Use of Microorganisms As Biofertilizers For Some Plants: June 2006

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USE OF MICROORGANISMS AS BIOFERTILIZERS FOR SOME PLANTS
Thesis · June 2006

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USE OF MICROORGANISMS AS BIOFERTILIZERS
FOR SOME PLANTS
BY
ALI SALAMA ALI SALAMA

B.Sc. Agriculture (Agricultural Genetics and Genetic Engineering),
Faculty of Agriculture, Zagazig University, (2001)
Under the Supervision of:
Prof. Dr. S. H. Salem ........................................................

Prof. of Agric. Microbiology, Agric. Microbiology Dept.
Faculty of Agriculture, Zagazig University
Prof. Dr. Fatma I. El-Zamik ............................................

Prof. and Head of Agric. Microbiology Dept.
Dr. Howaida M. L. Abd El-Basit ....................................

Assistant Prof. of Agric. Microbiology, Agric. Microbiology Dept.
2
Approval Sheet
USE OF MICROORGANISMS AS BIOFERTILIZERS

FOR SOME PLANTS
BY
ALI SALAMA ALI SALAMA

B.Sc. Agriculture (Agricultural Genetics and Genetic Engineering),
Faculty of Agriculture, Zagazig University, (2001)
This thesis for M. Sc. Degree has been
approved by:
Prof. Dr. S. H. Salem ........................................................

Prof. Dr. E. M. Gewaily ...................................................
Prof. Dr. Wedad T. Ewada ..............................................
Faculty of Agriculture, Ain Shams University
Prof. Dr. Fatma I. El-Zamik ............................................
Prof. and Head of Agric. Microbiology, Agric. Microbiology
Dept. Faculty of Agriculture, Zagazig University
Date of examination: / / 2006

3
ABSTRACT
This work was carried out in the laboratory and greenhouse
of Agric. Microbiology Dept. at the Faculty of Agriculture,
Zagazig University, Egypt, in order to isolate and select efficient
indigenous strains of Rhizobium leguminosarum bv. vicieae,
Azospirillium spp. and Azotobacter spp. which isolated from
different locations in Sharkia governorate to use them as bio-
fertilizers for broad bean (Vicia faba L.) and wheat (Triticum
aestivum L.) plants. Thirty two Rhizobium isolates were selected
as effective strains for nodulation on broad bean, 18 isolates of
Azospirillum, and 14 isolates of Azotobacter were efficient in
nitrogenase activity in liquid cultures. Five isolates showing high
values in nitrogen fixation of R. leguminosarum bv. vicieae and
the highest five effective isolates of each of Azotobacter spp. and
Azospirillum spp. were used for GA3, IAA and IBA production
in liquid cultures. The selected bio-fertilizers were examined on
the effect on growth and productivity of broad bean and wheat
plants which cultivated in both sandy and clay soils under
different levels of inorganic and organic N fertilizers in six
greenhouse experiments. The results showed that the isolate
RZ11 was the highest efficient isolate of R. leguminosarum bv.
vicieae in plant growth, nodulation, nitrogenase activity and
production of growth promoting substances. In addition the
isolate ZH21 of Azotobacter spp. and isolate ASH21 of
Azospirillum spp. reflected the highest activity of nitrogenase
enzyme and production of growth promoting substances in liquid
cultures. The inoculation of broad bean and wheat plants grown
on both sandy and clay soils with the selected bio-fertilizers
(R. leguminosarum bv. vicieae, Azotobacter spp and
zospirillum spp) is importance to obtain the best performance in
growth, yield and total nitrogen and mineral contents.
4
ACKNOWLEDGMENT
This work has been carried out under the supervision and
direction of Prof. Dr. Samir H. Salem Prof. of Microbiology,
Agric. Microbiology. Dept. Fac. Agric., Zagazig University. To
him I express my deepest gratitude for suggesting this problem,
valuable assistance, progressive criticism, keeping interest and
guidance.
I also wish to express my thanks and gratitude to Prof. Dr.
Fatma I. El-Zamik Prof. and head of Agric. Microbiology
Department, Fac. Agric., Zagazig University for her supervision,
unlimited help and effort during the preparation of this
manuscript.
Thanks are also due to Dr. Howaida M. L. Abd El-Basit
Assistant Prof. of Microbiology,in the same Department for
supervising this work and here valuable advise and guidance
Gratitude are also due to all the staff members and
colleagues in Agric. Microbiology. Dept. Fac. Agric., Zagazig
University for their encouragement and providing facilities
throughout this work.
5
CONTENTS
Title of contents Page
I-INTRODUCTION 1
II-REVIEW OF LITERATURE 4
III- MATERIALS AND METHODS 43
IV-RESULTS AND DISCUSSION 71
4.1. Testing the efficiency of different isolates of
Rhizobium, Azotobacter and Azospirillum..............................................
71
4.1.1. Testing the efficiency of Rhizobium isolates .........................................
71
4.1.1.1. Nodulation 72
4.1.1.2. Nitrogenase activity 73
4.1.1.3. Plant dry weight 74
4.1.1.4. Nitrogen content 75
4.1.1.5. The phage reaction of Rhizobium isolates .............................................
78
4.1.2. Testing the efficiency of N2-fixation by
Azotobacter and Azospirillum isolates in liquid
culture 79
4.1.3. Determination of growth promoting substances in
liquid cultures of Rhizobium, Azotobacter and
Azospirillum isolates 82
4.1.3.1. GA3, IAA and IBA in Rhizobium isolates .............................................
82
4.1.3.2. GA3, IAA and IBA in Azotobacter isolates ..............................................
83
4.1.3.3. GA3, IAA and IBA in Azaspirillium isolates ........................................
83
4.2. Response of broad bean plants to bio-fertilizers and
inorganic N-fertilizers under cultivation in sandy
and clay soils 86
4.3. Response of broad bean plants to bio and organic
fertilizers under cultivation in sandy and clay soil ....................................
109
6
4.4. Response of broad bean plants to bio-fertilizers

under cultivation in sandy and clay soils ....................................................
130
4.5. Effect of bio-fertilizers only or combined with
inorganic or organic N-fertilizers on GA3, IAA
and IBA content in roots of broad bean plants ...........................................
152
4.6. Comparison the response of broad bean plants to
application of biofertilizers only or combined with
organic or inorganic N-fertilizers with control
treatment in both sandy and clay soil ..........................................................
155
4.7. Response of wheat plants to bio-fertilizers and
and clay soils 159
4.8. Response of wheat plants to bio-and organic
fertilizers under cultivation in sandy and clay soils ..................................
181
4.9. Response of wheat plants to bio-fertilizers under
cultivation in sandy and clay soils 202
inorganic or organic N-fertilizers on GA3, IAA
and IBA content in roots of wheat plants .................................................
221
4.11. Comparison between the different responses of
wheat plants to application of bio-fertilizers only
or combined with organic or inorganic N-
fertilizers with control treatment in both sandy and
clay soil 224
V- SUMMARY AND CONCLUSION ..........................................................
228
VI- LITIRATURE CITED 233
VII- ARABIC SUMMARY
7
LIST OF TABLES
Table
No. Title of Tables page
1 The locations of soil samples selected for isolation

of rhizobia, azotobacter and azospirilla 47
2 physical and chemical analyses of the soil under
investigation 57
3 Shoot and root dry weight (g/plant), total nitrogen
content, nitrogenase activity and phage interaction
in nodulation test of broad bean plants, as affected
by inoculation with different isolates of rhizobia ......................................
73
4 physical and chemical analyses of soil in deffernt
locations of Sharkia governorate 77
5 Nitrogenase activity by Azotobacter and
Azospirillum isolates in liquid cultures 81
6 GA3, IAA and IBA in liquid culture of Rhizobium
leguminosarum bv. vicieae, Azotobacter and
Azospirillum isolates 84
7 Effect of bio-fertilizers and inorganic N-fertilizers
on number of nodules, dry weight of nodules
(mg/plant) and nitrogenase activity of broad bean
plants 87
on shoot, root and whole plant dry weight (g/plant)
of broad bean plants 91
on total nitrogen content in shoot, root and whole
plant (mg N/plant) of broad bean plants 96
on total phosphorus content in shoot, root and
whole plant (mg P/plant) of broad bean plants ..........................................
99
8

on total potassium content in shoot, root and whole
plant (mg K/plant) of broad bean plants 103
12 Effect of bio-fertilizers and inorganic N-fertilizers on
weight of 100 seeds (g), nitrogen % and protein % of
broad bean plants 106
13 Effect of bio and organic fertilizers on number of
nodules, dry weight of nodules (mg/plant) and
nitrogenase activity of broad bean plants ..................................................
110
14 Effect of bio and organic fertilizers on shoot, root
and whole plant dry weight (g/plant) of broad bean
plants 114
15 Effect of bio and organic fertilizers on total
nitrogen content in shoot, root and whole plant
(mg N/plant) of broad bean plants 118
phosphorus content in shoot, root and whole plant
(mg P/plant) of broad bean plants 121
potassium content in shoot, root and whole plant
(mg K/plant) of broad bean plants 125
18 Effect of bio and organic fertilizers on weight of
100 seeds (g), nitrogen % and protein % of broad
bean plants 128
19 Effect of bio-fertilizers on number of nodules, dry
weight of nodules (mg/plant) and nitrogenase
activity of broad bean plants 132
20 Effect of bio-fertilizers on shoot, root and whole
plant dry weight (g/plant) of broad bean plants .........................................
136
21 Effect of bio-fertilizers on total nitrogen content in
shoot, root and whole plant (mg N/plant) of broad
bean plants 139
9
22 Effect of bio-fertilizers on total phosphorus content

in shoot, root and whole plant (mg P/plant) of
23 Effect of bio-fertilizers on total potassium content
in shoot, root and whole plant (mg K/plant) of
24 Effect of bio-fertilizers on weight of 100 seeds (g),
nitrogen % and protein % of broad bean plants .........................................
149
25 Effect of bio-fertilizers only or combined with
inorganic or organic N-fertilizers on GA3, IAA and
IBA in root of broad bean plants 153
26 Comparing the response of broad bean plants to
application of bio and inorganic, bio and organic
and bio-fertilizers only in sandy soil 156
27 Comparing the response of broad bean plants to
and bio-fertilizers only in clay soil 158
on shoot, root and whole plant dry weight (g/plant)
in wheat plants 160
on nitrogenase and dehydrogenase activities
inrhizosphere of wheat plants 165
on total nitrogen content in shoot, root and whole
plant (mg/plant) in wheat plants 167
on total phosphorus content in shoot, root and
whole plant (mg/plant) in wheat plants 171
on total potassium content in shoot, root and whole
plant (mg/plant) in wheat plants 174
10

on weight of 1000 grains (g), nitrogen % and
protein % in wheat plants 178
34 Effect of bio and organic fertilizers on shoot, root
and whole plant dry weight (g/plant) in wheat
plants 182
35 Effect of bio and organic fertilizers on nitrogenase
and dehydrogenase activities in rhizosphere of
wheat plants 186
nitrogen content in shoot, root and whole plant
(mg/plant) in wheat plants 189
phosphorus content in shoot, root and whole plant
potassium content in shoot, root and whole plant
39 Effect of bio and organic fertilizers on weight of
1000 grains (g), nitrogen % and protein % in wheat
plants 200
40 Effect of bio-fertilizers on shoot, root and whole
plant dry weight (g/plant) in wheat plants .................................................
204
41 Effect of bio-fertilizers nitrogenase and
dehydrogenase activities in rhizosphere of wheat
plants 207
42 Effect of bio-fertilizers on total nitrogen content in
shoot, root and whole plant (mg/plant) in wheat
plants 209
in shoot, root and whole plant (mg/plant) in wheat
plants 212
11

in shoot, root and whole plant (mg/plant) in wheat
plants 216
45 Effect of bio-fertilizers on weight of 1000 grains
(g), nitrogen % and protein % in wheat plants ..........................................
218
46 Effect of bio-fertilizers only or combined with
IBA in root of wheat plants 222
47 Comparing the response of wheat plants to
and bio-fertilizers only in sandy soil 225
48 Comparing the response of wheat plants to
and bio-fertilizers only in clay soil 227
12
LIST OF FIGURES
Fig.
No. Title of Figure page
1 Map of Sharkia governorate showing localities of

isolation 44
number and dry weight of nodules in broad bean
plants grown in sandy and clay soils 88
dry weight of broad bean plants grown in both sandy and
clay soils 92
total nitrogen content (mg/plant) of broad bean
cultivated in both sandy and clay soils 97
total phosphorus content (mg/plant) of broad bean
total potassium content (mg/plant) of broad bean
grain yield, nitrogen (%) and protein (%) in seeds of
broad bean cultivated in both sandy and clay soils ...................................
107
8 Effect of bio and organic fertilizers on number and
dry weight of nodules in broad bean plants grown in
sandy and clay soils 111
9 Effect of bio and organic fertilizers on dry weight of
broad bean plants grown in both sandy and clay soils ..............................
115
10 Effect of bio and organic fertilizers on total nitrogen
content (mg/plant) of broad bean cultivated in both
13

phosphorus content (mg/plant) of broad bean
cultivated in both sandy and clay soils126 122
potassium content (mg/plant) of broad bean
13 Effect of bio and organic fertilizers on grain yield,
nitrogen (%) and protein (%) in seeds of broad bean
14 Effect of bio-fertilizers on number and dry weight of
nodules in broad bean plants grown in sandy and
clay soils 133
15 Effect of bio-fertilizers on dry weight of broad bean
plants grown in both sandy and clay soils .................................................
137
16 Effect of bio-fertilizers on total nitrogen content
(mg/plant) of broad bean cultivated in both sandy
and clay soils 140
and clay soils 143
and clay soils 147
19 Effect of bio-fertilizers on grain yield, nitrogen (%)
and protein (%) in seeds of broad bean cultivated in
both sandy and clay soils 150
dry weight of wheat plants grown in both sandy and
clay soils 167
total nitrogen content (mg/plant) of wheat cultivated
in both sandy and clay soils 168
total phosphorus content (mg/plant) of wheat
14

total potassium content (mg/plant) of wheat
24 Effect of bio-fertilizers and Inorganic N-fertilizers on
grain yield, nitrogen (%) and protein (%) in grains of
wheat cultivated in both sandy and clay soils ............................................
179
25 Effect of bio and organic fertilizers on dry weight of
wheat plants grown in both sandy and clay soils.......................................
183
26 Effect of bio and organic fertilizers on total nitrogen
content (mg/plant) of wheat cultivated in both sandy
and clay soils 190
phosphorus content (mg/plant) of wheat cultivated in
potassium content (mg/plant) of wheat cultivated in
29 Effect of bio and organic fertilizers on grain yield,
nitrogen (%) and protein (%) in seeds of wheat
30 Effect of bio-fertilizers on dry weight of wheat plants
grown in both sandy and clay soils 205
31 Effect of bio-fertilizers on total nitrogen content
(mg/plant) of wheat cultivated in both sandy and clay
soils 210
soils 213
soils 217
34 Effect of bio-fertilizers on grain yield, nitrogen (%)
and protein (%) in seeds of wheat cultivated in both
15
I. INTRODUCTION
The intensive agricultural farming system as practical in
Egypt, where the crop rotation consists of 2 or 3 crops per year,
are main reasons for the high consumption of chemical
fertilizers. Intensive methods of farming and food production are
having unfavorable consequences on the quality of food,
environment and on animal and human health. In these systems,
agriculture is treated like other industries, with an emphasis on
efficiency and maximum productivity regardless of their impacts
on human health and ecology. The amount of nitrogen applied to
the soil in intensive agricultural systems varies considerably,
depending upon the crop being grown, the soil type, and the
previous cropping history of the soil. Nowadays, the harmful
effects on the environment of heavy use of N fertilizers are
becoming more evident. Furthermore, the fossil fuels which are
used in the production of N fertilizers are becoming scarcer and
more expensive. Therefore, there is a great need to search for all
possible avenues to improve biological nitrogen fixation and its
use by farmers through bio-fertilization process (Hussien et al.,
1997).
Nitrogen fixation by the legume-Rhizobium symbiotic
partnership represents an inexpensive alternative to the use of
chemical nitrogen fertilizers in the production of food protein
and oil. The process requires that the host crop be adequately
nodulated by Rhizobium bacteria effective in nitrogen fixation.
Inoculation of legumes with suitable rhizobial strains is carrying
out in many countries to ensure nodulation (Brock et al. 2003).
16
A wide range of bacteria in rhizosphere can promote plant

growth, orchestrated by rhizosphere bacteria that communicate
with the plant using complex chemical signals, gibberellins,
glycolipids and cytokinins are now beginning to be fully
appreciated in terms of their biotechnological potential.
A critical process that occurs on the surface of the plant and
particularly in the root zone, is associative nitrogen fixation in
which the nitrogen fixing microorganisms is on the surface of the
plant root, the rhizoplane, as well as in the rhizosphere. This
process is carried out by representatives of the genera
Azotobacter and Azospirillum.
Recent evidence sugests that their majour contribution may
not be in nitrogen fixation but in production of growth-
promoting hormones that increase root hair development and
thus greater ability of the plant to take up nutrients. This is an
area of research that is particularly important in tropical
agricultural areas (Prescott et al. 2005).
Inoculation with indigenous Azospirillum strains is
important procedure when studying their inherent capacity to
benefit crops. In some cases, indigenous can perform better than
introduced strains in promoting the growth of crops due to their
superior adaptability to the environment (Gunarto et al. 1999).
On the other hand, application of organic fertilizers was shown
to enhance the incidence and activities of promoting plant
rhizobacteria and stimulating plant growth (Khamis and
Metwally, 1998).
For all reasons, there is a widespread interest in the use of
combination of mineral fertilizers and biofertilizers as an
17
alternative and cheap source for chemical fertilizers. From the

economic point of view the cost of inoculantes is not usually a
constraint to their use by farmers who outlay capital for seed.
Inoculant cost will seldom exceed 1 % of the seed cost (Bohlool
et al., 1992).
This study was conducted with the following objectives:
1. To isolate and select of efficient isolate of endigenous
Rhizobium leguminosarum bv. vicieae, Azospirillum spp
and Azotobacter spp from Sharkia governorate soils and
use them as bio-fertilizers in the next study.
2. To study the effect of selected bio-fertilizers with or
without different levels of inorganic N-fertilizers in two
soil textures on the growth of broad bean and wheat
plants.
3. To study the response of broad bean and wheat plants to
the selected bio-fertilizers with or without different levels
of organic amendment under cultivation in sandy and clay
soils.
4. To study the effect of selected bio-fertilizers only in two
soil textures on the growth of broad bean and wheat
plants.
5. To study the effect of bio-fertilizers only or combined
with organic or inorganic N-fertilizers on GA3, IAA and
IBA content in roots of broad bean and wheat plants.
18
II. REVIEW OF LITERATURE

The term "Biofertilizers" or more appropriately "microbial
inoculant" can be generally defined as preparations containing
live or latent cells of efficient strains of nitrogen-fixing,
phosphate solubilizing or celluloytic microorganisms used for
application to seed, soil or composting areas with the objective
of increasing the numbers of such microorganisms and
accelerating certain microbial processes to augment the extent of
the availability of nutrients in a form which can be easily
assimilated by plants. Subba Rao, 1993 added also that, in a
larger sense, the term may be used to include all organic
resources (manures) for plant growth which are rendered in an
available form for plant abstraction through microorganisms or
microorganisms plant associations or interactions.
Recently, Subba Rao (1999) mentioned that biofertilizers
are carried based preparations containing beneficial
microorganisms in available state intended for seed or soil
application and designed to improve soil fertility and help plant
growth by increasing the number and biological activity of
desired microorganisms in the root environment. Biofertilizers
are ecofriendly and cannot at any rate replace chemical fertilizers
that are indispensable for getting maximum yield of crops.
In general, biofertilizers are environment friendly, low cost
agricultural input with maximum output. These biofertilizers are
to play an important role in enhancing crop productivity through
nitrogen fixation, phosphate solubilization, plant hormone
productivity, ammonia excretion, siderophore formation and to
control various plant disease (Pankhurst and Lynch, 1995;
19
Pathak et al., 1997; Dadarwal et al., 1997 and Hedge et al.,

1999).
In Egypt, for instance, many types of peat based inoculants
of Rhizobium, Azospirillum and phosphate dissolving bacteria
are now produced and distributed to farmers under the
supervision of General Organization for Agriculture Equalization
Funds (GOAEF) which is belonged to Egyptian Ministry of
Agriculture and Land Reclamation (Abou El-Naga, 1994).
2.1. Biological nitrogen fixation
2.1.1. Symbiotic nitrogen fixers (Rhizobium)
The interaction of leguminous plant and bacteria of the
genera Rhizobium, Bradyrhizobium and Azorhizobium result in
the formation of root nodules, new organs in which the bacteria
are able to fix and reduce atmospheric nitrogen into ammonia
available for plant biosyntheses.
Legumes are commonly inoculated with efficient nitrogen-
fixing strains of rhizobia for maximizing crop productivity.
Nodule induction at high frequency by introduced inoculum
strains has been readily demonstrated in instances where
legumes have been grown on soil deficient in indigenous
rhizobia (Salem 1962, 1969; Bell and Nutman, 1971;
Roughley et al., 1976; Bromfield and Ayanaba, 1980;
Brockwell et al., 1987; Somasegaran et al., 1988).
Gewaily et al. (1981) carried out a pot experiment using
sterilized sand to test the growth and N content of soybean
inoculated with 6 different strains of Bradyrhizobium and broad
bean inoculated with 8 different strains of R. Leguminosarum bv.
vicieae only. They found that, strain No. 3409 belonging to
20
Bradyrhizobium out of six tested organisms, and four strains

belonging to R. Leguminosarum bv. vicieae out of 8 used were
categorized as efficient strains. Respiration rates of all these
strains of rhizobia in pentoses or hexoses sugars were highly
variable. When glycine was added, it stimulated the respiratory
rates of some of the strains only, which were in general found to
be quite efficient in N2-fixation in pot culture trials.
In addition Wagner and Zapata (1982) used various
reference crops and N15 A-value to estimate symbiotic
N2-fixation by inoculated broad bean. They found that N15
A-value for broad bean were higher than those for any of the non
N2-fixing crops, and the nitrogen fixed-estimated from A-value
was approximately 140 kg ha-1.
However, Abdallah et al. (1989) studied the biologically
fixed nitrogen by three cultivars of broad bean i.e., Giza 2, Giza
3 and Giza 402, grown on alluvial clay loam soil in the presence
of 20 kg N ha-1 and pH 8.1 using N15 dilution technique with
barley and wheat as reference crops. They found that amount of
N2- fixed varied according to the broad bean cultivar, the highest
fixed quantity was recorded by Giza 3 followed by Giza 2 and
Giza 402 being 113.9, 99.8 and 91.4 kg N ha-1, respectively.
Also Hassan et al. (1990) estimated the N2-fixed by broad
bean in three soil types namely clay, calcareous and sandy soils
by using A-value technique and wheat as reference crop. They
found that the amount of N2-fixed markedly differed depending
upon inoculation treatment, dose of N fertilizers applied and soil
type. They reported also that, the percentages of nitrogen derived
from atmosphere in uninoculated plants were 32.95-69.40 %
21
compared with 40.51-75.56 % in inoculated ones. On the other

hand, increasing dose of nitrogen fertilizer led to marked
decrease in nodulation of broad bean and amount of N2-fixed.
Also clay soil showed relatively lower percentage of nitrogen
derived from atmosphere (40.5-63.91%) as compared with both
calcareous (45.56-75-56%) and sandy (47.03-67.95%) soils.
Moreover, Hussein et al. (1997) reported that Rhizobium
inoculation significantly increased the number and dry weight of
nodules of broad bean cultivated in newly reclaimed soil of
Egypt. They showed that Rhizobium inoculation, combined with
the highest rate of P and K, produced the highest number
(51.7/plant) and dry weight (353.1 mg/plant) of nodules, dry
weight of shoot and N-content as well as protein yield.
The most important microorganisms used as inoculants,
today are the rhizobia that added to legume seeds to ensure
successful inoculation (Coyne, 1999). In early times, inoculation
methods involved the transfer of soil from the roots of
well-nodulated plants to the seeds at planting. However, during
the past few decades, different types of inoculants have been
developing and significant advances in formation technology and
delivery of Rhizobium inoculants are presented. In addition, there
is interest in developed co-inoculants containing other micro-
organisms which are able to improve legume growth (Mishra et
al., 1999 and Rodelas et al., 1999). These include rhizobacteria
which promote nodulation, nitrogen fixation, plant vigour and
yield via such mechanisms as phytohormones, antibiotic or
metal-binding compound production, bacteria or fungi which
protect against specific root phathogens and other which aid in
22
nutrient supply via phosphate solubilization. For instance,

Rodelas et al., (1999) pointed out that co-inoculants of broad
bean with R. Leguminosarum bv. vicieae plus plant growth
promoting Azotobacter and Azospirillum led to changes in total
content concentration and/or distribution of the macro and
micronutrients, K, P, Ca, Mg, Fe, B, Mn, Zn and Cu when
compared with plants inoculated with Rhizobium only.
Schulze et al., (2000) studied the efficiency of N2-fixation
in Vicia faba L. in combination with different R. leguminosarum
strains. They used three Rhizobium leguminosarum strains
inoculated in Vicia faba in a pot experiment during vegetative
and reproductive growth. Dry matter formation, N2 fixation and
the carbon (C) costs of N2 fixation were determined in
comparison with nodule free plants grown with urea. Nodule
number and the capacity of different respiratory chains in the
nodules were also measured. The C costs for N2 fixation were in
all cases significantly lower during reproductive growth
compared to vegetative growth. Neither the latter nor the
differences in C expenditure for N2 fixation between the
Rhizobium strains could be explained in terms of differences or
shifts in the capacity of different respiratory chains in the
nodules.
In addition, Hamaoui et al. (2001) studied the effects of
inoculation with Azospirillum brasilense on chick pea (Cicer
arietinum) and broad beans (Vicia faba) under different
conditions in greenhouse experiments with both legumes.
Inoculation with Azospirillum brasilense significantly enhanced
23
nodulation by native rhizobia and improved root and shoot

development when compared with non inoculated controls.
Moreover, Zaied et al. (2002) tested the response of broad
bean to inoculation with fungicide auxotrophic mutants induced
in Rhizobium leguminosarum bv. vicieae. They found that some
auxotrophic mutants induced significantly increased nodule
developments, dry matter yield and nodule dry weight, in
contrast, some other results in fewer nodule development.
On the other hand, Ma-Wenbo et al. (2002) showed the
strategies used by rhizobia to lower plant ethylene levels and
increase nodulation. They found that the phytohormone,
ethylene, acts as a negative factor in the nodulation process.
Recent discoveries suggest several strategies used by rhizobia to
reduce the amount of ethylene synthesized by their legume
symbionts decreasing the negative effect of ethylene on
nodulation.
Dakora, (2003) reported that defining new roles for plant
and rhizobial molecules in sole and mixed plant cultures
involving symbiotic legumes. Rhizobia (species of Rhizobium,
Bradyrhizobium, Azorhizobium, Allorhizobium, Sinorhizobium
and Mesorhizobium) produce chemical molecules that can
influence plant development including phytohormones, lipo-
chito-oligosaccharide Nod factors, lumichrome, riboflavin and
H2 evolved by nitrogenase. Very low concentrations of
lumichrome and H2 released by bacteroids promote plant growth
and increase biomass in a number of plant species grown under
field and glasshouse conditions.
24
Although, Humphry et al. (2003) studied the genotypic

and phenotypic characterization of the symbiotic properties of
Rhizobium leguminosarum strains isolated from Jordanian Vicia
faba. This study was conducted to compare Jordanian Rhizobium
leguminosarum bv. vicieae from Vicia faba to their British
counterparts to determine the relatedness of their chromosomal
and symbiotic genotypes. It was determined that Jordanian
isolates are genotypically distinct from the UK isolates, but from
anovel group with Rlv, on the basis of chromosomal (glutamine
synthetase gene) and nodulating V. sativa and V. cacca in
addition to V. faba also a gene (Nodx) responsiple for conferring
the ability to nodulate Pisum sativum cv. Afghanistan by R1v is
described.
More recently, Shumin et al. (2004) studied the enhancing
phosphorus and nitrogen uptake of broad bean inoculating
arbuscular mycorrhizal fungus and R. leguminosarum. They
found that the dual inoculation of broad bean with
R. leguminosarum and G. mosseae increased the plant height,
chlorophyll content and number and weight of nodules of broad
bean. The biomass of broad bean increased by 21.5 and 20.7 %
when inoculated with G. mosseae alone and combined with
R. leguminosarum, respectively. Mycorrhizal colonization
increased by approximately 12 % with the inoculation of
R. leguminosarum, the acid phosphatase and alkaline
phosphatase activities of broad bean increased compared with
the control when inoculated with both G. mosseae and
R. leguminosarum. Also, phosphorus and nitrogen uptake
increased by more than 50.9 and 22.0 %, respectively.
25
2.1.2. Non symbiotic nitrogen fixers

The association between N2-fixing bacteria and root of
non-legume plants, the organisms are present on rhizosphere and
rhizoplane of the root zones. Studying the bacterial inoculation
conditions such as, size of bacterium inoculum , time of
inoculation, amount and concentration of organic matter added,
mineral in soil and interaction between bacterial strains and host
plant an increases of 10-30 % were reported in grain and forage
yields with wheat, corn and sorghum after inoculation with
Azospirillum ( Subba Rao, 1993).
Döbereiner (1974) reported that contribution of biological
nitrogen fixation in Barzial may reach to 70 % for sugar cane
and up to 50 % in cereals through the activity of endophytic
diazotroph in non-legume.
2.1.2.1. Azospirillum
Azospirillum, an associative microaerophillic nitrogen fixer
commonly found in loose association with roots of cereals and
grasses which is of great interest. High nitrogen fixation
capacity, low energy requirement and abundant establishment in
the roots of cereals and tolerance to high soil temperature
(30-40 °C) are responsible for its suitability under tropical
conditions
(Hedge et al., 1999). Azospirilla are metabolically versatile and
can grow vigorously in presence of nitrogenous compounds
present in soil but as soon as the external combined nitrogen
supply is exhausted, the bacteria switch on to diazotrophy. The
ability to fix nitrogen is unaffected by presence of combined
nitrogen sources, and may account for the beneficial response of
26
Azospirillum inoculation in field receiving mineral fertilizers

(Fages, 1994; Bashan and Holguin, 1997 and Hedge et al.,
1999).
Of late, attention has been shifted from Azotobecter to
Azospirillum as an inoculant due to its widerspread distribution
in soil, association of crops grown on acidic to alkaline pH
range, easy to culture and identification because of its curved
form and type of motility and is relatively efficient in utilization
of carbon support N2-fixation. The application areas for
Azospirillum and Azotobacter are overlapping. However, in
general, Azospirillum is more appropriate for cereals and
azotobacter for non-food grain crops such as sugarcane, cotton,
potato and other vegetables (Chhonkar and Tilak 1997 and
Hedge et al., 1999).
The mechanism of the bacterization resulting in yield
increase with decrease or increase in N-concentration may be
attributed to enhanced N2-fixation or increased N assimilation by
plant (Aggarwal and Chaudhary, 1995 and Bashan and
Holguin, 1997) enhanced mineral uptake in the plant
(Stancheve et al., 1995) improved root growth and functions
(Sarig et al., 1992 and Fallik et al., 1994) nitrate production in
nitrate respiration (Bothe et al., 1992). In vitro Azospirillum
lipoferum produces siderophores may which improve iron-
nutrition of plant (Hedge et al., 1999) produces in vitro the
phytohormones IAA, gibberllins, cytokinin and ethylene. These
phytohormones, especially IAA play an essential role in plant
growth stimulation in general and in stimulating symbiosis
between legumes and rhizobia (Bashan and Holguin 1997) and
27
effects plant all metabolism from outside suggests that bacteria

are capable of excreting and transmitting a signal(s) which
crossed the plant cell wall and is recognized by plant membranes
and promoted nitrogen fixation. The modes of action of
Azospirillum proposed over the last three decades point to the
possibility that perhaps there is no major single mechanism
involved. The combined activities of all the involved
mechanisms may be responsible for the large measured effect of
Azospirillum spp. (Bashan and Holguin, 1997).
Egorenkova et al. (2000) noted the investigation of the
initial stages of interaction of the bacterium Azospirillum
brasilense with wheat seedling root adsorption and root hair
deformation. They showed that the adsorption of azospirilla on
root hair of soft spring wheat rapidly increased in the first hours
of incubation going then to a plateauphase.
In addition, Deaker and Kennedy (2001) studied the the
improved potential for nitrogen fixation in Azospirillum
brasilense SP7.5 associated with wheat nifH expression as a
function of oxygen pressure. They showed a strong correlation
between nitrogenase activity and nifH expression was found in
pure cultures. nifH expression was maximal at 0.5 % oxygen in
pure cultures of both the wild type SP7 and spontaneous mutant
SP7.5.
Moreover, Kaushik et al., (2001) tested the selection and
evaluation of Azospirillum barasilense strains growing at a
sub-optimum temperature in rhizocoenosis with wheat. They
showed that significant increase in plant growth parameters; the
overall response to inoculation was better in cultivar HD2285.
28
Also, Swedrzynska and Sawicka (2001) studied the effect

of inoculation with population numbers of Azospirillum bacteria
on winter wheat, oat and maize. They showed that the
inoculation of cereals with Azospirillum barsilense bacteria
contributed to the increase of their number in soil.
On the other hand, Pinheiro et al. (2002) studied the
adsorption and anchoring of Azospirillum strains to roots of
wheat seedlings. They showed that strains of A. brasilense,
originally isolated from surface sterilized wheat roots (SP 245,
SP 107) or with a proven ability to infect the interior of roots
(SP 245), showed no greater ability to anchor to the roots than
other Azospirillum strains isolated from wheat rhizosphere
(SP 246) or from the rhizosphere or rhizosphere soil of other
gramineae (SP 7, cd, S 82).
Recently, Amoo et al. (2003) investigated the effect of
Azospirillum inoculation on some growth parameters and yield
of three wheat cultivars. They showed that inoculation increased
growth parameters and yield of wheat cultivars, the effect being
directly dependent on the strain-cultivar combination.
Creus et al. (2004) studied the water relations and yield in
Azospirillum-inoculated wheat exposed to drought in the field.
They showed that the grains harvested from Azospirillum-
inoculated plants had significantly higher Mg, K and Ca than
non-inoculated plants. Neither drought nor inoculation changed
grain P, Cu, Fe and Zn contents.
2.1.2.2. Azotobacter
Azotobacter, a free-living heterotrophic, nitrogen-fixing
bacteria encountered in neutral to alkaline soils not provides the
29
nitrogen but produce a variety of growth promoting substances.

