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POTENTIAL OF RHIZOBIA AND FREE LIVING RHIZOBACTERIA
CONTAINING ACC-DEAMINASE FOR IMPROVING NODULATION AND

YIELD OF CHICKPEA (Cicer arietinum L.)

SHER MUHAMMAD SHAHZAD
M.Sc. (Hons.) Soil Science
A thesis submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
IN
SOIL SCIENCE
INSTITUTE OF SOIL AND ENVIRONMENTAL SCIENCES

UNIVERSITY OF AGRICULTURE
FAISALABAD, PAKISTAN
2009
The Controller of Examinations
University of Agriculture,
Faisalabad.
We, the supervisory committee, certify that the contents and the form of the thesis
submitted by Mr. Sher Muhammad Shahzad, Reg. No. 99-ag-1381, have been found
satisfactory and recommend that it be processed for the evaluation by the external examiners for
the award of degree.
SUPERVISORY COMMITTEE
CHAIRMAN : ___________________________
(DR. AZEEM KHALID)
MEMBER : ___________________________
(DR. MUHAMMAD ARSHAD)
MEMBER : ___________________________
(DR. KHALIL-UR-REHMAN)
DEDICATED
TO MY BELOVED SISTER
[TASLEEM KAUSAR]
TO THE ONE I ADORE AND TELL
MY MOST INNER THOUGHTS.
I THANK YOU.
FOR ALWAYS BEING THERE WHEN

I NEEDED A SHOULDER TO CRY ON.
YOU MAKE GLOOMY DAYS, BRIGHT!!
YOU MAKE A SAD SONG SEEM NOT SO SAD.

WHENEVER WE'RE TOGETHER THERE'S
ALWAYS A SMILE ON OUR FACES.
I HAVE A BEST SISTER AND
I AM GLAD IT'S YOU!!
IF I COULD BUY YOU THE WORLD,

I'D WRAP IT WITH GOLD AND A
RIBBON OF RAINBOWS.
BUT ALL I CAN GIVE YOU IS
A PART OF MY HEART OF LOVE
MY SISTER LIKE THE
TORCH I HAVE.
TO GO FORWARD WITH PURPOSE AND DETERMINATION

IT’S ALL BECAUSE OF YOU
YOU’ERE THE BEST!!!!
YOU'RE THE BEST!!!!!
SHE DIED ON 2ND OF JULY, 2007 (MAY SHE REST IN JANNATULFIRDOUS)

HER PRAYERS ARE AND ALWAYS WILL REMAIN WITH ME
ALLAH BLESS YOU IN HEAVEN

ACKNOWLEDGEMENTS
All the unfathomable analogies humble thanks giving are preferred to ALMIGHTY ALLAH, the
most Gracious and compassionate, whose blessing and exaltation flourished my thoughts and
thrived my ambitions to eventually shape up the cherished fruit of my modest endeavors to this
manuscript, my special praise for the HOLY PROPHET HAZRAT MUHAMMAD (P.B.U.H)
whose eternal teachings would remain a source of inspiration and guidance for the mankind for
ever, whose bounteous enabled me to perceive the higher ideals of life.
I deem it an utmost pleasure to be able to express the hearties gratitude and deep sense of
devotion to my reverend and worthy supervisor DR. AZEEM KHALID, Associate Professor,
Department of Environmental Sciences, PMAS University of Arid Agriculture, Rawalpindi,
Pakistan (Previously worked in the Institute of Soil and Environmental Sciences, University of
Agriculture, Faisalabad, Pakistan) for his skillful and affectionate guidance, unfailing patience and
generous guidance. I was condescended throughout the course of my studies as well as research
continuation up till accomplishment of the dissertation.
A deep and philosophic sagacity of appreciation is owed to DR. MUHAMMAD ARSHAD,
Professor, (T.I.), Institute of Soil and Environmental Sciences, for his cooperative attitude,
constant help, valuable suggestions and criticism towards the completion of this manuscript.
I am also thankful to DR. KHALIL-UR-REHMAN, Associate Professor, Department of
Biochemistry for his able guidance and valuable suggestions during the course of this research.
Special and particular thanks extended to DR. ZAHIR AHMAD ZAHIR, Associate
Professor, Institute of Soil and Environmental Sciences, for his cooperative and supportive attitude
for his help to accomplish this humble effort in its present form, and valuable suggestions towards
the achievement and completion of this script.
I am very thankful to MR. MUHAMMAD SADDIQUE (Lab attendant), Institute of Soil
and Environmental Sciences. Special thanks to special friends M/S TAUSEEF SULTAN,
MUHAMMAD SALEEM ARIF, HAFIZ MUHMAMMAD HAROON, MUHAMMAD
SALMAN, SHAHID RAZA, MUHAMMAD IRSHAD, MUHAMMAD ABUBAKAR
SIDDIQUE, MUHAMMAD ASIF RAZA, MUHAMMAD NAVEED MUHAMMAD,
SAJIDMEHMOOD NADEEM, AYYAZ ZAFAR and IJAZ MEHBOOB for their sincere help
and encouragement.
No acknowledgements could ever adequately express my obligations to my affectionate
and adoring PARENTS, MY SISTERS and BROTHERS, who always raised their hands in
prayers for me without whose moral and financial support; the present distinction would have
merely been a dream. I can only say I am here just due to prayers of family specially my mother.
Last but not the least, I want to express my gratitude to my beloved uncles and aunties,
who always motivate and pray for me and my future especially ABDUL HAMEED, MUKHTAR
AHMED, SHOUKAT ALI SHAHID and MUHAMMAD SARWAR.
Finally, I like to complement a stranger, for whom these words are nothing but something
especial from my heart and soul. I just want to say THANKS for everything you think and did for
me.
SHER MUHAMMAD SHAHZAD

