 36 36 36
% 75 75 100 100 100 100 100 100 100 100 100 100 100 100 100

- = No growth, + = Slight growth, ++= abundant growth

SW = South Wollo , WS = West Shoa

43
5. 3. 2. Salt tolerance

The isolates displayed differences in tolerance to grow on YEMA adjusted at different

NaCl concentrations (Table 6). All the isolates (100%) grew at a salt concentration of
0.5%. There is a progressive decrease in tolerance ranging from 1%-13%. However, the
number of isolates that managed to grow at NaCl concentration of 11, 12 and 13%
displayed up to 25%, 17% and 11% of tolerance, respectively. The most tolerant isolates
that were found to grow on all the tested salt media were AAUGR 37 from South Wollo
and AAUGR 48, AAUGR 59, and AAUGR 61 from West Shoa. On the other hand,
isolates AAUGR 9, AAUGR 22, AAUGR 32, AAUGR 43, AAUGR 46 and AAUGR 47
(South Wollo) and AAUGR 63, AAUGR 64, AAUGR 66, and AAUGR 68 from West
Shoa were found to be within a narrow range of salt tolerance.

44
Table 6. Tolerance of rhizobia nodulating grass pea at different level of salt concentrations
Isolates Site 0.5 1% 2 3% 4% 5% 6% 7% 8% 9% 10 11% 12 13%
% % % %
AAUGR-4 SW + ++ ++ + + + + - - - - - - -
AAUGR-9 SW + - - - - - - - - - - - - -
AAUGR-19 SW + + + + + + + + + + + + - -
AAUGR-21 SW + + + + + + + + - - - - -
AAUGR-22 SW + + + - - - - - - - - - - -
AAUGR-29 SW ++ ++ ++ + + + + + - - - - - -
AAUGR-31 SW + + + + + + + + + + - - - -
AAUGR-32 SW ++ ++ ++ + + + + + - - - - - -
AAUGR-34 SW ++ ++ ++ ++ ++ ++ ++ + + + + + + -
AAUGR-35 SW + + + + + + + - - - - - - -
AAUGR-37 SW ++ ++ ++ + + + + + + + + + + +
AAUGR-38 SW ++ ++ ++ ++ ++ + + + + + + - - -
AAUGR-39 SW + + + + + + + - - - - - - -
AAUGR-40 SW ++ ++ ++ + + + + + + + - - - -
AAUGR-41 SW ++ ++ ++ ++ ++ ++ ++ + + - - - - -
AAUGR-42 SW + + + + + + + - - - - - - -
AAUGR-43 SW + + + - - - - - - - - - - -
AAUGR-46 SW + - - - - - - - - - - - - -
AAUGR-47 SW + + - - - - - - - - - - - -
AAUGR-48 WS ++ ++ ++ + + + + + + + + + + +
AAUGR-49 WS ++ ++ ++ ++ ++ ++ ++ ++ - - - - - -
AAUGR-52 WS ++ ++ ++ ++ ++ + + + - - - - -
AAUGR-53 WS + + + + + + + + + + + - - -
AAUGR-54 WS + + + + + + + + + + + + - -
AAUGR-55 WS ++ ++ ++ + + + + + + + - - - -
AAUGR-56 WS + + + + + + + + + + - - - -
AAUGR-57 WS ++ ++ ++ ++ ++ + + + + + + - - -
AAUGR-58 WS ++ ++ ++ ++ ++ ++ ++ + + + + + - -
AAUGR-59 WS ++ ++ ++ ++ ++ ++ ++ + + + + + + +
AAUGR-60 WS + + + + + + + + + + + - - -
AAUGR-61 WS ++ ++ ++ + + + + + + + + + + +
AAUGR-63 WS + + + - - - - - - - - - - -
AAUGR-64 WS + + + - - - - - - - - - - -
AAUGR-66 WS + + + - - - - - - - - - - -
AAUGR67 WS ++ ++ ++ ++ ++ ++ ++ + + + + + + -
AAUGR-68 WS ++ + + + + + + + - - - - - -
TOTAL 36 33 31 26 26 26 25 22 18 15 13 9 6 4
% 100 92 86 72 72 72 69 61 50 42 36 25 17 11

- = No growth, + = Slight growth, ++= abundant growth , SW = South Wollo , WS = West Shoa

45
5. 3. 3. Temperature tolerance

The ability of the isolates to tolerate different incubation temperatures is shown in Table
7. All the tested isolates (100%) grew at temperatures between 15OC and 35OC. Likewise,
72%, 53% and 28% of the isolates were found to tolerate 40OC, 45 OC and 50 OC,
respectively. Whereas, 53% and 61% of the isolates were found to grow at 5 OC and 10
O
C, respectively. The isolates that tolerated all the tested range of temperature (5-50 OC)
include AAUGR19, 31, 35, 37 and 41 (South Wollo) and AAUGR 48, 55, 59, 61, and 67
from West Shoa. On the other hand, isolates that grew within narrow range of
temperature (15-45) were AAUGR 9, 43, 46, and 47 (South Wollo) and AAUGR 53, 63,
64, and 66 (West Shoa).

46
Table 7. Tolerance of grass pea rhizobia to different levels of temperature

Isolates Site 5 10 15 20 25 30 35 40 45 50O

O O O O O O O O O
C C C C C C C C C C
AUGR – 4 SW + + + + + + + + - -
AUGR – 9 SW - - + + + + + + - -
AUGR –19 SW + + + + + + + + + +
AUGR –21 SW + + + + + + + - - -
AUGR –22 SW + + + + + + + - - -
AUGR –29 SW + + + + + + + - - -
AUGR –31 SW + + + + + + + + + +
AUGR –32 SW + + + + + + + - - -
AUGR –34 SW - - ++ ++ ++ ++ ++ ++ + -
AUGR –35 SW + + + + + + + + + +
AUGR -37 SW + + ++ ++ ++ ++ ++ ++ + +
AUGR –38 SW + ++ ++ ++ ++ ++ ++ ++ + -
AUGR –39 SW - + + + + + + + + -
AUGR –40 SW - + ++ ++ ++ ++ ++ - - -
AUGR -41 SW + + ++ ++ ++ ++ ++ + + +
AUGR –42 SW + ++ ++ ++ ++ ++ ++ + + -
AUGR –43 SW - - + + + + + + - -
AUGR –46 SW - - + + + + + + - -
AUGR –47 SW - - + + + + + + - -
AUGR –48 WS + + + + + + + + + +
AUGR –49 WS ++ ++ ++ ++ ++ ++ ++ - - -
AUGR –52 WS ++ ++ ++ ++ ++ ++ ++ ++ + -
AUGR –53 WS - - + + + + + + - -
AUGR –54 WS + + + + + + + + + -
AUGR –55 WS ++ ++ ++ ++ ++ ++ ++ + + +
AUGR –56 WS + + + + + + + + + -
AUGR –57 WS - - + + + + + + + -
AUGR –58 WS - ++ ++ ++ ++ ++ ++ + + -
AUGR –59 WS + ++ ++ ++ ++ ++ ++ ++ + +
AUGR –60 WS - + + + + + + + + -
AUGR –61 WS + + + + + + + + + +
AUGR –63 WS - - + + + + + - - -
AUGR –64 WS - - + + + + + - - -
AUGR –66 WS - - + + + + + - - -
AUGR –67 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ +
AUGR –68 WS - - + + + + + + + -
Total 19 22 36 36 36 36 36 26 19 10
% 53 61 100 100 100 100 100 72 53 28

- = No growth, + = Slight growth, ++= abundant, SW = South Wollo, WS = West Shoa

47
5. 3. 4. Intrinsic antibiotic resistance

The isolates exhibited a wide range of resistance to different concentrations of different

antibiotics (Table 8). All the isolates tolerated and grew at the lowest concentrations (2.5
μg ml-1) in ampicillin, chloroamphenicol and Nalidixic acid. Three isolates from South
Wollo (AAUGR 31, AAUGR 37 and AAUGR 42) and ten isolates form West Shoa
AAUGR (48, 49, 55, 56, 57, 58, 59, 60, 61 and 67) were found to tolerate all the tested
antibiotics at different concentrations. The most sensitive isolates that grew within a
narrow range of antibiotic resistance include AAUGR (9, 21, 53, 63, 64 and 66). In
general, 36 % of the isolates tolerated all the tested antibiotics at different concentrations
whereas, 64 % showed fluctuation in range of tolerance. The antibiotic that showed an
inhibitory effect against the rhizobial isolates was erythromycin at 10.0 μg ml-1 .

48
Table 8. Antibiotic resistance of isolates after 5-7 days of incubation
Ampicillin Chloro- Erythro- Nali- Strepto-
amphenicol mycin dixic acid mycin
Isolates Site 2.5 5 10 2.5 5 10 2.5 5 10 2.5 5 10 2.5 5 10
AAUGR–4 SW + + - ++ + - + + + + + - ++ ++ ++
+
AAUGR-9 SW ++ ++ + + + + - - - + - - + + -

AAUGR-19 SW + + - + + + + + + + + + - - -
AAUGR 21 SW + - - + - - + + - + + - - - -
AAUGR 22 SW ++ + - + - - + + + + - - ++ ++ ++
AAUGR 29 SW ++ ++ + + + + + + + + + - + + -
AAUGR 31 SW + + + + + + + + + + + + + + +
AAUGR 32 SW + + + ++ + + + - - ++ ++ + + - -
+
AAUGR–34 SW + - - + + + - - - + + + ++ ++ +
AAUGR–35 SW + + + + + + + + + + + - + - -
AAUGR-37 SW ++ ++ + + + + + + + + + + + + +
AAUGR–38 SW ++ ++ + + + + + - - ++ ++ + + + +
AAUGR–39 SW ++ ++ + + + + + - - + + + + + +
AAUGR–40 SW ++ ++ + + + + + + + ++ + - ++ ++ +
AAUGR-41 SW ++ + + ++ + + + ++ ++ + - - + + +
+
AAUGR–42 SW ++ + + ++ + + + ++ ++ ++ ++ + + + +
+
AAUGR–43 SW + - - + + - + + + + + + - - -
AAUGR–46 SW + + + + - - + + - + + + - - -
AAUGR–47 SW ++ + + + + + + - - + + - + + +
AAUGR–48 WS ++ + + + + + + + + + + + + + +
AAUGR–49 WS ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
+
AAUGR–52 WS ++ ++ ++ ++ + ++ ++ + - + + + + + +
+
AAUGR–53 WS + - - ++ + - - - - + + + ++ + -
AAUGR–54 WS ++ + + ++ + ++ ++ ++ - + + + + + +
+
AAUGR–55 WS + + + ++ + ++ ++ ++ ++ + + + + + +
+
AAUGR–56 WS + + + + + + ++ ++ ++ + + + + + +
AAUGR–57 WS ++ ++ ++ + + + + + + + + + + + +
AAUGR–58 WS ++ ++ ++ + + + + + + + + + + + +
AAUGR–59 WS ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
+
AAUGR–60 WS ++ ++ ++ ++ + ++ ++ + + + + + + + +
+
AAUGR–61 WS ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
+
AAUGR–63 WS + + ++ + - - - - - + + - + - -
AAUGR–64 WS + + + + - - - - - + + + - - -
AAUGR–66 WS + + - + - - - - - + - - + + -
AAUGR–67 WS + + + ++ + ++ ++ ++ + + + + + + +
+
AAUGR–68 WS ++ + + ++ + ++ ++ + - + + + + + +
+
Total 36 32 28 36 3 26 30 26 21 36 32 25 30 26 22
0
% 100 89 78 100 8 72 83 72 58 100 89 69 83 72 61
3
- = No growth, + = Slight growth, ++= abundant growth, SW= South Wollo, WS= West Shoa