Some of these growth promoting substances are indole acetic
acid, gibberllins, B vitamins and antifungal substances. Another
important characteristic of Azotobacter association with crop
improvement is excretion of ammonia in the rhizosphere in the
presence of root exudates which helps in modification of nutrient
uptake by plant (Narula and Gupta, 1986). Solubilization of
different inorganic phosphates by A. chroococcum was also
reported (Tilak and Singh, 1994). The mechanisms by which
the plants inoculated with Azotobacter derive positive benefits in
terms of increased grain, plant bio mass and N uptake which are
attributed to small increase in N input from biological nitrogen
fixation, development and branching of roots, production of
plant growth hormones, enhancement in uptake of No3, NH4+,
H2Po4, K, Rb and Fe, improved water status of plants, increased
nitrate reductase activity and antifungal compounds (Wani et al.,
1988 and Hedge et al., 1999).
Several experiments conducted in temperate regions of the
world showed that nitrogen fixation in Azotobacter inoculated
soil is not more than 10 to 15 kg of N/ha/year, depending on the
availability of carbon source (Subba Raw, 1999).
On the other hand, Hedge et al. (1999), mentioned that in
many cases, inoculants increase plant yield and such increase is
statistically significant and also sometimes negative. Also,
experiments with Azotobacter and crop plants cultivars at the
Indian Agriculture Research Institute, New Delhi were carried
out and the obtained results indicated that significant increases in
30
growth and yield of wheat, rice and vegetable crops could be

obtained in pot trails.
However, under field conditions, such uniform trends
towards increases in yield are not always reproducible
(Subba Rao, 1999).
The benefits of Azotobacter inoculation in cereal
production are well documented in wheat (Lakshminarayana
et al., 1992). in barley (Tiwari et al., 1989); in sorghum (Lee
et al., 1994); in maize (Gill et al., 1993), in rice (Wani, 1990)
and in pearlmillet (Wani et al., 1988).
Ishac and Mostafa (1998) evaluated the interaction of
Azotobacter and vesicular arbuscular mycorrhizas and proposed
several mechanisms to mediate Azotobacter. VAM interactions:
hormonal effects, enhanced nutrients uptake, N2-fixation and
increased resistance to soil borne pathogenic fungi. They
reported that the synergistic interaction between Azotobacter and
VAM may lead to substantial increase in growth, mineral uptake
and yield of dually inoculated plants.
Kumar et al. (2001) studied that the effect of phosphate
solubilization strains of Azotobacter chroococcum on yield traits
and their survival in the rhizosphere of wheat genotypes under
field condition. They showed that seed inoculation of wheat
varieties with phosphate-solubilizing and phytohormone-
producing A. chroococcum showed a better response over
control.
Sharma et al. (2001) etsted the survival of Azotobacter
chroococcum in the rhizosphere of three different wheat crosses:
effect of AM fungi. They found that effect of wheat genotypes
31
and AM inoculation on the proliferation and survival

A. Chroococcum were prominent.
Behl et al. (2003) investigated the interactions among
mycorrhiza, Azotobacter chroococcum and root characteristics of
wheat varieties. They found that a positive association between
AMF infection in roots and Azotobacter survival in the
rhizosphere was apparent.
Vivek et al. (2004) studied the relative efficacy of
Azotobacter chroococcum on tall and dwarf wheats (Triticum
aestivum L.) in arid soils. They found that grain, straw yield and
root biomass were higher with mutants, which maintained higher
survival rate in rhizosphere during growth period of wheat crop.
2.2. Hormones and production of growth promoting
substance by nitrogen fixing bacteria
There is firm evidence that plant growth regulators like
auxine, gibberellins, cytokinins and ethylene produced by plant
are essential for their growth and development. There is also
evidence that plant growth hormones produced bacteria can
increase growth rates and improve yield of host plants
(Barea and Brown, 1974).
Azcon and Barea (1974) reported that culture supernatants
of Azotobacter vinelandii and Azotobacter beijerinkii contain
auxines, at least three of gibberellins-like substance and three of
cytokinins-like substances. They added that the amount of
hormones produced in these cultures are similar to those
produced by Azotobacter paspali and Azotobacter chroococcum.
32
Reynders and Vlassak (1979) found that all the tested

strains of Azospirillum converted tryptophane to indole-3-acetic
acid in pure culture.
Gaskin et al. (1977) reported that auxines produced by
Azospirillum stimulated the plant growth. Azospirillum produced
plant growth substance, namly indole acetic acid, indole lactic
acid, gibberellins and cytokinines like substance, such hormones
induced the proliferation of lateral roots and root hairs which
increase nutrient absorbing surfaces (Tien et al., 1979; Fouad,
1981 and Vose and Ruschel, 1981).
Shank and Smith (1984) reported that the increase of grass
crops after inoculation with Azospirillum spp is due to the
stimulation effect of phytohormons produced by bacteria.
Investigation about the mechanisms showed that the
beneficial effects of most plant growth promoting rhizobacteria
(PGPR) increased plant growth indirectly by changing the
microbial balance in the rhizospheres (Klopper and Schroth,
1981), by producing iron chelating siderophores and by
phytohormones or other plant growth enhancing compounds
(Schippers, 1988).
Kucey (1988) found that responses of wheat plants to
inoculation with Azospirillum barsilense and Bacillus C-11-25
were similar to those caused by addition of gebberellic acid in
growth pouches. Also, Taller and Wong (1989) reported that
Azotobacter vinelandii can produce cytokinine like substance in
culture medium.
Grozier et al. (1988) and Fallik et al. (1989) stated that the
isolate strains of Azospirillum are capable of producing some
33
hormones at very low levels namely indole lactic acid, indole-3-

Butyric acid, indole-3-ethanole, abscisic acid, and several
gibberellins and cytokinines
In addition, to the beneficial effects of N2-fixing bacteria
associated with roots of cereal crops, these bacteria are also
reported to produce growth promoting substance which help
increasing crop yield (De-Freitas and Germida, 1990).
Baca et al. (1994) stated the indole-3-acetic acid (IAA) is
excreted by different wild strains of Azospirillum spp. They
added that microorganisms can produce IAA during the late
stationary growth phase and significant increase in IAA
production occurred when tryptophan is added.
Strzelezyk et al. (1994) pointed out that some strains of
Azospirillum produced cytokinine-like substance (CLS) and all
tested strains of Azospirillum produced ethylene in presence of
different carbon source like methionine, malate, succinate and
pyruvate. Azospirillum can produce in-vitro the phytohormones
IAA, cytokinine and gebberellines (Rademcher, 1994;
Iosipenko and Iganov, 1995 and Patten and Glick, 1996).
Response of cereals and vegetables to inoculation with some
N2-fixing bacteria was attributed to the effect of plant growth
regulators released by such microorganisms (Beylar et al., 1997
and Salamone et al., 1997).
Amara and Dahodoh (1997) carried out a pot experiment
to study the effect of inoculating wheat grains with plant growth
promoting bacteria on yield and nutrients uptake. The inocula
used were different species of Azotobacter and Azospirillum and
Pseudomonas individually or in mixture and they improved grain
34
yield and total dry weight. Inoculation with mixture of these

strains showed the highest value of straw yield and increased the
uptake of N, P, K, Fe, Na, Zn, Mn and Cu.
Dobbelaera et al. (1999) reported that auxine produced by
Azospirillum is believed to play a major role in the plant growth
promoting effect. In addition, El-Sawah (2000) found significant
increase in N, P and K content of maize plant when seeds were
inoculated with Azospirillum brasilense and Bacillus
megatherium as well as low dose of mineral nitrogen fertilizers
was applied.
Zakharova et al., (2000) studied the effect of water-soluble
vitamins on the production of indole-3-acetic acid by
Azospirillum brasilense. They found that very low levels of
vitamin B which added at 10-100 μg 1-1 affected production of
indole-3-acetic acid in Azospirillum brasilense. The largest
release of these phytohormone was observed after amendment
with pyridoxine and nicotinic acid. Results of the study suggest a
role of these vitamins which may fulfill in the regulation of
indole-3-acetic acid synthesis in Azospirillum brasilense.
Dobbelaera et al., (2001) found that Azospirillum are able
to promote plant growth and increase yield in many crops, due to
the production of plant growth promoting substances, which
leads to an improvement in root development and increase in the
rate of water and mineral uptake.
Benizri et al., (2001) reported that certain rhizobacteria
referred to as "plant growth promoting rhizobacteria (PGPR)"
can contribute to the biological control of plant pathogens and
improve plant growth. These bacteria enhance root development
35
either directly by producing phytohormones or indirectly by

inhibiting pathogens through the synthesis of different
compounds.
Lapinskas (2001) studied the efficiency of combination of
phytohormones and Rhizobium leguminosarum bv. trifolii and
Sinorhizobium meliloti strains for Clover and Lucerne. They
determenated that the phytohormones beta-indoleacetic acid
(IAA, hetroauxin) and kinetin stimulated biomass growth
(size of colony) of some Rhizobium leguminosarum bv trifolii
and S. meliloti strains in field experiments, heteroauxine tended
to increase the symbiotic efficiency of all the investigated
Rhizobium leguminosarum bv trifolii strains. The dry matter
yield increase made up 0.70-1.05 ton/ha.
Yanni et al. (2001) evaluated the beneficial plant growth-
promoting association of Rhizobium leguminosarum bv. trifolii
with rice roots. They found that the inoculation increased total
protein quantity per hectare in field grain thereby, increasing its
nutritional value without altering the ratios of nutritionally
important proteins. Studies using a selected rhizobial strain
(E11) indicated that it produced auxine (indole acetic acid) and
gibberellin (Tentatively identified as gibberellin; GA3)
phytohormones representing two major classes of plant growth
regulators.
Sangeeta et al. (2002) studied the potential of homologous
and heterologous Azotobacter chroococcum strains as
bioinoculant for cotton. These varied a lot in their ability to
produce the phytohormone IAA and acetylene reduction activity.
The effect of heterologous and homologous strains of
36
Azotobacter chroococcum in improving yield of cotton was also

assessed. All the strains tested whether homologous or
heterologous, had positive effect on plant yield parameters.
There was instead, strain specific response of the crop to
inoculation more dependant on level of plant growth promoting
activity of the bioinoculant rather than the crop source of
isolation of the microbe.
The levels of the phytohormones auxin and gibberellin
were studied in the original Pea (Pisum sativum L.) cultivars
Rondo and Ramonskii 77 and in different types of symbiotic
mutants (non nodulating, with single nodules and supernodulating)
induced from them by Kholodar et al., (2001). They found that,
the levels of the phytohormones in the symbiotic mutants depend
on the plant's genotype, developmental phases and infection with
rhizobia. Two mutants were isolated whose phytohormonal
status markedly differed from the original forms.
Dey et al., (2004) tested the phytohormonal response in
enod 40-overexpressing plants of Medicago truncatula and rice.
They had compared the phytohormonal responses of mt enod
40-overexpressing and control plants in homologus legume back
ground (M. truncatula) and in the non legume rice. An enod
40-mediated root growth response induced by inhibition of
ethelene biosynthesis, was observed in both plant. On the other
hand, a significant differential effect of cytokinine was observed
only in rice plants.
Theunis et al. (2004) studied the flavonoids, NodD1,
NodD2 and Nod-NB15 modulate expression of the y4wEFG
locus that is required for indole-3-acetic acid synthesis in
37
Rhizobium spp. strain NGR234. They found that addition of

inducing flavonoids also stimulates the production of the
phytohormone indole-3-acetic acid (IAA) in several rhizobia.
Here, the molecular basis of IAA synthesis in Rhizobium
spp.NGR234 was investigated. Mass spectrometric analysis of
culture supernatants indicated that NGR234 is capable of
synthesizing IAA via three different pathways.
Sohail et al. (2004) studied the Rhizobium, Bradyrhizobium
and Agrobacterium strains isolated from cultivated legumes. The
study was conducted to isolate and characterize rhizobial strains
from root nodules of cultivated legumes, i.e., chickpea,
mungbean, pea and sirato. Preliminary characterization of these
isolates was done on the basis of plant infectivity test, acetylene
reduction assay, C-source utilization, phosphate solubilization,
phytohormones and polysaccharide production. The plant
infectivity test and acetylene reduction assay showed effective
root nodule formation by all the isolates on their respectivel
hosts. Except chickpea isolate Ca-18 that failed to infect its
orginal host. All strains showed homology to a typical
Rhizobium strain on the basis of growth pattern, C-source
utilizarion and polysaccharide production. The strain was
characterized by its phosphate solubilization and indole-3-acetic
acid IAA production.
2.3. Isolation of rhizobiophages from the soil
Rhizobiophages have bean reported to reduce rhizobial
populations in soil and also to affect the nitrogen fixing abilites
of rhizobia in root nodules (Kowalski et al., 1974).
Bacteriophages of lucerne nodule bacteria are present in root
38
nodules, roots and stems of old lucerne plants and also in the soil
surrounding the roots but not a distance for than about 35 cm
(Golebiowska et al., 1971).
Golebiowska et al., (1976) suggested that rhizobial phages
are present in the rhizosphere of legumes and are absent in
non-rhizosphere soils. In addition, Evans et al., (1979) stated
that the presence of a virulent bacteriophage in root zone of
clover growing in seedling agar under controlled environments
produced changes in the persistence and symbiotic effectiveness
of a susceptible strain of R. trifolii.
On surveying rhizobiophage distribution, a wide range of
phage types have been recognized from over 80% of the sites
examined (Kowalski et al., 1974 and Patel and Graig, 1984).
Also, they reported that bacteriophages of rhizobia occur
commonly in the rhizosphere of legumes and are often
associated with susceptible strains.
Also, in Egypt, rhizobiophages were found to be common
in the Nile valley soils cultivated with leguminous plants
(Emam et al., 1983; Othman, 1986; El-Didamony, 1990 and
Salama, 1992). Six phages were isolated by Emam et al., (1983)
from the rhizosphere of different legumes. These phages formed
morphologically different plaques and showed a relatively wide
host range. The tested Rhizobium strains were not lysogenic
when they were tested with mitomycin C.
Studying the distribution of 19 phages within the soil
samples taken from nine sites of pasture soils, Patel et al.,
(1985) found that phage ØWC 211 lysed the rhizobia strains
isolated from 11 sites, two sites (4, 8) only were free from 0W3
39
and rhizobia strains from three sites (2, 3, 4) were successful in

the isolation of phages (ØWC 10/Z, ØWC 10/4). However, the
distribution of six rhizobiophages in the soils was studied by
Dhar and Ramkrish (1987). Their results indicated that the
phage titer was high in the rhizospheric zone and around root
nodules .This region may be considered as a site for phage
multiplication because of the presence of metabolically active
rhizobial populations.
Early investigators reported that most of the rhizobiophages
studied were lytic strains (Kowalski and StanieWski, 1959,
Ley et al., 1972 and Dhar et al. 1978). Although, lysogeny is
common in rhizobia (Schwinghamer and Reinhardt, 1963 and
Kowalski 1966). Recently, Dhar et al. (1993) reported that two
lytic phages designated as H3V and R3V, specific for
R. leguminosarum bv. phaseoli were isolated and characterized.
These phages were frequently detected in healthy nodules of
french beans (Phaseolus vulgaris) at two different field locations
and no correlation between phage titer and nodule size or colour
was observed. Also, Radwan (1994) reported that out of
11 fields of broad beans, rhizobiophages were found in ten
fields, of R. leguminosarum bv. vicieae. No plaques were
detected in the plates inoculated with VS1, VH4, VZ7 or VY10
along with either of the eleven suspected rhizobiophage
suspensions. On the other hand, plaques were observed on plates
inoculated with Vicia faba isolates VA2, VE3, VM5, VW6,
VK8, VF9 or VL11, Also, the obtained results showed the
relation of four isolates of R. leguminosarum bv. trifolii and five
isolate of R. leguminosarum bv. vicieae (isolates from lentil
40
plants) with their homologous phages . He stated that only one

isolate from lentil showed no relation with any of the
11 suspensions of rhizobiophages, while the other eight isolates
were sensitive to one or more rhizobiophage isolates
(suspension) and were initially isolated from broad bean fields
El-Didamony (1995) found that seventeen rhizobiophages
infective against R. leguminosarum were isolated from field
grown Vicia faba in Al-Ibrahimia region, Sharkia governorate,
Egypt. In addition, El-Wafai et al., (1996) isolated three phages
namely; RV, RV3 and RV9, which were infectious to broad
bean fields in Sharkia governorate. Moreover, Zaghloul and
Abd El-Mageed (1996) found that the interaction effect of
inoculation of broad bean seeds with R. leguminosarum bv.
vicieae on damping - off and root rot caused by Rhizoctonia
solani and on viral infection caused by common bean mosaic
polyvirus was studied under sterilized and non- sterilized soil
conditions
Ali et al. (1998) isolated rhizobiophages against Rhizobium,
Bradyrhizobium and Sinorhizobium from the rhizosphere of
soybean plants, growing in a Webster clay loam soil and at the
early reproductive growth stage. Also, Dhar and Kumar (1998)
isolated bacteriophages active against Rhizobium spp nodulating
pigeon pea plants from their rhizospheric soils. They also
isolated phages infective on 3 cowpea Rhizobium strains and
6 local pigeon pea Rhizobium strains. Distributions of phages in
rhizosphere soil were different on indicator strains and had no
relationship with soil pH. The field with pigeon pea after wheat
contained phages infective more strains than fields with pigeon
41
pea after pigeon pea, phages were also found in nodule exudates
and maximum titer was 3.2 x 10 particles / g nodules mass.
Shahaby et al. (1999) studied the incidence and survival of
rhizobiophages in bacterial carriers and liquid culture media
under stress conditions as well as response of some legumes to
inoculation with rhizobial phage-resistant isolates. The response
of four legumes to inoculation with phage- resistant rhizobial
isolates and their homologous strains was also evaluated. The
phages were detected and isolated from both fine peat and Irish
peat carriers but not from vermiculite after enrichment with
YEM broth medium. The number of phages ranged from 9 to
18 and from 12 to 25 PFU/g carriers for fine peat and Irish peat,
respectively. When the three carriers were inoculated with
rhizobial strains, the Irish peat showed higher values of phage
plaques than fine peat, while the vermiculite carrier exhibited no
plaques. The numbers ranged from 1.8X102 to 4.6X102 and
1.6X102 to 2.8X102 PFU/g carrier for Irish peat and fine peat,
respectively. The isolated phages specific for rhizobial strains
showed no lysis for phage-resistant rhizobial isolates. Rhizobium
leguminosarum bv. trifolii isolates (ARC 102 and TAL 112) and
R. leguminosarum bv. vicieae isolates (ARC 207F and ICARDA
441) did not differ in appearance and showed no variations in
their colony morphology as compared to their parent strains,
while others were different. All tested phages were highly
sensitive to high temperatures.
Sharma et al., (2001) tested the diversity among
rhizobiophages from rhizospheres of legumes inhabiting three
ecogeographical regions of India. Diversity among phage
42
isolates was assessed by studying the plaque traits on their

indicator bacterial strains, and variation in morphology using
transmission electron microscopy. Except for plaque size, the
variation in the plaque traits was rather narrow. Based on the
variability pattern, the 64 phages were grouped into into
11 plaque types.
2.4. Effect of bio-fertilizers and inorganic N-fertilizers
on broad bean.
El-Ghandour and Galal (1997) evaluated biological
nitrogen fixation process by three cultivers of broad bean i.e.,
Giza 2, Giza 461 and Retablanka, using N15 dilution techniques
and wheat plants (Sakha 69) was used as reference crop.
Uninoculated and inoculated seeds with Rhizobium and/or VAM
were sown in sandy soil amended with labeled 15NH4NO3 (15 kg
N/fed, 4.68 % N15 a.e) or N15 rice straw (0.2 % w/w, 1.63 15N
a.e). Rock-P was added as source of P at a rate of 20 kg P fed-1.
They showed that combined inoculation with (Rhizobium +
VAM) increased shoot dry weight by about 51.6, 17.0 and 19.3
% for Giza 2, Giza 461 and Rentablanka, respectively under
organic N source. Nitrogen derived from air (Ndfa) were higher
in case of Rinta blanka compared with the other varieties even
under N15 rice straw or 15NH4NO3, but dual inoculation gave the
best result with organic residues treatment. Generally, they
concluded that application of N2-fixers and VAM in combined
inoculates for legumes can save about 80 % of N and 50 %
of P fertilizers.
Namdeo and Gupta (1999) searched the efficacy of
biofertilizers with different levels of chemical fertilizers on
43
pigeon pea. They used seed inoculated with Rhizobium and/or

phosphorus solubilizing bacteria PsB (Pseudomonas striata) and
given 50, 75 and 100 % of the recommended fertilizer rate
(20: 50 kg N P/ha). Rhizobium inoculation increased nodule
number and dry weight and shoot dry weight. Rhizobium, PsB
and Rhizobium + PsB with 100 % RFR produced 13.8, 9.9 and
20.4 % higher grain yield, respectively, than 100 % RFR alone.
Rhizobium + PsB with 75 % RFR gave a saving of 25 % of the
chemical fertilizer by producing yield equivalent to that of
100 % RFR alone. However, the highest net return was obtained
with 100 % RFR + Rhizobium + PsM.
Ayneabeba et al. (2001) studied the Rhizobium inoculation
and fertilizer treatment on growth and production of broad bean
(Vicia faba) in some yield deplet and yield sustained regions of
semien shewa. The results showed that shoot length, shoot dry
matter and nodules fresh weight were significantly affected
(P= 0.01) by soil type and different inoculation and fertilizer
treatment.
El-Ghandour et al. (2001) investigated the Rhizobium
inoculats and mineral nitrogen for growth, N2-fixation and yield
of broad bean. Application of inoculants showed remarkable
increase in dry matter accumulation of shoots and grain yield as
well as the N uptake by both components. Nitrogen fixation,
estimated by N difference method, was negatively affected by
the high level of fertilizer applied 40 kg N ha-1.
Abdalla, (2002) studied the effect of bio- and mineral
phosphorus fertilizer on the growth, productivity and nutritional
value of broad bean. Biofertilizer treatments improved plant
44
height, number of leaves and shoots, and dry matter content

compared to the control. Superphosphate at 150-200 kg/fed
improved plant growth. The contents of N, P, K, protein, total
soluble sugars and ascorbic acid increased with biofertilizer
treatments and with increasing rates of P from 100-200 kg
superphosphate/fed.
Jia YinSuo et al. (2004) evaluated the influence of
Rhizobium and arbuscular mycorrhizal fungi on nitrogen and
phosphorus accumulation by Vicia faba. The presence of
Rhizobium resulted in a significant decline in AMF colonization
levels irrespective of N supply. Without Rhizobium, AMF
colonization levels were higher in low N treatments. Presence or
absence of AMF did not have a significant effect on nodule mass
but high N with or without AMF led to a significant decline in
nodule biomass. Plants with the Rhizobium and AMF symbiotic
associations had higher photosynthetic rates per unit leaf area.
2.5. Effect of bio-fertilizers and organic fertilizers on
broad bean
Early experiment by Ishac et al. (1979) studied the effect of
addition of wheat straw at 0.3 and 0.6 ton/donum just before
sowing the soil with broad bean. They found that organic
supplements resulted in the enrichment of soil population in
general and the aerobic groups of cellulose-decomposers and the
non symbiotic nitrogen fixers in particular. As for dry weight
vegetation, compared with the control, it increased by 64 % for
roots; 60 % for stem; 16 % for leaves and 34 % for bods,
respectively.
45
Several authors (Elgamal andEl-Sheikh 1989; Das, 1991

and Arrese et al., 1991) detected that rhizobial-inoculation in
presence of organic manure showed significant positive effect
compared to inoculation with rhizobia alone in legume crops.
The results point to the possible influence of environmental
conditions and agronomic practices on the fate rhizobial-
inoculation.
Youssef (1993) found that inoculation with rhizobia plus
mixed population of associative diazotrophs had stimulative
effect on dry matter, crude protein crude fiber and ash yield of
berseem clover. He detected also, that application of organic
manure resulted in highly significant increases in shoot dry
weight, N-uptake and plant dry weight as compared with
rhizobia and associative diazotrophes inoculation treatment.
Moherram et al. (1998) in pot experiment, studied the
effect of application rates (0.0, 0.5, 1.0, 2.0 and 3.0 %) of
composted cattle dung and poultry manure mixture on soybean
and peanut inoculated with specific rhizobia and grown on newly
reclaimed sandy soil of West Samalout, Minia, governorate,
Egypt. They found that the application of the compost
significantly increased plant dry weight, N-yields, N-fixed, N %
derived from organic matter (% NDFO) and % fertilizer
utilization efficiency (% FUE) in both soybean and peanut
plants. They also, detected that the maximum nodules numbers
per plant were recorded at 2.0 % application rate of compost for
soybean and at 1.0 % for peanut.
Al-Kahal et al. (2001) studied the impact of olive oil waste
water (OOWW) and olive cake (OC) each were used at 1 , 2 and
46
3 % alone or in combination with Rhizobium leguminosarum

inoculation on nodulation and N2- fixation of Vicia faba cv. Giza
Blanca plants. They found that increasing OOWW and OC levels
alone resulted in decreases in the number and dry weight of
nodule, nitrogenase activity, shoot dry weight, and straw and
seed yield. When OOWW and OC were applied in combination
with R. Leguminosarum inoculation there was a significant
increase in nodulation, shoot dry weight and N and P contents
compared to uninoculated OOWW and OC treatments.
Abdelhamid et al. (2004) evaluated the effect of manuring with
rice straw composting with oilseed rape cake and poultry manure
on broad bean (Vicia faba L.) growth and soil properties. They
found that the addition of compost (20-200 g pot-1) improved soil
chemical (increased total N, total C and CEC), physical
(decreased particle density) and biological (increased soil
respiration rate) properties. Application of composts at a rate
of 20 g pot-1 significantly increased growth, yield, yield
components and total crude protein of broad bean plants. The
benefit of this compost without chemical fertilizer demonstrated
the validity and possibility of sustainable agronomic
performance of broad bean using locally available recycled
organic materials.
2.6. Effect of bio-fertilizers and inorganic N-fertilizers
on wheat
Goher et al. (1988) confirmed that biofertilization of wheat
plants, using N2-fixing Azotobacter and Azospirillum might
reduce the inorganic nitrogen requirement and/or increase the
inorganic nitrogen requirement and/or increase the yields. In
47
addition, application of phosphorus solubilizing bacteria may

render as sufficient quantity of phosphorus to be available for
wheat plant when rock phosphate is applied in wheat cropping.
El-Demerdash et al. (1993) studied the effect of
inoculation with symbiotic cowpea rhizobia and/or
R. leguminosarum bv. vicieae and/or asymbiotic N2-fixers
(Azotobacter chroococcum and Azospirillum lipoferum) on
nitrogenase activity in rhizosphere, endomycorrhizal infection,
growth and yield of wheat plants in field experiment. This was
carried out in the presence of half or full the recommended dose
of inorganic N-fertilizer and rock phosphate as P-fertilizer. Their
results showed that inoculation with a mixture of symbiotic and
asymbiotic N2-fixers gave better results of growth and yield of
wheat plants than using either of them only.
Galal and Elgandour (1994) carried out a pot experiment
to clarify the effects of Azospirillum spp and/or
R. leguminosarum on wheat growth. The experiment was divided
in two parts, one of them received 50 kg N fed-1 as labeled
ammonium nitrate and the other received 0.2 % N15 labeled rice
straw, in combined with different inoculates. They detected that
inoculation with either Azospirillum or Rhizobium increased
straw and grain yields as well as the total N uptake. Relative
increment was high in case of N15-rice straw applied as
compared with N15-labled ammonium nitrate. Also, Rhizobium
inoculated slowly gave a higher value of the above mentioned
parameters comparable to those obtained with either
Azospirillum solely or in mixture with rhizobia.
48
Rabie et al. (1995) showed that inoculation of wheat plants