E-Mail: crystalboy_uaf@yahoo.com
LIST OF CONTENTS
Chapter Title Page
1. Introduction 1
2. Review of Literature 7
2.1. Chickpea 7
2.2. Causes of low yield of chickpea in Pakistan 8
2.3. Ethylene biosynthesis and plant growth 9
2.3.1. Ethylene biosynthesis under stress conditions 12
2.3.1.1. High temperature 16
2.3.1.2. Salt toxicity and drought 17
2.3.1.3. Metals and organic contaminants 20
2.3.1.4. Soil compaction 22
2.3.1.5. Excessive moisture 22
2.3.1.6. Pathogen infection 23
2.3.2. Ethylene as an inhibitor of nodulation 24
2.4. Plant growth promoting rhizobacteria 25
2.4.1. Effect of plant growth promoting rhizobacteria on plant growth 26
2.4.2. Plant growth promoting rhizobacteria containing ACC-deaminase 26
2.4.2.1. Effect of bacteria containing ACC-deaminase on leguminous crops 27
2.4.2.2. Effect of bacteria containing ACC-deaminase on other crops 33
2.5. Rhizobia 35
2.5.1. Nodulation 36
2.5.2. Rhizobium inoculation of legumes 37
2.6. Co-inoculation with rhizobia and plant growth promoting bacteria
containing ACC-deaminase 38
2.7. Composted organic material and biological activity 42
3. Materials and Methods 44
3.1. Isolation of rhizobacteria containing ACC-deaminase and
rhizobia 44
3.2. Testing ability of rhizobacteria to use ACC as sole source of
nitrogen 45
3.3. Screening rhizobacteria and rhizobia for their abilities to
promote growth and nodulation of chickpea 47
3.3.1. Screening of rhizobacteria 48
3.3.1.1. Germination assay 48
3.3.1.2. Jar experiment 48
3.3.2. Screening of rhizobia 49
3.3.2.1. Jar experiment 49
3.3.2.2. Pouch experiment 50
3.4. Screening effective isolates of rhizobacteria and rhizobia for
co-inoculation (Jar and Pouch experiments) 50
3.5. Effect of single and co-inoculation on growth, yield and
Nodulation of chickpea under natural conditions 51
3.5.1. Pot experiments 51
Chapter Title Page
3.5.2. Field experiments 53
3.6. Characterization of rhizobacterial isolates for ACC-deaminase
activity 53
3.7. Classical triple response bioassay 54
3.8. Characterization of rhizobacteria and rhizobia for other traits 55
3.8.1. Phosphate solubilization assay 55
3.8.2. Chitinase activity 55
3.8.3. Siderophore production assay 55
3.8.4. Root colonization assay 56
3.9. Identification of selected isolates 56
3.10. Soil analyses 57
3.10.1. Soil textural class (Mechanical analysis) 57
3.10.2. Saturation percentage (SP) 57
3.10.3. pH of saturated soil paste (pHs) 58
3.10.4. Electrical conductivity (ECe) 58
3.10.5. Organic matter (OM) 58
3.10.6. Total nitrogen 58
3.10.7. Available phosphorus 59
3.10.8. Extractable potassium 59
3.11. Plant analysis 59
3.11.1. NP analysis 59
3.11.1.1. Nitrogen 61
3.11.1.2. Phosphorus 61
3.12. Statistical analysis 62
4.0 Results 63
4.1. Effect of rhizobacterial isolates on growth of chickpea
seedlings (Screening trials) 63
4.1.1. Utilization of ACC as source of N by rhizobacteria 63
4.1.2. Effect of rhizobacteria on germination of chickpea seeds 65
4.1.3. Effect of rhizobacterial isolates on of chickpea seedlings
under axenic conditions (Jar experiment) 65
4.1.3.1. Root growth 65
4.1.3.2. Shoot growth 70
4.2. Effect of rhizobial isolates on growth and nodulation of
chickpea under axenic conditions (Jar experiment) 70
4.2.1. Root growth 70
4.2.2. Shoot growth 72
4.3. Growth pouch experiment 74
4.3.1. Root growth 74
4.3.2. Shoot growth 77
4.4. Screening of effective isolates rhizobia and rhizobacteria for
co-inoculation of chickpea 80
Chapter Title Page
4.4.1. Jar experiment 80
4.4.2.3. Nodulation 83
4.4.2. Growth pouch experiment 83
4.4.2.3. Nodulation 87
4.5. Effect of inoculation and co-inoculation on nodulation, growth
and yield of chickpea under natural conditions 87
4.5.1. Pot experiments 90
4.5.1.1. Plant height 90
4.5.1.2. Fresh biomass 92
4.5.1.3. Number of pods per plant 92
4.5.1.4. Grain yield 94
4.5.1.5. 100-grain weight 94
4.5.1.6. Number of nodules per plant 94
4.5.1.7. Nodule dry weight per plant 96
4.5.1.8. Root length 96
4.5.1.9. Root dry weight 98
4.5.1.10. Nitrogen content in grain 98
4.5.1.11. Nitrogen content in straw 100
4.5.1.12. Phosphorous content in grain 100
4.5.1.13. Phosphorous content in straw 100
4.6. Pot experiment-II (2nd year) 101
4.6.1. Plant height 101
4.6.2. Fresh biomass 103
4.6.3. Number of pods per plant 103
4.6.4. Grain yield 104
4.6.5. 100-grain weight 104
4.6.6. Number of nodules per plant 104
4.6.7. Nodule dry weight per plant 105
4.6.8. Root length 105
4.6.9. Root dry weight 107
4.6.10. Nitrogen content in grain 107
4.6.11. Nitrogen content in straw 108
4.6.12. Phosphorous content in grain 108
4.6.13. Phosphorous content in straw 108
4.7. Field trials 110
4.7.1. First year trials 110
4.7.1.1. First trial: site-I (UAF) 110
4.7.1.1.1. Plant height 111
4.7.1.1.2. Fresh biomass 112
Chapter Title Page
4.7.1.1.3. Number of pods per plant 111
4.7.1.1.4. Grain yield 113
4.7.1.1.5. 100-grain weight 113
4.7.1.1.6. Number of nodules per plant 113
4.7.1.1.7. Nodule dry weight per plant 114
4.7.1.1.8. Root length 114