49
5. 3. 5. Utilization of carbon sources

All of the isolates were able to catabolize a large variety of carbon sources (Table 9). All the
isolates (100%) were found to catabolize glucose, fructose, maltose, mannose, galactose, xylose,
sucrose and mannitol. However, lactose, sorbitol, cellulobiose, arabinose, Na-citrate, trehalose,
starch and cellulose were utilized by 94, 92, 89, 78, 69, 69, 69, and 67%, respectively. Isolates
AAUGR 4, AAUGR 21, AAUGR 29, of South Wollo failed to grow on sorbitol whereas; all the
rest grew very well. Fifteen isolates (42% of the isolates) were found to catabolize and grew on
the basal medium containing all the 16 tested carbon sources while isolates AAUGR37, AAUGR
39, and AAUGR 40 of South Wollo and isolates AAUGR48, AAUGR 59, AAUGR 61 and
AAUGR 67 of West Shoa showed abundant growth on all tested carbon sources. On the other
hand, the isolates AAUGR 4 of South Wollo and AAUGR 49, AAUGR 52 and AAUGR 56 of
West Shoa utilized less number of carbon sources (69%) out of the tested carbon sources.

50
Table 9. Grass pea rhizobial utilization to different carbohydrate sources

Isolates

cellulose
Ffuctose

Lactose

Xylose

Manitol

starch

Na-citrate
Site

Trehalose
Glucose

Sorbitol
Arabinose

Maltose

Mannose

Galactose

Sucrose

Cellulobiose
AAUGR–4 SW ++ + + - + + + - + + + + + - - -
AAUGR-9 SW + + + ++ ++ ++ ++ ++ + + - + ++ + + +
AAUGR-19 SW + ++ ++ ++ + ++ + ++ + + + ++ - - - +
AAUGR 21 SW + + + - + + + + + + + + + + + +
AAUGR 22 SW ++ - + + + + + + + + ++ + + ++ ++ +
AAUGR 29 SW + + + - + + + + + + ++ ++ ++ - - -
AAUGR 31 SW + ++ ++ ++ + ++ + ++ + + ++ ++ + + + +
AAUGR 32 SW + + ++ ++ + + + + + + - + + ++ - -
AAUGR–34 SW ++ ++ ++ + + ++ + + + + ++ ++ ++ - + +
AAUGR–35 SW ++ ++ ++ ++ + + + + + + + + - + + +
AAUGR-37 SW ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AAUGR–38 SW ++ + + ++ + + + ++ + + ++ ++ - - + -
AAUGR–39 SW ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AAUGR–40 SW ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AAUGR-41 SW ++ + ++ + + + + + + + + + + + + +
AAUGR–42 SW ++ - ++ + + + + + + ++ ++ ++ - - + -
AAUGR–43 SW + ++ ++ + + + + ++ + + + + - + + +
AAUGR–46 SW + ++ + ++ ++ ++ ++ ++ + + ++ ++ + ++ + ++
AAUGR–47 SW + ++ + + + + ++ ++ + + ++ ++ ++ ++ ++ ++
AAUGR–48 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++
AAUGR–49 WS ++ - + ++ + + + + + + ++ ++ - - - -
AAUGR–52 WS ++ - + ++ + + + + + + ++ ++ - - - -
AAUGR–53 WS + + + + + + + + + + + + + + + +
AAUGR–54 WS + + + + + + + + + + + + - - - -
AAUGR–55 WS ++ - ++ + + ++ + ++ ++ + ++ + - - - +
AAUGR–56 WS + - + + + + + + + + + + - - - -
AAUGR–57 WS + + ++ + + ++ + ++ + + ++ + + ++ + +
AAUGR–58 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ - + - +
AAUGR–59 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AAUGR–60 WS ++ ++ ++ ++ ++ ++ + + + + + + + + + +
AAUGR–61 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AAUGR–63 WS + + + + + + + + + + - + + + + +
AAUGR–64 WS + + + + + + + + + + - + + + + +
AAUGR–66 WS + + + + + + + - + + + + + + + +
AAUGR–67 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AAUGR–68 WS ++ + ++ ++ ++ ++ + ++ ++ ++ + ++ ++ ++ ++ ++
Total 36 28 36 33 36 36 36 34 36 36 32 36 25 25 24 25
% 100 78 100 92 100 100 100 94 100 100 89 100 69 69 67 69

- = No growth, + = Slight growth, ++= abundant growth, SW= South Wollo, WS= West Shoa

51
5. 3 6. Amino acid utilization

Almost all of the isolates were able to catabolize a large variety of nitrogen sources
(Table 10). All the isolates (100%) utilized tyrosine, methionine, arginine and asparagine
likewise, 92, 94, 94, 89, and 83% of the rhizobial isolates catabolized alanine, lysine,
glutamate, leucine, tryptophan, respectively. Only 78% of the isolates utilized
phenylalanine which was found to be the most resistant amino acids.

Fifty percent (50 %) of the isolates that include AAUGR (9, 29, 31, 32, 34, 37, 39, 40 and
41) of South Wollo and isolates AAUGR (48, 49, 57, 58, 59, 61, 64, 67 and 68) from
West Shoa utilized 100% of the tested amino acids whereas, isolates AAUGR 42 of
South Wollo and AAUG 66 of West Shoa were found to be much fastidious with the
ability of catabolizing 70% of the tested amino acids.

52
Table 10. Utilization of grass pea rhizobia on different nitrogen sources
Site
Alan- Argi- Aspara- Gluta Leu- Lys- Methio- Phenyl- Trypto- Tyro-
Isolates ine nine gine mate cine ine nine alanine phane sine
AAUGR–4 SW +
+ + + + + + - + +
AAUGR-9 SW + ++ ++ ++ ++ ++ ++ ++ ++ ++
AAUGR-19 SW + + + + + + + - + +
AAUGR 21 SW - + + + + + + + + +
AAUGR 22 SW ++ ++ ++ ++ ++ - ++ ++ ++ ++
AAUGR 29 SW + + + + + + + + + +
AAUGR 31 SW ++ ++ ++ + ++ ++ ++ ++ ++ ++
AAUGR 32 SW + + + + + + + + + +
AAUGR–34 SW ++ ++ ++ + + + + ++ ++ ++
AAUGR–35 SW + + + + + ++ + - + +
AAUGR-37 SW + ++ ++ + + + ++ + ++ ++
AAUGR–38 SW + + + + - + + - + +
AAUGR–39 SW ++ ++ ++ ++ ++ ++ ++ + ++ ++
AAUGR–40 SW ++ ++ ++ ++ ++ ++ ++ + ++ ++
AAUGR-41 SW + + + ++ + + + + + +
AAUGR–42 SW - + + ++ - - + + + +
AAUGR–43 SW + + + - + + + + + +
AAUGR–46 SW ++ ++ ++ - ++ ++ ++ ++ ++ ++
AAUGR–47 SW ++ ++ ++ + ++ + + + - +
AAUGR–48 WS ++ ++ ++ + ++ + + + ++ ++
AAUGR–49 WS + + + + + + + + + +
AAUGR–52 WS + + + + + + + - - +
AAUGR–53 WS ++ ++ ++ + - + + - + +
AAUGR–54 WS
+ + + ++ + + + + - +
AAUGR–55 WS + + + + + + + + - +
AAUGR–56 WS + + + + + + + + - +
AAUGR–57 WS ++ ++ ++ + ++ + + + + ++
AAUGR–58 WS ++ ++ ++ + ++ + + + + ++
AAUGR–59 WS ++ ++ ++ + ++ + + + + ++
AAUGR–60 WS ++ + + + + + + - + ++
AAUGR–61 WS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
AAUGR–63 WS - + + + + + + + ++ ++
AAUGR–64 WS ++ ++ ++ + ++ ++ + + ++ ++
AAUGR–66 WS + + + + - + + - - +
AAUGR–67 WS + + + ++ + + + + + +
AAUGR–68 WS + + + + + ++ + + + +
Total 33 36 36 34 32 34 36 28 30 36
% 92 100 100 94 89 94 100 78 83 100

- = No growth, + = Slight growth, ++= abundant growth, SW= South Wollo, West Shoa

53
In all the eco-physiological tests undertaken isolates AAUGR 37, 48, 59, 61 and 67 were
found to be the most tolerant strains whereas, isolates AAUGR 43, 63, 64, and 66 were
also found to be the most sensitive. Among the most tolerant and sensitive isolates,
isolates AAUGR 37 and 43 were from the South Wollo whereas, all the rest were from
West Shoa (Table 11).