with Azotobacter chroococcum and/or Azospirillum brasilense
increased plant height, dry weight of shoots and roots, number of
the total tillers, as well as the percentage of fruitful tillers,
number of spikes, weight of spikes and grain yield/plant.
Densities of Azotobacter and azospirilla in the rhizosphere were
also increased. The results also showed that the inoculation
treatments increased significantly the level of auxin, gebberelline
and achieved remarkable in cytokinin activities in shoot and root
extracts during the different stages of wheat growth, comparing
to uninoculated plants.
Mitkees et al. (1996) inoculated wheat plants with mixture
of N2-fixing bacteria, Azospirillum, Bacillus, Klebsiella and
Azotobacter under different levels of mineral N-fertilizer in old
and new lands. Results indicated that biofertilizer could
compensate considerable parts or mineral N-fertilizer, it saves
about 50 kg N fed-1 in old land and 40 kg N fed-1 in new land.
Amara and Dahodoh (1997) found that inoculation with
mixture of Azotobacter and Azospirillum showed the highest
value of wheat yield.
Sarwar et al. (1998) reported that the application of
Azospirillum lipoferum inoculatant and three levels of nitrogen
fertilizers (10 kg/hr, 45 kg/hr and 90 kg/hr) gave high significant
increase in growth and yield of rice crop specially when nitrogen
was applied at a rate of 45 kg/hr.
Four experiments were conducted in El-Bostan district, on
newly reclaimed sandy soil, by Barakat and Gaber (1998) to
study the effect of inoculating tomato (cv. Castle) plants with
49
non-symbiotic N2-fixers of the genera Azotobacter, Azospirillum

and Klebsiella (alone or mixed inoculation) and applying
N-fertilizers at 0, 50, 100 to 150 kg/fed. On growth, fruit yield
and chemical composition of tomato plants, total fruit yield was
highest in both years when plants were inoculated with a mixture
of the three genera of N2-fixers and 100 kg N/fed was applied.
Sabry et al. (1998) evaluated the response of wheat cultivar
Giza 164 to inoculation with Azorhizbium caulinodans strain
IRBG314 and Azospirillum brasilense strain SP.245 under five
nitrogen levels (0, 25, 50, 75, 100 % of recommended dose) in
five locations of different soil types. The results indicated
significant difference between the means of experiments in the
five locations in grain yield, number of spikes per meter square,
number of kernels per spike, and 1000 kernels weight except
El-Nubaria. Grain yield and other yield components increased
gradually with the increase in chemical N-fertilizer regardless of
inoculation with A. caulinodans or A. brasilense. The highest
significant grain yield was obtained with 75 % N regardless of
type of inoculation.
De-Freitas et al. (2000) studied the effect of Azotobacter
chroococcum, Azospirillum brasilense and Bacillus polymyxa,
individually or in mixture on the yield of wheat. They found that
the response of plants inoculated with individual bacteria was
inconsistent and varied with treatment, while planted inoculated
with the mixture exhibited increase in shoot biomass.
Panwar (2000) reported that inoculation with Azospirillum
brasilense and Bacillus subtilis enhanced leaf area, chlorophyll
concentration, total biomass production and grain yield of wheat.
50
Sushila and Giri-Gajendra (2000) stated that biofertilization

by Azospirillum and Azotobacter had enhanced the growth and
yield of wheat.
Alam et al. (2001) reported that the inoculation with
mixture of several free living rhizobacteria, Azotobacter,
Bacillus and Enterobacter significantly increased acetylene-
reducing activity in roots of rice plant, total dry matter yield,
grain yield, N uptake, root length and leaf area.
El-Bakry et al. (2001) reported that inoculation with
Azospirillum caused the highest value of growth and yield in
sorghum, and an increase in P and N content in shoot and plant
dry weight.
Galal et al. (2001) mentioned that inoculation with
Azospirillum and Rhizobium caused higher dry matter
accumulation in shoots, N uptake and grain yield increased with
increasing N rate, but increasing N rate inhibited N-fixation in
plant. Inoculation of both bacteria increased growth and yield.
Samalieva et al. (2002) studied the economic evaluation of
wheat inoculation with Azospirillum in field conditions. The
effectivity of the use of bacterial suspension was studied with
4 levels of N (0, 8, 14 and 20 kg/daker). The greatest economic
effect was obtained at 14 kg N/ daker, where the increase in
production due to inoculation was 10.2 and 4.9 % (during the
first and second year, respectively) compared to the variants
without inoculation.
Galal and Thabet (2002) tested the effect of soybean
residues, Azospirillum, and fertilizer-N on nitrogen accumulation
and biological fixation in two wheat cultivars. N-fertilizer was
51
applied at 0 and 50 kg ha-1 labeled as ammonium sulfate applied

as a peat-based inoculum coating the wheat (cv. Giza 163 and
Giza 164). At the end of the vegetative period (90 days), shoot
dry weight was higher with organic amended and inoculated soil
than the control. Nitrogen uptake by either shoots or grains of
wheat plants was significantly increased by the application of
soybean residues and inoculation with Azospirillum sp.
Galal et al. (2003) searched the effect of inoculation by
mycorrhizae and Rhizobium on the growth and yield of wheat in
relation to nitrogen and phosphorus fertilization as assessed by
15 techniques. They found that the highest dry matter and
N-uptake by wheat shoot were obtained with the dual inoculation
(Rh+AM) at N2P2 rates of N and P fertilizers.
Recently, Singh et al. (2004) studied the performance and
gene effects for wheat under inoculation of arbuscular
mycorrhiza fungi and Azotobacter chroococcum. A mineral
fertilizer (80 kg N + 40 kg P + 18 kg ZnSO4) was applied in all
3 treatments. The application of bioinoculants, AMF and AMF +
AZC had a positive effect on plant height, peduncle length, grain
yield biological yield and harvest index in various populations of
all the crosses.
2.7. Effect of bio-organic fertilizers on wheat
The application of organic matter to clay and sandy soil
increased the counts on the non-symbiotic nitrogen fixing
bacteria and increased the efficiency of nitrogen fixation
correspondingly (Monib et al., 1970).
The data obtained by Peshakov et al. (1976) indicated that
the addition of green manure increased Azotobacter counts.
52
However, the rate of increase in the count of Azotobacter was

depending on the amount of green manure application.
The interaction between nitrogen fixers and organic
manuring was reflected on the total nitrogen content in the soil.
Khalil (1979) and Hashem (1980) found the total nitrogen
was increased considerably, as a result of supplementing soil
with different organic manures.
A pot experiment was designed by Hegazi et al. (1983) to
investigate the effects of inoculation with Azospirillum and/or
straw amendment on growth of maize plant grown in Giza soil.
They found that inoculation caused and increases in plant dry
weight (200 %) and total nitrogen content (157 %) of maize
plants.
Ishac et al. (1983) studied the effect of addition organic
amendments (0.2 % maize stalk) on nitrogen-fixation by a
symbiotic bacteria (Azotobacter and Azospirillum) associated
with wheat and maize plants. They detected that the greater
number of Azotobacter and azospirilla were recorded from maize
than from wheat plants. Although, the effect of organic additions
stimulated N2-fixation more markedly in the wheat plants than
maize plants. These data indicate that organic amendments may
be used in the agriculture in semitropical regions.
Salem et al. (1985 and 1986) investigated the effect of
organic matter addition (clover, sheep dung, barley straw and
conditioner, CMC) on the activities of the N2 fixing
microorganisms in sandy and calcareous soil. This study has
shown that the N2-fixers (Azotobacter and clostridia) were
greatly influenced with the organic matter addition where, their
53
counts and activities in N2-fixation have been increased

markedly, due to manuring of the soil.
Markus and Kramer (1988) determined if the N2-fixing
bacterial density and activity can by stimulated by organic
fertilizers. They reported that, in organic cultivated soil, low
number of Azospirillum spp could be found in the rhizoshphere
of wheat. While, common nitrogen-fixing bacteria, like
Azotobacter spp. appeared in much higher densities. The authors
also found that the Azospirillum spp number, after inoculation in
combination with organic fertilizers were higher than control
without organic fertilizer, specially in earlier growth stages of
wheat.
The growth of maize plants expressed as plant height, stalk
diameter number of leaves per plant as well as dry weight of
whole plants is considerably stimulated by inoculation with
Azospirillum strains in amended treatments with maize stalks at
the rate of 0.2 % was detected by El-Borollosy et al. (1988)
from the economical point of view. It can be stated that
inoculation with Azospirillum strains just before cultivation in
the presence of organic maize stalks resulted an increasing the
plant growth and reduced the requirements of inorganic nitrogen
fertilizers to half amount needed with inoculation.
A series of field experiments were conducted to elucidate
the potential of different plant rhizobacterial systems in sandy
and clay loam soils. Seeds of two major Egyptian groups wheat
and maize were inoculated with peat or sodium alginate based
inoculants contained numbers of Azotobacter chroococcum,
Azospirillum, Azorhizobium coluilodans, Bradyrhizobium
54
japonicum, Rhizobium leguminosarum, Rhizobium spp (cowpea),

Frankia, Pseudomonas fluorescens and pseudomonas spp. Also,
organic materials garbage compost or farmyard manure), rock or
superphosphate and mineral nitrogen (45, 67.5 and
90 kg N/acre) were manipulated by Ishac (1994) to evaluate
plant-rhizobacterial association under different conditions. Data
showed that N2-fixing bacteria were generally more effective
compared with other rhizobacteria or uninoculated treatment.
Where, all the tested rhizobacteria, except for Pseudomonas spp
promoted nitrogenase activity in rhizosphere of wheat plants.
Effective associations stimulated panicle initiation, number of
tillers and spikes in wheat as well as straw and grain yield in
maize plants.
Biomass, nitrogen and grain yield of wheat cvs. Sakha 69
and Giza 163 were determined to evaluate the response of the
graminous crop to diazotrophic inoculation (Azospirillum
brasilense and Bacillus polymyxa) and organic fertilization in
Ismailia sandy soil (Mohamed, 1997). She found that plants
amended with biogas manure with inoculation developed well
and showed better appearance than those only supplied with
N-fertilizers. Where the application of biogas manure was more
effective on grain production, protein accumulation in grain and
bacterial multiplication.
Six field trials were executed through out the winter
seasons from 1992 to 1996 by Hegazi et al. (1998) in Ismailia
and El-Arish desert soils these experiments were carried out to
evaluate the performance of the multi-strains inocula of
associative diazotrophs; Azop. brasilense, A. chroococcum,
K. pneumoniae, P. putida and Bacillus spp with or without yeast
55
and possible interaction with mineral and organic fertilization on

wheat (cvs. Sakha 69, Gemaza 1, Benisuif 1 and Giza 155) and
barley (cvs. Giza 123 and Giza 124) cereal crops. The organic
amendments include wheat straw (C/N 40: 1) and farmyard
manure (C/N 40: 1). They found that wheat plants inoculation
with associative diazotrophs when combined with dose of
N-fertilizer (60 kg N/acre) under sprinkler and rainfed irrigation
resulted in similar yield (1.9 kg/plot) to full dose of
N- fertilization 2.2 kg/plot. While, the application of organic
fertilizer did not result in a significant additional yield
1.2 kg/plot. Similar results in wheat were obtained with barley
plants in two experiments sites tested. The authors concluded
that the least yield may be due to the decomposition of organic
matter applied and nutrient release are impaired under that the
harsh and stressed environmental conditions.
Singh and Sharma (1999) studied the scope of
supplementing nitrogen requirement of wheat through organic
source and Azotobacter inoculation. Azotobacter inoculation
increased yield from 4.81 to 5.01 ton. The available nutrient
status of soil after harvest of the wheat crop was higher where
organic N was applied.
Ardakani et al. (2001) tested the effect of Azospirillum,
mycorrhiza and Streptomyces with manure utilization on yield
and yield component of wheat (Mahdavi var.).Their results
showed that, individually, Azospirillum and mycorrhiza caused
significant increases in spike/m2 and grain weight yield. Manure
increased most of the components specially when used with
Azospirillum and mycorrhiza.
56
Radwan et al. (2002) evaluated the response of wheat

plants to multistrains biofertilizers (phosphate-dissolving
bacteria + Azotobacter spp + Azospirillum spp + Pseudomonas
spp) and/or organic manure (fresh or composted farming
manure) on the dry weight, N and P content, grain, straw, and
biological yield of wheat cv. Giza 164 under different weed
control treatment. They found that association of biofertilizer
with composted farmyard manure and Bifenoherbicide
application or hand weeding, led to significant increases in grain,
straw and biological yield of wheat relative to the control.
Bacilio et al. (2003) studied the alleviation of noxious
effects of cattle rench composts on wheat seed germination by
inoculation with Azospirillum spp. They found that inoculation
of wheat seeds with two species of the plant growth promoting
bacteria Azospirillum brasilense cd and A. lipoferum JA4
(separately) prior to sowing in these amended soils improved
germination. Both of composted amendments increased both
height of wheat seedlings in range of 20-25 % and shoot dry
weight by 15-19 %, whereas, they were severally decreased root
dry weight by 51-54 %.
57
III. MATERIALS AND METHODS

This study was carried out in the laboratory and greenhouse
Zagazig University, Egypt, during the period of 2002 to 2005, in
order to study the use the isolated strains of Rhizobium,
Azotobacter and Azospirillum as biofertilizers for broad bean
(Vicia faba L.) and wheat (Triticum aestivum L.) plants.
3. Isolation and selection of indigenous Rhizobium

leguminosarum bv. vicieae, Azospirillum spp. and
Azotobacter spp. from Sharkia governorate.
3.1. Root-soil core samples
Root-soil core samples were collected following the root-
soil core procedure with roots of broad bean and wheat plants
grown in different locations in Sharkia governorate. These
locations were chosen as such it had guiding fields and a wide
spread fields of these crops. The collected samples represented
eight locations namely: Zagazig (Z), Fakous (F), Abo-Kibeer
(K), El-Hessenia (H), Kafr-Sakr (R), Abo-Hammad (A), Belbase
(B) and Salhia (S). Root nodules of broad bean plants grown on
each soil sample were used for Rhizobium-isolation and
rhizosphere soil used for Azotobacter isolation. Soil and roots of
wheat grown on each soil sample were used for isolation of
Azospirillum and Azotobacter. The isolation regions are
shown in Fig. 1.
58
Fig. (1) Map of Sharkia governorate showing localities of

isolation
Isolation regions
59
3.2. Isolation and purification

3.2.1. Isolation, purification and identification of rhizobia
Pure cultures of rhizobia (Rhizobium leguminosarum
bv. vicieae) were isolated according to the methods described by
Vincent (1970) and Somasegaran and Hoben (1985). Nodules
of each site were taken from the roots, washed thoroughly in
distilled water and immersed intactly for 3-5 minutes in 95 %
ethanol alcohol. Nodules were then transferred to 0.2 % (W/V)
solution of mercuric chloride for 3-5 minutes. Surface sterilized
nodules were then rinsed in five changes of sterile distilled water
and carefully crushed under aseptic conditions in sterile
phosphate buffer (pH 7.2). The suspension was streaked onto
plates of congo-red-yeast extract mannitol agar medium
(YEMA) and bromothymol blue yeast extract mannitol agar
medium, for differentiation of Rhizobium from any
contamination, (Somasegaran and Hoben, 1985). Selection of
single cell culture was carried out for subsequent studies. The
grown bacteria were plated and single colonies were inoculated
onto slopes of YEMA medium after microscopic testing of Gram
stained slides of the colonies. A total of 60 isolates of
R. leguminosarum bv. vicieae were isolated from all collected
soil samples. One rhizobial-isolate represent each soil sample
(Table, 1). These isolates were tested for their plant infection as
described by Vincent (1970) and Somasegaran and Hoben
(1985). Stock cultures of the rhizobial-isolates (32 isolates) were
maintained on slants of YEM agar at 4 °C and recultivated every
month.
60
3.2.2. Isolation, purification and identification of azospirilla

Azospirilla were isolated from soil and roots of wheat
plants according to the method described by Gunarto et al.
(1999). Soil and root samples were collected from different
locations in Sharkia governorate (Table, 1). Fresh root samples
were washed in rapidly running tap water for 20 minutes to
remove soil particles adhering to the root surface. The washed
roots were surface sterilized with 0.5 % sodium hypochlorite
(NaClO) solution for 30 minutes. The roots were rinsed in sterile
water at least 4 times, then cut into pieces (5-8 mm), which were
macerated with forceps and introduced into semi solid nitrogen
free broth (NFb) medium (Baldani and Döbereiner, 1980) and
were incubated at 30 °C for 2-4 days. Then the cultures were
streaked out on NF agar plates containing 0.5 g yeast extract L-1.
After one weak typical, small, white, dense and single colonies
were picked out and transferred into semi solid NFb medium.
Pellicle formation in this medium indicated successful
isolation. For final purification, these cultures were streaked out
on Potato infusion agar (BMS) medium (Baldani and
Döbereiner, 1980), and the typical pink, often wrinkled colonies
were transferred to an NFb agar slant containing 0.5 g yeast
extract L-1 without vitamin solutions for storage and use for
further experiments.
Isolation of Azospirillum spp from soil samples, one-ml from soil
dilution (10 g soil in 90 ml distilled water) was transferred to
each of 3 tubes containing 10 ml of selective NFM semi solid
medium (Day and Döbereiner, 1976).
61
Table (1): The locations of soil samples selected for isolation

of rhizobia, azotobacter and azospirilla
rhizobia azospirilla azotobacter
Total No. of Total No. of Total No. of

Locations No. of chosen Nomenation No. of chosen Nomenation No. of chosen Nomenation
isolates isolates isolates isolates isolates isolates
Zagazig RZ11, 21, 22,
9 6 9 2 ASZ11, 33 18 3 ZZ11, 13, 23
(Z) 23, 32, 33
Fakous RF12, 13, 21, ASF11, 12,

9 6 9 3 18 2 ZF22, 31
(F) 23, 31, 32 32
Abo- RK11, 12,

ASK21, 23,
Kibeer 9 7 13, 21, 23, 9 3 18 2 ZK12, 31
33
(K) 31, 32
El-
Hessenia 9 3 RH13, 23, 31 9 2 ASH21, 31 18 3 ZH11, 21, 31
(H)
Kafr-Sakr RR11, 13, 23,
9 4 9 1 ASR31 18 1 ZR22
(R) 31
Abo-
ASA12, 23,
Hammad 9 2 RA21, 33 9 3 18 2 ZA11, 31
33
(A)
Belbase
3 3 RB11, 12, 13 3 1 ASB11 6 - -
(B)
ASS11, 12,
Salhia (S) 3 1 RS13 3 3 6 1 ZS13
13
Tubes were incubated at 30 °C for 2-7 days. Positive tubes

were transferred several times into respective semi solid NFM
culture medium. For further purification, streaking on nutrient
agar plates, and single colony isolation were applied.
Sixty isolates were obtained from which only 18
representative isolates were selected for their nitrogenase
activities.
3.2.3. Isolation, purification and identification of azotobacter
Pure cultures of Azotobacter spp isolated from soil samples
collected from different locations in Sharkia governorate
62
(Table, 1) were obtained according to (Abd El-Malek and Ishac

1968). Soil samples were diluted (10 g soil in 90 ml distilled
water), then one-ml from soil dilution was transferred to each of
3 tubes containing 10 ml of selective NFM liquid modified
Ashby's medium. Tubes were incubated at 28 ± 2 for 7 days.
Positive tubes (which produce surface layer of growth with
chocolate pigment) were transferred several times into respective
modified Ashby's medium. For further purifications, the plate
method was used by successive streaking on modified Ashby's
agar medium. Incubation was carried out for 7 days at 30 °C.
Single colonies produced were microscopically examined for
pure isolates of Azotobacter spp, which were found that,
microbes are Gram negatives, large in size, oval or cocci, single
or pairs showing brown pellicle. The method of identification
was carried out according to Bergy's manual of determinative
Bacteriology (1994).
One hundred and twenty isolates were obtained from which
only 14 isolates were selected depending on their fast growth and
for their valuable nitrogenase activities.
3.3. Nitrogenase activity
The ability of azotobacter and azospirilla isolates to fix
atmospheric nitrogen (N2-ase activity), was assayed. The enzyme
activity was estimated using the acetylene reduction activity
technique (ARA) according to Somasegaran and Hoben
(1985). Using 24 hours old cultures (1 ml liquid medium) on
selective medium in test tubes, then the cotton plugs were
removed and replaced by serum stoppers. After inoculation, pure
acetylene C2H2 was injected into the test tubes to give acetylene
63
concentration 10 % (V/V) of the atmospheric air. Test tubes

were incubated for 48 hour then 1.2 ml of gas samples
withdrawn for the determination of ethylene C2H4 formed by
gas chromatograph using Hewlett Packard for chromatography
model HP (6890 GC) fitted with dual flame ionization detector
and 150 cm × 0.4 cm diameter stainless steel column fitted with
propa × R 100-120 mesh.
A standard curve was established before analyzing samples
using different concentrations of ethylene in air. The ethylene
into 1200 ml serum bottles, out of which serial dilution were
made one ml gas of each dilution was injected into gas
chromatograph at several attenuations. A linear relation between
ethylene concentration and ethylene peak heights was obtained
for each attenuation. Nitrogen was used at a flow rate of 30 ml
min-1 as a carrier gas. Hydrogen and air was used for the flame
flow rate was 30 and 300 ml min-1, respectively. The detector
temperature was 250 °C and the oven and injector part
temperatures were 80 °C and 100 °C respectively. Acetylene gas
used for the standard curve was purified by passing through
concentrated sulphuric acid followed by distilled water for
3 times before using. To calculate the ethylene amount in the gas
samples the peak high was measured in cm, and converted to the
equivalent μ mole/ C2H4 using multiplication factors derived
from the standard curve.
The concentration of ethylene in the samples (μ mole/ C2H4 /hr)
was then converted to μ mole nitrogenase enzyme by dividing
these values by the volume of the molecule weight of gas
64
(22.4 L). Nitrogenase activity was then calculated by the

following formula:
container volume (tube) 1 1 1
R    103
time of incubation inj.vol. D 22.4
Where
Inj. Vol. = injection volume
R= Reading of peak
D= Volume of the medium/ml
The results were presented as micromole C2H4 / ml culture/ hr.
3.4. Testing of different isolates of Rhizobium
leguminosarum bv. vicieae on nodulation and plant
growth of Vicia faba L. grown on greenhouse.
This experiment was conducted to study the effect of
inoculation with different isolates (32 isolates) of rhizobia
(Rhizobium leguminosarum bv. vicieae) on plant growth and
nodulation of broad bean (Vicia faba L.) plants.
Pots (20 cm) were filled with two kg of sandy soil
previously washed and sterilized for three successive days
(3 hr/day) at 121 °C. Sodium hypochlorite was used to disinfect
Vicia faba L. (Giza 843, from agronomy Dept. Agric. Res.
Center. Giza., Egypt) seeds and placed into each pot under
aseptic condition (5 seeds /pot). The pots were amended with
either of the following treatments:
1- control (without inoculation)
65
2- Inoculation with either of Rhizobium leguminosarum bv.

vicieae isolates at 1 × 108 cfu / ml (5 ml pot-1).
After 15 days, plants were thinned to 3 plants/pot and
watered daily with sterile nutrient solution free from nitrogen
(Somasegaran and Hoben, 1985). After 7 weeks from planting,
the stems were cut at the soil surface, and the plant tops were
dried at 70 °C, weighed and ground. The nitrogen content was
then determined colormetrically using Nessler solution
(Naguib, 1969).
Immediately after removal of tops, nodules number and
nodules dry weights were determined. The roots carrying the
formed nodules from each pot were gently separated from the
soil and washed, then nitrogenase activity was determined using
the acetylene reduction assay according to the method described
by Hardy et al., (1973). The experimental design was a
completely randomized with three replicates.
3.5. Assesment of nitrogenase activity in nodules
After 7 weeks of plantation, the vicia faba L. plants were
uprooted with great care to obtain root system intact as much as
possible. Immediately after removal of the tops, the obtained
root system was washed to be free of soil with water and placed
in a reaction jar fitted with a septum. The jar was amended with
10 % acetylene and incubated at 30 °C for 120 min. The
nitrogenase activity of nodules was calculated on the bases of
measurements of the intensity of acetylene reduction to ethylene
(C2H2  C2H4). The quantitative determination of the produced
66
ethylene was performed by means of gas chromatograph

(μ mole C2H4) per hour per gram dry weight of nodules using
gas chromatography system (Hewlett Packard- HP- 6890- Series,
USA).
3.6. Isolation of lytic phages from soil samples cultivated
with broad bean plants
In most cases the bacterial inoculation were attacked by
bacteriophage, for this reason came the importance of studying
the effect of bacteriophage on the Rhizobium strains in Sharkia
governorate soils.
Phages in soil which can infect Rhizobium leguminosarum
bv. vicieae were isolated by enrichment of soil samples from the
rhizosphere of broad bean plants in Sharkia governorate using
the method described by Patel and Graig (1984). The initial
isolation was connected by suspending 5 g of soil sample from
the rhizosphere of broad bean plants in 45 ml yeast mannitol
broth (Vincent, 1970) previously inoculated with fresh
representative rhizobial isolates and shaken in an incubator
shaker at 28 °C for 2-3 days, then centrifuged at 3000 rpm for
30 minute. The supernatant was filtrated through sterile millipore
membrane filter (0.45 μm). The resulting suspension was tested
for specific phages against growing rhizobia (Rhizobium
leguminosarum bv. vicieae) isolates. A volume of 0.1 ml from
each rhizobial isolates was mixed with 2.5 ml of molten YEMA
(0.7 % agar). The mixture was overlaid on solid YEMA plates
67
and spotted with a diluted phage suspension. Plates were

incubated at 28 °C for 48 hours. Positive plates were recorded.
3.7. Extraction and determination of phytohormones
(IAA, IBA and GA3) from bacterial isolates and
root plants
3.7.1. From bacterial isolates of rhizobia, azotobacter and
azospirilla
The chosen bacterial isolates were grown on 250 ml conical
flasks shaken at 150 rpm for 48 hours at 30 °C. Bacterial cultures
(100 ml) were centrifuged at 5000 rpm for 20 min. The
extraction was carried out according to method of Shindy and
Smith (1975). The supernatants (30 ml) of the stationary phase
culture were adjusted to pH 8.6 with 1 % NaOH.
3.7.2. From root plants
Fresh root samples (2 g) of each of broad bean and wheat
plants were extracted with 80 % cold methanol for 24 hr at 5 °C.
The extraction was carried out according to method of Shindy
and Smith (1975). The roots extract were reduced to 10 ml
under vacuum. Then were adjusted to pH 8.6 with NaOH.
The extraction separation for both bacterial isolates and
root plants was carried out with ethyleacetate for 3 times with
equal volumes. The ethyleacetate fractions (Basic fraction) were
evaporated to dryness and kept for further purification. The
aqueous phase were adjusted to pH 2.8 with 1 % HCl and
extracted 3 times with equal volume of ethyleacetate
(acid fractions) and the remaining aqueous phase was discarded.
The acidic ethyleacetate phase was reduced to 5 ml under
vacuum and determine this fraction containing acid hormones
68
such as IAA, IBA and GA3 were determined by high

performance liquid chromatography (HPLC).
The dried basic ethyleacetate fraction was dissolved in
80 % methanol, then methanol was evaporated under vacuum
leaving an aqueous phase which was adjusted to pH 2.8 and
extracted 3 times with 25 ml ethyleacetate. The ethyleacetate
was combined (neutral auxins) reduced to 5 ml and stored at
20 °C until determined.
3.7.3. Determination of IAA, IBA and GA3
Standard and extracts of hormonal compound were
fractionated with Isco model 2360 gradient liquid chromatograph
equipped with a V-UTS-250 UV. The column was a 4-6 min i-d.
25 cm. Altex ultrasphere-ODS C- 18 reverse phase column.
Solvent gradients were formed by a set of microprocessor-
controlled solvent pump. Solvents consisted of 95: 5 v/v; water
acetic acid (solvent A) and methanol (solvent B). Each
determination required approximately 10 min. Sample volume
was 15 μl and hormonal compounds in the effeluent were
monitored at 217 nm., IAA, IBA and GA3 were separated at
retention time (2.97, 3.37 min), respectively. This method
described by Frankenberger and Brunner (1983) and
Grolamys and Servando (1997).
3.8. Effect of selected biofertilizers on growth of broad
bean and wheat plants under different levels of
inorganic and organic N fertilizers in greenhouse
experiments
Six experiments were conducted in the greenhouse to
examine the effect of selected biofertilizers under different levels
of inorganic and organic N-fertilizers on the growth of the
69
mentioned plants cultivated in clay and sandy soil of Sharkia

governorate.
3.8.1. Inocula preparation
The most active three locally isolates of Rhizobium
leguminosarum bv. vicieae (RZ11), Azotobacter sp. (ZH21) and
Azospirillum sp. (ASH21) were used in this investigation.
Rhizobium isolate was inoculated in YEM broth
(Allen, 1959) and incubated in the dark at 28 ± 2 °C in rotary
shaker 120 rpm for 3-5 days. Azotobacter and Azospirillum
isolates were inoculated in modified Ashby's medium and NF
semisolid malate medium, respectively and incubated in the dark
at 28 ± 2 °C in rotary shaker 120 rpm for 7 days.
3.8.2. Soil types and analysis
Two different soil textures were used in these greenhouse
experiments, namely clay and sandy soils obtained from Zagazig
and Salhia, located in Sharkia governorate, Egypt. The
mechanical and physicochemical analysis of both soils were
described according to Jackson (1970) and Harrigan and
McCance (1966) and listed in Table (3). The soil was collected
from top layer (0-30 cm depth) then distributed in plastic pots
(25 cm diameter and 20 cm depth) at the rate of 6 kg per pot.
3.8.3. Inorganic fertilizers
The commercial ammonium sulphate (20.5 % N), calcium
superphosphate (15.5 % P2O5) and potassium sulphate (48 %
K2O) were used as N, P and K mineral fertilizers, respectively.
The recommended doses of the above mentioned inorganic
fertilizers for broad bean are, 100 kg fed-1, 75 kg fed-1
70
ammonium sulphate (20.5 kg N fed-1, 15.375 kg N fed-1); 200 kg

fed-1, 150 kg fed-1 calcium superphosphate (31 P2O5 kg fed-1,
23.25 kg P2O5 fed-1), for sandy and clay soil, respectively. While
potassium sulphate is 50 kg fed-1 (24 K2O fed-1) for both soils.
Calcium superphosphate and potassium sulphate were added just
before sowing in both soils for all treatments, while ammonium
sulphate was added in two equal doses, before cultivation and
after 15 days from planting in sandy soil, while in clay soil, it
was added after 10 days from planting.
The recommended doses for wheat are, 500 kg fed-1, 375 kg
fed-1 ammonium sulphate (20.5 % N) i.e., 100 kg N fed-1 and
75 kg fed-1, for sandy and clay soil, respectively. While, calcium
superphosphate is 100 kg fed-1 (15.5 P2O5 kg fed-1) and
potassium sulphate is 100 kg fed-1 (48 K2O fed-1) for both types
of soil. Calcium superphosphate and potassium sulphate were
added just before sowing in both soil textures for all
treatments, while ammonium sulphate was added in three equal
doses after 21, 60 and 90 days of plantation in sandy soil. In clay
soil ammonium sulphate was added in equal doses after 21 and
60 days.
3.8.4. Organic fertilizer
Garbage compost was provided from the organic fertilizers
company, Cairo, Egypt. It has an organic carbon of 30 %, total
nitrogen 1.0 %, moisture, 35 % and pH 7.5 ± 0.2. Garbage
compost was thoroughly mixed with soil 20 days before
cultivation in both sandy and clay soil [recommended dose is
5 ton fed-1 (0.5 %) in sandy soil and 2 ton fed-1 (0.2 %) in clay
soil].
71
Table (2): physical and chemical analyses of the soil under

investigation
Type of soil analysis Properties Salhia soil Zagazig soil
Physical analysis pH 8.12 7.6

-1
EC mmhos.Cm 3.10 2.67
Mechanical analysis Fine sand % 24.45 27.1

Coarse sand % 71.15 7.12
Silt % 2.00 12.97
Clay % 2.40 52.82
Type of soil Sandy Clay
Chemical analysis Cationes