4.7.1.1.9. Root dry weight 116
4.7.1.1.10. Nitrogen content in grain 116
4.7.1.1.11. Nitrogen contents in straw 118
4.7.1.1.12. Phosphorous content in grain 118
4.7.1.1.13. Phosphorous content in straw 118
4.7.1.2. Field trial-II: Site-II (Sajawal Farm) 120
4.7.1.2.4. Grain yield 122
4.7.1.2.5. 100-grain weight 122
4.7.1.2.8. Root length 125
4.7.1.2.9. Root dry weight 125
4.7.1.2.11. Nitrogen content in straw 126
4.7.2. Second year trials 128
4.7.2.1. First trial: site-I (UAF) 128
4.7.2.1.4. Grain yield 129
4.7.2.1.5. 100-grain weight 131
4.7.2.1.8. Root length 132
4.7.2.1.9. Root of dry weight 132
4.7.2.2. Second trial: site-II (Nawaz Farm, Jhang) 136
Chapter Title Page
4.7.2.2.4. Grain yield 139
4.7.2.2.5. 100-grain weight 139
4.7.2.2.8. Root length 140
4.7.2.2.9. Root of dry weight 140
4.9. Classical “triple” response 145
4.10. Identification and characterization of rhizobacteria and
rhizobial isolates 151
5. Discussion 153
5.1. Screening rhizobacteria and rhizobia for their potential to promote
growth and nodulation of chickpea under axenic conditions 153
5.1.1. Screening rhizobacteria containing ACC-deaminase for growth
promoting activity 153
5.1.2. Screening rhizobia for their ability to promote nodulation and
growth of chickpea 156
5.1.3. Effect co-inoculation on nodulation and growth of chickpea
under axenic conditions 157
5.2. Effect of inoculation and co-inoculation on chickpea
under natural conditions 158
Summary 164
References 170
LIST OF TABLES
Table Title Page
3.1. Bacterial isolates collected from different locations 46
3.2. Physico-chemical characteristics of soils used for field trials 60
4.1. Rhizobacterial isolates showing variable growth (measured as OD)
on the media containing ACC as sole N source 64
4.2. Germination percentage and root elongation of chickpea as affected
by inoculation with rhizobacterial isolates 66
4.3. Effect of rhizobacterial inoculation on root growth of chickpea
seedlings under axenic conditions (Jar experiment) 67
4.4. Effect of rhizobacterial inoculation on shoot growth of
chickpea seedlings under axenic conditions (Jar experiment) 71
4.5. Effect of rhizobial inoculation on root and shoot growth of chickpea
under axenic conditions (Jar experiment) 73
4.6. Effect of rhizobial inoculation on nodulation of chickpea under
axenic conditions (Jar experiment) 75
4.7. Effect of rhizobial inoculation on root and shoot growth of chickpea
under axenic conditions (Growth pouch experiment) 76
4.8. Effect of rhizobial inoculation on nodulation of chickpea under
axenic conditions (Growth pouch experiment) 78
4.9. Effect of co-inoculation with rhizobacteria and rhizobia on root and
shoot growth of chickpea seedlings under axenic conditions
(Jar experiment) 82
4.10. Effect of co-inoculation with rhizobacteria and rhizobia on
nodulation of chickpea seedlings under axenic conditions
(Jar experiment) 84
4.11. Effect of co-inoculation with rhizobacteria and rhizobia on root and
shoot growth of chickpea seedlings under axenic conditions
(Growth pouch experiment) 86
4.12. Effect of co-inoculation with rhizobacteria and rhizobia on
(Growth pouch experiment) 88
4.13. Effect of inoculation and co-inoculation with rhizobia and
rhizobacteria containing ACC-deaminase on growth and yield of
chickpea in presence and absence of P-enriched compost
(1st year pot trial) 91
4.14. Effect of co-inoculation with rhizobia and rhizobacteria containing
ACC-deaminase on nodulation and root growth of chickpea in
Presence and absence of P-enriched compost (1st year pot trial) 95
ACC-deaminase on N and P content in grain and straw of chickpea
in absence and presence of P-enriched compost (1st year pot trial) 99
Table Title Page
chickpea in presence and absence of P-enriched compost
(2nd year pot trial) 102
ACC-deaminase on nodulation and root growth of chickpea in
absence and presence of P-enriched compost (2nd year pot trial) 106
rhizobacteria containing ACC-deaminase on N and P content in
grain and straw of chickpea in absence and presence of P-enriched
compost (2nd year pot trial) 109
chickpea in absence and presence of P-enriched compost
(1st year field trial) 112
rhizobacteria containing ACC-deaminase on nodulation and root
growth of chickpea in absence and presence of P-enriched compost
compost (1st year field trial) 119
compost (1st year field trial) 127
(2nd year field trial) 130
Table Title Page
compost (2nd year field trial) 135
compost (2nd year field trial) 144
4.31. Classical “Triple” response of etiolated pea seedlings at
different concentrations of ACC (mM) 146
4.32. Comparative effect of inoculation with rhizobacteria containing
ACC-deaminase activity and cobalt (CO2+) on etiolated pea
seedlings in the presence of 10 mM ACC 149
4.33. Identification and characterization of selected strains of plant
growth promoting rhizobacteria and rhizobia isolated from chickpea 152
LIST OF FIGURES
Figure Title Page
2.1. Yang cycle and ethylene biosynthesis pathway in plant 11