Table 11. Summary of the most resistant and sensitive isolates to physiological
characters

NaCl% Temp. Carbohydr- Amino acid

ate
Site pH ( 0.5-13) 5-50 0C IAR utilization Utilization. utilization
4,9,19,21,
22,29,31,
32,36,37,
38,39,40, 9,29,32,34,
SW 41,42,47 34,37, 19,31,37, 37,42 37,39,40 37,39,40
48,49,52,
Most 53,55,56, 48,55,58,59, 48,49,57,
tolerant 58,59,60, 61,67 48,55,59, 48,49,55,56,57, 48,59, 58,59,61,
isolates WS 61 ,67,68 61,67 58,59,60,61,67 61,67 64,67,68
SW 34, 35, 9,22,31,
Most 38,43,46 43,46 43,46,47 9,21 43 42
sensitive WS 54,57, 49, 52,
isolates 64,66 63,64,66,68 63,64,66 53,63,64,66 52, 56 66

5. 4. Numerical analysis

The numerical analysis based on 76 phenotypic features allowed the grouping of all
isolates in to four similarity groups plus one ungrouped at 50% relative similarity
coefficient (Fig.3). Cluster I include 7 isolates AAUGR (4, 19, 29, 32, 35, 54, and 56)
with two separated sub- clusters in which they were separated at 80% level of relative
similarity. Cluster II again consist of 7 isolates AAUGR (38, 41, 42, 49, 52, 55 and 67)
that displayed two sub–clusters at 70% level of relative similarity. Cluster III which is the
biggest cluster encompassed 13 isolates AAUGR (9, 22, 31, 37, 39, 40, 47, 48, 57, 58,
60, and 61) that were separated further in to two sub-clusters at 60 % level of similarity.
Cluster IV comprised of 6 isolates AAUGR (21, 43, 53, 63, 64, and 66) that were further

54
 divided in to two sub-clusters at 64% level of similarity. Isolate AAUGR 68 had been left
out of the clusters at 50% level of similarity (Fig.3).

II

III

IV

Fig.3. Dendrogram highlighting the phenotypic diversity of grass pea rhizobial

isolates

5. 5. Soil analysis

The soil brought from Awash Bolto was analyzed for its pH, available phosphorous, total
nitrogen and organic carbon (Table 12). The soil was almost neutral with lowest N (0.09)
and relatively high P (12.88).

55
Table 12. Physical and chemical analysis of selected soils

parameters  Value 
pH  7.1
Total nitrogen (%)  0.09
Organic carbon (%)  0.87
Available phosphorous 
(ppm)  12.88

5. 6. Symbiotic effectiveness test on pouch culture for preliminary

screening

The pouch culture study on nodulation and symbiotic effectiveness of isolates showed
marked variations among the isolates in nodule number, nodule dry weight and shoot dry
weight at P=0.05(Table 13).

56
Table 13. Symbiotic effectiveness of isolates of grass pea rhizobia from different sampling sites
Isolates Nodule number per Nodule dry Wt. per Shoot dry Wt. % SE Effectiveness
plant plant(g) per plant (g)
AAUGR 4 35 ± 22ce 0.054 ± 0.005a-g 0.173 ± 0.047 a-d 68 E
AAUGR 9 24 ± 5c-e 0.047 ± 0.009 a-g 0.131 ± 0.089 b-d 52 E
AAUGR 19 36 ± 10c-e 0.040 ± 0.014b-g 0.144 ± 0.144b-d 57 E
AAUGR 21 38 ± 27c-e 0.035 ± 0.022c-g 0.133 ± 0.112b-d 52 E
AAUGR 22 33 ± 13c-e 0.027 ± 0.002 d-g 0.128 ± 0.047b-d 50 E
AAUGR 29 28 ± 6c-e 0.029 ± 0.016 d-g 0.145 ± 0.026b-d 57 E
AAUGR 31 90±22a-e 0.062 ±0.007a-f 0.266 ± 0.055 a-d 108 HE
AAUGR 32 59 ± 7a-e 0.047 ± 0.010 a-g 0.160 ± 0.006b-d 63 E
AAUGR 34 49 ± 10a-e 0.046± 0.007a-g 0.181± 0.095a-d 71 E
AAUGR 35 44 ± 8b-e 0.054 ±0.018a-g 0.152± 0.024b-d 60 E
AAUGR 37 135±57a 0.086±0.012a 0.296± 0.034a 120 HE
AAUGR 38 67±6a-e 0.036±0.003c-g 0.164± 0.042b-d 65 E
AAUGR 39 42 ± 25b-e 0.038±0.012b-g 0.143±0.126b-d 56 E
AAUGR 40 75 ± 18a-e 0.061 ± 0.012 a-g 0.169 ± 0.047a-d 67 E
AAUGR 41 83±27a-e 0.066 ± 0.018 a-f 0.264 ± 0.018a-d 104 HE
AAUGR 42 73±6a-e 0.059 ± 0.006 a-g 0.151 ± 0.077b-d 59 E
AAUGR 43 66 ± 6a-e 0.043 ± 0.016 a-g 0.160 ± 0.066b-d 63 E
AAUGR 46 62 ± 23a-e 0.065 ± 0.005 a-g 0.127 ± 0.064b-d 50 E
AAUGR 47 78 ± 23a-e 0.070 ± 0.017 a-d 0.216 ± 0.040a-d 85 HE
AAUGR 48 130±55ab 0.082±0.013ab 0.287 ± 0.062ab 116 HE
AAUGR 49 53 ± 6a-e 0.043 ± 0.005 a-g 0.215 ± 0.105a-d 85 HE
AAUGR 52 73 ± 8a-e 0.058 ± 0.005 a-g 0.220 ± 0.066a-d 87 HE
AAUGR 53 55 ± 37a-e 0.036 ± 0.012 c-g 0.198 ± 0.116a-d 78 E
AAUGR 54 26 ± 20c-e 0.025 ± 0.011 e-g 0.135 ± 0.074b-d 53 E
AAUGR 55 65 ± 28a-e 0.062 ± 0.012 a-f 0.167 ± 0.075a-d 66 E
AAUGR 56 48 ± 11a-e 0.041 ± 0.010 a-g 0.149 ± 0.073b-d 59 E
AAUGR 57 48 ± 4a-e 0.048 ± 0.008 a-g 0.168 ± 0.055a-d 66 E
AAUGR 58 68 ± 25a-e 0.035±0.028a-g 0.158 ± 0.033b-d 62 E
AAUGR 59 116±59a-c 0.075±0.013a-c 0.275 ± 0.047a-c 112 HE
AAUGR 60 57±16a-e 0.051 ± 0.001 a-g 0.249 ± 0.027a-d 98 HE
AAUGR 61 111±82a-d 0.073 ±0.011a-c 0.267 ± 0.048a-d 108 HE
AAUGR 63 73 ± 11a-e 0.059 ± 0.013 a-g 0.153 ± 0.067b-d 60 E
AAUGR 64 45 ± 12a-e 0.055 ± 0.003 a-g 0.178 ± 0.031a-d 70 E
AAUGR 66 48 ± 32a-e 0.069 ± 0.005 a-e 0.259 ± 0.176a-d 104 HE
AAUGR 67 108±46a-e 0.064 ± 0.005a-f 0.269 ± 0.023a-c 108 HE
AAUGR 68 65 ± 40a-e 0.053 ± 0.001 a-g 0.152 ± 0.111b-d 60 E
- ve control - - 0.082 ± 0.010d 32
+ ve control - - 0.254 ± 0.013a-d 100
Note: Levels not followed by the same letter/letters are significant at p=0.05 (Tukey’s HSD test)

57
The physical appearances of inoculated plants were clearly differentiated from the
negative control. The control plants appeared shorter whereas, the inoculated plants
showed deeper green leaves and long shoots with many branches. The inoculated plants
also had pink colored nodules (data not shown).

With regard to the number of nodule of the tested plants, the highest nodule number of
135/plant was recorded from plants inoculated with isolate AAAUGR 37 followed by
130 nodules/plant displayed by isolate AAUGR 48 (Table 13). Likewise, the smallest
nodule number was displayed by AAUGR 8 with 9 nodules/ plant followed by AAUGR
15 with16 nodules/plant. Regarding nodule dry weight, the highest nodule dry weight
(86mg/plant) was recorded from plants inoculated with isolate AAUGR 37 whereas, the
least record (11mg/plant) was from isolate AAUGR 6.

The data also showed variations amongst the isolates with regard to shoot dry matter
accumulation. The highest shoot dry matter accumulation of 296mg/plant was recorded
from the plants inoculated with isolates AAUGR 37 followed by isolates AAUGR (41,
31, 61, 67, 59 and 48) that induced nodulation on the host with shoot dry weight ranging
from 264mg/plant to 287mg/plant. The least effective isolates were isolates AAUGR (6,
8, 16, 25, 26 and 45) that were found to accumulate 66-76mg/plant in shoot dry weight
that were comparable to the uninoculated negative control (82mg/plant). Based on the
relative plant dry matter accumulation of the inoculated plants with uninoculated and
nitrogen fertilized control (positive control), 18% (12 isolates) were highly effective (85
– 120 effectiveness), 43 % (27 isolates) were effective (50-78% effectiveness) and 29%
(18 isolates) were lowly effective (35-49% effectiveness) whereas, 6 isolates (10%) were
ineffective (26-33 effectiveness). In general, 61% of the isolates were found to be
effective compared to the 39% of the isolates that were lowly effective and ineffective.

In this study, five isolates (8%) that include AAUGR (6, 8, 16, 25, 26 and 45) that were
all from South Wollo were much more ineffective than the negative control. On the other
hand, 8 isolates (13%) that include AAUGR(31, 37, and 41) (South Wollo) and isolates

58
AAUGR (48, 59, 61, 66, and 67) from West Shoa displayed much more effectiveness as
compared to uninoculated and nitrogen fertilized control. The data also showed a pattern
of effectiveness on the basis of site of collection. Accordingly, 95% of the isolates from
West Shoa and 47% of the isolates from South Wollo induced effective nodulation and
nitrogen fixation and shoot dry weight accumulation (Table 14). Most of the effective
isolates were recorded from West Shoa.

Table 14. Rate of effectiveness of isolates by sampling regions

Sampling HE E
regions %HE %E Total %LE %IE Total isolates isolates
31,37,41,47 2,4,9,19,21,22,29,32,34,
SW 10 37 47 17 6 23 35,38,39,40,42,43,46
48,49,52,59, 51,53,54,55,56,57,58,
WS 40 55 95 5 _ 5 60,61,66,67 62,63,64,,68

5. 7. Symbiotic effectiveness of selected isolates on potted soil

After pouch culture experiment, based on the shoot dry matter, the top four isolates from
West Shoa and three isolates form South Wollo were selected based on their performance
among the isolates. These include AAUGR 31, AAUGR 37, and AAUGR 41 of South
Wollo and AAUGR 48, AAUGR 59, AAUGR 61 and AAUGR 67 from West Shoa.
Grass pea, “Wasse” variety grew on potted neutral (7.1) Awash Bolto soil.