Ca++ 0.19 0.54
++
Mg 0.30 0.66
+
Na 0.79 1.49
+
K 0.06 0.07
Aniones
HCo3- 0.31 0.48
-
Cl 0.43 1.28
So4-- 0.61 1.52
Co3-- Traces Traces
Total nitrogen % 0.015 0.14

72
3.9. Experimental design and treatments study on the

selected biofertilizers on growth of broad bean
plants under different levels of N-application:
3.9.1. First experiment:
The first greenhouse experiment was carried out to examine
the effect of using biofertilizers with or without inorganic
fertilizers in two soil textures namely clay and sandy soils. Pots
(25 cm) were filled with 6 kg of non-sterile sandy or clay soil.
The sterilized seeds of broad bean (Giza 843, from Agronomy
Dept. Agric. Res. Center. Giza, Egypt) were soaked in
Rhizobium-suspension at the rate of 1 × 109 cfu for two hours. In
addition to control seeds were soaked in distilled water. The
seeds were sown on the 2nd of November 2003 and then 5 ml of
cell suspension was applied over the seeds in each treated pot
and covered with soil. Pots were amended with the following
treatments:
1- Control (uninoculated and without inorganic N-fertilizer) +
5 ml. broth medium.
2- Uninoculated + normal field dose of inorganic N-fertilizer.
3- Inoculated with Rhizobium leguminosarum bv. vicieae
(RZ11).
4- Inoculated with RZ11 + normal field dose of inorganic
N-fertilizer.
5- Inoculated with RZ11 + quarter the normal dose of inorganic
N-fertilizer.
6- Inoculated with RZ11 + half the normal dose of inorganic
N-fertilizer.
73
7- Inoculated with RZ11 + three quarter the normal dose of

inorganic N-fertilizer.
8- Inoculated with RZ11, Azotobacter (ZH21) and Azospirillum
(ASH21) + half the normal dose of inorganic N-fertilizer.
Inorganic fertilizers of phosphate and potassium were
applied as proviousy mentioned in both experiments. The
experimental design was completely randomized with
8 treatments and 3 replicates.
3.9.2. Second experiment
The second greenhouse experiment was carried out to
examine the effect of selected biofertilizers with or without
organic fertilizers in two soil textures on the growth of broad
bean plants. Pots (25 cm) were filled with 6 kg of none sterile
sandy or clay soil. The sterilized seeds of broad bean (Giza 843
cultivar) were soaked in Rhizobium-suspension at the rate of 1 ×
109 cfu for two hours. In addition, control seeds were soaked in
distilled water. The seeds were sown on the 2nd of November
2003 and then 5 ml of cell suspension was applied over the seeds
in each treated pot and covered with soil. Pots were amended
with the following treatments:
1- Control (uninoculated and without organic N-fertilizer) +
5 ml. broth medium.
2- Uninoculated + normal field dose of organic N-fertilizer.
(RZ11).
4- Inoculated with RZ11 + normal field dose of organic
N-fertilizer.
5- Inoculated with RZ11 + half normal field dose of organic
N-fertilizer.
74
6- Inoculated with RZ11 + double normal field dose of organic

N-fertilizer.
7- Inoculated with RZ11, Azotobacter (ZH21) and Azospirillum
(ASH21) + normal dose of organic N-fertilizer.
The experimental design was completely randomized with
3.9.3. Third experiment
The third greenhouse experiment was carried out to study
the effect of selected biofertilizers alone on growth of broad bean
plants in two soil textures namely sandy and clay soils. The
sterilized seeds of broad bean (Giza 843 cultivar) were soaked in
Rhizobium-suspension at the rate of 1 × 109 cfu. In addition,
control seeds were soaked in distilled water. The seeds were
sown on the 2nd of November 2003 and then 5 ml of cell
suspension was applied over the seeds in each treated pot and
covered with soil. Pots were amended with the following
treatments:
1- Control (uninoculated) + 5 ml. broth medium.
(RZ11).
3- Inoculated with RZ11 + Azotobacter (ZH21).
4- Inoculated with RZ11 + Azospirilum (ASH21).
5- Inoculated with RZ11 + Azotobacter (ZH21) +
Azospirilum (ASH21).
In all these three experiments, after ten days, plants were
thinned to three plants/pot and watered daily with tap water.
75
After 55 days from planting, plants were uprooted. Plant tops

were dried at 70 °C, weighed and ground. The nitrogen content
was then determined colorimetrically using Nessler solution
according the method described by Naguib (1969). Immediately
after removal of the tops, response samples of the roots
containing nodules from each plant of the respective treatment
were taken, transferred to the reaction jars for determining the
nitrogenase activity using the acetylene reduction assays method
(Hardy et al., 1973). Nodules were then counted and separated
from root, dried and weighed. Nitrogenase activity was estimated
on bases of per hours per gram weight of nodules. Total
N content in roots was also determined (Naguib, 1969).
Total phosphorus content in root and shoots was
determined colorimetrically by the hydroquinone method as
described by Snell and Snell (1954). Total potassium content in
roots and shoots using a carlzeiss flame-photometer with
acetylene burn described by Brown and Lilleland (1946) was
also determined. Determination of phytohormones (IAA, IBA,
GA3) in fresh roots were determined (Shindy and Smith, 1975).
At maturity the rest of all plants were harvested and seed
index and total nitrogen were determined.
3.10. Effect of selected biofertilizers on growth of wheat
plants under different levels of N-application
3.10.1. First experiment:
The first greenhouse experiment was carried out to examine
the effect of using biofertilizers with or without inorganic
fertilizers in two soil textures. Pots (25 cm) were filled with 6 kg
of non sterile sandy or clay soil. The grains of wheat (Giza
168 cultivar), were obtained from Wheat Research Section, Field
76
Groups Research Institute, ARC, Giza, Egypt. The wheat grains

were surface sterilized according to Vincent, (1970). The
sterilized grains were soaked in Azospirillum isolate suspension
at the rate of 1 × 109 cfu for two hours. The seeds were sown on
the 20th of November 2003 and then 5 ml of cell suspension was
applied over the grains in each treated pot and covered with soil.
Pots were amended with the following treatments:
1- Control (uninoculated and without inorganic N-fertilizer)
+5 ml. broth medium.
2- Uninoculated + normal field dose of inorganic N-fertilizer.
3- Inoculated with Azospirillm spp (ASH21).
4- Inoculated with ASH21 + normal field dose of inorganic
N-fertilizer.
5- Inoculated with ASH21 + quarter the normal dose of
6- Inoculated with ASH21 + half the normal dose of inorganic
N-fertilizer.
7- Inoculated with ASH21 + three quarter the normal dose of
inorganic N-fertilizers.
8- Inoculated with ASH21, Azotobacter (ZH21) and Rhizobium
leguminosarum bv. vicieae (RZ11) + half the normal dose of
Inorganic fertilizers of phosphate and potassium were
applied as proviousy mentioned in both experiments. The
experimental design was completely randomized with
77
3.10.2. Second experiment:

The second greenhouse experiment was carried out to
examine the effect of selected biofertilizers with or without
organic fertilizers in two soil textures on the growth of wheat
plants. Pots (25 cm) were filled with 6 kg of non sterile sandy or
clay soil. The sterilized grains were soaked in Azospirillum
suspension at the rate of 1 × 109 cfu for two hours. The seeds
were sown on the 20th of November 2003 and then 5 ml of cell
suspension was applied over the grains in each treated pot and
covered with soil. Pots were amended with the following
treatments:
1- Control (uninoculated and without organic N-fertilizer) +
5 ml. broth medium.
2- Uninoculated + normal field dose of organic N-fertilizer.
3- Inoculated with Azospirillm (ASH21).
4- Inoculated with ASH21 + normal field dose of organic
N-fertilizer.
5- Inoculated with ASH21 + half normal field dose of organic
N-fertilizer.
6- Inoculated with ASH21 + double normal field dose of
organic N-fertilizer.
7- Inoculated with ASH21, Azotobacter (ZH21) and Rhizobium
leguminosarum bv. vicieae (RZ11) + normal dose of organic
N-fertilizer.
78
3.10.3. Third experiment:

The third greenhouse experiment was carried out to study
the effect of selected biofertilizers alone on growth of wheat
plants in two soil textures. The sterilized grains were soaked in
Azospirillum suspension at the rate of 1 × 109 cfu for two hours.
The seeds were sown on the 20th of November 2003 and then
5 ml of cell suspension was applied over the grains in each
treated pot and covered with soil. Pots were amended with the
following treatments:
1- Control (uninoculated) + 5 ml. broth medium.
2- Inoculated with Azospirillm (ASH21).
3- Inoculated with ASH21 + Azotobacter (ZH21).
4- Inoculated with ASH21 + Rhizobium (RZ11).
5- Inoculated with ASH21 + Azotobacter (ZH21) + Rhizobium
leguminosarum bv. vicieae (RZ11).
In all the above three experiments, after ten days, plants
were thinned to 5 plants/pot and watered daily with tap water.
After 60 days from planting, the following parameters were
determined.
1- Dry weight of different parts of wheat plants (shoots and
roots) were determined by drying the plant parts at
70 °C for 24 hours (Somasegaran and Hoben, 1985).
2- Acetylene reduction assay: the acetylene reduction
activity (ARA) of rhizosphere soil was measured using
the method of Hardy et al. (1973).
79
3- Dehydrogenase activity was assayed according to

Casida et al. (1964)
4- Determination of phytohormones (IAA, IBA, GA3) in
fresh root (Shindy and Smith, 1975).
5- Total nitrogen content: total nitrogen content in roots
and shoots was determined colorimetrically using
Nessler solution according to the method described by
Naguib (1969).
6- Total phosphorus content in root and shoots
determinated colorimetrically by the hydroquinone
method as described by Snell and Snell (1954).
7- Total potassium content in root and shoots using a
carlzeiss
flame-photometer with acetylene burn described by
Brown and Lilleland (1946).
8- At harvesting time
a. Weighing of 1000 grains.
b. Total nitrogen content: total nitrogen content in
grains was determined colorimetrically using
Nessler solution according to the method described
by Naguib (1969).
c. Determination of crude protein in grains.
3.11. Media and solutions
The following media and solutions were used for isolation,
purification and growing of the tested Rhizobium, Azotobacter
and azospirillum isolates used in the present study.
80
3.11.1. Yeast extract mannitol (YEM, Allen, 1959)

Mannitol 10 g
Yeast extract 0.5 g
K2HpO4 0.5 g
MgSO4.7H2O 0.2 g
NaCl 0.1 g
Distilled water 1000 ml
pH 6.8
pH was adjusted to 6.8 then the medium was autoclaved at
121 °C for 15 minute. To prepare YEMA 15-20 g agar / liter
were added to the above mentioned broth medium (Allen, 1959;
Vincent, 1970 and Somasegaran and Hoben, 1985).
3.11.2. Congo-red yeast extract mannitol agar
The medium was prepared by adding 10 ml of 0.25 %
aqueous solution of Congo-red to each liter of YEMA as
previously described (Somasegaran and Hoben, 1985).
3.11.3. Bromothymol blue yeast extract mannitol agar
This media was prepared by adding 5 ml stock solution of
brom othymol blue (0.5 g / 100 ml ethanol to each liter of
YEMA medium) (Somasegaran and Hoben, 1985).
3.11.4. N. free nutrient solution
This nutrient solution was prepared as recommended by
Somasegaran and Hoben, (1985) as follows:
Stock solutions from g/l
1 CaCl2.2H2O 297.10
2 KH2PO4 136.10
3 Fe.citrate 6.7
MgSO4.7H2O 123.30
81
K2SO4 87.00
MnSO4 0.338
4 H3BO3 0.247
ZnSO4.7H2O 0.288
CuSO4.5H2O 0.10
CoSO4.7H2O 0.056
Na2MoO2.2H2O 0.048
For each 10 liters of full strength culture solutions, 5 ml
each of solution 1-4 were taken then added to 5 liter of water,
then diluted to 10 liters. NaOH (1.0 N) was used to adjust the pH
to 6.6 – 6.8.
3.11.5. Nutrient agar (Difco, 1985)
Beaf extract 3.0 g
Peptone 5.0 g
Agar-agar 15.0 g
Tap water 1000 ml
pH 7.00
3.11.6. Semi solid malate medium (Döbereiner et al., 1976)
Malic acid 5g
NaCl 0.1 g
K2HPO4 0.1 g
KH2PO4 0.4 g
FeCl3.6H2O 0.01 g
MgSO4.7H2O 0.2 g
CaCl2.2H2O 0.02 g
Na2MoO2.2H2O 0.002 g
Bromothymol blue (0.05 % in ethanol) 2.0 ml
82
KOH 4.0 g
Agar-agar 1.75 g
pH 7
3.11.7. N-free semi solid malate medium (NFb medium)
(Baldani and Döbereiner, 1980)
Malic acid 5g
K2HPO4 0.5 g
MgSO4.7H2O 0.2 g
NaCl 0.1 g
CaCl2 20 mg
KOH 4.0 g
Bromothymol blue (0.05 % in ethanol) 2.0 ml
Trace elements 2 ml
Vitamin solutions 1 ml
Fe.EDTA 4 ml
Agar 1.75 g
pH 6.8
The trace-element solution containing:
Na2MoO2.2H2O 200 mg
MnSO4.H2O 235 mg
H3BO3 280 mg
ZnSO4.7H2O 24 mg
CuSO4.5H2O 8 mg
83
The vitamin solution contained:

Biotin 10 mg
Pyridoxine 20 mg
3.11.8. Yeast extract semi solid malate medium (Day and
Döbereiner, 1976)
Malic acid 5.0 g
K2HPO4 0.5 g
NaCl 0.1 g
CaCl2 0.1 g
FeSO4.H2O 0.02 g
MnSO4.H2O 0.01 g
KOH 4.0 g
Yeast extract 0.5 g
pH was adjusted to 6.8 with KOH
3.11.9. Potato infusion agar (BMS medium, Baldani and
Dobereiner, 1980)
Potatos 200 g
Malic acid 2.5 g
KOH 2g
Sucrose 2.5 g
Vitamin solution 1 ml
pH was adjusted to 7 with KOH
84
Bromothymol blue (0.05 % in ethanol) 2 ml

Washed potatos were poiled for 30 minute and solution was
filtered through cotton gauze
3.11.10. Modified Ashby's medium (Abd El-Malek and Ishac,
1968)
Sacrose 10 g
Mannitol 10 g
K2HPO4 0.5 g
CaCO3 5g
NaCl 0.2 g
CaSO4 0.1 g
FeCl3 0.005 g
MnSO4.H2O 0.005 g
pH 7.00
3.11. Statistical analysis
The data were subjected to statistical analysis as a complete
randomized design according to Snedecor and Cochran (1980).
85
IV. RESULTS AND DISCUSSION

4.1. Testing the efficiency of different isolates of
Rhizobium, Azotobacter and Azospirillum
A total of 32 isolates of Rhizobium leguminosarum bv.
vicieae were isolated from roots of broad bean plants growing in
different locations in Sharkia governorate. In addition,
14 isolates of Azotobacter spp. and 18 isolates of Azospirillum
spp. were also isolated from soil and roots of wheat plant
growing in different locations in Sharkia governorate.
The collected isolates were tested for their efficiencies to
fix atmospheric nitrogen by using different methods, in order to
use the efficient Rhizobium, Azotobacter and Azospirillum
isolates as biofertilizers for broad bean (Vicia faba L.) and wheat
(Triticum aestivum L.) plants in the experiments carried out
in this study.
4.1.1. Testing the efficiency of Rhizobium isolates
Thirty two isolates of R. leguminosarum bv. vicieae were
tested in the greenhouse in order to study their efficiencies on
plant growth, nodulation and N2-fixation of broad bean
(Vicia faba L.) plants.
Data in Table 3 show the effect of inoculation with
32 isolates of R. leguminosarum bv. vicieae plus control
(without inoculation) on plant dry weight, nodulation,
nitrogenase activity, nitrogen content and phage interaction in
broad bean plants.
86
4.1.1.1. Nodulation
Data in Table 3 indicat that inoculation of broad bean
seedlings with different 32 isolates of R. leguminosarum bv.
vicieae resulted in formation of root-nodules with different
numbers depending on the efficiency of rhizobial- isolates. The
mean number of nodules per plant after 7 weeks from cultivation
ranged between 10.3 (in case of inoculation with isolate RH21)
and 126.7 (in case of isolate RZ33), while the uninoculated
control treatment did not form nodules because of the use of
autoclaved soil.
In addition, isolates RZ32, RK13, RZ23 and RZ22 of
R. leguminosarum bv. vicieae had the highest nodules dry weight
per plant, being 163.33, 150.0, 143.3 and 140.0 mg / plant,
respectively.
4.1.1.2. Nitrogenase activity
The specific nitrogenase activity of broad bean root
nodules, measured as μ mole C2H4/h/g nodules dry weight,
as affected by inoculation with different isolates of
R. leguminosarum bv. vicieae is also shown in Table 3.
Nitrogenase activity ranged between 7.206 (for isolate RZ11)
and 0.564 (for isolate RK11) μ mole C2H4/h/g nodules dry
weight. The results show that shoot nitrogen content of broad
bean plants was highly correlated with nitrogenase activity and
nodule mass in most of inoculated treatments.
These results are in agreement with those reported by
Salem et al. (1981), Narendra et al., (1996) and Hussein et al.
87
Table (3): Shoot and root dry weight (g/plant), total nitrogen
content, nitrogenase activity and phage interaction
in nodulation test of broad bean plants, as affected
by inoculation with different isolates of rhizobia
Mean
Mean dry Nitrogenase Mean Mean
Mean no. of shoot Mean root Phage
weight of activity root dry shoot N
Isolates nodules dry N content inter-
nodule µ mol/h/g weight content
/plant weight (mg/plant) action
(mg/plant) dry nodule (g/plant) (mg/plant)
(g/plant)
control 0.00 0.00 0.000 0.58 0.19 12.41 2.28 0
RR11 43.30 86.66 2.945 1.43 0.60 55.63 12.30 VI
RR13 36.70 103.33 2.917 1.04 0.28 34.32 5.12 VI
RR23 116.70 110.00 2.590 1.49 0.34 53.49 6.22 V
RR31 47.00 116.66 1.892 0.84 0.33 31.50 6.37 II
RF12 45.00 123.33 1.509 1.32 0.33 45.14 5.78 VI
RF13 36.00 123.33 3.955 1.36 0.45 55.62 10.44 VI
RF21 57.70 73.33 6.015 1.55 0.34 64.64 9.28 V
RF23 64.00 100.00 3.605 1.19 0.34 44.15 7.31 III
RF31 66.00 26.67 3.846 1.24 0.33 47.00 6.93 IV
RF32 83.30 16.66 3.225 1.04 0.34 40.77 6.63 VI
RH13 87.70 123.33 2.410 1.21 0.44 41.38 8.58 V
RH21 10.30 50.00 2.465 0.97 0.41 35.60 7.18 IV
RH31 92.00 93.33 3.833 0.95 0.27 36.67 5.94 V
RA21 25.70 116.66 2.494 0.93 0.28 31.81 5.40 V
RA33 35.00 130.00 1.219 1.28 0.53 43.26 8.90 VI
RZ11 46.70 90.00 7.206 1.65 0.46 69.96 12.56 IV
RZ21 48.70 126.67 1.707 1.25 0.55 41.75 9.63 VI
RZ22 56.70 140.00 5.008 1.47 0.49 61.30 11.27 IV
RZ23 69.30 143.33 1.273 1.37 0.56 44.25 9.46 IV
RZ32 43.00 163.33 0.766 0.68 0.21 19.86 3.34 V
RZ33 126.70 126.66 3.923 0.87 0.38 35.58 8.47 IV
RK11 71.70 73.33 0.564 0.86 0.20 23.74 3.06 VI
RK12 49.00 70.00 4.532 1.31 0.43 53.58 9.59 VI
RK13 42.30 150.00 1.563 0.96 0.32 35.23 5.60 IV
RK21 89.70 123.33 1.355 1.35 0.34 44.55 5.88 V
RK23 101.70 116.66 1.273 1.02 0.33 32.33 5.31 IV
RK31 41.70 30.00 1.760 0.98 0.24 33.52 4.39 IV
RK32 56.70 83.33 2.365 1.10 0.37 40.37 6.22 V
RS3 61.00 46.66 2.779 1.12 0.30 43.90 5.58 IV
RB1 66.00 113.33 1.043 1.19 0.42 44.74 8.06 IV
RB2 50.00 70.00 4.331 1.37 0.34 57.13 7.89 III
RB3 43.30 76.66 3.978 1.29 0.33 51.21 6.77 0
88
(1997) who reported that Rhizobium-inoculation significantly

increased the number and dry weight of nodules of broad bean
cultivated in newly reclaimed soil of Egypt.
They showed that Rhizobium-inoculation, combined with
the highest rate of P and K, produced the highest number and dry
weight of nodules, dry weight of shoot and N-content as well as
protein yield.
4.1.1.3. Plant dry weight
Inoculation of broad bean seedlings with either of
32 isolates of R. leguminosarum bv. vicieae increased both
shoots and roots dry weight as compared with the uninoculated
control (Table, 3). Inoculation with isolates RZ11 and RF21
gave the highest shoots dry weight, i.e., 1.65 and 1.55 g/plant,
respectively. On the other hand, isolates RR11 and RZ23 had the
highest roots dry weight, which recorded 0.60 and 0.56 g/plant,
respectively. However the uninoculated control treatment
yielded only 0.58 g shoots/plant and 0.19 g roots/plant. This
result demonstrated that broad bean plants when inoculated with
isolate RZ11 showed the highest plant dry weight as compared to
the other isolates, irrespective with the number and weight of
nodules which didn't show the highest parameters. It was also
observed that their nodules were large enough to fix more
nitrogen which reflected on the plant dry weights. Previous
investigations showed that the number of nodules is not
necessarry be correlated with their efficiencies for symbiotic
N2-fixed (Salem, 1962, 1969 and Mishra et al., 1999).
89
4.1.1.4. Nitrogen content

The effect of inoculation of broad bean seedlings with
different isolates of R. leguminosarum bv. vicieae on nitrogen
content of shoots and roots show variation in their response
(Table, 3). Isolates RZ11 and RF21 showed the highest shoots
nitrogen contents being 69.96 and 64.64 mg N/plant,
respectively. In addition, isolates RZ11 and RR11 showed also
the highest roots nitrogen contents being 12.56 and 12.30 mg
N/plant, respectively, giving the same general trends like those
of plant dry weight.
From these results it can be concluded that inoculation with
isolate RZ11 gave the highest nitrogen contents in shoots and
roots of broad bean plants as compared to plants inoculated with
other strains. This could also emphasize the phenomena of that
legumes responded differently when inoculated with different
rhizobial-strains isolated from different localities showing
varying efficiencies towards the symbiotic N2-fixation (Salem,
1962 and 1969;Hussein et. al.,1997and Brock et al. 2003).
In general conclusion, inoculation of broad bean
(Vicia faba) seedlings with R. leguminosarum bv. vicieae isolates
increased nodulation, plant dry weight, nitrogen content and
nitrogenase activity as compared with uninoculated control
treatment. The percent of increases differed from rhizobial-
isolate to another depending on the eco-systems of the isolates
and the physicochemical properties of the soil (Alexander 1977;
Russel Jones and 1975; Salem et al., 1981and Pathak, et al.
1997). The highest five isolates of R. leguminosarum bv. vicieae
in nitrogen fixation were RZ11, RZ22, RF21, RB2 and, RK12
90
which were isolated from nodules of plants collected from

Zagazig (Z), Fakous (F), Belbase (B) and Abo-Kibeer (K),
respectively. From data in Tables 3 and 4 it could be observed
that the more efficient rhizobial-isolates were isolated from
nodules of plants grown on soils characterized by being fertile
clay soil with normal salinity having EC ranging between
0.93 – 6.40 dsm-1 (Table 4). In this respect Hassan et al., (1990)
estimated the N2-fixed by broad bean plants grown on three soil
types. They found that the amount of N2-fixed differed markedly
depending upon inoculation treatment, dose of N-fertilizers
applied, and soil type. Also, in this respect, inoculation of broad
bean plants with isolate no. RH21 (isolated from El-Hessenia)
resulted in the lowest No. of nodules (10.3 nod./plant) with
relatively low values of shoot dry weight (0.97 mg/plant),
nitrogenase activity (2.465 μ mole C2H4/h/g nodule dry weight)
and shoot nitrogen content (35.6 mgN/plant). The latter
rhizobial-isolate had been isolated from plants grown on
El-Hessenia soil of high salinity (EC 9.4 dsm-1). Hwoever broad
bean plants inoculated with rhizobial-isolate No. RH31 showed
higher values of nodules number and dry weight, nitrogenase
activity and total nitrogen content of the plant, although the
inoculated Rhizobium was isolated from soil of relatively higher
salinity (EC 19.7 dsm-1) than RH21. This suggest that isolate
RH31 is relatively tolerant to soil salinity and could be used as
inoculant in such saline soil.
91
Table (4) physical and chemical analyses of soil in deffernt

locations of Sharkia governorate
Chemical analysis
locations physical analysis Mechanical analysis
cations anions
E.C Sand Clay

Locations sites pH O M.% Silt % Type of soil Ca++ Mg++ Na+ K+ Hco3 Cl- So4--
dsm-1 % %
1 7.86 2.67 2.43 33.22 13.91 52.89 Clay 0.55 0.60 1.65 0.07 0.28 0.81 1.70
Zagazig (Z) 2 7.45 0.93 2.79 43.55 13.10 43.31 Clay 0.33 0.31 0.45 0.03 0.41 0.32 0.07
3 8.09 1.45 2.50 35.0 30.2 43.7 Clay 0.19 0.45 1.31 0.3 0.54 0.42 0.68
Clay loam
1 7.51 4.3 1.68 25.5 40.1 34.2 5.7 6.9 15.9 .31 3.6 13.4 11.7
Sandy
Fakous (F) 2 7.50 5.6 0.87 55.1 25.5 19.6 10.3 12.8 31.7 .32 3.0 29.8 22.8
loam
3 8.00 6.4 1.82 38.8 5.6 55.6 10.7 11.5 43.5 .54 3.0 32.6 30.6
Clay
1 7.75 4.8 1.22 58.0 8.1 33.8 S.clay loam 6.8 6.8 34.8 0.4 3.6 8.6 3.9
Abo-Kibeer
2 7.95 1.2 1.59 35.0 30.2 34.7 Clay loam 7.1 5.5 3.5 0.5 4.1 41.3 51.7
(K)
3 8.01 8.8 1.52 70.8 7.6 21.6 S.clay loam 11.4 13.8 71.7 0.36 3.1 8.0 3.2
1 7.85 1.5 2.08 18.22 7.8 74.00 Clay 5.7 2.7 4.4 0.36 3.1 8.0 3.2
El-Hessenia
2 7.85 9.4 1.89 9.58 10.33 80.09 Clay 27.81 26.2 60.9 0.08 2.5 43.2 65.0
(H)
3 7.95 19.7 1.98 34.32 12.97 52.82 Clay 40.0 27.8 119.6 0.5 2.7 132.4 52.8
9.08
1 8.20 1.95 36.0 20.1 43.8 Clay 26.2 23.2 60.9 0.08 2.5 4.32 12.1
Kafr-Sakr 17.2
2 7.55 1.22 40.1 35.2 24.7 Loam 51.5 24.0 7.5 3.2 3.6 9.6 75.7
(R) 0
3 7.60 2.13 38.8 7.7 53.5 Clay 29.9 37.8 163.0 1.3 3.5 148.8 79.6
32.8
Abo- 1 7.75 4.8 1.8 32.2 27.6 39.6 Clay loam 6.8 6.3 34.8 0.41 3.4 28.4 16.2
Hammad 2 7.5 2.8 1.33 35.0 31.6 33.3 Clay loam 14.8 3.3 3.01 1.9 2.2 8.1 4.2
(A) 3 8.1 8.8 1.96 15.4 24.2 60.1 Clay 11.4 13.8 71.7 0.50 4.1 41.3 51.7
Sandy
Belbase (B) 1 7.1 7.0 1.22 87.2 1.6 11.2 26.2 22.1 34.8 0.46 2.4 38.4 42.7
loam
Salhia (S)
1 7.75 4.2 1.33 89.0 5.7 5.1 Sandy 27.1 15.3 20.1 0.31 2.3 43.3 18.1
th
92
4.1.1.5. The phage reaction of Rhizobium isolates

Phage typing is a common technique used to discriminate
between various strains of Rhizobium (Kowalski et al. 1974).
Phage typing potentially is more discriminatory than the use of
serology and as such can be used to type Rhizobium beyond the
level of antigenic reaction. The 32 isolates of Rhizobium
legumenosarum bv. vicieae were shown to fall into six categories
when typed with phage isolated from the rhizosphere of broad
bean plants in Sharkia governorate. The six categories were: no.
susceptibility (RB3 isolate), susceptible two (RR31), three
(RF23, RB2), four (RF31, RH21, RZ33, RZ11, RZ23, RK13,
RK31, RK23, RS3 and RB1), five (RR23, RF21, RH31, RH13,
RA21, RZ32, RK21, RK32) and the rest with all six phages.
No trend was observed between phage infection and ability
of the isolates to nodulate broad bean and fix atmospheric
nitrogen.
Rhizobiophages have been detected in soils of many
countries and are usually associated with legumes (Vincent,
1977). Some reports suggested that rhizobiophages are present in
the rhizosphere of legumes and are absent in non-rhizosphere
soils, (Golepiowska et al., 1971 and 1976). Also, in Egypt
rhizobiophage were found to be common in the Nile Valley soils
cultivated with leguminous plants (Emam et al., 1983; Othman,
1986; El-Didamony, 1990 and Salama, 1992) The presence of
rhizobiophage in fields, therefore, suggest that they could play
an important role in selection, propagation or elimination of
Rhizobium genotypes in nature (Vincent, 1977).
93
4.1.2. Testing the efficiency of N2-fixation by Azotobacter and

Azospirillum isolates in liquid culture
Acetylene reduction assay was set up in liquid culture to measure
the efficiency of nitrogenase activities of 14 different isolates of
Azotobacter and 18 isolates of Azospirillum (Table, 5).
Nitrogenase activity of Azotobacter isolates ranged between 0.69
(in case of isolate ZR22) and 52.44 μ mole C2H4/m1/h (in case of
isolate ZH21). Isolates ZH21, ZZ23, ZZ13 and ZH31 showed
the highest nitrogenase activity being 52.44, 40.27, 33.83 and
29.32 μ mole C2H4/m1/h, respectively. On the other hand,
isolates ZR22, ZZ11 and ZH11 resulted in the lowest nitrogenase
activity being 0.69, 12.04, and 13.64 μ mole C2H4/m1/h,
respectively. The highest five isolates of Azotobacter spp.
in nitrogen fixation were ZZ23, ZS3, ZH31, ZZ13 and ZH21
(Table, 4). It seams that the efficiencies of the isolated
Azotobacter differed from one isolate to another depending on
the environmental conditions prevailing the location from which
they were isolated. For example Azotobacter isolates ZZ23 and
ZZ13 which showed high efficiency for N2-fixation were
isolated from fertile clay soil from Zagazig region having
relatively high organic matter percentages (2.79 % and 2.43 %
respectively). This could be the reason for isolation of
Azotobacter of high N2-fixation efficiency from such soil
because of the impact of eco-system and synergistic effect of
other microbes execreting growth promoting substances that
proliferate high densities of Azotobacter. Alexander, (1977)
stated that a number of environmental factors govern the rate and
94
magnitude of non-symbiotic N2-fixation, and transformation is

markedly affected by the physical and chemical characteristics of
the habitat.
Another eco-factor could affect the proliferation of
Azotobacter and their efficiencies for N2-fixation is that of the
Ca-content of the soil used for isolation. For example isolate
ZH21 of Azotobacter which showed the highest nitrogenase
activity (52.44 µ mole c2H4/h /ml) was isolated from El-Hessenia
soil having high Ca content (27.81 %). In this regard Alexander,
(1977) reported that a requirement for calcium has been
demonstrated during N2-assimilation by the blue green alge and
some species of Azotobacter, but the calcium can sometimes be
replaced by strantium. On the other hand, the eco-factor could
also deleteriously affect the efficiency of the isolate if salinity is
prevailing the soil used for it's isolation (Russel and Jones
1975; Bashan and Holguin 1997 and Subba Rao, 1999). In
this respect, isolate ZR22 of Azotobacter which showed lower
nitrogenase activity was isolated from Kafr Saker soil which is
also characterized by being saline soil (EC 17.2 dsm-1) with
relatively low organic matter percentage (1.22 %).
Concerning Azospirillum, isolates nitrogenase activity
ranged between 0.01 (isolate ASS3) and 0.76 μ mole C2H4/h/m1
(isolate ASH21). Isolates ASH21, ASF32 and ASF11 showed
the highest nitrogenase activity values being 0.76, 0.62 and 0.51
μ mole C2H4/h/m1, respectively. However, isolates ASS3, ASS2
and ASS1 reflected the lowest values of nitrogenase activity
being 0.01, 0.02 and 0.03 μ mole C2H4/m1/h, respectively.
95
Table (5): Nitrogenase activity by Azotobacter and