2.2. Ethylene biosynthesis pathways 13
2.3. Plant ethylene production as a function of time following an
environmental stress 15
4.1. Classical “Triple” response of etiolated pea seedlings at
different concentrations of ACC (mM) 147
4.2. Comparative efficacy of inoculation with rhizobacteria containing
ACC-deaminase activity and cobalt (Co2+) on etiolated chickpea
seedlings in the presence of 10 mM ACC concentration 150
Co-inoculation of Chickpea Abstract
POTENTIAL OF RHIZOBIA AND FREE LIVING RHIZOBACTERIA CONTAINING

ACC-DEAMINASE FOR IMPROVING NODULATION AND YIELD OF
CHICKPEA (Cicer arietinum L.)
ABSTRACT
There is a beneficial association (symbiotic) between rhizobia and leguminous plants, which
consequences in the development of nodules (N-fixing sites in plant roots) on the roots of legumes.
Besides to other features, Phytohormones have an imperative function for increasing number of
nodules. Some free living rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase
(ACCD) enzyme activity promotes nodulation in plant by regulating endogenous biosynthesis
ethylene in roots. Thus, co-inoculation of rhizobia and rhizobacteria containing ACC-deaminase
were evaluated along with P-enriched compost to improve nodulation and yield of chickpea. A
series of studies were conducted under axenic, pot (wire house) and natural conditions to evaluate
the effectiveness of co-inoculation plus enriched compost on nodulation in chickpea. A number of
isolates of free living rhizobacteria and rhizobia were isolated from soil and nodules samples of
chickpea plants by using minimal salt medium with ACC as sole source of nitrogen and Yeast
Extract Mannitol (YEM) medium respectively. The isolated rhizobacteria were screened for their
ability. The collected rhizobial strains were screen for their ability to cause nodulation and PGPR
strains for their growth promoting activity under gnotobiotic conditions. All possible combinations
of the selected rhizobial and PGPR isolates were tested under gnotobiotic conditions for improving
nodulation to screen the best combinations. The role of the selected combinations and rhizobial and
PGPR strains included in the combinations, was evaluated for improving nodulation and growth of
chickpea through co-inoculation in pot and field trials. The selected rhizobial as well as plant
growth promoting rhizobacterial strains containing ACC-deaminase were characterized for plant
growth promoting attributes. It was observed that inoculation with rhizobia or PGPR containing
ACC-deaminase alone improved nodulation/growth of chickpea over control under natural
conditions. Moreover, co-inoculation with rhizobia and ACC-deaminase containing plant growth
promoting rhizobacteria further improved nodulation and growth of chickpea as compared to single-
strain-inoculation. In a classical triple response study, it was also observed that the selected PGPR
containing ACC-deaminase diluted the effects of exogenously applied ACC.
1
Co-inoculation of Chickpea Introduction
INTRODUCTION
The plant rhizosphere is a remarkable ecological environment where numerous
microorganisms colonize in, on, and around the roots of growing plants. The diverse
groups of bacteria are associated with the root systems of all higher plants (Khalid et al.,
2006a). These bacteria are considered as efficient microbial competitors in the root zone,
and the net effect of plant-microbe associations on plant growth could be positive, neutral
or negative (Kennedy, 2005; Nadeem et al., 2006; Patel et al., 2008; Khalid et al., 2009).
Such bacteria in close association with roots and capable of stimulating plant growth by
any mechanism (s) of action are referred to as plant growth promoting rhizobacteria
(PGPR) (Kloepper et al., 1986; Kijne et al., 1992; Arshad and Frankenberger, 1998;
Salamone, 2000; Asghar et al., 2004; Khalid et al., 2004a; Zahir et al., 2004; Kremer,
2006; Bohm et al., 2007). They affect plant growth and development directly or indirectly
either by releasing plant growth regulators (PGRs) or other biologically active
substances, altering endogenous levels of PGRs, enhancing availability and uptake of
nutrients through fixation and mobilization, reducing harmful effects of pathogenic
microorganisms on plants and/or by employing multiple mechanisms of action for plant
growth promotion (Kloepper et al., 1991, 1992; John, 2001; Nadeem et al., 2006;
Mansoor et al., 2007; Patel et al., 2008). A diverse array of bacteria including species of
Rhizobium, Bradyrhizobium, Pseudomonas, Azospirillum, Azotobacter, Bacillus,
Klebsiella, Enterobacter, Xanthomonas, Serratia and many others, have been shown to
1
facilitate plant growth by various mechanisms (Lugtenberg et al., 2002; Raj et al., 2003;
Guo et al., 2004; Khan, 2005; Greenberg et al., 2006a, b; Akhtar and Siddiqui, 2009).
Biological N2 fixation by rhizobia is one of the widely studied mechanisms by
which plants benefit from the association of interacting partners (Sindhu et al., 2002; Bai
et al., 2003; Baudoin et al., 2003; Orhan et al., 2006; Figueiredo et al., 2008; Afzal and
Bano, 2008; Khalequzaman and Hossain, 2008). The bacteria benefit the plants by fixing
N2 in exchange for fixed carbon either provided directly to the bacteria or indirectly
releasing carbon as root exudates. A range of bacteria participates in interactions with
different plants, significantly increasing their vegetative growth and grain yields.
However, obtaining maximum benefits from microbial inoculants under field conditions
requires a systematic strategy to fully utilize all beneficial factors for increasing crop
yields.
In the recent years, the PGPR have received worldwide importance for
agricultural benefits as they are the potential tools for sustainable agriculture and have
shown significant increases in growth and yield of agricultural crops both under
greenhouse and field conditions (Kennedy, 2004; Khalid et al., 2004a, 2006b; Kumar et
al., 2007; Gravel et al., 2007; Zhuang et al., 2007; Arshad et al., 2008; Banchio et al.,
2008; Contesto et al., 2008; Figueiredo et al., 2008; Mubeen et al., 2008; Naiman et al.,
2008). Beside promoting plant growth, PGPR ensure the availability of nutrients and
enhance the nutrient use efficiency, and mitigate biotic and abiotic stresses (Huang et al.,
2004, 2005; Khan et al., 2005; Nomura et al., 2005; Kausar and Shahzad, 2006; Arshad et
al., 2007; Saleem et al., 2007; Ahmad et al., 2008; Amor et al., 2008; Dell’Amico et al.,
2008; Kohler et al., 2008; Lebeau et al., 2008; Masoud and Abbas, 2008).
2
Recently, some PGPR have shown great potential to enhance plant growth by
altering the synthesis of endogenous phytohormones through the production of specific
enzymes. Among these enzymes, bacterial 1-aminocyclopropane-1-carboxylate (ACC)
deaminase plays a significant role in the regulation of a plant hormone, ethylene, and
thus, modify the growth and development of plants (Primose, 1979; Mattoo and Suttle,
1991; Noel, 1992; Arshad and Frankenberger, 2002; Glick, 2005; Khalid et al., 2009).
Ethylene (C2H4) is a potent plant hormone and its presence at extremely low
concentration could have tremendous impact on plant growth (Primose, 1979; Arshad and
Frankenberger, 1990a, b; Khalid et al., 2006b). Ethylene is required for seed germination
by many plant species, and the rate of ethylene production increases during germination
and seedling growth (Abeles et al., 1992). Low levels of ethylene (as low as 10 µg L-1)
have been found to enhance root initiation and growth while the higher levels (as high as
25 µg L-1) may lead to inhibition of the root growth (Mattoo and Suttle, 1991; Ma et al.,
1998). Ethylene in higher concentration has also been known as an inhibitor of
nodulation in various legumes, including both those that produce indeterminate nodules
and those that produce determinate nodules (Nukui et al., 2000; Arshad and