The in vivo symbiotic test of isolates on soil culture showed differences in nodule
number, and weight, shoot dry weight and nitrogen fixation (Table 15). The highest
nodule number was recorded by plants inoculated with isolate AAUGR 37 (165
nodule/plant) followed by isolate AAUGR 48 (147 nodules/plant) whereas, the least
number of nodule (93 nodule/plant and 97 nodule/plant) was recorded from isolates
AAUGR 41 and 61, respectively. There was a significant difference in nodule number
amongst the isolates. Regarding nodule dry weight, the isolates AAUGR 37 and AAUGR
48 showed the highest nodule mass of 1.0g/plant and 0.98g/plant that were significantly

59
different from the nodule dry weight recorded by the other isolates. The least nodule
mass of 0.70g/plant was displayed by isolate AAUGR 61.

The highest shoot dry matter accumulation of the rhizobia treated plants was recorded
from the inoculation of plants with isolate AAUGR 37 followed by isolates 48, 59 and
67 with shoot dry mass of 1.95g/plant, 1.79g/plant, 1.67g/plant, and 1.67g/plant,
respectively. The least shoot dry weight of 1.47g/plant was recorded from isolate 41 that
was significantly different from the uninoculated negative control (0.93g/plant). The
shoot dry matter accumulation by the selected isolates on pot trial was found to be much
higher than pouch culture (Table 16). The nitrogen content of all the tested isolates was
also within the range of 2.6-3.9 with one another (Table 15). The highest nitrogen content
of 3.9% was recorded by isolate AAUGR 37 whereas, the least (2.6%) was displayed by
isolate AAUGR 41. The nitrogen content of the inoculated plants was found to be twice
more than the nitrogen content of the uninoculated negative control (1.2%).

Table.15. Symbiotic effectiveness of selected isolates of grass pea rhizobia on potted

soil
Isolate Nodule number Nodule dry Shoot dry weight Total nitrogen
per plant weight per per plant(g) per plant (%)
plant(g)
AAUGR 31 117± 1d 0.073 ± 0.3d 1.51 ± 0.2e 2.8 ± 0.1f
AAUGR 37 165± 1a 1.011 ± 0.9a 1.95± 0.6a 3.9 ± 0.3a
AAUGR 41 93± 1f 0.071 ± 0.4de 1.47 ± 0.3g 2.6 ± 0.8g
AAUGR 48 147± 1b 0.098 ± 0.8b 1.79 ± 0.5b 3.7 ± 0.5b
AAUGR 59 135± 1c 0.081± 0.7c 1.67 ± 0.4 c 3.3 ± 0.2d
AAUGR 61 97± 1e 0.070 ± 0.4e 1.50 ± 0.7f 3.2 ± 0.6d
AAUGR 67 133± 1c 0.079 ± 0.6c 1.67 ± 0.7d 3.5 ± 0.7c
- ve control 19 ± 3g 0.007 ± 0.1f 0.93 ± 0.8i 1.2 ± 0.9h
+ ve control 17 ± 1g 0.004 ± 0.2g 1.31 ± 0.1h 3.1 ± 0.4e

Note: Levels not followed by the same letter/letters are significant at p=0.05 (Tukey’s HSD test)

60
The in vivo test that was undertaken in the greenhouse both on pouch and soil culture
showed significant differences in nodule number, nodule dry weight and shoot dry
weight. The comparative percentage of effectiveness between soil and pouch was 82-
85%. Accordingly, the isolates accumulated much shoot dry weight on soil culture than
on pouch culture (Table 16).

Table 16. Shoot dry weight and %SE of selected isolates on both pouch and pot
cultures

On pouch culture On potted soil Comparative

%
effectiveness
Shoot Shoot dry between soil
Isolates dry wt./p SE (%) wt./plant SE (%) /pouch
AAUGR31 108 1.51 115 82
0.27
AAUGR37 0.3 120 1.95 149 85
AAUGR41 0.26 104 1.47 112 82
AAUGR48 0.29 116 1.79 137 84
AAUGR59 0.28 112 1.67 127 83
AAUGR61 0.27 108 1.51 115 82
AAUGR67 0.27 108 1.67 127 84
-VE 91
CONTROL 0.08 32 0.93 71
+VE 81
CONTROL 0.25 100 1.31 100

The result of the physiological and symbiotic tests of this study showed that grass pea
rhizobial isolates were more tolerant and effective than their cross inoculation group of R.
leguminosarum var viceae. Consequently, the isolates of this study were found to tolerate
high salt, high temperature and high pH and performed better symbiotic effectiveness on
sand culture as compared to other rhizobial isolates from faba bean and field pea (Table
17).

61
Table 17. Comparative analysis of maximum tolerance and effectiveness of different
R.leguminosarum var viceae isolates from faba bean and field pea

% of Maximum % of % of
Maximum isolates Temp. isolates Maximum isolates % SE on
Researcher pH Grown. (0C) grown NaCl (%) grown sand % TN
Aregu
Amsalu
(2007) 10 82 40 10 6 33 133 2.93

Gebremeskel
Gebremariam
(2007) 9.5 85 45 20 6 35 85 2.80
Zerihun
Belay

(2006) 9.0 100 40 10 5 5 96 3.9

Getaneh
Tesfaye
(2008) 10 23 40 13 7 5 91.4 3.19
This work 12 100 50 25 13 11 120 3.9

62
6. Discussion

In this study, root nodule rhizobia from grass pea (Lathyrus sativus) were collected from
four districts of South Wollo and three districts of West Shoa. They were characterized
on the basis of their different phenotypic and symbiotic characteristics. Out of the 68
isolates, 63 of them were authenticated as root nodule bacteria according to Vincent
(1970). According to Brockwell (1998), the ability to form nodules along with the
subsequent capacity of fixing nitrogen are widely used means to evaluate the inherent
links between the rhizobia and their host plant. Although failure to nodulate could
emanate from loss of symbiotic plasmids (Sym-plasmids) that govern the symbiotic
interaction between the two partners (Zhange et a.l, 2001), the failure of these five
isolates to re-nodulate their parent host may be due to other intruding bacteria that
penetrated the nodule (Johnston and Beringer, 1976) or they may not originally be a
rhizobial isolate.

The majority of the colonies (78%) showed large mucoid (LM) whereas, 22% of them
displayed large watery colonies (LW). Fifty eight percent (58%) of the isolates displayed
large colony diameters of (2-4 mm) while 42% exhibited very large ones (4-6 mm) after
five days of inoculation. The largest colony diameter (6.0mm) was displayed by AAUGR
35, AAUGR 41, AAUGR 47 from South Wollo and AAUGR 49 and AAUGR 55 from
West Shoa. Likewise, 25% of the isolates (9 isolates) showed the smallest colony
diameter of 2.0mm. Almost all isolates were fast growers with excessive production of
exopolysaccharides as described by Jordan (1994).

All the authenticated isolates changed the BTB –YEMA media to yellow which showed
that they were acid producers and fast growers (Jordan, 1984; Lupwayi and Haque,
1994). With the exception of isolates AAUGR43, 46, and 64, all the isolates showed
doubling times of less than 4hrs characteristic of fast growing root nodule bacteria
(Jordan, 1984). The fact that most of the isolates displayed fast doubling times (1.24-
4hrs), had larger colonies (2-6mm) and changed the BTB-YEMA media to yellow
showed that they were fast growing rhizobia (Jordan,1984). Since the isolates nodulated

63
grass pea (Lathyrus sativus), they can safely be categorized into R. leguminosarum var
viceae.

The result revealed that the different rhizobial isolates grew at a different range of pH
(pH 4-12) (Table 5). The majority of the isolates were able to tolerate higher pH and nine
of the isolates (25% of the isolates) failed to grow at pH 4.0 and 4.5. All of the isolates
(100%) were found to grow at pH 5- pH 12 which is consistent with (Graham et al.,
1982; Surange et al., 1997; Merih et al., 2006) that reviewed the tolerance of rhizobia
that nodulate alfalfa and narrow leafed lupins (L. angustifolius) to higher pH (pH 8-12).
In this study, the rhizobial isolates that nodulated grass pea (Lathyrus sativus) were found
to be highly resistant to higher and lower acidity and alkalinity than R. leguminosarum
that nodulated other cool season legumes (Zerihun, 2006; Aregu, 2007; Gebremeskel,
2007 and Getaneh, 2008) (Table 17).

The inhibitory effect of different salt concentrations varied among isolates (Table 6). The
isolates showed tolerance to 0.5%-13% NaCl. The result indicated that the percentage of
tolerance continued to decrease as the concentrations of NaCl increases. Fifty percent of
the isolates grow at 8% concentrations whereas, 4 (11%) of the isolates were found to
tolerate 13% concentration of NaCl. Likewise, some isolates which included AAUGR 37
(SW) and AAUGR 48, AAUGR 59 and AAUGR 61 (WS) were found to grow at all
tested salt levels of concentrations as described in Boredleau and Prevost (2002) that
reported that some rhizobia can survive in the presence of extremely high levels (12%) of
salt concentrations both in culture and in soil while some others can tolerate salinities that
are equivalent to sea water. Almost all the isolates of this study were found to be highly
tolerant to higher levels of NaCl concentrations in contrast to the R. leguminosarum of
other cool season food legumes (Zerihun, 2006; Aregu, 2007; Gebremeskel, 2007 and
Getaneh, 2008) (Table 17).

The isolates grew within varied range of temperature 5-50 OC (Table 7). All the isolates
(100%) grew well between 15OC and 35OC as reported in (Getaneh, 2008; Zerihun, 2006

64
Aregu, 2007). 72%, 56% and 28% of the isolates were also found to grow at a
temperature of 40OC, 45OC and 50OC, respectively. This result is still in harmony with
(Hungria and Vargas, 2000; Zahran , 1999; Al-Falih, 2002) that reviewed the tolerance of
some strains at 40 OC, 47 OC and 50 OC. The high temperature (50OC) tolerant strains of
this work were AAUGR (19, 31, 35, 37 and 41) of South Wollo and AAUGR (48, 55, 59,
61, and 67) of West Shoa. The result of this work is again unique in that several isolates
tolerated a temperature of 45 OC and 50 OC in contrast to earlier works on other cool
season food legumes (Zerihun, 2006; Aregu, 2007; Gebremeskel, 2007 and Getaneh,
2008) (Table 17).