Azospirillum isolates in liquid cultures
Nitrogenase Nitrogenase
Azotobacter Azospirillum
activity µ mole activity µ mole
isolates isolates
C2H4/h/ml C2H4/h/ml
ZH21 52.44 ASH21 0.76

ZZ23 40.27 ASF32 0.62
ZZ13 33.83 ASF11 0.51
ZH31 29.32 ASA12 0.47
ZS3 28.03 ASZ11 0.41
ZK31 22.83 ASA23 0.39
ZA11 16.41 ASK23 0.31
ZK12 15.26 ASK33 0.08
ZF22 25.64 ASB1 0.06
ZF31 27.94 ASZ33 0.06
ZA31 26.98 ASH32 0.16
ZH11 13.64 ASA33 0.09
ZZ11 0.69 ASF12 0.10
ZR22 0.69 ASR31 0.06
ASS2 0.02
ASS3 0.01
ASK21 0.04
ASS1 0.03
The highest five isolates of Azospirillum spp in nitrogen

fixation were ASH21, ASF32, ASF11, ASA12 and ASZ11
(Table, 5).
Azospirillum isolates showed also some correlations
between location of their isolation and their efficiencies in fixing
96
nitrogen in liquid culture. It was also observed that the efficient

isolates were isolated from fertile clay soil of either Zagazig
region (ASZ11) or Fakous (ASF11 and ASF32) or El-Hessenia
(ASH21).
Here, also an additional proof that efficiency for N2-fixation
of Azospirillum isolates is correlated to some extent with the
ecosystem and habitat of these organisms, in addition to the
physico-chemical properties of the soil they survived,
(Döbreiner, 1974; Okon, 1984 and Okon and Itzigsohn 1995).
4.1.3. Determination of growth promoting substances in
liquid cultures of Rhizobium, Azotobacter and
Azospirillum isolates
The highest five N2-fixing isolates of each of
R. leguminosarum bv. vicieae, Azotobacter spp. and
Azospirillum spp were used for determination of GA3, IAA and
IBA in liquid culture.
4.1.3.1. GA3, IAA and IBA in Rhizobium isolates
Data in Table 6 indicate that the highest GA3content in
culture filtrate was obtained by rhizobial-isolate RZ11
(48.398 μg/100 m1), followed by RK12 isolate (25.825 μg/100
m1). The lowest content of GA3 in culture filtrate was obtained
by RF21 isolate (5.865 μg/ 100 m1). In addition, the highest
content of IAA in culture filtrate was obtained by RK12 isolate
(0.22 μg/100 m1) followed by RZ22 isolate (0.17 μg / 100 m1).
While, the lowest content of IAA in culture filtrate was obtained
by RF21 isolates (0.00 μg/ 100 m1). On the other hand, isolates
RF21 and RZ22 produced high concentration of IBA being 29.96
97
and 18.93 μ g/100 m1, respectively. However, IBA was not

detected in culture filtrate of RK12, RB2 and RZ11.
4.1.3.2. GA3, IAA and IBA in Azotobacter isolates
Data in Table 6 indicated that production of GA3 in
Azotobacter spp. isolates generally, ranged between 0.933 and
17.735 μg/100 m1. The highest content of GA3 in culture filtrate
was obtained by ZH21 (17.735 μg/100 m1) followed by ZZ13
isolate (10.712 μg/100 m1). The lowest content was obtained by
ZS3 isolate (0.933 μg/ 100 m1).
The culture filtrate content of IAA produced by
Azotobacter isolates generally, ranged between 0.04 and
0.15 μg/100 m1. The highest content of IAA in culture filtrate
was obtained by isolate ZH21 (0.15 μg/ 100 m1). The lowest
content in culture filtrate was obtained by isolates ZZ23, ZZ13
and ZH31 (0.04 μg/100 m1of each). However, the culture
filtrates of all examined Azotobacter isolates were IBA-free.
In this respect Taller and Wong (1989) reported that
Azotobacter vinelandii can produce cytokinine like substances in
culture medium.
4.1.3.3. GA3, IAA and IBA in Azaspirillium isolates
Data in Table 6 indicate that production of GA3 in
Azospirillum spp. isolates generally, ranged between 16.208 and
118.416 μg/ 100 m1. The highest value was obtained by ASF 32
isolate (118.416 μg/ 100 m1), followed by isolate ASH21
(48.518 μg/ 100 m1), while, the lowest content of GA3 in culture
filtrate was obtained by isolate ASF 11 (16.208 μg/ 100 m1).
IAA of culture filtrate in Azospirillum isolates generally, ranged
between 0.15 and 1.91 μg/ 100 m1. The highest content was
obtained by isolates ASH21 (1.91 μg/ 100 m1), followed by
98
ASF32 (1.07 μg/ 100 m1), while the lowest content was obtained
by isolate ASF 11 (0.15 μg/ 100 m1).
Table (6): GA3, IAA and IBA in liquid culture of Rhizobium
leguminosarum bv. vicieae, Azotobacter and
Isolates GA3 (μg/100ml) IAA (μg/100ml) IBA (μg/100ml)
Rhizobium isolates
RF21 5.865 0.00 29.96

RZ22 10.614 0.17 18.93
RK12 25.825 0.22 0.00
RB2 9.507 0.12 0.00
RZ11 48.398 0.10 0.00
Azotobacter isolates
ZZ23 2.164 0.04 0.00

ZS3 0.933 0.06 0.00
ZH31 1.071 0.04 0.00
ZZ13 10.712 0.04 0.00
ZH21 17.735 0.15 0.00
ASF32 118.416 1.07 362.48

ASA12 17.973 0.93 22.20
ASZ11 17.943 1.05 111.42
ASF11 16.208 0.15 1480.28
ASH21 48.518 1.91 1517.48
However, the culture filtrate content of IBA generally,

ranged between 22.20 and 1517.48 μ g/100 m1. The highest
content was obtained by isolate ASH21 (1517.48 μ g/100 m1)
followed by ASF11 (1480.228 μ g/ 100 m1). However, the
99
lowest content of IBA was obtained by isolate ASA12

(22.20 μ g/ 100 m1).
In this respect Tien et al. (1979) reported that Azospirillum
in culture produces plant growth promoting substances.
Hartmann et al. (1983) obtained IAA over producing mutants
of Azospirillum in culture media. Baca et al. (1994) stated that
indole-3-acetic acid (IAA) is excreted by different wild strains of
Azospirillum spp. They added that microorganisms can produce
IAA during the late stationary growth phase and significant
increase in IAA production occurred when tryptophan is added.
Also, Dobbelaera et al. (1999) reported that auxine produced by
Azospirillum is believed to play a major role in the plant growth
promoting effect.
These results suggest that isolate RZ11 of
R. leguminosarum bv. vicieae was the highest efficient isolate in
terms of plant growth, nodulation, nitrogenase activity and
production of growth promoting substances. In addition, isolate
ZH21 of Azotobacter sp. reflected the highest nitrogenase
activity and production of growth promoting substances.
Moreover, isolate ASH21 of Azospirillum sp. showed also the
highest nitrogenase activity and production of growth promoting
substances.
The three isolates RZ11, ASH21 and ZH21 of
R. leguminosarum bv. vicieae , Azospirillum sp and Azotobacter
sp, respectively were chosen as the best indigenous isolates to be
used as bio-inoculants for seedling of broad been and wheat
plants in the next exeperiments .
Utilization of plant-growth promoting bacteria in order to
minimize fertilizer application should be recommended both
100
economically and also to avoid environmental pollution by

excessive applications of industrially produced fertilizers to
cultivate fields.
4.2. Response of broad bean plants to bio-fertilizers and
and clay soils
A pot experiment was conducted to evaluate the response of
broad bean (Giza 843) grown on sandy and clay soils under
different N-fertilizers levels to inoculation with
R. leguminosarum bv. vicieae (RZ11) alone or tri-mixture
inoculants of R. leguminosarum bv. vicieae (RZ11) +
Azotobacter spp. (ZH21) + Azospirillum spp. (ASH21).
The nodulation, nitrogenase activity, plant dry weight, and total
nitrogen, phosphorus, and potassium contents as well as grain
yield of broad bean plants were determined.
4.2.1. Nodulation
Data in Table 7 and illustrated by Fig. 2 show the effect of
bio-fertilization and inorganic N-fertilizers application on
number of nodules, dry weight of nodules and nitrogenase
activity in broad bean plants grown on both sandy and clay soils.
The results showed that the number of nodules of broad bean,
generally ranged between 8.00 and 24.33 nodules/plant. The
highest number of nodules was obtained by treatment the plants
with 1/2 N (10 kg nitrogen / fed) + inoculation with isolate RZ11
in sandy soil and treatment receiving only 1/4 N (5 kg nitrogen / fed)
+ inoculation with isolate RZ11 in clay soil being 24.33 and
14.67 nodules/plant, respectively. The uninoculated treatment
(control) gave the lowest number of nodules in both soils being
Table (7): Effect of bio-fertilizers and inorganic N-fertilizers on number of nodules, dry weight of
nodules (mg/plant) and nitrogenase activity of broad bean plants
Soil textures Number of Nodules dry weight Nitrogenase activity µ mole
nodules/plant (mg/plant) C2H4/h/g dry nodule
Fertilization treatments Sandy Clay Av. Sandy Clay Av. Sandy Clay
Control 4.67 0.00 2.33 6.33 0.00 3.17 0.128 0.000
Inoculation by RZ11 * 17.33 16.67 17.00 16.00 42.33 29.17 4.040 3.905
Recommended nitrogen dose 5.00 0.00 2.50 6.67 0.00 3.33 2.137 0.000
N + RZ11* 16.67 0.00 8.33 23.33 0.00 11.67 4.817 0.000
1/4 N + RZ11* 16.33 14.67 15.50 18.00 70.67 44.33 3.489 2.377
1/2 N + RZ11* 24.33 8.33 16.33 53.33 36.33 44.83 5.446 1.264
3/4 N + RZ11* 16.67 14.33 15.50 15.33 20.33 17.83 3.362 0.082
**
1/2 N+ RZ11+ZH21+ASH21 19.67 8.00 13.83 35.00 16.67 25.83 7.423 1.427
Soil texture av. 15.08 7.75 21.75 23.29 - -
LSD for Soil texture Fertilization S × F Soil texture Fertilization S × F Soil texture Fertilization
0.05 2.14 4.27 6.04 NS 23.07 NS - -

0.01 3.09 6.18 8.75 NS NS NS - -
* Inoculation with Rhizobium leguminosarum bv. vicieae (RZ11).
** Inoculation with Rhizobium leguminosarum bv. vicieae (RZ11), Azotobacter spp. (ZH21) and Azospirillum
spp. (ASH21).
N: Recommended dose of nitrogen (20 kg N in sandy soil and 15 kg N in clay soil).
25 Sandy
No. of nodules/plant
20 Clay
15
10
0
1 2 3 4 5 6 7 8
Treatments
80 Sandy
70
Nodule dry weight
60 Clay
(mg/plant)
50
40
30
20
10
0
1 2 3 4 5 6 7 8
Treatm ents
Fig (2): Effect of bio-fertilizers and inorganic N-fertilizers on

number and dry weight of nodules in broad bean
plants grown in sandy and clay soils
1: Control 2: Inoculation by RZ11 3: N dose

4: N + RZ11 5: 1/4 N + RZ11 6: 1/2 N + RZ11
7: 3/4 N + RZ11 8: 1/2 N+ RZ11+ZH21+ASH21.
103
4.67 and 0.00 nodules/plant in sand and clay soils, respectively.

The differences among treatments were highly significant
(P ≤ 0.01).
In addition, the data indicate also that sandy soil recorded
highly significant number of nodules (P ≤ 0.01) being
15.08 nodules/plant as compared to the clay soil which had
7.75 nodules/plant. Also, the interaction between fertilization
treatments and soil textures were highly significant (P ≤ 0.01).
On the other hand, the data in Table 7 and Fig. 2 indicate
that nodules dry weight (mg/plant) generally, ranged between
15.33 and 70.67 mg/plant. The differences among treatments
were significant (P ≤ 0.05). The highest nodules dry weight on
broad bean plants were obtained by receiving 1/2 N dose +
inoculation with R. leguminosarum bv. vicieae isolate (RZ11)
under growing in sandy soil and the treatment received 1/4 N
dose + inoculation with isolate RZ11 in plants grown on clay soil
by 53.33 and 70.67 mg/plant, respectively. The differences
between soil textures were not significant, as well as the
interaction between fertilization treatments and soil textures.
The specific nitrogenase activity of broad bean root nodules
(μ mole C2H4/g nodules dry weight) as affected by rhizobial-
inoculation and application of different levels of inorganic
N-fertilizers in both sandy and clay soils is also given in Table 7.
The values ranged from 0.082 to 7.423 μ mole C2H4/h/g nodules
dry weight. The highest value was obtained by treatment of the
plants with 1/2 N dose + inoculation with tri-mixture inoculants
of RZ11, ZH21 and ASH21 in sandy soil, showing 7.423 μ mole
C2H4/g nodules dry weight. The lowest value of nitrogenase
activity (0.128 μ mole C2H4/h/g nodules dry weight) was
104
observed in uninoculated plants in sandy soil. In clay soil,

however the plants did not form nodules and hence no
nitrogenase activity was detected. This could be attributed to the
high amount of nitrogen in clay soil as seen in chemical analysis
(Table, 2) which could be deleteriously affecting the nodule
formation in such soil.
These results are in agreement with those obtained by
Hassan et al. (1990). who found that the increase of dose of
nitrogen fertilizer led to marked decrease in nodulation of broad
bean and amount of N2-fixed. Also clay soil showed relatively
lower percentage of nitrogen derived from atmosphere as
compared with both calcareous and sandy soils. In this respect,
Narendra et al. (1996) and Hussein et al. (1997) and Hamaoui
et al. (2001) demonstrated that inoculation with Azospirillum
brasilense significantly enhanced nodulation by native
Rhizobium and improved root and shoot development of
chickpea and broad bean plants when compared to the
uninoculated control.
Our results concluded that inoculation with tri-mixture
inoculants of RZ11, ZH21 and ASH21 isolates plus the
application of 1/2 dose inorganic N gave the best results with
regard to number of nodules, nodules dry weight and nitrogenase
activity in broad bean plant cultivated in sandy and clay soils.
4.2.2. Plant dry weight:
Data in Table 8 and illustrated by Fig. 3 show the effect of
bio-fertilizers and inorganic N-fertilizers on plant dry weight
(g/plant) of broad bean plants grown on both sandy and clay
Table (8): Effect of bio-fertilizers and inorganic N-fertilizers on shoot, root and whole plant dry weight
(g/plant) of broad bean plants
Soil textures Shoot dry weight Root dry weight Whole plant dry
g/plant g/plant weight g/plant
Fertilization treatments Sandy Clay Av. Sandy Clay Av. Sandy Clay Av.
Control 1.30 1.13 1.22 0.24 0.22 0.23 1.54 1.35 1.44
Inoculation by RZ11 * 2.07 1.99 2.03 0.55 0.42 0.48 2.61 2.40 2.51
Recommended nitrogen dose 2.23 1.38 1.81 0.43 0.35 0.39 2.66 1.73 2.20
N + RZ11* 2.16 1.52 1.84 0.48 0.33 0.41 2.64 1.85 2.25
1/4 N + RZ11* 1.67 1.64 1.65 0.31 0.32 0.32 1.98 1.96 1.97
1/2 N + RZ11* 2.25 1.74 1.99 0.43 0.47 0.45 2.68 2.20 2.44
3/4 N + RZ11* 1.80 1.91 1.86 0.45 0.45 0.45 2.25 2.36 2.31
1/2 N+ RZ11+ZH21+ASH21 ** 2.15 2.00 2.08 0.48 0.50 0.49 2.63 2.50 2.56
Soil texture av. 1.95 1.66 0.42 0.38 2.37 2.04
LSD for Soil texture Fertilization S×F Soil texture Fertilization S×F Soil texture Fertilization S×F
0.05 0.23 NS NS NS 0.09 NS 0.24 0.49 NS

0.01 NS NS NS NS 0.13 NS NS NS NS
spp. (ASH21).
2.5 Sandy
Shoot dry weight
(g/plant) 2 Clay
1.5
0.5
0
1 2 3 4 5 6 7 8
Treatm ents
0.6 Sandy
0.5
Root dry weight
Clay
(g/plant)
0.4
0.3
0.2
0.1
0
1 2 3 4 5 6 7 8
Treatm ents
3 Sandy
Whole plant dry weight (g/plant)
2.5 Clay
2
1.5
1
0.5
0
1 2 3 4 5 6 7 8
Treatm ents

dry weight of broad bean plants grown in both sandy
and clay soils
4: N + RZ11 5: 1/4 N + RZ11 6: 1/2 N + RZ11
7: 3/4 N + RZ11 8: 1/2 N+ RZ11+ZH21+ASH21.
107
soils. The results showed that shoot dry weight generally ranged
from 1.13 to 2.25 g/plant. The lowest value of shoot dry weight
was obtained by uninoculated treatment (control) in both sandy
and clay soils, being 1.30 and 1.13 g/plant, respectively. The
highest value of shoot dry weight was obtained by treatment
receiving 1/2 N dose + inoculated with RZ11 in sandy soil and
treatment receiving 1/2 N dose + inoculated with
tri-mixture inoculants of RZ11, ZH21 and ASH21in clay soil,
being 2.25 and 2.00 g/plant, respectively. The differences
between soil textures were significant (P ≤ 0.05), while the
differences among treatments and the interaction between soil
textures and treatments were not significant.
In addition, the root dry weight (g/plant) ranged from 0.22
to 0.55 g/plant. The differences among treatments were highly
significant (P ≤ 0.01). While, the differences between soil
textures and the interaction between soil textures and fertilization
treatments were not significant. The highest average root dry
weight (0.49 g/plant) was recorded for plants treated with 1/2 N
dose + inoculated with tri-mixture inoculants of RZ11 + RZ21
and ASH21 as compared with the averages of all other
treatments of both sandy and clay soils.
Regarding the effect of bio-fertilization and inorganic
N-fertilizers on the whole plant dry weight, it generally, ranged
between 1.35 and 2.68 g/plant. The differences among
fertilization treatments and between soil textures were significant
(P ≤ 0.05). However, the interaction between soil textures and
treatments was not significant. The lowest dry weight of the
whole plant was obtained in case of the control treatment in both
108
sandy and clay soils, being 1.54 and 1.35 g/plant, respectively.
The highest value of whole plant dry weight was obtained by the
treatment receiving 1/2 N dose + inoculation with isolate RZ11
in sandy soil and treatment receiving 1/2 N dose + inoculation
with tri-mixture inoculants of RZ11 + ZH21 and ASH21 in clay
soil, showing 2.68 and 2.50 g/plant, respectively. It is clear that,
whole plant dry weight of broad bean plants grown on sandy soil
was higher than those grown on clay soil, reflecting average
values of 2.37 and 2.04 g/plant, respectively.
The obtained date are in agreement with those reported by
Hassan et al., (1990), El-Ghandour and Galal (1997),
Namdeo and Gupta (1999) and Anyeabeba et al. (2001). The
latter revealed that inoculation of broad bean plants with bio-
fertilizers and applied inorganic N-fertilizer gave the best results
with regard to shoots, roots and whole plant dry weights.
In addition, El-Ghandour et al. (2001) studied the effect of
Rhizobium inocultion and mineral nitrogen for growth,
N2-fixation and yield of broad bean. They found that the
application of inoculants showed remarkable increase in dry
matter of shoot and grain yield as well as the N-uptake by both
components.
These data concluded that inoculation with tri-mixture
inoculants of RZ11, ZH21 and ASH21 plus the application of
1/2 N-inorganic gave the highest value with regard to shoots,
roots and whole plant dry weights of broad bean plants cultivated
in sandy and clay soils.
109
4.2.3. Total nitrogen content

Data presented in Table 9 and illustrated by Fig. 4 show
that application of bio-fertilizers plus inorganic N-fertilizer to
broad bean plants increased the total nitrogen content in shoots,
roots and the whole plants (mgN/plant) comparing with the
uninoculated control treatment. The highest value of total
nitrogen content in shoots of broad bean plants was recorded by
the treatment received 1/2 N dose + inoculation with tri-mixture
inoculants of RZ11 + ZH21 + ASH21 under growing in sandy
soil, giving 95.64 mgN/plant as compared with the control which
showed 37.71 mgN/pant. The highest value of total nitrogen
content in shoots of broad bean plants grown on clay soil was
recorded by the treatment inoculated with RZ11 only being
78.70 mgN/plant as compared with the control which showed
33.73 mgN/plant.
The total nitrogen content in roots of broad bean plants
showed nearly the general trend like shoots. The statistical
differences among fertilization treatments were highly
significant (P ≤ 0.01).Concerning the total nitrogen valuses of
the whole plant, they ranged from 37.19 to 105.83 mgN/plant.
The statistical differences among fertilization treatments were
highly significant (P ≤ 0.01). Also, the statistical differences
between soil textures were significant (P ≤ 0.05). The treatment
received 1/2 N dose + inoculatied with tri-mixture inoculants of
RZ11 + ZH21 and ASH21 gave the highest value of total
nitrogen content in whole plant as compared to the control being
105.83 and 41.34 mgN/plant in sandy soil and 85.46 and
37.19 mgN/plant in clay soil, respectively.
Table (9): Effect of bio-fertilizers and inorganic N-fertilizers on total nitrogen content in shoot, root and
whole plant (mg N/plant) of broad bean plants
Soil textures Total N/shoot Total N/ root Total N/plant
(mg/plant) (mg/plant) (mg/plant)
Sandy Clay Av. Sandy Clay Av. Sandy Clay Av.
Fertilization treatments
Control 37.71 33.73 35.72 3.63 3.47 3.55 41.34 37.19 39.27
N + RZ11* 79.79 66.03 72.91 9.41 7.19 8.30 89.20 73.22 81.21
1/4 N + RZ11* 63.96 56.98 60.47 5.83 6.82 6.33 69.79 63.81 66.80
1/2 N + RZ11* 88.85 67.53 78.19 12.63 10.62 11.63 101.48 78.15 89.82
3/4 N + RZ11* 68.47 70.20 69.34 9.01 9.86 9.44 77.48 80.06 78.77
1/2 N+ RZ11+ZH21+ASH21 ** 95.64 76.25 85.95 10.19 9.21 9.70 105.83 85.46 95.65
Soil texture av. 74.75 62.72 8.79 8.00 83.54 70.72
0.05 9.60 19.19 NS NS 2.15 NS 10.14 20.28 NS

0.01 NS 27.79 NS NS 3.11 NS NS 29.37 NS
spp. (ASH21).
100 Sandy
80 Clay
Total N/shoot
(mg/plant)
60
40
20
0
1 2 3 4 5 6 7 8
Treatm ents
14 Sandy
12
Clay
Total N/root
(mg/plant)
10
8
6
4
2
0
1 2 3 4 5 6 7 8
Treatm ents
120 Sandy
100 Clay
Total N/plant
(mg/plant)
80
60
40
20
0
1 2 3 4 5 6 7 8
Treatm ents

total nitrogen content (mg/plant) of broad bean
cultivated in both sandy and clay soils
4: N + RZ11 5: 1/4 N + RZ11 6: 1/2 N + RZ11
7: 3/4 N + RZ11 8: 1/2 N+ RZ11+ZH21+ASH21.
112
These results are in harmony with those obtained by Salem,

(1962) and (1969); Hassan et al., (1990); Hussein et. al., (1997);
El-Ghandour et al., (2001) and Brock et al. (2003). In addation,
Rodelas et al. (1999), pointed out that co-inoculants of broad
bean with Rhizobium leguminosarum bv. vicieae + Azotobacter
and Azospirillum led to changes in total N content concentration
and/or distribution of macro and micronutrients, K, P, Ca, Fe,
Mg, Mn, B, Zn, and Cu when compared with plants inoculated
with Rhizobium only.
4.2.4. Total phosphorus content
the effect of bio-fertilizers and inorganic N-fertilizer on total
phosphorus content in shoot, root and whole plant (mg P/plant)
in broad bean plants cultivated in both sandy and clay soils. The
results showed that the total phosphorus content in shoot of
broad bean plants, generally, ranged from 3.35 to 14.27 mg
P/plant. The total phosphorus content of broad bean plants of
shoots grown on both soils increased due to application of bio-
fertilization on the expense of inorganic N-fertilizer. The highest
total phosphorus content in broad bean plant shoots was obtained
by the treatments receiving 1/2 N dose + inoculated with tri-
mixture inoculants of RZ11, ZH21 and ASH21 in plants grown
under both sandy and clay soils, giving 14.27 and 8.64 mg
P/plant, respectively. While, the uninculated treatment (control)
gave the lowest total phosphorus content in broad bean shoots in
sandy and clay soils, being 4.41 and 3.35 mg P/plant,
respectively. The statistical differences among fertilization
Table (10): Effect of bio-fertilizers and inorganic N-fertilizers on total phosphorus content in shoot, root
and whole plant (mg P/plant) of broad bean plants
Soil textures Total P/shoot Total P/root Total P/plant
Control 4.41 3.35 3.88 0.41 0.34 0.38 4.82 3.69 4.25
N + RZ11* 12.24 7.29 9.77 1.24 0.80 1.02 13.48 8.09 10.78
1/4 N + RZ11* 10.84 6.52 8.68 0.62 0.70 0.66 11.45 7.22 9.34
1/2 N + RZ11* 11.78 6.61 9.20 0.95 1.06 1.00 12.73 7.67 10.20
3/4 N + RZ11* 9.37 8.08 8.72 0.96 1.18 1.07 10.33 9.26 9.79
1/2 N+ RZ11+ZH21+ASH21 ** 14.27 8.64 11.46 1.25 1.13 1.19 15.52 9.77 12.65
Soil texture av. 10.75 6.51 0.94 0.84 11.70 7.35
0.05 1.15 2.31 NS NS 0.20 NS 1.19 2.38 NS

0.01 1.67 3.34 NS NS 0.29 NS 1.72 3.44 NS
spp. (ASH21).
16 Sandy
14
Clay
Total P/shoot
12
(mg/plant)
10
8
6
4
2
0
1 2 3 4 5 6 7 8
Treatm ents
1.4 Sandy
1.2
Clay
Total P/root
(mg/plant)
1
0.8
0.6
0.4
0.2
0
1 2 3 4 5 6 7 8
Treatm ents
16 Sandy
14
Clay
Total P/plant
12
(mg/plant)
10
8
6
4
2
0
1 2 3 4 5 6 7 8
Treatm ents

total phosphorus content (mg/plant) of broad bean
4: N + RZ11 5: 1/4 N + RZ11 6: 1/2 N + RZ11
7: 3/4 N + RZ11 8: 1/2 N+ RZ11+ZH21+ASH21.
115
treatments and between soil textures were highly significant

(p ≤ 0.01), but the interaction between treatments and soil
textures was not significant.
Concerning the total phosphorus content in broad bean
roots (Table, 10), almost similar results were obtained as found
in shoots of broad bean plants. It was found that, the application
of bio-fertilizers combined with inorganic N-fertilizer increased
the total phosphorus content of broad bean roots grown on both
sandy and clay soils, as compared to the uninoculated treatment
(control). The differences among fertilization treatment were
highly significant (p ≤ 0.01).
Consequently similar results were also found in the total
phosphorus content in whole broad bean plant. Application of
1/2N dose and inoculation with tri-mixture inoculants of RZ11,
ZH21 and ASH21 gave the highest values in both sandy and clay
soil, being 15.52 and 9.77 (mg P/plant), respectively. The lowest
values were obtained by the uninoculated treatment (control),
being 4.82 and 3.69 mg P/plant in both soils, respectively. The
statistical differences among fertilization treatments and between
soil textures were highly significant (p ≤ 0.01). These results are
in agreement with those obtained by Rodelas et al. (1999).
In general, the results concluded the importance of
inoculation of broad bean plants grown either in sandy or clay
soils with Rhizobium leguminosarum bv. vicieae (RZ11),
Azotobacter sp. (ZH21) and Azospirillum sp. (ASH21)
to realize the highest plant growth containing high value of
phosphorus in the whole plant.
116
4.2.5. Total potassium content

The effect of bio-fertilizers and inorganic N-fertilizer on
total potassium content in shoot, root and whole plant of broad
bean cultivated in both sandy and clay soils is shown in Table 11
and illustrated by Fig. 6 .The results show that the treatment
inoculated with R. leguminosarum bv vicieae (isolate RZ11)
gave the highest values of total potassium content in shoot of
broad bean plants grown on sandy and clay soils, as compared to
uninoculated treatment (control). These values were 95.76 and
90.46 (mg K/plant) in inoculated treatment, and 41.15 and 38.52
mg K/plant in uninoculated treatment (control) in both sandy and
clay soils, respectively. Application of inorganic N-fertilizers
combined with inoculation by bio-fertilizers had lower values of
potassium content in shoot of broad bean plants as compared to
the inoculated treatment only without inorganic N-fertilizers.
The statistical differences among treatment were highly
significant (P≤0.01).
Regarding the total potassium content in roots of broad
bean plants, ranged between 3.47 and 15.11 mgK/plant in both
soils.
Concerning the total potassium content in whole plant
(Table 11 and Fig. 6), similar results were found as given in
shoot of broad bean plants in both soils. The treatment
inoculated with isolate RZ11 gave the highest total potassium
content in whole plant of broad bean as compared to the other
treatments. The statistical differences among fertilization
Table (11): Effect of bio-fertilizers and inorganic N-fertilizers on total potassium content in shoot, root
and whole plant (mg K/plant) of broad bean plants
Soil textures Total K/shoot Total K/root Total K/plant
Control 41.15 38.52 39.84 3.55 3.47 3.51 44.70 41.99 43.35
*
Inoculation by RZ11 95.76 90.46 93.11 15.11 10.29 12.70 110.87 100.75 105.81
N + RZ11* 93.18 65.61 79.40 11.96 6.84 9.40 105.14 72.44 88.79
1/4 N + RZ11* 64.90 64.63 64.77 6.54 6.26 6.40 71.45 70.88 71.17
1/2 N + RZ11* 88.58 82.89 85.74 8.63 12.09 10.36 97.20 94.98 96.09
3/4 N + RZ11* 73.10 81.84 77.47 8.06 13.69 10.87 81.16 95.53 88.34
1/2 N+ RZ11+ZH21+ASH21 ** 89.89 84.92 87.41 10.01 9.86 9.94 99.91 94.78 97.35
Soil texture av. 78.98 72.18 8.97 8.69 87.95 80.87
Soil Soil
LSD for texture
Fertilization S×F
texture
Fertilization S×F Soil texture Fertilization S×F
0.05 NS 19.47 NS NS 2.01 2.84 NS 20.48 NS