Frankenberger, 2002).
Any factor/stimulus which causes a change in ethylene levels of a plant either by
changing the ethylene synthesis endogenously or in the close vicinity of the root can
modify growth (Arshad and Frankenberger, 2002; Khalid et al., 2006b; Shaharoona et al.,
2007a). Seed or root inoculation with PGPR containing ACC-deaminase decreases
endogenous ethylene levels and promotes root growth (Glick et al., 1998; Ghosh et al.,
2003; Shaharoona et al., 2006a, b, 2007a, b, 2008). Lowering of endogenous levels of
ethylene occurs because of hydrolyses of ACC (an immediate precursor of ethylene) in
3
the rhizosphere by the ACC-deaminase of a bacterium. Furthermore, the ACC from seeds
or plant roots along with other small molecules exudes in the rhizosphere and thus the
endogenous production of ethylene reduces in response to decreased ACC levels within
the plant (Penrose and Glick, 2001). Bacterial strains with ACC-deaminase can at least
partially eliminate the stress-induced ethylene-mediated negative impact on plants by
converting the germinating seed/root’s ACC into α-ketobutyrate and ammonia (Glick et
al., 1998). Numerous species of Gram negative and Gram positive bacteria have been
reported to produce ACC-deaminase (Belimov et al., 2005; Pandey et al., 2005; Sessitsch
et al., 2005; Blaha et al., 2006; Madhaiyan et al., 2006; Stiens et al., 2006; Shaharoona et
al., 2006a, b, 2007a, b, 2008; Shahzad et al., 2008).
Plants grown under natural (soil) conditions are, generally, exposed to numerous
environmental stresses and more ethylene is produced by the plant in response to any
kind of stress (Jacobson et al., 1994; Glick et al., 1998; Grichko and Glick, 2001a, b;
Arshad and Frankenberger, 2002; Mayak et al., 2004b; Reed and Glick, 2005; Saleem et
al., 2007). Since ACC-deaminase lowers the level of C2H4 in plants, this activity is
important for normal plant development because it protects plants from the deleterious
effects of environmental stresses (Hontzeas et al., 2004a, b; Reed and Glick, 2005). It is
very likely that the use of rhizobacteria with ACC-deaminase could stimulate plant
growth by promoting either proliferation of primary roots or lateral roots or by increasing
nodule formation through their ACC-deaminase activity. Studies have shown that
chemical inhibitors of ethylene synthesis (aminoethoxyvinylglycine and cobalt) and
action (silver) promoted nodulation of various legumes (Zaat et al., 1989; Ligero et al.,
1991; Guinel and LaRue, 1992; Penrose et al., 2001; Grichko and Glick, 2001a; Dodd et
al., 2004). Recently, Shaharoona et al. (2007a) reported that inoculation with
4
Psedumonas putida containing ACC-deaminase diluted the ACC-imposed effect on
etiolated pea seedlings most likely by lowering ethylene through ACC-deaminase activity
while the inoculation with Acinetobacter calcoaceticus or Pseudomonas fluorescens in
the presence of L-MET caused a stronger classical “triple” response in etiolated pea
seedlings most likely by producing ethylene from L-MET. The authors conluded that
rhizobacteria with ACC-deaminase can act as biological inhibitor like the chemical
inhibitor Co2+, which inhibits the activity of ACC-oxidase and prevents the conversion of
ACC into ethylene. Thus, plants grown in association with such bacteria should have
better roots and shoots, and be more resistant to growth inhibition by a variety of ethylene
inducing stresses (Glick et al., 1998; Mayak et al., 1999; Shaharoona et al., 2006a, b;
Contesto et al., 2008).
Root growth promotion is one of the major markers by which the beneficial effect
of PGPR is measured. Stimulation in root growth and biomass production of different
plant species by inoculation with PGPR conatining ACC-deaminase activity has been
well documented (Belimove et al., 2001, 2005; Van Loon and Glick, 2004; Safronova et
al., 2006; Nadeem et al., 2006; Arshad et al., 2008; Patel et al., 2008). Rapid growth and
establishment of roots, whether by elongation of primary roots, is advantageous for
young seedlings as this increases the ability of the plant to obtain more water and
nutrients from the surrounded environment.
Chickpea is one of the most important dry land field crops. Its per hectare yield in
Pakistan is much lower than its yield potential, because of suboptimal growth conditions
faced by this crop during different growth stages. It is very likely that root growth and
nodulation could be affected due to production of higher levels of ethylene in roots under
field conditions. Since the bacterial enzyme ACC-deaminase lowers the level of ethylene
5
in roots, inoculation with PGPR containing ACC-deaminase could be very effective in
improving growth and nodulation of chickpea.
The use of rhizobacteria containing ACC-demainase in co-inoculation with
rhizobia could be more beneficial due to their multiple effects on plant through different
growth enhancing mechanisms. Furthermore inoculation benfitscan be enhanced by
maintaining high densities of effective bacteria around the roots. Application of humus
like organic material in the soil could provide a niche that might have positive effect on
plant growth most likley by supporting activities of PGPR in the rhizosphere of a plant
throughout its life cycle (Pooran et al., 2002; Baipai et al., 2002; Cheuk et al., 2003;
Hameeda et al., 2006). Thus, integrated use of PGPR, rhizobia and composted organic
waste could be highly effective in improving yield and nodulation of chickpea crop.
Keeping in view the above discussion, the present study was designed with the
following objectives:
Isolation and screening of effective rhizobia and PGPR with ACC-deaminase
activity for promoting growth and nodulation of chickpea under axenic conditions
Testing potential of selected rhizobia and PGPR strains alone as well as in
combination (co-inoculation) for improving growth, yield and nodulation of
chickpea under pot and field conditions
Evaluating effectiveness of selected rhizobia and PGPR inoculants for further
improving yield and nodulation in chickpea grown in the soil amended with P-
enriched compost.
Identification and characterization of selected rhizobia and PGPR containing
ACC-deaminase
6
Co-inoculation of Chickpea Review of Literature
REVIEW OF LITERATURE
Crops which belong to family Leguminosae are generally referred to as legumes or
pulses. These crops in South Asian countries are mainly cultivated in barani or rainfed
areas by small and medium scale farmers. In most of the areas, yield of legumes is low
while demand for these crops has increased dramatically due to rapid increase in
population in the last few decades. In addition to the abiotic stresses such as low rainfall
and high temperature, the crops are attacked by a number of diseases, insects and weeds
causing major yield losses.
2.1. Chickpea
Chickpea (Cicer arietinum L.) is one of the important legume crops, and being
rich in protein content its seeds are used as a vegetable and dry bean. In fact, it a
multipurpose crop used in human diets, animal fodder and industrial purposes. The world
production of chickpea is roughly three times that of lentil and world consumption is
second only to dry bean among pulses crops marketed as a human food (ICARDA, 1993).
During 2006-2007, worldwide production of chickpea was 8.24 million tons from
an area of 9.4 million hectares and average production of 0.77 t ha-1. While, contribution
of Asia was 7.36 million tons (89.4%) (http://faostat.fao.org/). However, Australia,
Canada, Ethiopia, India, Iran, Mexico, Myanmar, Pakistan and Turkey collectively
contribute 93.1% of the global chickpea production. But North and Central America and
Oceania together share only 6.2% of the world chickpea production.
7
Chickpea can thrive under good moisture conditions with daytime temperature
between 21 to 29 oC and night time temperature near 20 oC (Siddique et al., 1999;