The isolates showed diversity in intrinsic antibiotic resistance on the tested antibiotics
(Table 8). All the isolates (100%) were able to resist ampicillin, chloroamphenicol and
nalidixic acid that were adjusted at a concentration of 2.5 μg ml -1. However, no isolate
was found to display such resistance (Aregu, 2007; Zerihun, 2007; Getaneh, 2008).
Although some isolates were sensitive to erythromycin and streptomycin particularly at a
-1 -1
concentration of 5μg ml and 10μg ml , the percent of tolerance was higher as
compared to the results of ( Aregu, 2007; Zerihun, 2007; Getaneh, 2008). 72 and 61% of
grass pea rhizobial isolates were able to tolerate streptomycin at concentrations of 5μg ml
-1
and 10μg ml,-1 compared to 52 and 32 % of the isolates from field pea in Aregu
(2007). Thus, grass pea rhizobial isolates were found to develop better resistance to
different antibiotics at different levels of concentrations and thereby could have better
coexistence with antibiotic producing soil microorganisms as reviewed in Muller et al.,
(1988).

Isolates AAUGR 31, AAUGR 37, AAUGR 42, of South Wollo and AAUGR 48,
AAUGR 49, AAUGR 55, AAUGR 56, AAUGR 57, AAUGR 59, AAUGR 60, AAUGR
61 and AAUGR 67 from West Shoa exhibited the broadest antibiotic resistance on all the
tested antibiotics at all concentrations. On the other hand, isolates AAUGR 21 of South
Wollo and AAUGR 53, AAUGR 63, AAUGR 64 and AAUGR 66 from West Shoa
displayed the narrowest range of antibiotic resistance in all tested antibiotics (Table XI).

65
The tested isolates showed little variation in their range of carbohydrate utilization (Table
9). All isolates (100%) grew on D-glucose, D-fructose, maltose, D-mannitol, galactose,
xylose, sucrose and mannose and greater than 75% of the isolates were found to grow on
arabinose, sorbitol, lactose and cellobiose. This result is consistent with (Stowers, 1985;
Amargar et al, 1997; Hungria and Vargas, 2000) which described that the greater
proportion of carbon sources were utilized by most of the isolates and rhizobia has the
ability to utilize a wide variety of carbon sources for growth and energy with several
pathways for carbon metabolism. In this study, 69%, 69%, 69%, and 67% of the isolates
utilized starch, Na-citrate, and trehalose and cellulose, respectively. Upon utilization of
Na-citrate only two isolates (Gebremeskel, 2007), eleven isolates (Aregu, 2007) and no
isolates (Getaneh) whereas, 25 (69%) of grass pea rhizobial isolates were found to utilize
Na-citrate. Isolates AAUGR 4 from South Wollo and isolates AAUGR 49, AAUGR 52
and AAUGR 56 from West Shoa showed a narrow range of carbohydrate utilization. On
the other hand, isolates AAUGR (31, 37, 39, 40, 41, 46 and 47) from South Wollo and
AAUGR (48, 53, 57, 59, 60, 61, 67 and 68) from West Shoa displayed wide range of
utilization of all the tested carbohydrate sources (Table 11) whereas, 15% of the isolates
from faba bean exhibited such broad range of utilization (Getaneh, 2008). The results of
grass pea rhizobia was in harmony with Sukrita et al. (1981) that concluded that rhizobia
catabolize a wide range of substrates and have ecological advantage over those with
specific carbon sources. Similarly, Jordan (1984) reported that fast growing rhizobia
utilize wide range of carbohydrates as sole sources of carbon.

All the isolates (100%) were able to utilize L-Arginine, L-Asparagine, L-Methionine and
L-Tyrosine and greater than 80% of the tested isolates were also found to catabolise L-
tryptophan, L-lysine, L-glutamate and L-alanine. This result agreed with (Sessisch et al.,
1997) that reported most of the nitrogen sources were utilized by rhizobia nodulating faba
bean. This result is inconsistent with other cool season legumes nodulated by R.
leguminosarum var viceae (Zerihun, 2007 and Getaneh, 2008). On the other hand, 78%
of the tested isolates showed less capacity of utilization to phenylalanine as described in
Jordan (1984). Eighteen isolates (50% of the isolates) displayed wide range of utilization

66
of nitrogen sources. Whereas, two isolates AAUGR 42 (South Wollo) and AAUGR 66
(West Shoa) showed less range of nitrogen utilization relatively (Table 10).

A cluster analysis on grass pea rhizobia was carried out to evaluate the phenotypic
relationships among the isolates of the different districts of South Wollo and West Shoa.
The dendrogram displayed the occurrence of a high level of diversity among the isolates.
At 50% similarity coefficient, the thirty – six isolates grouped in to four clusters and one
unclustered isolate. Each cluster and sub-cluster encompasses isolates of different
sampling areas. The dendrogram indicated that relatedness of the isolates decrease as
similarity coefficient level increase. As similarity coefficient increase, the number of
clusters increased. This shows that similarity analysis of 76 phenotypic characteristics
could be helpful to determine the genetic diversity among the isolates.

The preliminary screening for infectiveness (ability to form nodules) and effectiveness
(ability to form enough nitrogen) of the isolates were evaluated on the basis of nodule
number, nodule dry weight and shoot dry weight. Although there was a discrepancy in
nodule number and weight among the treated plants, a significant difference existed with
respect to their shoot dry matter at P=0.05. The result of the symbiotic effectiveness of
the isolates revealed that 61% (39 isolates) rated highly effective and effective by
accumulating 50 - 120% symbiotic effectiveness whereas, with respect to sites of the
samples, 95% and 47% of the isolates that were found to record both highly effective and
effective were from West Shoa and South Wollo, respectively. Although the isolates that
were able to display high effectiveness were isolates AAUGR (31, 37, and 41) from
South Wollo and AAUGR (48, 49, 52, 59, 60, 61, 66 and 67) from West Shoa, the
isolates of West Shoa performed better than isolates of South Wollo without any
ineffective isolate (Table 14). The shoot dry matter accumulated by these highly effective
isolates was also too much greater than that accumulated by both the inoculated
treatments and the uninoculated controls.

67
The shoot dry matter accumulated in the infected host plant was used to evaluate the
relative symbiotic effectiveness of the isolate. According to Lupwayi and Haque (1994);
peoples et al. (2002) and Mulongoy (2008), shoot dry matter is a good indicator of
relative isolate effectiveness and there exists a sound correlation between the nitrogen
fixing capacity of legumes and their shoot dry matter accumulation. In general, a
significant difference (P=0.05) in shoot dry matter was recorded among inoculated grass
pea and the controls and also between the positive control (0.25g/p) and the negative
control (0.08g/p).

The soil experiment which was done using Awash Bolto soil with pH 7.1 for ascertaining
infectivity and effectivity displayed marked differences among the isolates in nodule
number, shoot dry matter and plant total nitrogen. Most of the selected isolates (5 of 7
isolates) showed slight increase in nodule number on soil experiment (93-165
nodules/plant) than on pouch culture (83-135 nodules/plant). The controls, both positive
and negative were also able to form a few nodules that were small in size and white in
colors from the indigenous rhizobia. The inoculated isolates formed nodule ranging from
93-165 while the positive and negative control formed nodules 17 and 19 respectively.
This result can lead us to a conclusion that inoculation increased the amount of nodules
formed.

Shoot dry matter accumulation of the rhizobial-inoculated plants revealed differences on

the performance of the isolates in soil. The isolates were found to increase shoot dry
matter by 82-85% in soil than on pouch cultures (Table 16). As Ayeneabeba et al. (2002)
reported a 45-50% increase in shoot dry matter between sand culture and soil culture
experiments on faba bean production in the Northern Shoa. Kang and Mills (2004) also
reviewed that the high nitrogen content of the soil, additional nodulation by the
background indigenous strains of the tested soil and other rhizosphere effects on plant
growth might have contributed to the increased shoot dry matter on the soil culture as
compared to the sand culture.

68
In this experiment, a significant difference in total nitrogen among the effective isolates
was also recoded at P = 0.05. The isolates were able to accumulate 2.6-3.9% total
nitrogen. On the other hand, the negative control was able to accumulate 1.2% total
nitrogen. The total nitrogen accumulated by grass pea rhizobial isolates was found to be
higher than that accumulated by other cool season legumes (Aregu, 2007; Gebremeskel,
2007 and Getaneh, 2008) (Table 17). Thus, grass pea associated microsymbionts are to
some extent more effective in nitrogen fixing efficiency.

69
7. Conclusion and Recommendations
7.1. Conclusion

This particular study that focused on the phenotypic and symbiotic characterization of
rhizobia nodulating grass pea in major growing areas of South Wollo and West Shoa has
been done for the first time in Ethiopia. The result of plant shoot dry matter, total
nitrogen and literatures supported that grass pea has greater intrinsic potential to enrich
the nitrogen deficient soil. Apart from this, grass pea grows in poor soils, in water
logging and drought conditions and in general, in soils at extreme conditions. Thus, its
cultivation best suits the agricultural conditions of the country.

In this study, all isolates changed the YEMA-BTB medium to yellow colour. With the
exception of few isolates, all isolates showed a mean doubling time of less than four (4)
hrs, and induced nodulation on grass pea. All these and other characteristics can qualify
these isolates of the cross-inoculation group of Rhizobium leguminosarum var viceae.

Most of the isolates were tolerant for most of the environmental factors like high pH and
low pH, high and low temperature, low and high salt concentrations. The majorities of
the isolates were able to utilize a wide variety of carbon and nitrogen sources and also
displayed a considerable resistance to many of the tested antibiotics. When the
physiological test result of grass pea rhizobia is compared with the result of other cool
reason legumes that were carried out in the past few consecutive years, the tolerance of
grass pea rhizobia to these extreme abiotic factors was found to be slightly better than
other isolates from other cool season legumes.

This capacity may favor the establishment of the rhzobia in the soil and may represent an
advantage to be used as inoculants for grass pea. This gives an ecological advantage in
colonizing the soil or rhizosphere as compared to strains having limited preference and
competitive advantage for the isolates when they live as saprophytes in the soil. This is an

70
essential component in strain selection and for production of inoculants. The fact that
61% of the isolates were effective in screening trials indicates that some of the grass pea
growing areas evaluated mostly in South Wollo are in need of inoculants that could
enhance the symbiotic performance of the crop.

7.2. Recommendations

On the basis of the above conclusion, the following recommendations are suggested:

¾ The isolates were taken from two sampling sites of South Wollo and West Shoa.
More should be studied from different grass pea growing areas of the country in
order to reveal the true character of grass pea rhizobia.

¾ In order to reveal the true diversity of grass pea rhizobia, phenotypic

characterization should be supplemented with polyphasic approach that includes
genetic characterization.

¾ The highly effective isolates that were tested in soil culture in the greenhouse
should be re-tested under field conditions to ascertain their performance in vivo.