0.01 NS 28.19 NS NS 2.90 4.11 NS 29.65 NS
spp. (ASH21).
N: Recommended dose of nitrogen ( 20 kg N in sandy soil and 15 kg N in clay soil).
100 Sandy
80 Clay
Total K/shoot
(mg/plant)
60
40
20
0
1 2 3 4 5 6 7 8
Treatm ents
16 Sandy
Total K/root (mg/plant)
14
Clay
12
10
8
6
4
2
0
1 2 3 4 5 6 7 8
Treatments
120 Sandy
100 Clay
Total K/plant
(mg/plant)
80
60
40
20
0
1 2 3 4 5 6 7 8
Treatm ents

total potassium content (mg/plant) of broad bean
4: N + RZ11 5: 1/4 N + RZ11 6: 1/2 N + RZ11
7: 3/4 N + RZ11 8: 1/2 N+ RZ11+ZH21+ASH21.
119
treatments were highly significant (P≤0.01), but the differences

between soil textures and the interaction between treatment and
soil type were not significant.
In this concern, these findings verify the results found by
Rodelas et al. (1999) and El-Sawah (2000), since they found
significant increase in N, P and K content of maize plant when seeds
were inoculated with Azospirillum brasilense and Bacillus
megatherium as well as low dose of mineral nitrogen fertilizers was
applied.
These results concluded that the inoculation of broad bean
plants grown on sandy and clay soils with bio-fertilizers led to an
increase in the total potassium content in shoots, roots and whole
plants of broad bean.
4.2.6. Biomass yield and nitrogen and protein percentages
Data in Table 12 and illustrated by Fig. 7 present the
biomass yield (g/100 seeds), nitrogen % and protein % of broad
bean plants grown on both sandy and clay soils as affected by
bio-fertilization under different levels of inorganic N-fertilizers.
Results indicated that there were highly significant (P≤0.01)
differences among fertilization treatments, between soil textures
and the interaction between them in biomass yield of bread bean
plants. The highest value was obtained by the treatment
receiving 1/2 N dose + inoculation with isolate RZ11
(75.70 g/100 seeds) in clay soil, as compared with the control
In addition, data also show that inoculation of broad bean
with tri-mixture inoculants of isolates RZ11, ZH21 and ASH21
in both sandy and clay soils resulted in the highest nitrogen (%)
Table (12): Effect of bio-fertilizers and inorganic N-fertilizers on weight of 100 seeds (g), nitrogen % and
protein % of broad bean plants
Soil textures
Weight of 100 seeds (g) N% in seeds Protein % in seeds
Fertilization treatments Sandy Clay Av. Sandy Clay Av. Sandy Clay Av.
Control 37.25 49.15 43.20 3.18 3.20 3.19 19.83 19.97 19.90
*
Inoculation by RZ11 57.20 74.10 65.65 3.57 3.82 3.69 22.28 23.81 23.05
N + RZ11* 59.20 65.45 62.33 3.65 3.65 3.65 22.81 22.79 22.80
1/4 N + RZ11* 49.10 66.40 57.75 3.55 3.57 3.56 22.15 22.30 22.23
1/2 N + RZ11* 58.00 75.70 66.85 3.80 3.73 3.77 23.75 23.32 23.53
3/4 N + RZ11* 54.00 73.15 63.58 3.71 3.76 3.73 23.19 23.52 23.35
1/2 N+ RZ11+ZH21+ASH21 ** 59.20 74.05 66.63 3.81 3.81 3.81 23.78 23.79 23.79
Soil texture av. 53.65 67.08 3.61 3.63 22.53 22.76
0.05 0.93 1.85 2.62 NS 0.08 0.11 NS 0.40 0.56

0.01 1.34 2.68 3.79 NS 0.11 NS NS 0.57 NS
spp. (ASH21).
80 Sandy
70
Clay
weight of 100
seeds (g) 60
50
40
30
20
10
0
1 2 3 4 5 6 7 8
Treatm ents
4 Sandy
3.5
Nitrogen (%) in
3 Clay
2.5
seeds
2
1.5
1
0.5
0
1 2 3 4 5 6 7 8
Treatm ents
25 Sandy
20 Clay
Protein (%) in
seeds
15
10
0
1 2 3 4 5 6 7 8
Treatm ents

grain yield, nitrogen (%) and protein (%) in seeds of
broad bean cultivated in both sandy and clay soils
4: N + RZ11 5: 1/4 N + RZ11 6: 1/2 N + RZ11
7: 3/4 N + RZ11 8: 1/2 N+ RZ11+ZH21+ASH21.
122
123
and protein (%) in seeds of broad bean plants. The nitrogen

values were 3.81 % N in plants grown on both soils. The protein
values were 23.78 and 23.79 % in seeds of plants grown on
sandy and clay soils, respectively. The values of the
uninoculated control treatments had lower nitrogen and protein
percentages in seeds of broad bean plants being 3.18 and 3.20 %
N in plants grown on sandy and clay soils, respectively. Protein
values were 19.83 and 19.97 % in the respective soils. This
emphasizes the impact of bio-inoculation of broad bean plants
grown on both sandy and clay soils. There were highly
significant (P≤0.01) differences among fertilization treatments in
both nitrogen (%) and protein (%) in seeds of broad bean plants,
while the differences between soil textures were not significant.
Dual inoculation of legumes with Azospirillum and
Rhizobium has been found to increase several plant-growth
variable when compared with single inoculantion. Azospirillum
is considered a Rhizobium helper by stimulating nodulation,
nodule function and possibly plant metabolism. phytohormones
produced by Azospirillum promote epidermal-cell differentiation
in root hairs that increase the number of potential sites for
rhizobial-infectoion
(Andreeva et al., 1993).
These results also emphasize the role of bio-fertilizers
application as inoculants for increasing the seeds yield of broad
bean plants grown on both sandy and clay soils (Hassan et al.,
1990; Hussien et al. 1997; and EL-Ghandour et al. 2001)
124
In this respect, Namdeo and Gupta (1999) found that

Rhizobium inoculation increased nodule number and dry weight
and shoot dry weight of pigeon pea plants. Inoculation with
Rhizobium, PsB (Pseudomonas striata) and Rhizobium + PsB
with 100 % RFR (recommended fertilizer rate) produced 13.8,
9.9 and 20.4 % higher grain yield, respectively, than 100 % RFR
alone. Rhizobium + PsB with 75 % RFR gave a saving of 25 %
of the chemical fertilizer by produced yield equivalent to that of
100 % RFR alone. However, the highest net return was obtained
with 100 % RFR + Rhizobium + PsM.
4.3. Response of broad bean plants to bio and organic
fertilizers under cultivation in sandy and clay soil
broad bean plants (Giza 843) grown on sandy and clay soils to
bio-fertilizers under different garbage compost levels as an
organic fertilizers. The plants were inoculated with either
R. leguminosarum bv. vicieae (RZ11) alone or with tri-mixture
inoculants of R. leguminosarum bv. vicieae (RZ11) +
Azotobacter sp. (ZH21) + Azospirillum sp. (ASH21). The
response of the plants was evaluated by determining the nodule
formation on the root system, the plant dry weight, nitrogenase
activity, and total nitrogen, phosphorus and potassium contents
as well as the grain yield of broad bean plants.
4.3.1. Nodulation
that application of bio-fertilizers combined with organic
fertilizers increased the number of nodules/plant, dry weight of
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127
nodules (mg/plant), and nitrogenase activity in broad bean plants

grown on both sandy and clay soils. The results show that
number of nodules of broad bean generally, ranged from 8.33 to
20.0 nodules /plant. The highest number of nodules
(20.0 nodules/plant) was obtained by the treatment receiving
compost (recommended dose) and inoculated with
R. leguminosarum bv. vicieae (RZ11) in sandy soil. The
uninoculated treatment (control) gave the lowest numbers of
nodules in both soils, showing 4.67 on plants grown on sandy
soil and nodules were not formed on plants grown on clay soil.
The differences among fertilization treatments were highly
significant (P≤0.01).
The data indicate also that the sandy soil recorded highly
significant number of nodules (P≤0.01), being 15.05 odules/plant
as compared to the clay soil which had 10.62 nodules/plant. It
seems that the used inoculum reflected more response by the
plants grown on sandy soil than those growing in clay soil
(Hassan et al. 1990). Also, the interaction between treatments
and soil textures were highly significant (P≤0.01). The obtained
data are in agreement with Salem et al. (1981); and
Abdelhamid et al. (2004).
Regarding the dry weight of nodules (Table 13 and Fig. 8).
The results show that inoculation of broad bean plants grown on
both sandy and clay soils with isolate RZ11 or with tri-mixture
inoculants of RZ11, ZH21 and ASH21 and combined with
organic fertilizers increased the nodules dry weight (mg/plant) in
both soils with relatively high values for the last as compared to
uninoculated treatment (control). The treatment inoculated with
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RZ11 only gave the highest values of nodules dry weight of

broad bean in clay soil, being 36.67 mg/plant, while the lower
value was recorded in control plants in, being 6.33 mg/plant. The
significant (P≤0.01), while, the differences between soil textures
and the interaction between soil textures and fertilization
treatments were not significant.
Concerning the nitrogenase activity of broad bean plants
(Table 13), as representative for nitrogen fixation, it generally,
ranged from 0.128 to 8.096 μ mole C2 H4 /h/g dry nodules in
sandy soil. The highest value was recorded by the same
treatment receiving compost (5 ton/fed.) and inoculated with
tri-mixture inoculants of RZ11, ZH21 and ASH21 in sandy soil,
being 8.096 μ mole C2H4 /h/g dry nodules.Organic matter
stimulates the inoculant for symbiosis with the plants fixing
more nitrogen (Subba Rao 1999).
These results verify the finding reported by Moherram
et al., (1998), Subba Roa (1999) and Abdelhamid et al. (2004).
These results referred to the importance of organic fertilizer
and bio-fertilizers for enhancement of nodulation and
nitrogenase activity in broad bean plants, specially in sandy soil.
4.3.2. Plant dry weight
Data in Table 14 and Fig. 9 show the effect of
bio-inoculants and organic fertilizers on shoot, root and whole
plant dry weight, of broad bean plants grown on both sandy and
clay soils. The results showed that the application of
bio-fertilization increased the shoot dry weight of broad bean
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plants over the uninoculated treatment (control) or the

application of organic fertilizer only. The shoot dry weight
values, ranged between 1.13 and 2.54 g/plant. The highest shoot
dry weight value was obtained by treatment receiving compost
and inoculated with tri-mixture inoculants of RZ11 + ZH21 and
ASH21 in sandy soil, giving 2.54 g/plant, compared to the
unionculated treatment (control) which gave 1.30 g/plant. Only
the statistical differences among fertilization treatments were
significant (P≤0.05). The root dry weights, showed the same
ternd. The statistical differences among fertilization treatments
were highly significant (P≤0.01).
Regarding the whole plant dry weight, it ranged between
1.35 and 3.07 g/plant. The statistical differences among
fertilization treatments were highly significant (P≤0.01). The
application of compost combined with bio-fertilization increased
the whole plant dry weight of broad bean as compared to the
averages of all other treatments in both sandy and clay soils. The
impact of organic manure amendment to the Rhizobium-
inoculated broad bean increased also the productivity of the plant
which is very clear in our work. In the same aspect, Abdelhamid
et al. (2004) found that maturing of broad bean plants with rice
straw composting with oil seed of rape coke and poultry manure
increased growth yield, yield components and total crude protein
of the plants.
The obtained data are in harmony with those reported by
Moherram et al. (1998) in upper Egypt who reported that
application of composted cattle dung and poultry manure
mixture on soybean and peanut inoculated with specific
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Rhizobium and grown on newly reclaimed sandy soil

significantly increased plant dry weight N-yields, N2-fixed, N %
derived from organic matter in both plants.
These results concluded that inoculation with
R. leguminosarum bv. vicieae (RZ11) and / or Azotobacter sp.
(isolate ZH21) plus Azospirillum sp.(isolate ASH21)
significantly increased the shoot, root and whole plant dry
weight of bread bean grown on sandy and clay soils.
The effect of bio-inoculant and organic fertilizers on total
nitrogen content in shoot, root and whole plant in broad bean is
presented in Table 15 and illustrated by Fig. 10. Data show that
application of bio and organic fertilizers increased the total
nitrogen content in shoots, roots and whole plant in broad bean
plants comparing with the control treatment. The highest value
of total nitrogen content in shoots of broad bean plant was
obtained by the treatment inoculated with tri- mixture inoculants
of RZ11 + ZH21 + ASH21 combined with compost
(recommended dose) in sandy soil, being 97.88 mg N /plant, as
compared with the control treatment which showed 37.71 mg
N /plant. The statistical differences among fertilization
treatments were highly significant (p ≤ 0.01) However, both soil
textures under investigation gave almost similar average and the
differences in soil textures were not significant.
Concerning the total nitrogen content in roots of broad bean
plants, data showed the same general trend like shoot parts. The
highest values were observed in treatment inoculated with
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tri–mixture inoculants of RZ11 + ZH21 + ASH21 combined with

compost in both sandy and clay soils, giving 12.40 and 10.91 mg
N /plant, respectively, as compared with the lowest values of the
nitrogen which were observed in control treatment in both sandy
and clay soils, being 3.63 and 3.47 mg N /plant, respectively.
The statistical differences among treatments were highly
significant (p ≤ 0.01). Total nitrogen contents in whole plant of
broad bean, ranged from 37.19 mg N /plant in control treatment
of clay soil to 110.28 mg N /plant in treatment inoculated with
tri–mixture inoculants of RZ11 + ZH21 + ASH21 combined with
compost in sandy soil. The statistical differences among
fertilization treatments were highly significant (p ≤ 0.01).
However, the differences between soil textures were not
significant. This confirmed the previous results of nodulation,
dry weights and N2-ase activityof broad bean plants indicating
the clear impact of bio-fertilizers and organic manure on the
productivity of the plants especially in sandy soil. Moherram
et al. (1998), Subba Roa (1999) and Abdelhamid et al. (2004)
came also to the same general conclusion.
These results, in general, concluded that inoculation with
bio-fertilizers combined with organic fertilizers is very important
to reach highest figures regard to total nitrogen contents in
shoots, roots and whole plant of broad bean grown on sandy and
clay soils.
Data presented in Table 16 and illustrated in Fig. 11 show
the effect of bio and organic fertilizers on total phosphorus (mg
P /plant) of broad bean plants grown on both sandy and clay
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138
soils. The results indicated that the total phosphorus in shoots of

broad bean plant increased due to inoculation with
bio-fertilization combined with application of organic
fertilization. Data showed also that the highest total phosphorus
content in shoots of broad bean plants was obtained by the same
treatment which was inoculated with tri–mixture inoculants of
RZ11, ZH21 and ASH21 combined with application of compost
in both sandy and clay soil, being 13.34 and 13.42 mg P /plant,
respectively. The control treatment however, had the lowest total
phosphorus content in shoots in both soils, being 4.41 and 3.35
mg P /plant, respectively.The statistical differences among
fertilization treatments were highly significant (p ≤ 0.01).
Similar results were obtained in almost treatments of total
phosphorus content in roots as found in shoots of broad bean
plants. It was found that the inoculation with bio-fertilizers
combined with compost as organic fertilizers increased the total
phosphorus content in roots of broad bean grown on both sandy
and clay soils, as compared with the uninoculated treatment
(control). The statistical differences among treatments were
highly significant (p≤0.01). Meanwhile, the differences between
soil textures were not significant.
Concerning the total phosphorus in whole broad bean plant.
The treatment received compost and inoculated with tri-mixture
inoculants of RZ11, ZH21 and ASH21 gave the highest values in
both sandy and clay soils, being 14.51 and 14.54 mg P /plant,
respectively. The uninoculated treatment (control), however, had
the lowest values in both sandy and clay soils, being 4.82 and
3.69 mg P /plant, respectively. The differences among
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fertilization treatments were highly significant (p ≤ 0.01), but the

differences between soil textures were not significant.
These results verify the finding reported by AL-Kahal
et al. (2001). They found that application of organic fertilizers as
olive oil waste water (OOWW) and olive cake (OC) in
combination with R. leguminosarum inoculation of broad bean
plants, led to a significant increase in nodulation, shoot dry
weight and N and P content as compared to uninoculated
OOWW and OC treatments.
The obtained results referred to the importance of
application of bio and organic fertilizers to achieve the highest
plant growth with total phosphorus content in broad bean plants
grown on both sandy and clay soils. These results confirmed
those obtained by Abdelhamid et al. (2004).
Data presented in Table 17 and illustrated in Fig. 12 show
the effect of bio and organic fertilizers on total potassium content
(mg K /plant) of broad bean plant grown on both sandy and clay
soils. The results showed also that the same treatment in which
application of compost as organic fertilizers combined with
inoculation by tri-mixture inoculants of RZ11, ZH21 and ASH21
gave the highest values of total potassium content in shoots of
broad bean plants in both sandy and clay soils, being 112.46 and
101.96 mg K /plant, respectively, as compared with control
treatment which showed the lowest values of total potassium
content in shoots of broad bean plant in both sandy and clay
soils, being 41.15 and 38.52 mg K /plant, respectively.
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(p ≤ 0.01) while the differences between soil textures were not
significant.
The same trend was also found in roots of broad bean
plants. The values ranged from 3.47 to 15.11 mg K /plant. The
statistical differences among fertilization treatments were highly
significant (p ≤ 0.01).
Also total potassium content in whole plant gave similar
results as found in shoot of broad bean plants in both soils. The
treatment inoculated with tri-mixture inoculants of RZ11, ZH21
and ASH21 had the highest values in both sandy and clay soils,
being 126.09 and 112.07 mg K /plant, respectively.
These results concluded that application of biofertilizers in
broad bean plants cultivated in sandy and clay soils increased the
total potassium content in whole parts of the plants.
The effect of bio and organic fertilizers on biomass yield
(g/100 seeds), nitrogen % and protein % of broad bean plants
grown on both sandy and clay soils is given in Table 18 and
illustrated by Fig. 13. Results showed that there were highly
significant (p ≤ 0.01) differences among fertilization treatments
in biomass yield (g/100 seeds) of broad bean plants grown on
both sandy and clay soils. The highest value was obtained by the
treatment inoculated with RZ11 combined with compost as
organic fertilizers in clay soil, being 78.70 g/100 seeds. Result of
plants grown on clay soil had higher average values than sandy
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soil, being 68.05 and 52.35 g/100 seeds for the first and second
soil, respectively. The differences between soil textures were
highly significant (p ≤ 0.01).
In addition, data in Table 18 and Fig 13 show also that
inoculation of broad bean plants with R. leguminosarum bv.
vicieae (RZ11) combined with compost gave the highest
nitrogen (%) and protein (%) in seeds of broad bean plants
grown on both soils. The values were 3.84 and 3.91 % N and
23.94 and 24.43 % protein in seeds of broad bean plants grown
on both sandy and clay soils, respectively. The statistical
differences in nitrogen (%) and protein (%) among fertilization
(p ≤ 0.01). Abd EL-Ghany (1996) showed o that, the 100 seeds
weight/plant of soyabean and number of pods/plant were
positively influenced by seed inoculation with Azotobacter
chroococcum, Azospirillum lipoferum and its mixtures (1:1) as
compared with uninoculated treatments. Also, Sarwar et al.
(1998) demonstrated that, the percentage of increase in yield due
to the inoculation with specific Bradyrhizobium ranged from
15-30% in cowpea to 75:200 % in soybean.
These results emphasize the importance of bio-fertilizers
inoculation of broad bean plants either growing in sandy or clay
soils with bio-fertilizers to obtain the highest biomass yield and
nitrogen percentage in seeds.
4.4. Response of broad bean plants to bio-fertilizers
under cultivation in sandy and clay soils
broad bean plants (Giza 843) grown on sandy and clay soils to
146
bio-fertilizers alone. Plants were inoculated with R.

leguminosarum bv vicieae (RZ11) alone or with Azotobacter sp.
(ZH21) or with Azospirilum sp. (ASH21) or with tri-mixture
inoculants of RZ11+ZH21+ASH21.The response of the plants
was evaluated by determining the nodule formation on the root
system, the plant dry weight, nitrogenase activity, nitrogen,
phosphorus and potassium contents as well as the grain yield of
broad bean plants.
4.4.1. Nodulation
It is evident from the data in Table 19 and Fig. 14 that the
number of nodules/plant significantly increased by inoculation of
broad bean seeds with either of nitrogen fixing bacteria isolates
(RZ11, ZH21 and ASH21) compared to the uninoculated seeds
in both sandy and clay soils. Inoculated broad bean seeds with
tri-mixture inoculants of RZ11, ZH21 and ASH21 significantly
produced the highest values of nodule number (average
20.33 nodules/plant), followed by the treatment inoculated with
both RZ11 + ZH21 (average 19.00 nodules/plant). The
significant (p≤0.01).
Concerning the nodule dry weights they ranged from
6.33 to 42.33 mg/plant. The highest value of nodule dry weight
(mg/plant) was obtained by the treatment inoculated with
RZ11alone in clay soil (42.33 mg/plant), followed by the
treatment inoculated with RZ11 + ZH21 (38.33 mg/plant) in
sandy soil. The uninoculated treatment (control) did not form
nodules in clay soil.
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149
In addition, data in Table 19 indicate also that the

nitrogenase activity of broad bean plants ranged from 0.128 to
5.342 μ mole C2 H4 / h/g dry nodules. The highest value of
nitrogenase activity was obtained by treatment inoculated with
RZ11 + ZH21 giving 5.342 μ mole C2 H4 / h/g dry nodules
followed by the treatment inoculated with tri-mixture inoculants
of RZ11, ZH21 and ASH21 (showing 4.635 μ mole C2H4 / h/g
dry nodules) in sandy soil. The nitrogenase activities of the
different treatments were in parallel with those obtained by
nodule numbers and dry weight of nodules.These results
confirmed those reported by Arisha et al. (1998). They found
that the inoculation with R. leguminosarum bv. vicieae
(USDA 2500 strain) of pea plants grown on sandy soil
(El-Khattara) significantly increased number of nodules per
plant, dry weight of shoots, roots and whole plant as well as
acetylene reduction activity. Abdelhamid et al. (2004).
demonestrated that Rhizobium inoculation significantly increased
the number and dry weight of nodules of broad bean cultivated
in newly reclaimed soil of Egypt. In addition, there was
a beneficial effect due to applicatooin of co-inoculants
containing other micro-organisms, which are able to improve
legume growth and nodulation (Mishra et al., 1999 and
Rodelas, 1999). These include rhizobacteria which promote
nodulation, nitrogen fixation and yield via such mechanisms as
phytohormones, antibiotic or metal-binding compound
production bacteria, which aid in nutrient supply via phosphate
solubilization. (Subba Rao, 1999).
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4.4.2. Plant dry weight:

The effect of different isolates of bio-fertilizers on dry
weight of broad bean plants grown on both sandy and clay soils
is given in Table 20 and Fig. 15. The results showed that
application of bio-fertilization increased the shoots dry weight of
broad bean plants over the uninoculated plants
(control treatment) grown on both sandy and clay soils. The
treatment inoculated with tri-mixture inoculants of RZ11,ZH21
and ASH21, gave average of 2.47 g/plant, while, the lowest
average of shoot dry weight value was obtained by the
uninoculated treatment (control), being 1.22 g/plant. The
differences among bio-fertilization treatments were highly
significant (p≤0.01).
Regarding the root dry weight, it ranged from 0.22 to
0.63 g/plant. The highest values of root dry weights were
obtained also by plants inoculated with RZ11 combined with
ASH21 in both sandy and clay soils, being 0.56 and 0.63 g/plant,
respectively. While, the lowest values were obtained by the
uninoculated treatment (control), being 0.24 and 0.22 g/plant in
both sandy and clay soils, respectively. The statistical differences
among bio-fertilization treatments were highly significant
(p≤0.01)
The data showed also that the whole plant dry weight
average ranged from 1.44 to 2.97 g/plant. The statistical
differences among bio-fertilization treatments were highly
significant (p≤0.01). The application of bio-fertilization
increased the whole plant dry weight of broad bean plants as
compared to the average of uninoculated treatment.
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153
The obtained results agreed with those reported by Salem

et al. (1981); Hassan et al. (1990) and Hassan (1997), who
found that, the average values of plant fresh and dry weights of
snap bean significantly increased by seed inoculation with
Rhizobium as compared with the control.
These results concluded that inoculation of broad bean
plants with R. leguminosarum bv. vicieae (RZ11) combined with
Azotobacter sp.(isolate ZH21) plus Azospirilum sp. (isolate
ASH21) significantly increased the shoot, root and whole plant
dry weight of broad bean plants grown on sandy and clay soils.
The effect of bio-fertilizers on total nitrogen content in
shoots, roots and whole plant of broad bean is given in Table 21
and Fig. 16. Data show that application of bio-fertilizers
significantly increased the total nitrogen content in shoots, roots
and whole plant comparing with the uninoculated treatment
(control). The highest values of total nitrogen content in shoots
in broad bean plants were also obtained by the treatment
inoculated with tri-mixture inoculants of RZ11, ZH21 and
ASH21, showing 88.77 and 109.61 mgN/plant in both sandy and
clay soils, respectively. While, the lowest values of total nitrogen
content in shoots were recorded in the uinoculated treatment
(control), giving 37.71 and 33.73 mgN/plant in both sandy and
clay soils, respectively. The statistical differences among
treatments were highly significant (p≤0.01).
Concerning the total nitrogen content in roots of broad bean
plants, the treatment inoculated with RZ11 and ASH21 gave the
highest value, being 13.40 mgN/plant in clay soil, followed by
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the treatment inoculated with RZ11(11.39 mgN/plant) in sandy

soil. The lowest values of total nitrogen content were obtained
by uninoculated treatment (control), being 3.63 and
3.47 mgN/plant in both sandy and clay soils, respectively.
(p≤0.01).
Regarding the total nitrogen content in whole plant of broad
bean, the application of bio-fertilizers significantly increased the
total nitrogen content as compared to the uninoculated treatment
(control). The same general trend was obtained like those of
shoots and roots of the plants. The treatment inoculated with
tri-mixture inoculants of RZ11, ZH21 and ASH21 isolates had
the highest values, being 97.48 and 121.76 mg/plant in both
sandy and clay soils, respectively. The statistical differences
among treatments were highly significant (p≤0.01). Similar
results were obtained by Salem et al. (1981); Hassan et al.
(1990); Hamaoui et al., (2001); Brock et al. (2003) and
Abdelhamid et al. (2004).
The results in this work, in general, concluded that
inoculation with bio-fertilizers improved the total nitrogen
content in shoots, roots and whole plant of broad bean grown on
both sandy and clay soils when compared with uninoculated
treatment (control).
4.4.4 Total phosphorus content
The total phosphorus content in shoots of broad bean plants
grown on both soils significantly increased due to the application
of bio-fertilization as compared to uninoculated treatment
(control) (Table 22 and Fig. 17). The highest total phosphorus
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content in broad bean shoots was obtained by the treatment

ASH21 isolates, being 14.71 mg P/plant in clay soil. The
uninoculated treatment (control) gave the lowest values, being
4.41 and 3.35 mg P/plant in both sandy and clay soils,
Concerning the total phosphorus content in roots of broad
bean plants (Table, 22 Fig,17), almost similar results were
obtained as those found in shoots.However, the highest values of
total phosphorus content in roots were obtained by the treatment
inoculated with RZ11 plus ASH21 isolates, being
1.65 and 1.52 mg P /plant in both sandy and clay soils,
respectively. While, the lowest values of total phosphorus
content in roots were obtained by the uninoculated treatment
(control), being 0.41 and 0.34 mg P /plant in both sandy and clay
soils, respectively. The statistical differences among fertilization
Regarding the total phosphorus content in whole plant of
broad bean plants, it was found that inoculation with RZ11 alone
or combined with ZH21 and / or ASH21 gave almost similar
results as those in shoot and roots P contents. The highest value
(16.18 mg P /plant) was obtained by the treatment inoculated
with tri-mixture inoculants of RZ11, ZH21 and ASH21 in clay
soil. The uninoculated treatment (Control) had the lowest value
(4.82 and 3.69 mg P /plant) in both sandy and clay soils,
treatments were highly significant (p≤0.01). These results
160
confirmed the findings reported by Rodelas et al., (1999) and

Abdelhamid et al. (2004)
In general, the obtained results concluded the importance of
inoculation of broad bean plants cultivated in sandy or clay soils
with R. leguminosarum bv. vicieae (RZ11), Azotobacter sp.
(ZH21) and Azospirillum sp. (ASH21) to obtain the high figures
of the total phosphorus content in shoot, root and
whole plant.
4.4.5. Total potassium content:
The effect of bio-fertilizers on total potassium content in
shoot, root and whole plant (mg K/plant) in broad bean plants
cultivated in both sandy and clay soils is given in Table 23 and
illustrated by Fig. 18. The results show a great variation due to
the effect of inoculation with different bio-fertilizers on total
potassium content in shoot, root and whole plant of broad bean
grown on both soils. The application of RZ11 plus ZH21 isolates
gave the highest value (101.21 mgK/plant) shoot in sandy soil.
While, the application of tri-mixture inoculants of RZ11, ZH21
and ASH21 gave the highest value (136.34, mg K /plant) in clay
soil. The uninoculated treatment (control) had the lowest values
of total potassium content in shoots namely 41.15 and
38.52 mg K/plant in both sandy and clay soils, respectively. The
significant (p ≤ 0.01). Also, the differences between soil textures
were significant (p ≤ 0.05).
Similar variation was observed in the total potassium
content in root inoculation with RZ11 only giving the highest
value (15.11 mg K/plant) in sandy soil, while the application of
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163
RZ11 plus ASH21 isolates had the highest value