Srinivasan et al., 1998, 1999). Length of crop maturity depends on available heat and
moisture, but is usually in the range of 95-110 days depending on type of chickpea
species (Hamblin, 1985; Wery et al., 1994; Croser et al., 2003). Due to long tap root
system, chickpea is relatively drought tolerant. They are not well adapted to high
moisture areas, saline soil, soils which are slow to warm in spring and wet or waterlogged
soils (Russell and Goss, 1974; Smith et al., 1989; Petrie and Hall, 1992; Singh et al., 1998;
Pardo et al., 2000). Chickpea production works well in rotation with cereal grains. As a
member of the family Leguminosae, chickpea can fix nitrogen from the atmosphere and
nitrogen fertilizers are usually not required. To improve the nitrogen fixation ability, seed
should be inoculated with the bacterial strains of nitrogen fixing inoculants of chickpea.
2.2. Causes of low yield of chickpea in Pakistan
There are several factors which contribute to its low productivity in Pakistan.
These include exhaustion of soil nutrients, shortage of water as well as poor quality
ground water, buildup of pathogenic fungal diseases, temperature stress during
reproductive stage, a bad soil structure, excessive moisture, ineffectiveness of Rhizobium
species and severe drought stress at initial stages of growth (Singh et al., 1998;
Yamaguchi and Blumwald, 2005; Toker et al., 2007a, b; Canci and Toker, 2009).
Ethylene is produced by almost all plants and it mediates a range of different plant
responses and development stages (Arshad and Frankenberger, 2002; Akhtar et al., 2005;
Khalid et al., 2006b; Saleem et al., 2007). When plants are exposed to the conditions that
threaten their ability to survive under biotic and abiotic stresses, the same mechanism that
8
produces ethylene for normal development of plant instead functions to produce “stress”
ethylene. When “stress” ethylene synthesis increases, it causes stress senescence response
in the plant that may lead to physiological changes in those cells that are at or near the
site of stress. Production of excessive amounts of ethylene in plants causes inhibition of
seed germination and root elongation (Suzuki and Taylorson, 1981; Nojavan-Asghari and
Ishizava, 1998; Matilla, 2000; Mergemann and Sauter, 2000; Armstrong and Drew, 2002;
Beaudoin et al., 2002; Ghaddemain et al., 2002; Bialeca and Kepczynski, 2003b;
Norastehnia et al., 2007).
It is very likely that any check on the accelerated ethylene production in plants
exposed to environmental and biological stresses could be helpful in minimizing the
negative impact of stress on plant growth and development (Arshad and Frankenberger,
2002; Balaota et al., 2004; Owino et al., 2006; Arshad et al., 2008). It is hypothesized that
the co-inoculation of rhizobia with plant growth promoting bacteria carrying the trait of
ACC-deaminase may enhance plant growth by regulating the endogenous level of ACC
by lowering ethylene in plant under suboptimal growth conditions.
2.3. Ethylene biosynthesis and plant growth
Ethylene is a simplest unsaturated hydrocarbon which regulates many metabolic
and developmental processes within plant body (Arshad and Frankenberger, 2002;
Belimove et al., 2002). The history of its discovery as a plant hormone was documented
by Abeles et al. (1992). Chemical evidence that plants generate ethylene was made
available by Gane (1934), who studied the gases release by 60 lbs of ripening apples.
Therefore, a stage was set to investigate the function of ethylene as an endogenous single
molecule in plants.
9
Ethylene is generated by most of the plant tissues. Biosynthesis of this hormone
begins with the compound S-adenosylmethionine (SAM) that is also required as the
precursor in many other pathways and is, therefore, abundant within plant tissues. The
ethylene pathways (Figure 2.1), along with the Yang Cycle (Yang and Hoffman, 1984)
initiate with the enzyme ACC-synthase that converts SAM to 1-amonocyclopropane-1-
carboxylic acid (ACC) and 5V methylthioadenosine (MTA), which is recycled to L-
methionine. This permits levels of L-methionine to remain relatively unchanged even
during high rates of ethylene production (Abeles et al., 1992). It has been considered to
be the main step in the ethylene biosynthesis pathway, since the extremely labile ACC-
synthase enzyme has been shown to be rate limiting and to rise proportionally to ethylene
within the tissue of certain plants (Abeles et al., 1992). The next step is the conversion of
ACC to ethylene by ACC-oxidase, which is present in most tissues at very low levels.
Other than in ripening fruit, it appears that both ACC-synthase and ACC-oxidase are
inducible enzymes. Thus, defining the rate-limiting step in ethylene biosynthesis may be
somewhat complex in that it may vary from one plant to another, from one tissue to
another and from one actuating signal to another.
A huge increase in ethylene production is activated by the exposure to exogenous
ethylene with the activation of ACC-synthase and/or ACC-oxidase (Suttle and Kenede,
1980; Liu et al., 1985; Inaba and Nakamura, 1998; Wang et al., 2002). Stimulation of
ethylene by IAA, also occurs in etiolated pea seedlings, via a rapid increase in the
buildup of ACC-oxidase a transcript proceeded by that of ACC-synthase (Peck and
Kenede, 1995).
10
HPO2- 4 HCOOH-
+NH3
MTR – 1- P KMBA |
PPi + Pi RCHOO-
MTR-kinase O
║
RCOO-
ATP
YANG CYCLE L-methionine
MTR
Adenine SAM -synthase ATP
MTA-nuclosidase SAM
MTA
PPi + Pi
ACC-Synthase
ACC N-malonyl transferase γ- glutmyl transpeptidase
MACC GACC
ACC
Malonyl Co A γ- glutmyl moiety of glutathione
O2 ACC
oxidase
HCNN CO2
ETHYLENE
Figure 2.1: Yang cycle and ethylene biosynthesis pathway in plant (Yang and Hoffman, 1984)
ACC : 1-aminocyclopropane-1-carboxylic acid
GACC : 1-(glutamyl)-ACC
KMBA : 2-keto-4-methylthiobytyric acid
MACC : 1-(malonyl)-ACC
MTA : 5Vmethylthioadenosine;
MTR : 5V-methylthioribose
MTR-1-P : 5V-methylthioribose-1-phosphate
SAM : S-adenosyl-methionine.
11
It is well known that ethylene action in plants resulting from huge endogenous and
exogenous ethylene production; encourage certain stress mediated growth response.
The effects of ethylene may be inhibitory or stimulatory depending upon its
concentration, the nature of physiological process and growth phase of plant. Its presence
in extremely low concentration could have tremendous effect on plant growth (Arshad
and Frankenberger, 2002; Khalid et al., 2006b). It is required for seed germination
(Abeles et al., 1992), to enhance root initiation and growth (Gosh et al., 2003), however,
when present in high levels it could be damaging for plants, leading to epinasty, shorter
roots and premature senescence (Holguin and Glick, 2001; Ma et al., 1998; Shaharoona et
al., 2007a).
2.3.1. Ethylene biosynthesis under stress conditions
Ethylene is also known as “stress” hormone and its accelerated production is
associated with both biotic and abiotic stresses (Morgan and Drew, 1997; Arshad and
Frankenberger, 2002; Arshad et al., 2008). The term “stress” ethylene was coined by
Abeles (1973). When, plants are confronted to conditions that affect their ability to
survive, the same mechanism that produces ethylene for normal development functions to
produce “stress” ethylene (Figure 2.2). Since different plants respond differently to stress
factors and also have a range of sensitivities to ethylene, it is difficult to exactly know the
the functioning of stress ethylene. Furthermore, there is a complex type of relations
between ethylene and other plant hormones that varies greatly. Nevertheless, plants are
exposed to various types of stresses invariably show increased ethylene levels, leading to
increased damage to the plant (Hyodo, 1991).
12
Spd/Spm
Inhibited by : DFMO
Spd/Spm Synthase
DFMA
Orinthine
Polyaminase
ODC
Putrescine Arginine
dSAM ADC
SAMDC
Lignification/Betaine
STRESS Cell damage
L-Methionine SAMS Ethylene