¾ Among the highly effective isolates, isolate AAUGR 37 (SW) and isolates
AAUGR 48, 59, 61 and 67 from West Shoa are strongly suggested to be used as
inoculums in field conditions.

71
8. References

Aarons, S. R. and P. H., Graham (1991). Response of Rhizobum legumionsarum bv.

Phaseoli to acidity. Plant soil 134: 145-151.
Adugna Negere and Shelemew Woldemariam (2001). An overview of grass pea
(Lathyrus sativus) production in Ethiopia. Ministry of Agriculture, Addis
Ababa, Ethiopia. PP. 65-70.
Ahmad, M. H., Rafique Uddin, M. and McLaughlin, W. (1984). Characterization of
indigenous rhizobia from wild legumes. FEMS. Microbiol. Letters 24: 197-203.
Al- Falih, A. M. K. (2003). Factors affecting the efficiency of symbiotic nitrogen fixation
by Rhizobium. Pakistan J. Biol. Sci. 5: 1277-1293.
Al- Niemi, T. S., Kahn, M. L. and McDermott, T. R. (1997). P metabolism in the bean
Rhizobum tropici symbiosis. Plant physiol. 113: 1233-1242.
Alexander, M. (1997). Introduction to soil microbiology, 2nd ed. John Willy and Sons,
Inc,USA. PP. 467.
Allen, O.N. and Allen, E.K. (1981). The leguminosae. A source book of characteristics,
use and Nodulation. Macmillan, London. PP. 812.
Amargar, N., Macheret, V. and Aguerre, G. (1997). Rhizobum gallicum sp. Nov, and
Rhizobum giardinii sp. Nov. from Phaseolus vulgaris nodules. Int. J. syst.
Bacteriol. 47: 996-1006.
Angaw Tsige and Asnakew Woldeab. (1994). Fertilizer response trials on highland food
legumes of Ethiopia. In: Cool season food legumes of Ethiopia (Asfaw, Tilaye,
Geletu, Saxena, M.C. and Solh, M.B. eds), ICARDA, Addis Ababa, Ethiopia.
PP.279-292.
Aregu Amsalu (2007). Symbiotic and phenotrpic characterization of Rhizobium
leguminosarum biovar viciae isolates of field pea (Pisum sativum) form different
pulse growing regions of Ethiopia. M.SC. Thesis. Addis Ababa University,
Ethiopia.
Arrese – Igor, C., Minchin, F. R., Gordon, A. J. and Nath, A. K. (1997). Possible causes
of the physiological decline in soybean nitrogen fixation in the presence of
nitrate. J. exp. Bot.48: 905-913.

72
Asgelil Dibabe, Wasse Haile, Asmare Ayalew and Wondimagegn Syoum (2003). The
status of grass pea (Lathyrus staivus) research and production in Ethiopia.
Institute of Agricultural Research, Adet Research Centre, Bahir Dar, Ethiopia.
PP. 46-60.
Asmare Ayalew, Asgelil Dibabe, Wolde Amlak Araia, Bekele Hundie and Yeshanew
Ashagrie (2000). The status of grass pea (Lathyrus Sativus) research and
production in Ethiopia. Institute of Agricultural Resaerch Centre, Adet Research
Centre, Bahir Dar, Ethiopia. PP. 47.
Atkins, C. A. (1984). Efficiencies and inefficiencies in legume / Rhizobum symbiosis. A
review plant soil 82: 273-284.
Ayneabeba Admasu, Fasil Assefa, Asfaw Hailemariam and Endashaw Bekele (2001).
Studies of Rhizobium inoculation and fertilizer treatment on growth and
production of faba bean (Vicia faba ) in some yield depleted and yield sustained
regions of Semien Showa. SINET. Ethiop. J. Sci. 24: 197-211.
Badaruddin, M. and Meyer, D. W. (1994). Grain legume effects on soil nitrogen grain
yield and nitrogen nutrition of wheat. Crop Sci. 34: 1104-1309.
Ballen, K. G., Graham, P. H., Jones, T. and Bowers, J. H. (1998). Acidity and calcium
interaction affecting cell envelop stability in Rhizobium. Can. J. microbiol.
44: 582-587.
Beijerinck, M. W. (1888). Rhizobial systematic. Bot. Ztg. 46: 796-804.
Berhanu M. Abegaz, Redda T. Haimanot, Valerie, S. P. and Peter, S. S. (2000).
Proceedings of the second International Lathyrus / Lathyrism Conference in
Ethiopia under the International Net work for the improvement of Lathyrus
sativus and the Eradication of Lathyrism. Plant Genetic Resource Centre, A. A.
Ethiopia. PP 1-9.
Bernal, G. and Graham, P. H. (2001). Diversity in the rhizobia associated with Phaseolus
vulgaris L. in Ecuadore and comparisons with Mexican bean rhizobia. Can. J.
Microbiol. 47: 526-534.
Bohlool, B. B., Ladha, J. K., Garrity, D. P. and George, T. (1992). Biological nitrogen
fixation for sustainable agriculture: A perspective plant and soil 141: 1-11.

73
Bordeleau, L. M. and Prevost, D. (1994). Nodulation and nitrogen fixation in extreme
environments. Plant and soil 161: 115-125.
Bostford, J. L. and Lewis, J. A. (1990). Osmoregulation in Rhizobum melitoti: production
of glutamic acid in response to osmotic stress. Appl. Environ. Microbiol. 56:
188-194.
Bremer, J. M. (1965). Total nitrogen In: Methods of soil analysis. Part II (Black, C. A.
ed.) PP 1149-1178.
Brockwell, J. (1998). Matching rhizobia and temperate species of Africa. In: Recent
development in Acacia planting. (Turn bull, L. W., Crompton, H. R. and
Pinyopusarerk, K., eds). ACIAR. Canberra, Australia. PP. 264-273.
Broughton, W. J., Samery, U. and Stanley, J. (1987). Ecological genetics of Rhizobum
meliloti: symbiotic plasmid transfer in the Medicago sativa rhizospere. FEMS.
Microbiol. Letters 40: 251-255.
Burdas, D. (2000). Rhizobium, Root nodules and nitrogen fixation: society for general
microbiology. Appl. Environ. Microbiol. 25: 187-196.
Burris, R. H. and Roberts, G. P. (1993). Biological nitrogen fixation. Annu. Rev. Nurt.
13: 317-335.
Caetano – Anollen, G., Lagares, A. and Favelukes, G. (1989). Adsorption of Rhizobium
meliloti to alfalfa roots: dependence on individual cations and pH. Plant
soil 117: 67-74.
Campo, R. J. (1995). Residual effects of aluminum on Bradyrhizobium japonicum in
defined medium and soil solution. PhD. Thesis. The University of Reading.
Cassman, K. G., Munns, D. N and Beck, D. P. (1981). Phosphorous nutrition of
Rhizobum japonicum strain differences in phosphate storage and utilization. Soil
Sci. Soc. Am. J. 45: 517.
Chen, H., Richardson, A. E. and Rolfe, B. G. (1993). Studies on the physiological and
genetic basis of acid tolerance in Rhizobum leguminosarum biovar trifolii. Appl.
Environ. Microbiol. 59:1798-1804.
Cordovilla, M. P., Ligero, F. and Lluch, C. (1994). The effect of salinity on N fixation
and assimilation in Vicia faba. J. Exp. Bot. 45: 1483-1488.

74
Cordovilla, M. P., Ocana, A., Ligero, F. and Lluch, C. (1995). Salinity effects on growth
analysis and nutrient composition in four grain legumes – rhizobium
symbiosis. J. plant Nutr. 18:1595-1609.
Correa, O. S. and Barneix, A. J. (1997). Cellular mechanisms of pH tolerance in
Rhizobium loti. World J. Microbiol. Biotechnol. 13: 153- 157.
Crews, T. E. and Peoples, M. B. (2004). Legume versus fertilizer sources of nitrogen
ecological trade offs and human needs. Agric. Econ. Environ. 102: 279-297.
Dawit Tadesse (1991). Grass pea (Lathyrus Sativus): Genetic resources conservation and
utilization in Ethiopia. Proceedings of the second international lathyrus
/Lathyrism conference in Ethiopia. Plant Genetic Resources Centre, A.A.
Ethiopia. PP. 62.
De-Faria, S.M., Lewis, G.P., Sperennt, J. I. and Southerland, J. M. (1989). Occurrence of
nodulation on Leguminosae. New phytol. 11: 607-609.
Dean, D. R. and Jacobson, M. R. (1992). Biochemical genetics of nitrogense. In:
Biological nitrogen fixation. (Stacy, G., Burris, R. H. and Evans, H.
J. eds). Chapman Hall, New York. PP. 763-831.
Delgado, M. J. F,. Ligero and C., Lluch (1994). Effects of salt stress on growth and
nitrogen fixation by pea faba bean common bean and soy bean plants. Soil
boil. Biochem. 26: 371-376.
Dilworth, M. J. and Glenn, A. R. (1991). Biology and biochemistry of nitrogen fixation.
Elsevier, Amsterdam. PP. 83.
El–Hamdaoui, M., Redonda –Nieto, M., Rlvilla, R., Bonilk, I. and Bolanos, L. (2003).
Effect of boron and calcium nutrition on the establishment of the Rhizobum
leguminosarum pea (Pisum sativum) symbiosis and nodules development under
salt stress. Plant cell and environ. 26: 1003-1011.
El- Shinnawi, M. M., El- Saify, A. N. and Waly, J. M. (1989). Influence of the ionic form
of mineral salts on growth of faba bean and Rhizobium leguminosarum. World
J.Microbiol. Biotec. 5: 247-254.
El-Sheikh, E. A. E., and M., Wood. (1990). Salt effects on survival and multiplication of
chick pea and soy bean rhizobia. Soil boil. Biochem.22:343-347.