(15.08 mgK/plant) in clay soil. The uninoculated treatment
(control) had the lowest values in both sandy and clay soils,
being 3.55 and 3.47 mg K/plant, respectively. The statistical
significant (p ≤ 0.01).
The total potassium content in whole plant of broad bean
plants cultivated in both sandy and clay soils behaved the same
trend like those of shoots and roots of the plant. Inoculation with
RZ11 only gave the highest value (110.87 mgK/plant) in sandy
soil, while inoculation with both RZ11 plus ASH21 gave the
highest value (137.39 mgK/plant) in clay soil. The uninoculated
treatment (control) had the lowest values, being 44.70 and
41.99 mgK/plant in both sandy and clay soils, respectively. The
significant (p ≤ 0.01). These results verify the findings reported
by Patel et al. (1985); Rodelas et al., (1999); and El-Sawah
(2000)
These results, in general, concluded that the inoculation of
broad bean plants grown on sandy and clay soils with different
bio-fertilizers (RZ11, ZH21 and ASH21) led to an increase in the
total potassium content of shoot, root and whole plant.
the effect of bio-fertilizers on biomass yield (g/100 seeds),
nitrogen percentage and protein percentage of broad bean plants
grown on both sandy and clay soils. The results indicated that
there were highly significant differences among fertilization
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166
treatment (p≤0.01) in biomass yield (g/100 seeds) in both sandy

and clay soils. The highest fertilization treatments average for
both soils was 70.00 g/100 seeds. While, the uninoculated
treatment (Control) had the lowest average, being 43.13 g/100
seeds. The clay soil had significantly higher yield (72.42 g/100
seeds) than sandy soil (53.69 g/100 seeds). Application of
bio-fertilizers of RZ11, ZH21 and/ or ASH21 isolates increased
the nitrogen and protein percentages- in seeds of broad bean
plants grown on both sandy and clay soils. The highest values
were 3.75 and 3.87 % N and 23.40 and 24.14 % protein in seeds
of broad bean plants grown on both sandy and clay soils,
respectively. Both the statistical differences among fertilization
(p ≤ 0.01). The clay soil had higher nitrogen and protein
percentages than the sandy soil. The uninoculated treatment
(Control) had the lowest values of nitrogen (3.18 and 3.20 %)
and protein (19.84 and 19.97 %) in seeds of broad bean plant
grown on sandy and clay soils, respectively. In this respect,
Pathak et al. (1997); Dadarwal et al. (1997) and Hedge et al.
(1999) explained the role of bio-fertilizers in leguminous crops.
They reported that these bio-fertilizers play an important role in
enhancing crop productivity through nitrogen fixation, phosphate
solubilization, plant hormone productivity, ammonia excretion
and to control various plant diseases.
The obtained results emphasize the role of bio-fertilizers
application as inoculants for increasing the crop yield and
improvement of nitrogen and protein contents in seeds of board
bean plants grown on both sandy and clay soils.
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IBA content in roots of broad bean plants
Data in Table 25 show the effect of application of
bio-fertilization only or combined with inorganic or organic
N-fertilizers on G ِ ِ ِ ِ A3, IAA and IBA content in roots of
broad bean plants grown on both sandy and clay soils. The
highest values of gibberellins (GA3) were obtained by the
treatment inoculated with tri-mixture inoculants of
R.Leguminosarum bv. vicieae (RZ11), Azotobacter sp. (ZH21)
and Azospirillum sp. (ASH21) combined with the recommended
compost dose in sandy soil, being 101.05 μ g/g fresh roots,
followed by the treatment inoculated with RZ11 combined with
½ dose of inorganic
N-fertilizers in clay soil, being 74.88 μ g/g fresh roots.
The uninoculated treatment (control) however showed very
low values of GA3 being 5.71 and 13.11 μ g/g fresh roots in
plants grown on sandy and clay soils, respectively.
Concerning the Indole acetic acid (IAA) in roots of broad
bean plants cultivated in both sandy and clay soils (Table 25),
the highest values of IAA were obtained by the treatment
ASH21 combined with the recommended compost dose in sandy
soil, being 1.27 μ g/g fresh roots and the treatment inoculated
with RZ11 only in clay soil, being 1.35 μ g/g fresh roots.
However, the uninoculated treatment (control) of the plants
grown on both sandy and clay soils, showed low figurs being
0.4 and 0.62 μ g/g fresh roots.
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169
Regarding the IBA in roots of broad bean plants cultivated

in both sandy and clay soils, the results show that the highest
values of IBA in roots were obtained by the treatment inoculated
with R. leguminosarum bv. vicieae (RZ11), rely in both sandy
and clay soils, being 344.15 and 513.01 μ g/g fresh roots,
respectively. The lowest values of IBA were obtained by the
uninoculation treatment (control) in both sandy and clay soils,
being 17.58 and 4.61 μ g/g fresh roots, respectively.
The obtained results are in agreement with those reported
by Mishra et al. (1999) and Rodelas et al. (1999). They found
that the interest in developed Co-inoculants containing other
micro-organisms, which are able to improve legume growth,
included rhizobacteria which promote nodulation, nitrogen
fixation, plant vigor and yield via such mechanisms as play to
hormones, antibiotic or metal binding compound production.
In the same subject Dakora (2003) found that new roles
for plant and rhizobial molecules had been detected in soil and
mixed plant cultures involving symbiotic legumes Rhizobium
(species Rhizobium, Bradyrhizobium, Azorhizobium,
Allorhizobium, Sinorhizobium and Mesorhizobium) produce
chemical molecules that can influence plant development
including phytohormones, lipo-oligosaccharide, Nod factors,
lumichrome, riboflavin and H2 evolved by Nitrogenase. Theunis
et al. (2004) found that addition of inducing flavonoids also
stimulates the production of the phytohormone indole-3-acetic
acid (IAA) in several Rhizobium. The molecular basis of IAA
synthesis in Rhizobium spp.NGR234 was investigated. Mass
spectrometric analysis of culture supernatants indicated that
NGR234 is capable of synthesizing IAA via three different
pathways.
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4.6. Comparison the response of broad bean plants to

application of biofertilizers only or combined with
organic or inorganic N-fertilizers with control
treatment in both sandy and clay soil
4.6.1. In sandy soil
Data presented in Table 26 show comparison among the
average response of broad bean plants to application of different
biofertilizers only, bio and inorganic N-fertilizers and bio and
organic fertilizers with the untreated (control) treatment in sandy
soil. It is clear that the application of bio-fertilizers alone or
combined with organic or inorganic N-fertilizers gave the
highest average values of all traits studied as compared to
untreated (control) treatment.
The response of broad bean plants cultivated in sandy soils
and inoculated with bio-fertilizers only, combined with inorganic
N-fertilizers or combined with organic fertilizers was
inconsistent and varied with experiments. The application of
broad bean plants with bio-fertilizers only gave superior
response in the performance of some traits studied, while the
application of bio-fertilizers combined with inorganic or organic
fertilizers gave superior response in the performance of other
traits. In some traits the application of the three experiments
(bio-fertilizers only, combined with inorganic or combined with
organic fertilizers) gave similar response.
The average value of different traits studied were increased
from 114.1 to 4128.8 % as the result of the application of
bio-fertilizers, bio and organic or bio and inorganic N fertilizers
as compared to untreated control treatment (100 %). The results
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referred to the importance of inoculation of broad bean plants

grown on sandy soils with bio-fertilizers (R. leguminosarum bv.
vicieae, Azotobacter and Azospirillum) to obtain the best
performance in growth, nodulation and total nitrogen and
mineral contents.
4.6.2. In clay soil
average response of broad bean plants to application of different
bio-fertilizers only, bio and organic and bio and inorganic
N fertilizers with untreated treatment (control) in clay soil. The
data show that the application of bio-fertilizers alone or
combined with organic or inorganic N fertilizers had the highest
average values of all traits studied as compared to untreated
treatment (control).
The response of broad bean plants cultivated in clay soil
and inoculated with bio-fertilizers only, combined with inorganic
or organic fertilizers was inconsistent and varied with
experiments. The application of broad bean plants with
bio-fertilizers only gave superior response in the performance of
some traits studied. While, the application of bio-fertilizers
combined with inorganic or organic fertilizers gave superior
response in the performance of other traits. In some traits, the
performance of the three experiments (bio-fertilizers only,
combined with organic or combined with inorganic N fertilizers)
gave similar response. The average values of different traits
studied were increased from 116.8 % in protein in seeds to
348.5 % in total P/root mg/plant as a results of the application of
bio-fertilizers, bio and organic or bio and inorganic N fertilizers
as compared to untreated treatment (control).
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The results focused on the importance of inoculation of

broad bean plants grown on clay soil with bio-fertilizers
(R. leguminosarum bv. vicieae, Azotobacter and Azospirillum) is
great importance to obtain the best performance in growth,
nodulation and total nitrogen and mineral contents.
4.7. Response of wheat plants to bio-fertilizers and
and clay soils
A pot experiment was conducted to evaluate the response
of wheat plants (Giza 168) grown on sandy and clay soils under
different N-fertilizer levels and inoculation with Azospirillum
spp (ASH21) alone or tri-mixture inoculants of Azospirillum sp.
(ASH21) + Azotobacter sp.(ZH21) + Rhizobium leguminosaram
bv. vicieae (RZ11).
The plant dry weight, total nitrogen, total phosphorus and
total potassium contents as well as grain yield of wheat plants
and nitrogenase and dehydrogenase activity in rhizosphere of
wheat plants were determined.
Data in Table 28 and illustrated by Fig. 20 show the effect
of bio-fertilizers and inorganic N-fertilizers on shoot, root and
whole plant dry weight (g/plant) of wheat plants grown on both
sandy and clay soils. The results showed that shoot dry weight
generally, ranged from 0.14 to 1.39 g/plant. The lowest values of
shoot dry weight (0.14 and 0.69 g/plant) were obtained by the
uninoculated treatments (control) in both sandy and clay soils,
respectively. The highest value of shoot dry weight
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(1.39 g/plant) was obtained by treatment receiving 1/2 N dose

(recommended N dose =100 kg N fed-1 in sandy soil and 75 kg N
fed-1 in clay soil) + inoculation with tri-mixture inoculants of
ASH21, ZH21 and RZ11 in sandy soil, whereas, the highest
shoot dry weight (1.23 g/plant) was found in clay soil treated
with nitrogen dose only without biofertilizers. The differences
between soil textures were not significant, while the differences
between fertilization treatments were highly significant
(P ≤ 0.01), meanwhile the interaction between soil textures and
fertilization treatments were not significant.
In addition, data show also that root dry weight values
behavied the same general trend like those of the shoots. The
values ranged from 0.09 to 0.73 g/plant. The highest average
root dry weight (0.59 g/plant) was recorded for plants treated
with 1/2 N+ inoculation with tri-mixture inoculants of ASH21,
ZH21 and RZ11 as compared with the averages of all other
treatments of both sandy and clay soils. The differences among
fertilization treatments were highly significant. The differences
between soil textures and the interaction between soil textures
and fertilization treatments were also significant (P ≤ 0.05).
Regarding, the effect of biofertilizers and inorganic
N-fertilizers on whole plant dry weight, the same trend was also
observed. The values, generally, ranged between 0.23 to
1.84 g/plant. The lowest value of whole plant dry weight was
obtained by control treatment in both sandy and clay soils, being
0.23 and 0.93 g/plant, respectively. While the highest value of
whole plant dry weight was obtained by the treatment receiving
1/2 N + inoculation with tri-mixture inoculants of ASH21, ZH21
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and RZ11 in sandy soil and treatment receiving nitrogen dose in

clay soil, being 2.12 and 1.68 g/plant, respectively. Data show
also that the whole plant dry weight values of wheat plants
grown on sandy soil were lower than those grown on clay soil,
where the average showed 1.39 and 1.42 g/plant in both soils,
respectively. Statistical analysis showed also that the differences
between soil textures were not significant, while the differences
between fertilization treatments were highly significant
(P ≤ 0.01). The differences in the interaction between soil
textures and fertilization treatments were significant (P ≤ 0.05).
The obtained data are in agreement with those reported by
Goher et al. (1988) who reported that bio-fertilization of wheat
plants, using N2-fixing Azotobacter and Azospirillum reduced the
inorganic nitrogen requirement and increased the biomass yields.
Also in the same aspect Mitkess et al. (1996) concluded that
application of bio-fertilizers (Azospirillum spp. + Bacillus +
Klebsiella + Azotobacter) for wheat plants could compensate
considerable parts of mineral N-fertilizers, since it saves about
50 Kg N fed-1 in old land and 40 Kg fed-1 in new land. Sarwar
et al. (1998) reported also that the application of Azospirillum
lipoferum inoculants and three levels of nitrogen fertilizers
(10 Kg/hr, 45 Kg/hr and 90 Kg/hr) to rice plant gave highly
significant increase in growth and yield of the crop specially
when nitrogen was applied at rate 45 Kg/hr.
In general the results concluded that the inoculation with
tri-mixture inoculants of ASH21, ZH21 and RZ11 plus the
application of inorganic N-fertilizers gave the best results of
wheat plants either cultivated in sandy or clay soils.
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4.7.2. Nitrogenase and dehydrogenase activities in

rhizosphere soil of wheat plants
Data presented in Table 29 show the effect of bio-fertilizers
and inorganic N-fertilizers on nitrogenase and dehydrogenase
activities in rhizosphere of wheat plants grown either in sandy or
clay soils. The results showed that the nitrogenase activity
generally, ranged from 0.298 to 29.645 μ mole C2H4 /h/g dry
soil. Treatment receiving 1/2 N + inoculation with tri-mixture
inoculants of ASH21, ZH21 and RZ11 in clay soil, gave the
highest record for this enzyme activity, being 29.645 μ mole
C2H4 /h/g dry soil, comparing with the control (1.556 μ mole
C2H4 /h/g dry soil). In sandy soil, however, the same previous
treatment gave also the highest figure for N2-ase in the
rhizosphere of wheat plants with relatively lower values.
The specific dehydorgenase activities in rhizosphere of
wheat (μ g hydrogen/g dry soil) as affected by bio-inoculation
and inorganic N-fertilizers in both sandy and clay soils are given
in Table 29. The values ranged from 3.242 to 11.142 μ g H2/g
dry soil. Here also the same treatment received 1/2 N +
inoculation with tri-mixture inoculants of ASH21, ZH21 and
RZ11 showed the highest record in both sandy and clay soils,
being 11.142 and 9.274 μ g H2/g dry soil, respectively as
compared with the control values which were 3.242 and 3.252
μg H2/g dry soil in sandy and clay soils, respectively. The
obtained values of nitrogenase and dehydrogenase activities
were in concomitant with the values of dry weight shoots and
roots of the plants.
These results are in harmony with those reported by
El-Demerdash et al. (1993). Who showed that inoculation with
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a mixture of symbiotic and asymbiotic N2-fixers gave better

results of growth and yield of wheat than using either of them
only. Also, Alam et al. (2001) reported that the inoculation with
mixture of several free living rhizobacteria, Azotobacter,
Bacillus and Enterobacter significantly increased acetylene-
reducing activity in roots of rice plant, total dry matter yield,
grain yield, N uptake, root length and leaf area. On the same
subject Galal et al. (2001) reported that inoculation with
Azospirillum and Rhizobium gave higher dry matter
accumulation in shoots, N uptake and grain yield, but increasing
N rate inhibited N-fixation in plant.
These results focused and gave more evidence for the
importance of application of bio-fertilizers for enhancement of
nitrogenase and dehydrogenase activities in rhizosphere of wheat
plants.
that the application of bio-fertilizers plus inorganic N-fertilizers
increased the total nitrogen content (mg N/plant) in the whole
parts of wheat plants comparing with the uninoculated treatment
(control). The highest values of total nitrogen content in shoots
of wheat plants were recorded by treatment receiving 1/2 N +
RZ11 in both sandy and clay soils, being 34.15 and 33.17 mg
N/plant, respectively. Where the lowest values of total nitrogen
content were recorded by the control treatments in both soils,
being 1.88 and 8.73 mg N/plant, in sandy and clay soils,
respectively. The differences between soil textures were
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significant (P≤0.05). The differences between fertilization

treatments were also highly significant (P≤0.01). Meanwhile, the
interaction differences between soil textures and fertilization
Similar results were found in total nitrogen content of roots
of wheat plants. The statistical differences between soil textures,
and among fertilization treatments and the interaction between
soil textures and fertilization treatments were highly significant
(P≤0.01).
Concerning the total nitrogen contents in whole plant, the
values ranged from 2.84 to 43.99 mg N/plant. In addition, plant
grown on clay soil gave higher values of total nitrogen contents
in wheat plants than sandy soil, namely 28.56 and 26.09 mg
N/plant, respectively.The statistical differences among
fertilization treatments were highly significant (P≤0.01), while
the differences between soil textures and the interaction
differences between soil textures and fertilization treatments
were not significant.
Galal and Elgandour (1994) who stated that inoculation with
either Azospirillum or Rhizobium increased straw and grain
yields as well as the total N uptake. Similar results were also
obtained by Alam et al. (2001) in rice plants and Galal et al.
(2001) in wheat plants.
Here also, the results confirmed that the application of
tri-mixture inoculants of Azospirillum sp. (ASH21), Azotobacter
sp. (ZH21) and R. leguminosarum bv. vicieae (RZ11) plus the
application of inorganic N-fertilizers gave the best results in total
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nitrogen content of wheat plants either cultivated in sandy or

clay soils.
that application of bio-fertilizers plus inorganic N-fertilizers
increased the total phosphorus content in shoots, roots and whole
plant (mg P/plant) in wheat plants grown on both sandy and clay
soils comparing with uninoculated treatment (control). The
highest values of total phosphorus content in shoots of wheat
plants were recorded by the same treatment receiving 1/2 N +
inoculation with tri-mixture inoculants at ASH21, ZH21 and
RZ11, being 4.14 and 5.86 mg P/plant in sandy and clay soils,
respectively. The respective values of the control treatment in
both soils were 0.22 and 1.11 mg P/plant in sandy and clay soils,
respectively. The statistical differences between soil textures and
among fertilization treatments were highly significant (P≤0.01),
while the statistical differences in the interaction between soil
textures and fertilization treatments were not significant.
In addation, the highest value of total phosphorus content
in roots of wheat plant (mg/plant) was recorded also by the same
treatment (1/2 N + inoculation with tri-mixture inoculants, of
ASH21, ZH21 and RZ11), showing 0.70 mg P/plant for those
grown on sandy soil. The statistical differences among
fertilization treatments were highly significant (P≤0.01). The
differences between soil textures and the interaction between soil
textures and fertilization treatments were also significant
(P≤0.05).
Concerning the values of the total phosphorus content in
whole plant of wheat they ranged from 0.28 to 6.26 mg P/plant.
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In clay soil, the treatment received 1/2 N + inoculation with

tri-mixture inoculants of ASH21, ZH21 and RZ11 gave the
highest value of total phosphorus content in whole plants as
compared to the control, being 6.26 and 1.23 mg P/plant. In
sandy soil, however, treatment received 1/2 N + inoculation with
ASH21 only gave the highest value of total phosphorus content
in whole plant as compared to the control, being 4.97 and
0.28 mg P/plant, respectively. In addition, clay soil gave higher
value of total phosphorus content in whole plant of wheat plants
than those of sandy soil, being 4.40 and 3.38 mg/plant,
treatments and the differences between soil textures were highly
significant (P≤0.01), but the interaction differences between soil
textures and fertilization treatments were not significant.
The obtained data are in harmony with those reported by
Goher et al. (1988) on wheat plants. El-Bakry et al., (2001) also
reported that inoculation with Azospirillum gave the highest
value of growth and yield in sorghum, and increased the P and
N content in shoot and whole plant. The same results found also
by Abdelhamid et al. (2004) in broad bean.
The results concluded that inoculation with ASH21, ZH21
and RZ11 plus the application of 1/2 inorganic N-fertilizers gave
the best results on total phosphorus contents of wheat plants
cultivated in sandy and clay soils, which were also in
concomitant with the other previous parameters.
The effect of bio-fertilizers and inorganic N-fertilizers on
total potassium contents in wheat plants cultivated in both sandy
and clay soils are shown in Table 32 and illustrated by Fig. 23.
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The results show that the treatment inoculated with tri-mixture

inoculants of Azospirillumsp. (isolate ASH21), Azotobacter sp.
(isolate ZH21) and R. Leguminosarum bv. vicieae (RZ11)
combined with 1/2 N dose increased the potassium content of
wheat plants giving the highest values in shoots of wheat plants
grown on sandy and clay soils, as compared with the untreated
treatment (control). The values were 35.29 and 34.45 mg K/plant
in inoculated treatment, and 2.20 and 8.69 mg K/plant in
untreated treatment (control) in both sandy and clay soils,
respectively.
Total potassium content in roots of wheat plants ranged
between 1.11 and 11.56 mg K/plant in both sandy and clay soils.
The highest values of potassium content in roots of wheat were
obtained also by the same treatment inoculated with tri-mixture
inoculants of ASH21, ZH21 and RZ11combined with
1/2 inorganic N-fertilizers in sandy soil and treatment receiving
only N dose without bio-fertilization in clay soil. The values
were 11.56 and 6.99 mg K/plant in both sandy and clay soils,
respectively. The uninoculated treatment (control) showed only
1.11 and 2.65 mg K/plant in sandy and clay soils, respectively.
In the whole plants, however similar results of potassium
contents were found as given in shoots of wheat plants in both
soils. The treatments inoculated with tri-mixture inoculants of
ASH21, AH21 and RZ11 combined with 1/2 N dose gave the
highest values of total potassium content in whole plant of wheat
plants, being 46.85 and 40.44 mg/plant in sandy and clay soils,
respectively as compared with other treatments. The statistical
significant (P≤0.01), but the differences between soil textures
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and interaction differences between soil textures and fertilization

The obatained data are in harmony with those reported by
Rodelas et al. (1999) in broad bean. In this respect,
Panwar (2000) reported that inoculation with Azospirillum
brasilense and Bacillus subtilis significantly enhanced leaf area,
chlorophyll concentration, total biomass production, a grain
yield and total mineral content of wheat.
Data in Table 33 illustrated by Fig. 24 present the biomass
yield (g/1000 grains), nitrogen % and protein % of grains of
wheat plants grown on both sandy and clay soils as affected by
bio-fertilizers under different levels of inorganic N-fertilization.
Results indicated that there were highly significant (P≤0.01)
differences among fertilization treatments, soil textures and the
interaction between fertilization treatments and soil textures in
biomass yield (g/1000grains) of wheat plants grown on sandy
and clay soils. The highest value was obtained by treatment
receiving N-dose + inoculation with ASH21 in sandy soil
reaching 38.60 g/1000 grains and treatments receiving N-dose
only without bio-inoculants and 1/2 N dose + inoculants of
ASH21 + ZH21 + RZ11 in clay soil, being 48.25 and 46.70
g/1000 grains, respectively, comparing with the lowest value
obtained by the uninoculated treatment (control) in sandy and
clay soils.
In general, results show that the average values of biomass
(g/1000 grains) of wheat plants grown on sandy soil were lower
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than those obtained from wheat plants grown on clay soil, being
29.52 and 41.62 g/1000grains, respectively. In addition, there
were highly significant differences (P≤0.01) among fertilization
treatments, soil textures and interaction between soil textures and
fertilization treatments in nitrogen (%) and protein (%) of wheat
plants grown on both sandy and clay soils. The highest value was
obtained by the treatment received 1/2 N + inoculation with
tri-mixture inoculants of ASH21, AH21 and RZ11 in sandy soil
being, 2.19 N% and 12.60% protein in grains. However, in clay
soil treatment received N-dose without bio-fertilization and
treatment received 3/4 N dose + inoculation with ASH21 only
gave also highest figures namely 2.15 and 2.14 % N and
12.36 and 12.33 % protein in grains, respectively. The
uninoculated treatment (Control) in sandy soil did not form any
grains in plant.
El-Demerdash et al., (1993), Galal and Elgandour (1994) and
Galal and Thabet (2002). They revealed that the inoculation
with bio-fertilizers and biomass applied inorganic N-fertilizers of
wheat plants gave the best results with regard to yield and
N uptake in shoot and grains of wheat plants.
Inoculation with associative N2-fixers particularly
Azotobacter and Azospirillum have a great importance to
improve growth and increase yield of cereal crops as well as the
mineral contents of shoot, root and whole plants not only due to
high N2-fixation activity, but also the ability of these bacteria to
produce antibiotics, growth-promoting substances (phytohormones),
siderophores and the ability to solubilize phosphate (Pandey and
Kumar, 1989)
196
In conclusion, inoculation of wheat plants seedlings with

Azospirillum alone or mixed with Azotobacter and
R. leguminosarum bv. vicieae with applied different levels of
inorganic N-fertilizers increased plant dry weight, nitrogen,
phosphorus and potassium contents in wheat plants increased
biomass yield and protein percentage in grains of wheat plants.
This also in harmony with Goher et al. (1988), Galal and
Elgandour (1994), El-Bakry et al. (2001), Galal et al. (2001).
4.8. Response of wheat plants to bio-and organic
fertilizers under cultivation in sandy and clay soils
A pot experiment was conducted to evaluate the response
of wheat plants (Giza 168) grown on sandy and clay soils under
different garbage compost levels as an organic fertilizers and
inoculation with inoculants from Azospirillum spp. (ASH21)
alone or tri-mixture inoculants of Azospirillum sp. (ASH21),
Azotobacter sp. (ZH21) and R. leguminosarum bv vicieae
(RZ11).
The plant dry weight, nitrogenase and dehydrogenase
activities, total nitrogen content, total phosphorus content, total
potassium content and grain yield of wheat plants were
determined.
Data in Table 34 and illustrated by Fig. 25 show the effect
of bio-fertilizers and organic amendment on dry weight of wheat
plants grown on both sandy and clay soils. The results showed
that shoot dry weight, generally, ranged from 0.14 to
0.97 g/plant. Application of bio-fertilizers combined with bio or
organic amendment increased the shoot dry weight of wheat
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plants over the uninoculated treatment (control) or treatment

received organic N-fertilizers only. The highest shoot dry weight
values were obtained by treatment receiving compost
(recommended dose of compost 5 ton fed-1 in sandy soil and
2 ton fed-1 in clay soil) and inoculation with tri-mixture
inoculants of ASH21, ZH21 and RZ11 in sandy and clay soils,
giving 0.79 and 0.97 g/plant, respectively. While the lowest
values of shoot dry weight were obtained by the uninoculated
treatment (control) in both sandy and clay soils, which were
0.14 and 0.69 g/plant, respectively. The statistical differences
among fertilization treatments and between soil textures were
highly significant (P≤0.01), but, the interaction between soil
textures and fertilization treatments were not significant. Similar
results were also obtained in the data of root dry weights.
The statistical differences among fertilization treatments and
between soil textures were highly significant (P≤0.01). On the
other hand, the statistical differences between soil textures and
the interaction between soil textures and fertilization treatment
were not significant.
Regarding the whole plant dry weight, generally, ranged
between 0.23 and 1.37 g/plant. The statistical differences
between soil textures and among fertilization treatments were
highly significant (P≤0.01), but the interaction between soil
textures and fertilization treatment, were not significant. The
application of compost combined with bio-fertilization of
tri-mixture inoculants ASH21, ZH21 and RZ11 increased the
whole pant dry weight of wheat plants as compared to the
average of all other treatments in both sandy and clay soils. The
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lowest values of whole plant dry weight were obtained by

uninoculated treatment (control) in both sandy and clay soil,
giving 0.23 and 0.93 g/plant respectively. The stimulatory effect
may be due to application of compost as an organic material
which can also provide the microorganisms with suitable sources
of nutrients, growth factor and energy to survive and persist in
the rhizosphere of plants (Molla et al., 2001).
Hegazi et al. (1983) who found that inoculation of maize plants
with Azospirillum caused increases in plant dry weight (200 %)
and total nitrogen content (157 %). Also, Mohamed (1997)
found that wheat plants amended with biogas manure together
with inoculation by Azospirillum brasilense and Bacillus
polymyxa in sandy soil of Ismailia region developed well and
showed better appearance and increasing grains production and
protein accumulation in grain than those only supplied with
N-fertilizers.
The results concluded that inoculation with ASH21, ZH21
and RZ11 compared with compost significantly increased the
shoots, roots and whole plant dry weight of wheat plants grown
on either sandy or clay soils.
The effect of bio-fertilizers and organic amendments on
nitrogenase and dehydrogenase activities in rhizosphere of wheat
plants is presented in Table 35. Results showed that the
application of bio-fertilizers and different levels of organic
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fertilizers increased the nitrogenase activity (μ mole C2 H4 /h/g

dry soil) in rhizosphere of wheat plants grown on both sandy and
clay soils comparing with the control treatment. The highest
values of nitrogenase activity in rhizophere of wheat plants
grown on sandy and clay soils, were obtained by the treatment
receiving compost plus inoculation with tri-mixture inoculants of
ASH21, ZH21 and RZ11 being 48.79 and 161.53 μ mole
C2H4 /h/g dry soil, respectively. The control treatment had the
lowest values of nitrogenase activity in rhizosphere of wheat
plants grown on both soils, being, 0.45 and 1.56 μ mole
C2H4 /h/g dry soils in sandy and clay soils, respectively. The
obtained data are in agreement with those reported by
Ishac et al. (1983) who studied the effect of applied organic
amendments (0.2 % maize stalk) on nitrogen-fixation by
asymbiotic bacteria (Azotobacter and Azospirillum) associated
with wheat and maize plants. They detected that the greater
number of azotobacters and azospirilla were recorded from
maize than from wheat plants. They also indicated that organic
amendments may be used in the agriculture in semitropical
regions. In this respect, Salem et al., (1985 and 1986) reported
that the activities of N2-Fixing microorganisms in sandy and
calcareous soils were greatly influenced with the organic matter
addition where, their counts and activities in N2-Fixing have
been increased markedly, due to manuring of the soil. Also,
Markus and Kramer (1988) found that counts of Azospirillum
spp exceeded those of the Azospirillum spp numbers, in the
rhizosphere of wheat plants after inoculation in combination with
organic fertilizers as compared with that of the control without
organic fertilizer, especially in earlier growth stages of wheat.
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Data also show that the dehydrogenase activity (μg H2/g

dry soil) in rhizosphere of wheat plants grown on sandy and clay
soil, generally, ranged from 3.242 to 16.713 μ g H2/g dry soil.
The results also behaved the same general trend like those of
nitrogenase. The respective values of the treatment received
compost plus inoculation with tri-mixture inoculants of ASH21,
ZH21 and RZ11 gave the highest values of dehydrogenase
activity in the rhizosphere of wheat plants grown on both sandy
and clay soils, being 16.713 and 11.269 μ g H2/g dry soil,
respectively. The respective values for the control treatment
were 3.242 and 3.252 μ g H2/g dry soil. The proliferation and
activation of microorganisms in rhizosphere of the inoculated
plants may explain the observed increase in the dehydrogenase
activity. In this respect, a good correlation between microbial
biomass and soil dehydrogenase activity has been demonstrated
by Carlile et al. (2004).
The effects of bio-fertilizers and organic amendment on
total nitrogen content of wheat plants are presented in Table 36
and illustrated by Fig. 26. Data show that application of
bio-fertilizers and different levels of organic fertilizers increased
the total nitrogen content in shoots, roots and whole plant in
wheat plants comparing with the uninoculated treatment
(control). The results of total nitrogen contents of wheat plants
were in harmony with those of the plant dry weight. The highest
values of total nitrogen content in shoots of wheat plants grown
on both sandy and clay soils were obtained by the same
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treatment inoculated with tri-mixture inoculants of ASH21,