ACCS ACCO
SAMS
Inhibited by
Induced by Fruit ripening AVG, AOA,
IAA; Ethylene Rhizobitoxine Induced by Inhibited by
Ca-cytokinin Ripening Anaerobiosis,
Physical wounding wounding Uncouplers cobalt/S
Chilling injury, Drought stress, Temperature 350C
Anaerobiosis, Flooding Free ROS scavengers
Figure 2.2: Ethylene biosynthesis pathways:
ACCO : 1-aminocyclopropane-1-carboxylic acid oxidase

ACCS : 1-aminocyclopropane-1-carboxylic acid synthase
ADC : Arginine decarboxylase
AOA : Aminooxyacetic acid
AVG : Aminoethoxyvinylglycine
DFMA : DL-difluorometyl arginine
DFMO : DL-difluorometyl ornithine
ODC : Ornithine decarboxylase
SA : Salicylic acid
SAM : S-adenosyl-L-methionine
SAMDC : SAM decarboxylase
SAMS : SAM syhthase
Spd : Spermidine
Spm : Spermine
13
Contradictory effects of ethylene on plants have also been reported. Stress
ethylene may alleviate or intensify some of the effects of pathogen infection, depending
upon the plant species, its age and nature of the pathogen (Abeles et al., 1992; Arshad
and Frankenberger, 2002; Van Loon and Glick, 2004). A model that explains these
contradictory effects of stress ethylene on plants has been proposed (Stearns and Glick,
2003; Pierik et al., 2006; van Loon et al., 2006). In one description of this model, there is
an initial small peak of ethylene close in time, usually a few hours after, to the onset of
the stress and then a second much larger peak some time later, usually one to three days
(Figure 2.3). The first peak is only a small fraction of the magnitude of the second peak
and is thought to initiate a prospective reaction by the plant, such as record of
pathogenesis associated genes and obtained resistance (Ciardi et al., 2000; Van Loon and
Glick, 2004). The first small wave of ethylene production is thought to consume the
existing pool of ACC within plant tissues (Robison et al., 2001a). While second ethylene
peak is so large that processes such as senescence, chlorosis and abscission are initiated,
the overall effect of which is generally inhibitory to plant survival. Thus, following a
severe infection by pathogens, a large portion of the damage that occurs to a plant is due
to autocatalytic ethylene production and not from direct action of pathogen (van Loon,
1984). In this regard, not only can exogenous ethylene increase the severity of a pathogen
infection but as well, inhibitors of ethylene production or its action can decrease the
severity of a bacterial or fungal infection. The second peak of ethylene synthesis occurs
as a result of increased transcription of ACC-synthase gene activated by developmental
and environmental cues (Yang and Hoffman, 1984). Increased levels of ethylene
14
Deleterious peak
Ethylene
Beneficial peak
Time
Deleterious peak
Ethylene
Beneficial peak
Time
Figure 2.3: Plant ethylene production as a function of time following an environmental stress.
A : In the absence of any exogenous bacteria.