75
Food and Agricultural organization of United Nations (FAO) (1994). Production year
book. Rome, Italy. PP. 151.
Ford, C. W. (1984). Accumulation of low molecular weight solutes in water stressed
tropical legumes. Phytochem. 23: 1007-1015.
Frank, B. (1889). Rhizobial systematic. Beru Devt. Bot. Ges. 7: 322-346.
Fred, E. B., Baldwin, I. L. and Mc Coy, E. (1932). Root nodule bacteria and leguminous
plants, Madison University of Wisconsin press. PP. 343.
Freiberge, C. Rosenthal, A., Fellay, R. W., Broughton, J., perret, X. and Bairoch, A.
(1997). Molecular basis of symbiosis between rhizobium and leumes. Nature
387: 394-401.
Frink, C. R, Waggoner, P. E. and Ausubel, S. H. (1991). Nitrogen fertilizer: retrospect
and prospect. Proc. Natl. Acad. Sci. 96:1175-1180.
Fujihara, S. and Yoneyama, T. (1993). Effects of pH and osmotic stress on cellular
polyamine contents in the soybean rhizobia Rhizobium fredii P 220 and Brady
rhizobium japonicum A 1017. Appl. Environ. Microbiol. 59: 1104-1109.
Gebremeskel Gebremariam (2007). Characterization and evaluation of nitrogen fixation
of Rhizobium leguminosarum bv. Vicia isolates collected from different parts
of Ethiopia. M.SC. Thesis. Addis Ababa University, Ethiopia.
Getaneh Tesfaye (2008). Symbiotic and phenotypic diversity of rhizobial isolates
nodulating Vicia faba from western Shoa and Hararghe, Ethiopia. M.SC.
Thesis. Addis Ababa University, Ethiopia.
Gibson, A. H. (1971). Factors in physical and biological environment affecting
nodulation and nitrogen fixation by legumes. Plant soil 35: 139-152.
Giller, K. E. (2001). Nitrogen fixation in tropical cropping systems (2nd ed.). PP 57-70,
CABI, international, Wallingfork, UK. PP. 57-70.
Giller, K. E. and Wilson, K. J. (1991). Nitrogen fixation tropical cropping systems. C. A.
B. international, Wallingford, USA. PP. 423.
Gittoni, N. E. and M. A., Bueno (1996). Changes in cellular content of trehalose in four
pea nut rhizobia strains cultured under hypersalinity. Symbiosis 20: 117-127.
Graham, P. H. (1992). Stress tolerance in Rhizobium and brady rhizobium and nodulation
under adverse soil conditions. Can. J. Microbiol. 38: 475-484.

76
Graham, P. H. and Vance, C. P. (2003). Legumes: importance and constraints to greater
utilization. Plant physiol. 131:827-887.
Graham, P. H., Ferrey, M. L., Conroy, M. J. and Hammer, B. E. (1994). Acid pH
tolerance in strains of Rhizobium and Brady rhizobium and initial studies on
the basis acid tolerance of rhizobium tropici UMR 1899. Can. J. microbiol. 40:
198-207.
Graham, P. H., Viteri, S. E., Mackie, F., Vargas, A. T. and Palacios, A. (1982). Variation
in acid soil tolerance among strains of Rhizobium phaseoli. Field crops res. 5:
121-128.
Hart, A. L. (1989). Nodule phosphorous and nodule activity in white clover N. Z. J.
Agric. Res. 32: 145-149.
Haward, J. B. and Rees, D. C. (1996). Structural basis of biological nitrogen fixation.
Chem.Rev. 96: 2965-2982.
Herridge, D. F. and Rose, I. A. (2000). Breeding for enhanced nitrogen fixation in crop
legumes. Field crops res. 65: 229-248.
Hickey, E. W., and Hirshifield, I.N. (1990). Low-pH induced effects on patterns of
protein synthesis and on internal pH in Escherichia coli and Salmonella
typhimurium. Appl. Environ. Microbiol. 56: 1038-1045.
Howieson, J. G., O’Hara, G. W. and Carr, S. J. (2000). Changing roles of legumes in
Mediterranean agriculture, developments from an Australian perspective.
Field res. 65: 107-122.
http://www.Hydra.Org. UK.Anonymous (2001).
Hubbell, D. H and Kidder, G. (2003). Biological nitrogen fixation. Food and agric.
Sci. 16: 1-4.
Hungria, M. and Franco, A. A. (1993). New sources of high temperature tolerance
rhizobia for Phaseous vulgaris L. Plant soil 149: 103-109.
Hungria, M. and Stancy, G. (1997). Molecular signals exchanged between host plants and
rhizobia: basic aspects and potential application in agriculture. Soil boil.
Biochem. 29: 519-830.

77
Hungria, M. and Vargas, M. A. T. (2000). Environmental factors affecting N2 fixation in
grain legumes in tropics, with an emphasis on Brazil. Field crops res. 65:
151-164.
Hungria, M., A. A. Franco, and J. I. Sprent (1989). New sources of high temperature
tolerant rhizobia for Phaselous vulgaris L. Plant soil 149: 103-109.
Israel, D. W. (1987). Investigation of the role of phosphorous in symbiotic dinitrogen
fixation. Plant physiol. 84: 835-840.
Jayasundara, H. P. S., Thomson, B. D. and Tang, C. (1988). Response of cool season
legumes to soil abiotic stress. Adv. Agro. 63: 77-151.
Jennifer, S. M. (2003). A brief history of grass pea and its use in crop improvement.
Lathyrus/ Lathyrism news letter 3: 18-20.
Johnson, J. F., Allan, D. L., Vance, C. P. and Weiblen, G. (1996). Root carbondioxide
fixation by phosphorous deficient Lupnus albus – contribution to organic acid
exudation by proteoid roots. Plant physiol.112: 19-30.
Johnston, A.W. B. and Beringer, J. E. (1976). Pea root nodules containing more than one
Rhizobium species. Nature. 264: 502-504.
Jongruaysup, S., Dell, B., R. W., O’Hara, G. W. and Bradley, J. S. (1997). Effect of
molybdenum and inorgainic nitrogen on molybdenum redistribution in
black gram (Vagina mungo L. Hepper) with particular reference of seed
fill. Ann. Bot.79: 67-74.
Jordan, D. C. (1984). Family III. Rhizobiaceae. In: Berges manual of systematic
bacteriology. (Chrieg, N. R. and Hot, J. G. eds). Williams and
Willikins, Baltimore. PP. 234-256.
Kang, S. and Mills, A.L. (2004). Soil bacterial community structure changes following
disturbance of the overling plant community. Soil sci. 169: 55-65.
Kim, M. M., Asher, C. J., Edwards, D. G. and Dater, R. A. (1985). Aluminum toxicity:
effects on growth and nodulation of subterranean clover. Proceedings of the 15th
International grass lands congress, Kentuky. PP. 501-503.
Kouchi, H., Akao, S and Yoneyama, T. (1986). Respiratory utilization of “C- labelled
photosynthate in nodulated root systems of soybean plants. J. Exp. Bot. 37:
987-993.

78
Latta, R. A and Carter, E. D (1998). Increasing production of an annual medic – wheat
rotation by grazing and grass removal with herbicides in the Victorian mallee.
Aust. J. Exp. Agr. 36: 211-217.
Leigh, G. J. (2002). Nitrogen fixation in the millennium. Elsevier, Amsterdam. PP. 115-
175.
Lewis, W. E. (1967). Legume inoculation: what it is – what it does? Farmers bulletin
number. 2003: 1-10.
Lie, J. A. (1981). Environmental physiology of legume – Rhizobum symbiosis. In:
Nitrogen fixation. (Broughton, W. J. ed.). Clarendon press, oxford. Ecol.1: PP.104-
134.
Lupwayi, N. Z. and Haque, I. (1994). Working document: legume-rhizobium technology
manual. Environmental science division international livestock center for
Africa, Addis Ababa, Ethiopia. PP 1-40.
Maatallah, J., Berraho, E. B., Sanjuan, J. and Lunch, C. (2002). Phenotypic
characterization of rhizobia isolates from chick pea (Cicer arrientium)
growing in Morroccan soils. Agronomic 22: 321-329.
Martinez- Romero, E., Segovia, L., Mercante, F. M., Franco, A. A., Graham, P. and
Pardo, M. A. (1991). Rhizobium tropici, a novel species nodulating Phaseolus
vulgaris L. beans and Leucaena sp trees. Int. J. Syst. Bacteriol. 41: 417-426.
Maschner, H. (1995). Mineral nutrition of higher plants. 2nd ed. Harcourt science and
technology company, New York, London, Sydney, Tokyo, Boston. PP. 889.
Mcklay, I. A. (1993). Production and excretion of nod metabolites by Rhizobium
leguminosarum biovar trifolli are disrupted by the same environmental factors
that reduce nodulation in the field. Appl. Environ. Microbiol.59: 3385-
3392.
Miller, K. J. and Wood, J. M. (1996). Osmoadaplation by rhizosphere bacteria. Annual
Rev. Micorbiol. 50: 101-136.
Miller, R. (2008). Legume inoculation. Food and Agri. Sci. 20: 1-5.
Montanez, A. (2000). Overview and case studies on biological nitrogen fixation:
perspectives and limitations. Field crop res. 26: 1-11.

79
Muehlbauer, F. J and Abebe, Tullu (1997). Lathyrus staivus L. Department of Agronomy
and Farming system, university of Adelaide. PP. 1-5.
Muller, J. G., Sjiper, H. D. and Shipe, E. R. (1988). Intrinsic antibiotic resistance in
Brady rhizobium japnoicum. Soil boil. Biochem. 20: 879-882.
Mulongoy, K. (2004). Technical paper 2: Biological nitrogen fixation. Can. J. Microbiol.
21:1-19.
Negales, J. Campos, R., Banabdelkhalek, H., Olivares, J., Lluch, C. and Sanjuan, J.
(2002). Rhizobium tropic genes involved in free living salt tolerances are
required for the establishment of efficient nitrogen fixing symbiosis with
Phaseolus vulgaris. Mol. Plant- Microbe Interact. 15: 225-232.
Norris, D. O. (1965). Legume-rhizobium association. Plant soil 22: 143-146.
Nour, S. M., Cleyet – Marel, J., Beck, D., Effosse, A. and Fernandez, M. P. (1994).
Genotypic and phenotypic diversity of rhizobium isolated from chick pea (Cicer
ariethinum L.). Can. J. Microbiol. 40: 345-353.
O’ Hara, G. W., Boonkerd, N. and Dilworth, M. J. (1988). Mineral constraints to nitrogen
fixation. Plant soil 108: 93-110.
O’ Hara, G. W., Hartzook, A., Bell, R. W. and Loneragan, J. F. (1993). Differences
between Brady rhizobium strains NC92 and TAL 1000 in their nodulation and
nitrogen fixation with peanut in iron deficient soil. Plant and soil 155: 333-336.
Odee, D. W., Haukka, K., Mclnroy, S. G., Sprent, J. I, Sutherland, J. M. and Young, J. P.
W. (2002). Genetic and symbiotic characterization of rhizobia isolated form free
and herbaceous legumes grown in soils from ecologically diverse sites in
Kenya. Soil biol. Biochem. 34: 801-811.
Peoples, M. B., Herridge, D. F. and Ladha, J. K. (1995). Biological nitrogen fixation: an
efficient source of nitrogen for sustainable agricultural production. Plant soil
174: 3-28.
Pereira, P. A. A. and Bliss, F. A. (1989). Selection of common bean (Phaseolus Vulgaris
L.) for N2 fixation at different levels of available phosphorous under field and
environmentally controlled conditions. Plant soil 115: 75-82.
Peters, J. W., Fisher, K. and Dean, D. R. (1995). Nitrogenase and function: a biochemical
genetic perspective. Ann. Rev. Microbiol. 49: 335-336.