ZH21 and RZ11 combined with compost in both sandy and clay
soils, being 18.8 and 21.23 mg N/plant, respectively, as
comparing with the respective values of the control treatment
reflecting 1.88 and 8.73 mg N/plant. The statistical differences
among fertilization treatments and between soil textures were
highly significant (P≤0.01), but the interaction differences
between soil textures and fertilization treatments were
not significant.
Concerning the total nitrogen content in roots of wheat
plants, the values behaved the same general trend like those of
shoot system. The values ranged between 0.96 and 5.49 mg
N/plant, showing also the highest figures of total nitrogen
content in roots of wheat plants by the same treatment which
received compost and inoculated with tri-mixture inoculants of
ASH21, ZH21 and RZ11 in both sandy and clay soils.
Regarding to total nitrogen content in whole plant,
generally, ranged from 2.84 to 25.95 mg/plant. The statistical
differences between soil textures and among fertilization
treatments were highly significant (P≤0.01). While, the
interaction between soil textures and fertilization treatments
were not significant. The application of compost combined with
bio-fertilization of tri-mixture inoculants of ASH21, ZH21 and
RZ11 increased the total nitrogen content of wheat plants as
compared to the average of all other treatments in both sandy
and clay soils. The lowest values of nitrogen content in whole
plants were obtained by uninoculated treatment (control) in both
sandy and clay soils, being 2.84 and 11.29 mg/plant,
207
respectively. This ensures the stimulative impact of

bio-fertilization on the growth and nutrition status of wheat plants.
Khalil (1979) and Hashem (1980) who found that the total
nitrogen of wheat plants was increased considerably, as a result
of supplementing soil with different organic manures. Similar
results obtained by Hegazi et al. (1983) in maize plants.
The results concluded that the inoculation with
Azospirillumsp. (isolate ASH21) only or mixed with Azotobacter
sp. (isolate ZH21) and R. leguminosarum bv. vicieae (RZ11)
combined with compost significantly increased the total nitrogen
content in shoots, roots and whole plant of wheat plant grown on
sandy and clay soils.
The effects of bio-fertilizers and organic amendments, on
total phosphorus content in shoot, root and whole plant in wheat
plants are presented in Table 37 and Fig. 27. Data showed that
application of bio and organic fertilization increased the total
phosphorus content in shoots of wheat plants comparing with
control treatment. The highest average value of total phosphorus
content (3.19 mg P/plant) in shoots of wheat plants grown on
both sandy and clay soils was obtained by treatment inoculated
with tri-mixture of ASH21, ZH21 and RZ11 and combined with
the recommended dose of compost. On the other hand, the
plants grown on clay soil achieved the highest P content
(2.23 mg P/plant) in shoot of wheat plants as compared to the
plants grown on sandy soils. Regarding, the effect of interaction
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209
210
between fertilization treatments and soil textures, it appears that

fertilization with compost + inoculation with tri-mixture
inoculants of ASH21 + ZH21 + RZ11 gave the highest P content
in shoot of wheat plants (3.31 and 3.07 mg P/plant) in both sandy
and clay soils, respectively.
The fertilization treatment affected the P content in roots of
wheat plants significantly. For instance, the highest (0.37 and
0.36 mg P/plant) were gained by 1/2 compost + inoculation with
ASH21 and compost + inoculation with tri-mixture inoculants,
respectively. The statistical differences between soil textures and
the interaction between fertilization treatments and soil textures
were insignificant.
Moreover, data in Table 37 and fig. 27 disclose that total
P content/plant, generally ranged between 0.28-3.60 mg P/plant.
Regarding, fertilization treatment effect, the data clarified its
significance on total P content/plant. The highest P content
(3.55 mg P/plant) was obtained by compost + inoculation with
tri-mixture inoculants as compared with all other treatments.
Concerning the soil texture effect, the data indicated its
significance on total P content in whole plant. As such, the
highest total P content/plant (2.51 mg P/plant) was obtained
by clay soil.
The interaction between fertilization treatment and soil
texture effects on total P content/plant was also significant.
Meanwhile, the highest values were obtained by compost +
inoculation with tri-mixture inoculants showing 3.69 and
3.41 mg P/plant in both sandy and clay soils, respectively.
211

Radwan et al. (2002) who found that association of multi strains
of bio-fertilizers (phosphate dissolving bacteria + Azotobacter
spp + Azosperillum spp + Pseudomonas spp) with compost
farming manure, led to significant increases in grain, straw,
N and P content and biological yield of wheat plants.
The results, generally, concluded that the inoculation with
mixed inoculants of ASH21, ZH21 and RZ11 combined with
compost led to significant increase on total phosphorus content
in shoots, roots and whole plants in wheat plant grown on sandy
and clay soils.
The effects of bio and organic fertilizers on total potassium
content in wheat plants are presented in Table 38 and illustrated
by Fig. 28. Data showed that application of bio-fertilizers and
organic amendment increased the total potassium content in
shoot, root and whole plant of wheat plants grown on both sandy
and clay soils. The results showed that the treatment receiving
compost plus inoculation with tri-mixture inoculants of ASH21,
ZH21 and RZ11 gave the highest values of total potassium
content (19.84, 6.81 and 26.15 mg K/plant) in shoot, root and
whole plant, respectively. The uninoculated treatment (control)
had the lowest values of total potassium content in shoot, root
and whole plant of wheat plants in both sandy and clay soils.
As for soil texture effect, clay soil significantly increased total
potassium content in both shoot and whole plant, 15.46 and
19.82 mg K/plant respectively as compared with sandy soil.
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213
214
Interaction between fertilization treatments and soil texture

concerning potassium content in shoot, root and whole plants
was insignificant.
In this respect, Pandey and Kumar (1989) showed that
inoculation with associative N2-fixers particularly Azotobacter
and Azospirillum have a great importance to improve growth and
increase yield of cereal crops as well as the mineral contents of
shoot, root and whole plants not only due to high N2-fixation
activity, but also the ability of these bacteria to produce
antibiotics, growth-promoting substances (phtohormones),
siderophores and the ability to solubilize phosphate. Similar
results were found by Radwan et al. (2002) in wheat plants.
These results concluded that application of bio-fertilizers in
wheat plants cultivated in sandy and clay soils significantly
increase the total potassium content in shoot, root and whole
plant of wheat plants.
The effects of bio-fertilizers and organic amendments on
biomass yield (g/1000 grains), nitrogen (%) and protein (%) of
wheat plants grown on both sandy and clay soils are given in
Table 39 and illustrated by Fig. 29. Results obviously show that
weight of 1000 grains, nitrogen and protein percentage were
significantly affected by the studied fertilization treatment. The
highest average weight of 1000 grains (35.85 g), nitrogen
(1.93 %) and protein (11.10 %) in grains were obtained by
treatment received compost + tri-mixture inoculants of ASH21 +
ZH21 + RZ11 as compared with the other treatments.
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216
217
As for the soil texture, clay soil achieved the significantly

highest value of 1000 grain weight, nitrogen and protein
percentage in grains as compared to the sandy soil.
Interaction between the two studied factors concerning
weight of 1000 grain and nitrogen and protein percentage were
significant as such the highest values usually obtained from
compost + inoculation with tri-mixture inoculants in clay soil.
The obtained data are in agreement with those obtained by
Singh and Sharma (1999) who found that wheat plants
inoculated with Azotobacter increased yield from 4.81 to
5.01 ton. The available nutrient status of soil after harvest of the
wheat crop was higher where organic fertilization was applied.
Ardakani et al. (2001) showed that, individually Azospirillum
and mycorrhiza caused significant increases in spike/m2 and
grain weight yield of wheat plants. Moreover, manure increased
most of yield components specially when used with Azospirillum
and mycorrhiza.
The results concluded that the inoculation with tri-mixture
inoculants of ASH21, ZH21 and RZ11 combined with compost
led to significant increases in grains weight and N and protein
percentage in grains of wheat plants grown on sandy and
clay soils.
4.9. Response of wheat plants to bio-fertilizers under
cultivation in sandy and clay soils
A pot experiment was carried out to study the effect of
selected bio-fertilizers alone on the response of wheat plants
cultivated in both sandy and clay soils. Plants were inoculated
with either Azospirillum sp. (ASH21) alone or with Azotobacter
218
sp. (ZH21) or with Rhizobium leguminosarum bv. vicieae

(RZ11) or with tri-mixture inoculants of the three inoculants
ASH21 + ZH21 + RZ11.
The plants dry weight, nitrogenase and dehydrogenase
activities in rhizosphere of wheat plants, total nitrogen content,
total phosphorus content, total potassium content and biomass
yield of wheat plants were determined.
The effects of bio-fertilizer on shoot, root and whole plant
dry weight (g/plant) of wheat plants grown on both sandy and
clay soils are given in Table 40 and illustrated by Fig. 30. The
results show that application of bio-fertilization significantly
increased shoot, root and whole plant dry weights of wheat
plants as compared to the uninoculated treatment (control). As
for the effect of soil texture, clay soil significantly increased both
shoot (0.89 g/plant) and whole plant (1.23 g/plant) dry weights
as compared to sandy soils (0.57 and 0.89 g/plant) in shoot and
whole plant, respectively.
Interaction between two studied factors on shoot, root and
whole plant dry weight of wheat plants was generally
insignificant.
by Rabie et al. (1995) and Amoo et al. (2003). They showed
that inoculation of wheat plants with Azotobacter chroococcum
and/or Azospirillum brasiliense increased growth parameters and
yield of wheat cultivars, the effect being directly dependent on
the strain cultivar combination.
219
220
221
These results concluded that inoculation of wheat plants

with Azospirillum sp. (ASH21) combined with Azotobacter sp.
(ZH21) and R. leguminosarum bv. vicieae (RZ11) significantly
increased the shoots, roots and whole plants dry weigh of wheat
plants grown on both sandy and clay soils.
Results in Table 41 showed a great variation in the effect of
inoculation with different bio-fertilization on nitrogenase and
dehydrogenase activities in rhizosphere of wheat plants grown
on both sandy and clay soils. The nitrogenase activities
generally, ranged from 0.45 to 63.08 n mole C2H4/h/100g dry
soil . The highest values of nitrogenase activities in rhizosphere
of wheat plants were obtained by treatment inoculated with
tri-mixture inoculants of ASH21 + ZH21 and RZ11, showing
11.84 and 63.08 n mole C2H4/h/100g dry soil in both sandy and
clay soils, respectively. The lowest values of nitrogenase activity
were obtained by uninoculated treatment (control) in both sandy
and clay soils, being 0.45 and 1.56 n mole C2H4/h/100g dry soil,
respectively. Concerning the dehydrogenase activity, it generally
ranged from 1.586 to 11.543 µ mole H2/h/g dry soil. The highest
values of dehydrogenase activity were obtained by the treatments
inoculated with tri-mixture inoculants of ASH21 + ZH21 and
RZ11 and inoculation with ASH21 in both sandy and clay soils.
In the second rank came plants inoculated with ASH21 + ZH21
in both soil textures. Control plants and those inoculated with
RZ11 recorded the lowest values for dehydrogenase activity in
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223
both soil textures. The proliferation and activation of

microorganisms in rhizosphere of the inoculated plants may
explain the observed increase in the dehydrogenase activity. In
this respect, a good correlation between microbial biomass and
soil dehydrogenase activity has been demonstrated by Carlile
et al. (2004).
The obtained results are in harmony with those reported by
El-Demerdash et al. (1993) in wheat plants. Döbereiner (1974)
reported that contribution of biological nitrogen fixation in
Brazil may reach to 70 % for sugar cane and up to 50 % in
cereals through the activity of endophytic diazotroph in
non-legume. Similar results were obtained by Alam et al. (2001)
and Galal et al. (2001).
The effect of bio-fertilization on total nitrogen content in
shoot, root and whole plant of wheat plants are given in Table 42
and illustrated by Fig. 31. Data showed that application of
bio-fertilizers significantly increased the total nitrogen content in
shoot, root and whole plant comparing with the uninoculated
treatment. The highest average values of total nitrogen content in
shoot, root and whole plant of wheat plants were obtained by the
treatments which inoculated with tri-mixture inoculants of
ASH21, ZH21 and RZ11 and those treated with ASH21 + ZH21
without significant differences between them.As for the effect of
soil textures, the total nitrogen content was significantly
increased in clay soil (13.57 mg N/plant) as compared to sandy
soil (10.79 mg N/plant).
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225
226
The interaction between the two tested factors, concerning

total nitrogen content in shoot, root and whole plant of wheat
plants was insignificant.
The stimulatory effect of bio-fertilization on the plant
growth and total nitrogen contents of all parts of wheat plants
could be attributed not only to N2-fixation activity but also to the
production of growth promoting substances such as
phytohormones, siderophores, which in turn affected the plant
growth. Oken (1984) reported that inoculation of wheat and corn
plants with Azospirillum increased their biological nitrogen
fixation mainly after flowering and increased plant growth
promoting substances. He stated that Azospirillum preferentially
adsorbs and proliferates on and in roots of wheat and corn plants
concerning improvement in root function which in turn results in
faster accumulation of dry matter in the plants and in many cases
higher crop yield. In the same subject Antoun et al. (1998)
found that rhizobia and bradyrhizobia are capable of producing
phytohormones and sidrophores which improved plant growth.
Alam et al. (2001) and Creus et al. (2004). They reported that
the inoculation with mixture of several free living rhizobacteria,
Azotobacter, Bacillus and Enterobacter significantly increased
acetylene reducing activity in roots of rice plants, total dry
matter yield, grain yield, N uptake, root length and leaf area.
the effect of bio-fertilizers on total phosphorus content in shoot,
root and whole plant (mg/plant) of wheat plants grown on both
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228
229
sandy and clay soils. The results show that the total phosphorus
content in shoot, root and whole plant of wheat plants were
significantly increased by the application of bio-fertilization as
compared to the uninoculated treatment (control). The highest
values of total phosphorus content namely 3.51, 0.36 and
3.87 mg P/plant in shoot, root and whole plant, respectively,
were obtained by the treatment inoculated with tri-mixture
inoculants of ASH21, ZH21 and RZ11. The lowest values of
total phosphorus content in shoot, root and whole plant of wheat
plants were obtained by uninoculated treatment (Control). In this
respect, the effect of soil texture was insignificant. On contrast,
the interaction between the two tested factors reveal that
RZ11 in sandy soil induced the highest total phosphorus content,
being 4.67 and 5.07 mg P/plant in both shoot and whole plant
respectively.
by El-Bakry et al., (2001) in sorghum and Radwan et al.,
(2002) in wheat.
The results concluded that inoculation of wheat plants with
Azospirillum sp. (isolate ASH21) combined with Azotobacter sp.
(isolate ZH21) and R.Leguminosarum bv. vicieae (isolate RZ11)
significantly increased the total phosphorus content in shoot, root
and whole plant dry weight of wheat plants grown on both sandy
and clay soils.
The effect of bio-fertilizers on total potassium content in
shoot, root and whole plant (mg/plant) in wheat plants cultivated
230
in sandy and clay soils is given in Table 44 and illustrated by

Fig. 33. The results show that bio-fertilizers treatments
significantly affected total potassium content in wheat plants. In
this respect inoculation with the tri-mixture inoculants induced
the highest potassium content reaching 19.87, 6.12 and 25.99 mg
K/plant in shoot, root and whole plant, respectively, without
significant differences with those treated with ASH21 + AH21.
The lowest values of total potassium content in shoot, root and
whole plant were obtained by uninoclated treatment (control) in
both sandy and clay soils. Patel et al. (1985) revealed that the
application of Azotobacter chroococcum on wheat increased N,
P and K uptake.
Data in Table 45 and illustrated by Fig. 34 indicated that
the bio-fertilization treatment had significant effect on weight of
1000 grains, nitrogen and protein percentage in grains. The
highest values (35.33 g and 1.95 % and 11.20 %) for the
previous three parameters, respectively, were recorded by plants
inoculated with tri-mixture inoculants of ASH21, ZH21 and
RZ11. in the second rank, came plant treated with inoculation of
ASH21 + ZH21 followed by those treated with ASH21 + RZ11
and those treated with ASH21 alone. While, uninocualtion
treatment (control) had the lowest values.
Moreover, soil textures had significant effect on weight of
1000 grains and nitrogen and protein percentage in grains. The
highest values were obtained by plants grown on clay soil as
compared with those grown on sandy soil.
231
232
233
234
235
Interaction between two tested factors was significant. The

highest values represented as 40.70 g, 2.02 % and 11.59 % were
recorded for the weight of 1000 grains, nitrogen and protein
percentage, respectively with the plants treated with tri-mixture
inoculants of ASH21, ZH21 and RZ11 in clay soil, followed by
those treated with ASH21 + ZH21 in clay soil without
significant differences between them in most cases. The lowest
values were recorded for the uninoculated treatment (control).
In this respect, Panwar (2000) and Sushila and Giri-
Gajendra (2000) stated that bio-fertilization by Azospirillum
and Azotobacter enhanced the growth and yield of wheat.
Recently, Vivek et al. (2004) studied the relative efficacy of
Azotobacter chroococcum on tall and dwarf wheat
(Triticum aestivum L.) in arid soils. They found that grain, straw
yield and biomass of root were higher with mutants, which
maintained higher survival rate in rhizosphere during growth
period of wheat crop.
Inoculation with associative N2-fixers particularly
Azotobacter and Azospirillum have a great importance to
improve growth and increase yield of cereal crops as well as the
mineral contents of shoot, root and whole plants not only due to
high N2-fixation activity, but also the ability of these bacteria to
produce antibiotics, growth-promoting substances
(phtohormones), siderophores and the ability to solubilize
phosphate (Pandey and Kumar, 1989).
These results concluded that application of bio-fertilizers in
wheat plants cultivated in sandy and clay soils significantly
increase the shoot, root and whole wheat plants.
236
The results concluded that inoculation of wheat plants with

Azospirillum sp. (isolate ASH21) combined with Azotobacter sp.
(isolate ZH21) plus R. leguminosarum bv. vicieae (isolate RZ11)
significantly increased total nitrogen, phosphorus and potassium
contents as well as biomass yield and nitrogen and protein
percentages in grains of wheat plants grown on sandy and
clay soils.
IBA content in roots of wheat plants
Data presented in Table 46 show the effect of application
of bio-fertilizers only or combined with inorganic or organic
N-fertilizers on GA3, IAA and IBA content in roots of wheat
plants cultivated in both sandy and clay soils. The results show
that the highest values of GA3 content were obtained by
treatment inoculated with tri-mixture inoculants of Azospirillum
sp. (ASH21), Azotobacter sp. (ZH21), and R. leguminosarum bv.
vicieae (RZ11) combined with 1/2 N inorganic fertilizers in both
sandy and clay soils, being 50.42 μg/ g fresh weight of roots. The
lowest values were obtained by uninoculated treatment (control)
in both sandy and clay soils, being 10.43 and 7.86 μg/ g fresh
weight of roots, respectively.
Concerning the IAA content (μg/ g fresh weight of roots),
in wheat plants grown on both sandy and clay soils the results
show that the highest values of IAA content were obtained by
the same treatment inoculated with tri-mixture inoculants of
ASH21, ZH21 and RZ11 combined with 1/2 inorganic
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238
N-fertilizers in both sandy and clay soils, being 0.78 and

1.06 μg/ g fresh weight of roots, respectively. The lowest values
of IAA were obtained by the uninoculation treatment (Control)
in both sandy and clay soils, being 0.01 and 0.03 μg/g fresh
weight of roots, respectively.
Regarding the IBA content in roots of wheat plants grown
on both sandy and clay soils the results indicated that the
treatment inoculated with ASH21 and combined with 1/2 N
inorganic fertilizers in both sandy and clay soils, being
326.42 μg/ g fresh weight of roots, in both soils. The obtained
results are in harmony with the results obtained by De-Freitas
and Germida (1990). They reported that the beneficial effect of
N2-fixing bacteria are also due to production of growth
promoting substance which help increasing crop yield.
In addition Yanni et al. (2001) studied the beneficial
effected on plant growth promoting association of Rhizobium
leguminosarum bv. trifolli with rice roots. Studies using selected
Rhizobium strain (E11) indicated that it produced auxine
(indole acetic acid IAA) and gibberellins GA3 phytohormones
representing two major classes of plant growth regulators.
Also, Dobbelaera et al. (2001) found that Azospirillum are
able to promote plant growth and increase yield in many crops.
The production of plant growth promoting substance, which, led
to an improvement in root development and increase in the rate
of water and mineral uptake.
239
4.11. Comparison between the different responses of

wheat plants to application of bio-fertilizers only
or combined with organic or inorganic N fertilizers
with control treatment in both sandy and clay soil
4.11.1. In sandy soils
average response of wheat plants to application of different
bio-fertilizers only, bio and inorganic N-fertilizers and bio and
organic fertilizers with untreated (control) treatment in sandy
soil. It is clear that the application of bio-fertilizers alone or
combined with organic or inorganic N-fertilizers gave the
highest average values of all traits studied as compared to
untreated (control) treatment.
The response of wheat plants cultivated in sandy soil and
inoculated with bio-fertilizers only, combined with inorganic or
combined with organic fertilizers was inconsistent and varied
with experiments. The application of wheat plants with
bio-fertilizers combined with inorganic N-fertilizers gave
superior response in the performance of some traits studied.
The average value of different traits studied were increased
from 117.3 % (in protein % in grains) to1875.6 %
(in nitrogenase activity n mole C2H4/h/100g dry soil ) as the
result of the application of bio-fertilizers, bio and organic or bio
and inorganic N fertilizers as compared to untreated control
treatment (100 %).
The results concluded that inoculation of wheat plants
grown on sandy soil with bio-fertilizers (Azospirillum spp,
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241
Azotobacter spp and R. leguminosarum bv. vicieae) is important

to obtain the best performance in growth and total nitrogen and
mineral contents.
4.11.2. In clay soils
Data presented in Table 48 show comparing among the
average response of wheat plants to application of different
bio-fertilizers only, bio and organic and bio and inorganic
N-fertilizers with untreated treatment (control) in clay soil. Like
those observed for plants grown on sandy soil, data show that the
application of bio-fertilizers alone or combined with organic or
inorganic N-fertilizers had the highest average values of different
traits studied as compared to the untreated treatment (control).
The response of wheat plants cultivated in clay soil and
inoculated with bio-fertilizers only, combined with inorganic or
organic fertilizers was also inconsistent and varied with
experiments. The application of wheat plants with bio-fertilizers
only gave superior response in the performance of some traits
studied. While, the application of bio-fertilizers combined with
inorganic or organic fertilizers gave superior response in the
performance of other traits. In some traits studied, the average
values of different traits studied were increased from 117.3 % in
protein of grains to 2275.3 % in nitrogenase activity µ mole
C2H4/g dry soil as a results of the application of bio-fertilizers,
bio and organic or bio and inorganic N-fertilizers as compared to
the untreated one (control 100 %).
The results concluded that the inoculation of wheat plants
grown on clay soil with bio-fertilizers (Azotobacter spp,
Azospirillum spp and R. leguminosarum bv. vicieae) is important
to obtain the best performance in growth, total nitrogen and
mineral contents.
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243
V. SUMMARY AND CONCLUSION

This work was carried out in the laboratory and greenhouse
Zagazig University, Egypt, in order to isolate and selecte
efficient indigenous strains of Rhizobium leguminosarum bv
vicieae, Azospirillum and Azotobacter. from different locations
in Sharkia governorate to use them as biofertilizers for broad
bean (Vicia faba L.) and wheat (Triticum aestivum L.) plants to
minimize the contamination of the environment with chemical
fertilizers. Therefore, this study was carried out as followes:
1- Sixty isolates of Rhizobium were obtained from nodule roots
of broad bean plants and 60 isolates of Azotobacter were
isolated from rhizosphere of these plants
2- Sixty isolates of azospirilla and 60 isolates of Azotobacter
were isolated from roots and rhizosphere of wheat plants.
3- Thirty two isolates of Rhizobium were selected for
nodulation test, nitrogen fixation and effect on growth of
broad bean plants
4- 18 isolates of azospirilla and 14 isolates of Azotobacter were
selected to efficient nitrogenase activity in liquid media.
5- Only five isolates of Rhizobium which showing high values
in nodulation, nitrogen fixation and affect the growth of
broad bean were used to determine production of growth
promoting substances (GA3, IAAand IBA) in liquid media.
6- The highest five effective isolates in nitrogenase activity of
each azospirilla and Azotobacter were used for production of
growth promoting substances (GA3, IAAand IBA) in liquid
media.
244
7- The highest efficient isolate of Rhizobium on nodulation,

nitrogen fixation and production of growth promoting
substances and the highest isolates of azospirilla and
Azotobacter in nitrogenase activities and production of
growth promoting substances were used as biofertilizers for
broad bean (Vicia faba L.) and wheat (Triticum aestivum L.)
plants.
The selected bio-fertilizers were examined on growth and
productivity of broad bean and wheat plants which cultivated in
both sandy and clay soils under different levels of inorganic and
organic N fertilizers in six greenhouse experiments as follow:
1. Examine the effect of selected bio-fertilizers with or without
different levels of inorganic N-fertilizers in two soil textures
on the growth of broad bean plants.
different levels of organic N-fertilizers in two soil textures on
the growth of broad bean plants.
3. Examine the effect of selected bio-fertilizers only in two soil
textures on the growth of broad bean plants.
different levels of inorganic N-fertilizers in two soil textures
on the growth of wheat plants.
different levels of organic N-fertilizers in two soil textures on
the growth of wheat plants.
6. Examine the effect of selected bio-fertilizers only in two soil
textures on the growth of wheat plants.
245
The obtained results could be summarized as follows:

1. The isolate RZ11 was the highest efficient isolate of
R. leguminosarum bv. vicieae on plant growth, nodulation,
nitrogenase activity and production of growth promoting
substances.
2. The isolate ZH21 of Azotobacter. and isolate ASH21 of
Azospirillum. reflected the highest activity of nitrogenase
enzyme and production of growth promoting substances.
3. Effect of selected bio-fertilizers with or without inorganic
and organic N-fertilizers on broad bean plants was as
follows:
3.1. Inoculation with tri-mixture inoculants of RZ11, ZH21 and
ASH21 isolates plus the application of 1/2N dose inorganic
N-fertilizer gave the best results with regard to number of
nodules, nodules dry weight and nitrogenase activity, shoot,
root and whole plant dry weight, total nitrogen, total
phosphorus and total potassium contents in shoot, root and
whole plant of broad bean plant cultivated in sandy soils.
3.2. Inoculation with RZ11 and / or ZH21 and ASH21 isolates
and/or application of 1/2N dose inorganic N-fertilizer
significantly increased the biomass yield and N % and
protein % in seeds of broad bean grown on clay soils
3.3. Inoculation with tri-mixture inoculants of isolates RZ11,
ZH21 and ASH21 receiving recommended dose of compost
gave the highest values of nodules dry weight, total nitrogen,
phosphorus and potassium contents in shoots, roots and
whole plant of broad bean plants grown on both soils.
246
3.4. Inoculation with R. leguminosarum bv. vicieae

(isolate RZ11) receiving recommended dose of compost
gave the highest number of nodules, biomass yield, nitrogen
(%) and protein (%) in seeds of broad bean plants growing
in both soils.
3.5. Inoculation with tri-mixture inoculants of RZ11, ZH21 and
ASH21 isolates gave the best results with regard to number
of nodules, nodules dry weight and nitrogenase activity,
shoot, root and whole plant dry weight, total nitrogen, total
whole plant and biomass yield, nitrogen (%) and protein (%)
in seeds of broad bean plants grown on both soils.
The results concluded that the inoculation of broad bean
plants grown on both sandy and clay soils with bio-fertilizers
(R. leguminosarum bv. vicieae, Azotobacter and Azospirillum)
is important to obtain the best performance on growth, total
nitrogen and mineral contents.
4. Effect of selected bio-fertilizers with or without inorganic and
organic N-fertilizers on wheat plants was as follows:
4.1. Inoculation with tri-mixture inoculants of ASH21, ZH21 and
RZ11 isolates plus the application of 1/2N dose of
recommended inorganic N-fertilizer gave the best results
with regard to, shoot, root and whole plant dry weight,
nitrogenase and dehydrogenase activity in rhizosphere, total
nitrogen, total phosphorus and total potassium contents in
shoot, root and whole plant, biomass grain yield, nitrogen
(%) and protein (%) in grain of wheat plants grown on both
sandy and clay soils.
247
4.2. Inoculation with ASH21, ZH21 and RZ11 combined with

compost significantly increased the shoot, root and whole
plant dry weight, nitrogenase and dehydrogenase activity in
rhizosphere of wheat plants grown on sandy and clay soils.
4.3. Inoculation with ASH21 alone or mixed with ZH21 and
RZ11 combined with compost led to significant increase in
total nitrogen, total phosphorus and total potassium content
in shoots, roots and whole wheat plants grown on sandy and
clay soils.
4.4. Inoculation with ASH21 alone or mixed with ZH21 and
RZ11 combined with compost led to significant increases in
grains weight and N %, protein % of wheat plants growing
in sandy and clay soils.
4.5. Inoculation with tri-mixture inoculants of ASH21, ZH21 and
RZ11 isolates gave the best results with regard to, shoot,
root and whole plant dry weight, nitrogenase and
dehydrogenase activity in rhizosphere, total nitrogen, total
whole plant, grain yield, nitrogen (%) and protein (%) in
grain of wheat plants growing in both sandy and clay soils.
The results concluded that the inoculation of wheat plants
grown on sandy and clay soils with bio-fertilizers (Azospirillum,
Azotobacter and R. leguminosarum bv. vicieae) is important to
obtain the best performance on growth, total nitrogen and
mineral contents.
These results, in general, concluded that inoculation with
indigenous strains is an important procedure when studing there
inherent capacity to benefit cropes in some cases, indigenous
strains can perform better than introduced strains in promoting
the growth of the plants due to their superior adaptability to the
environment.
248
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USE OF MICROORGANISMS AS BIOFERTILIZERS FOR SOME PLANTS

Thesis · June 2006

Ali Salama Ali Salama

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ALI SALAMA ALI SALAMA

Under the Supervision of:

Prof. Dr. S. H. Salem ........................................................

Prof. Dr. Fatma I. El-Zamik ............................................

Dr. Howaida M. L. Abd El-Basit ....................................

USE OF MICROORGANISMS AS BIOFERTILIZERS

ALI SALAMA ALI SALAMA

This thesis for M. Sc. Degree has been

Prof. Dr. S. H. Salem ........................................................

Date of examination: / / 2006

4.4. Response of broad bean plants to bio-fertilizers

1 The locations of soil samples selected for isolation

11 Effect of bio-fertilizers and inorganic N-fertilizers

22 Effect of bio-fertilizers on total phosphorus content

33 Effect of bio-fertilizers and inorganic N-fertilizers

44 Effect of bio-fertilizers on total potassium content

1 Map of Sharkia governorate showing localities of

11 Effect of bio and organic fertilizers on total

23 Effect of bio-fertilizers and inorganic N-fertilizers on

A wide range of bacteria in rhizosphere can promote plant

alternative and cheap source for chemical fertilizers. From the

II. REVIEW OF LITERATURE

Pathak et al., 1997; Dadarwal et al., 1997 and Hedge et al.,

Bradyrhizobium out of six tested organisms, and four strains

compared with 40.51-75.56 % in inoculated ones. On the other

nutrient supply via phosphate solubilization. For instance,

nodulation by native rhizobia and improved root and shoot

Although, Humphry et al. (2003) studied the genotypic

2.1.2. Non symbiotic nitrogen fixers

Azospirillum inoculation in field receiving mineral fertilizers

effects plant all metabolism from outside suggests that bacteria

Also, Swedrzynska and Sawicka (2001) studied the effect

nitrogen but produce a variety of growth promoting substances.

growth and yield of wheat, rice and vegetable crops could be

and AM inoculation on the proliferation and survival

Reynders and Vlassak (1979) found that all the tested

hormones at very low levels namely indole lactic acid, indole-3-

yield and total dry weight. Inoculation with mixture of these

either directly by producing phytohormones or indirectly by

Azotobacter chroococcum in improving yield of cotton was also

Rhizobium spp. strain NGR234. They found that addition of

and rhizobia strains from three sites (2, 3, 4) were successful in

plants) with their homologous phages . He stated that only one

isolates was assessed by studying the plaque traits on their

pigeon pea. They used seed inoculated with Rhizobium and/or

height, number of leaves and shoots, and dry matter content

Several authors (Elgamal andEl-Sheikh 1989; Das, 1991

3 % alone or in combination with Rhizobium leguminosarum

addition, application of phosphorus solubilizing bacteria may

Rabie et al. (1995) showed that inoculation of wheat plants

non-symbiotic N2-fixers of the genera Azotobacter, Azospirillum

Sushila and Giri-Gajendra (2000) stated that biofertilization

applied at 0 and 50 kg ha-1 labeled as ammonium sulfate applied

However, the rate of increase in the count of Azotobacter was

counts and activities in N2-fixation have been increased

japonicum, Rhizobium leguminosarum, Rhizobium spp (cowpea),

and possible interaction with mineral and organic fertilization on

Radwan et al. (2002) evaluated the response of wheat