80
Postgate, J. R. (1998). The fundamentals of nitrogen fixation. 3rd edition. Cambridge
university press, Cambridge UK. P. 112.
Rao, D. C. N. and Sharama, P. C. (1995). Effectiveness of rhizobial strains for chickpea
under salinity stress and recovery of nodulation on desalinization. Int. J. Exp.
Boil. 33: 500-504.
Richardson, A. E., Simpson, R. J., Djordjevie, M. A. and Rolfe, B. J. (1998). Expression
of nodulation genes in Rhizobium leguminosarum biovar trifolic is affected
by low pH and by Ca2+ and Al ions. Appl. Environ. Micriobiol. 54:
2541- 2548.
Roughley, R. J. (1980). Environmental and cultural aspects of the management of
legumes and Rhizobium. In: Advances in legume sciences. PP. 97-103.
Sahgal, M. and Johri, B. N. (2003). The changing face of rhizobial systematic. Curr. Sci.
84: 43-48.
Sahlemedhin Sertsu and Taye Bekele (2000). Procedures for soil and plant analysis.
National Soil Research Centre. EARO. PP. 70-76.
Sharma, R.N. and Pandey, R.L. (2001). Seed and seedling vigor of grass pea (Lathyrus
sativus L.) in relation to moisture stress for better performance under relay
cropping with paddy rice. Lathyru/lathyrism Newsletter 2: 99-100.
Shaw, B.D. (1983). None coordinated regulation of Rhizobium nitrogenase synthesis by
oxygen: studies with bacterioids from nodulated Lupins angusifilius. J. Gen.
Microbiol. 129: 849-853.
Shenbagarathi, R. (1993). Isolation and characterization of mutants of rhizobum SBS–R
100 symbiotic with Sesbania procumbent. Soil boil. Biochem. 25: 1339-1342.
Singleton, P. W. and Bohlool, B. B. (1983). Effect of salinity on the functional
components of the soybean Rhizobium japonicum symbiosis. Crop sci. 23: 29-35.
Smil, V. (2000). Cycles of life. Scientific American library, New York. PP. 276-298.
Smith, D. C. and Douglas, A. E. (1987). The biology of symbiosis. Edward Arnold,
London. PP. 64-88.
Somasegaren, P. and Hoben, H. J. (1994). Hand book for rhizobia. Methods in legume-
rhizobium technology. Springer – verlag, New York. PP. 1-441.

81
Stowers, M. D. (1985). Carbohydrate metabolism in rhizobium species. Ann. Rev.
Microbiol. 39: 89-108.
Sukrita, C., Lee, M. S. and Gibson, A. H. (1981). Diversity in the nutritional
requirements of strains of various rhizobum species. Soil biol. Biochem.
13: 349-354.
Surange, S., Wollum, A. G., Kumar, N. and Nautiyal, S. C. (1997). Characte rization of
Rhizobium from root nodules of leguminous trees growing in alkaline soils.
Can. J. Microbiol. 43: 891-894.
Tang, C., Robson, A. D. and Dilworth, M. J. (1992). The role of iron in the (Brady)
Rhizobium legume symbiosis. J. Plant nut. 15: 2235-2252.
Tate, R. L. (1995). Soil microbiology (symbiotic nitrogen fixation). John Wiley and Sons,
Inc, New York. N. Y. PP. 307-333.
Thies, J. E., Holmes, E. M. and Vachot, A. (2001). Application of molecular techniques
to studies in rhizobium ecology: a review Aust. J. Exp. Agr. 41: 299-319.
Tilak, K. V. B. R., Ranganayaki, N. K. K., Saxena, A. K., Nautial, C. S,
Mittal, S., Tripathi, A. K. and Johri, B. N (2005). Diversity of plant growth and
soil health supporting bacteria. Curri. Sci. 89: 136-140.
Vance, C. P. (1997). Enhanced agricultural sustainability through biological nitrogen
fixation. In: Biological fixation of nitrogen for ecology and sustainable
agriculture. (Legocki, A., Bothe, H. and Puhler, A. eds.). Springer
–Verlag, Berlin, Headelberg. PP. 179-186.
Vance, C. P. (1998). Legume symbiotic nitrogen fixation: agronomic aspects. In: The
rhizobiaceae. (Spaik, H. P., Kondorosi, A. and Hooykass, P. J. J.,
eds). Kluwer academic press: Dordecht the Netherlands. PP. 509-530
Vargas, A. A. T. and Graham, P. H. (1998). Phaseolus vulgaris cultivar and Rhizobium
strain variation in acid pH tolerance and nodulation under acid conditions. Field
crops res. 19: 91-101.
Vassileva, V. G., Milanov, G., Ignatov, A. and Nikolov, B. (1997). Effect of low pH on
nitrogen fixation of common bean grown of various calcium and nitrate
levels. J. Plant Nutr. 20: 279-294.

82
Vincent, J. M (1970). A manual for the practical study of root nodule bacteria. Blackwell
sci. publ. oxford. PP.164.
Waughman, G. J. (1977). The effect of temperature on nitrogenase activity. J. Exp. Bot.
28: 949-960.
White, D. (1995). The physiology and Biochemistry of prokaryotes. Oxford University
Press. PP. 34-46.
Willems, A. (2006). The taxonomy of rhizobia: an overreview. Plant and soil 287: 3-14.
Wuletaw Tadesse and Endashaw Bekele (2001). Factor analysis of components of yield
in grass pea (Latyrus Sativus L). Lathyrus lathyrism news letter 2: 91-93.
Zahran, H. H. (1999). Rhizobium – legume symbiosis and nitrogen fixation under severe
conditions and in an arid climate. Microbiol. Mol.Biol. Reviews 63: 968 – 989.
Zahran, H. H., Moharram, A. M. and Mohammad, H. A. (1992). Some ecological and
physiological studies on bacteria from salt –affected soils of Egypt. J. basic.
Microbiol. 32: 405-413.
Zerihun Belay (2006). Symbiotic and phenotypic diversity of Rhizobium leguminosarum
var isolates (vicia faba) from Northern Gonder. M.SC. Thesis, Addis Ababa
University, Addis Ababa, Ethiopia.
Zhang, X., Harper, R. Karsisto, M. and Lindstrom, K. (1991). Diversity of Rhizobum
bacteria isolated from the root nodules of leguminous trees. Int. J. Syst.
bacteriol.41: 104-113.
Zhange, X., Kosier, B. and Priefer, U. B. (2001). Symbiotic plasmid rearrangement in
Rhizobium leguminosarum bv. Viciae VF39SM. J. Bacteriol. 183: 2141-2144.
Zahran, H. H. (1991). Conditions for successful rhizobium-Legume symbiosis in salinity
environments. Biol. Ferti. Soils 12: 73-80.

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9. Appendices
Appendix 1. Inoculated and control plants after 45 days of growth on pouch culture in
greenhouse

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Appendix 2. Inoculated and control plants after 45 days of growth on potted soil in
greenhouse

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Appendix 3. Authenticated and non-authenticated isolates that that are not
characterized.

Isolates Nodule number per Nodule dry Wt. per Shoot dry Wt. % SE Effectiveness
plant plant(g) per plant (g)

AAUGR1 - - - - Non-authenticated
AAUGR2 46±21a-e 0.039±0.012b-g 0.135±0.049b-d 53 E
AAUGR3 22±10de 0.024±0.016fg 0.116±0.095cd 46 LE
AAUGR5 - - - - Non-authenticated
AAUGR6 23±15de 0.011±0.008g 0.066±0.057d 26 IE
AAUGR7 - - - - Non-authenticated
AAUGR8 9±3e 0.028±0.008fg 0.075±0.057d 30 IE
AAUGR10 35±10c-e 0.035±0.022c-g 0.125±0.051b-d 49 LE
AAUGR11 44±26b-e 0.050±0.005a-g 0.117±0.056cd 46 LE
AAUGR12 22±11de 0.021±0.013fg 0.096±0.079d 37 LE
AAUGR13 31±6c-e 0.017±0.007g 0.104±0.041cd 41 LE
AAUGR14 16±9e 0.043±0.010a-g 0.111±0.028cd 41 LE
AAUGR15 27±27c-e 0.043±0.016a-g 0.122±0.097cd 44 LE
AAUGR16 25±13c-e 0.023±0.004fg 0.085±0.033d 33 IE
AAUGR17 19±10de 0.020±0.004fg 0.119±0.061cd 47 LE
AAUGR18 - - - - Non-authenticated
AAUGR20 31±24c-e 0.016±0.008g 0.099±0.078d 39 LE
AAUGR23 30±13c-e 0.025±0.011fg 0.121±0.040cd 48 LE
AAUGR24 27±18c-e 0.025±0.002fg 0.117±0.047cd 46 LE
AAUGR25 20±6de 0.022±0.008fg 0.076±0.014d 30 IE
AAUGR26 26±20c-e 0.010±0.002g 0.069±0.068d 27 IE
AAUGR27 34±21c-e 0.038±0.005c-g 0.122±0.044cd 48 LE
AAUGR28 31±13c-e 0.023±0.010fg 0.099±0.014d 39 LE
AAUGR30 51±8a-e 0.059±0.015a-g 0.124±0.050cd 49 LE
AAUGR33 30±23c-e 0.024±0.019fg 0.089±0.013d 35 LE
AAUGR44 28±7c-e 0.023±0.010fg 0.093±0.043d 37 LE
AAUGR45 29±12c-e 0.022±0.005fg 0.067±0.025d 26 IE
AAUGR50 74±16a-e 0.060±0.003a-g 0.114±0.050cd 45 LE
AAUGR51 44±49b-e 0.035±0.024c-g 0.138±0.037b-d 54 E
AAUGR62 60±39a-e 0.061±0.003a-g 0.158±0.030b-d 62 E
AAUGR65 - - - - Non-authenticated
Note: Levels not followed by the same letter/letters are significant at p=0.05 (Tukey’s HSD test)

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