B : In the presence of an ACC-deaminase producing plant growth promoting
bacterium.
In both cases, there is an initial small peak of ethylene that is thought to activate transcription of plant
defense genes, which is often difficult to detect, followed some time later by a much larger ethylene peak
that can cause adverse responses in the plant. The amount of ethylene produced in response to an
environmental stress is related to the plant age as well as the nature and severity of the stress
15
production in plants on exposure to various types of stresses including heat, chilling,
pathogen, wounds, infection and nutritional stresses have been reported (Hyodo, 1991;
Stearns and Glick, 2003).
2.3.1.1. High temperature
Ethylene accumulation in soil is increased with rising temperature in the
mesophillic range. Otani and Ae (1993) found positive correlation between ethylene level
and soil temperature. Similarly, Sextone and Mains (1990) reported much more ethylene
evolution from different horizons of Spruce Mountains and Gaudinner Knob soils
incubated at 65 oC than 55 oC. Ethylene production was 2.3 to 3.8 fold greater at 50 oC
than at ambient (22 oC) conditions (Frankenberger and Phelen, 1985). Similarly,
increased ethylene evolution was observed in two soils when heated up to 80 oC (Smith
and Cook, 1974). Smith and Dowdwell (1974) documented that concentration of ethylene
increased logarithmically with soil temperature, nearly 20 folds over the range of 4 to 11
o
C. Smith and Restall (1971) showed that temperature below 10 oC resulted in
substantially lower rate of ethylene evolution and higher temperature (30 to 35 oC)
prompted its increase up to 2.0 fold. The increase in ethylene content of soil with rising
temperature might be due to increased enzymatic activity responsible for biological
ethylene production or due to thermal decomposition of organic matter and adsorbed of
ethylene.
Mitcham and Mc Donaled (1993) reported an increase in ethylene production in
plants in response to heat. Antunes and Sfakiotakis (2000) observed a pronounced
stimulatory effect of heat on ethylene biosynthesis. Likewise, Ievinsh et al. (2000) found
that temperature is responsible for an increase in ACC-oxidase activity, resulting in
16
increases in ethylene production of Pinus sylvestris needles. Fl-abd et al. (1994) recorded
similar results for an increase in ethylene production in tomato plants as the temperature
was increased. Apelbaum et al. (1981) observed rapid conversion of ACC to ethylene
with increased temperature. While, Wang and Adams (1982) reported that chilling
stimulated ethylene production in cucumber fruits. They observed low ACC level, ACC-
synthase activity and ethylene production rates in the cucumber fruits when held at
chilling temperature while increased rapidly upon transfer to warm temperature.
2.3.1.2. Salt toxicity and drought
Salt concentration can negatively affect the growth and yield of chickpea. Many
hypotheses have been described the harmful effect of salt on biological nitrogen fixation
in leguminous plants, lessen supply of photosynthates to nodule of plants (Bekki et al.,
1987; Georgiev and Atkins, 1993), reduced respiratory substrates supply to bacteroids
(Delgado et al., 1994) and modifications in the diffusion barrier of oxygen (Serraj et al.,
1994). In fact, salinity can badly change the photosynthetic carbon metabolism,
chlorophyll content of leaf, in addition to photosynthetic efficiency (Seeman and
Critchley, 1985; Sharkey et al., 1985). In the hot and dry areas of the world, the soils are
normally saline with low agricultural potential. In these areas most crops are grown under
irrigation, and to exacerbate the problem, inadequate irrigation management leads to
secondary salinization that influences 20% of irrigated land all over the world (Mayak et
al., 2004b).
El- Iklil et al. (2002) conducted an experiment to study the effect of salt stress on
epinasty in relation to ethylene synthesis and water relation in tomato pant. Three
Lycopeersicon esculentum cultivars were subjected to four different levels of salt stress at
17
the roots 0, 50, 100 and 200 mM NaCl. Epinasty improved with increasing salinity levels
that depending upon genotype and salt stress. Relatively ethylene synthesis by tomato
petioles also enhanced with concentration of salt stress, genotype, leaf age and salt
tolerant varieties showed less epinasty and lower comparative ethylene synthesis. In this
study three weeks old plants were treated with 0, 25, 50 or 100 mM NaCl and studied
role of ethylene in the responses of peas to saline tress. Hence diametric growth,
measured in 5 days old seedlings of peas. Results exhibited increase in ethylene
production with increasing concentration of NaCl (Fl-Abd et al., 1994).
In recent years, considerable awareness has been directed towards genetically
engineering plants to be more salt tolerant, with reasonable success (Apse et al., 1999). A
substitutive approach to overcoming some of the problems concerned with growing
plants in saline soils involves employing an ACC-deaminase activity containing bacteria
(Mayak et al., 2004b). This strain dramatically lowered the level of ethylene and prevents
inhibition of growth of tomato plants grown in the presence of high concentration of salts
(Mayak et al., 2004b). The same bacterial strain lowered the ethylene level and
significantly reduced the growth inhibition of peppers and tomatoes from drought stress
(Mayak et al., 2004a).
Salts stress enhance ethylene synthesis which in most of the cases acts as stress
hormone in plant (Feng and Barker, 1992; O’ Donell et al., 1996). Current studies reveal
the potential of plants inoculated with plant growth promoting rhizobactertia containing
ACC-deaminase thrive through the salinity menace while representing normal growth
pattern (Mayak et al., 2004a). They investigated that plant growth promoting
rhizobactertia containing ACC-deaminase activity significantly promoted the fresh and
18
dry weight of tomato seedlings grown up to 172 mM NaCl salt. Bacteria reduced the
biosynthesis of ethylene by tomato seedlings, which were stimulated when seedlings
were bared to rising salt concentrations, but did not decrease the content of sodium. But
slightly it increased the uptake of P and K, which might have involved in part to
activation of processes that played role in the alleviation of the salt effects. Bacteria also
improved the water use efficiency in saline conditions and facilitated in alleviating the
salt inhibition of photosynthesis. Similarly, Nadeem et al. (2006) reported that
rhizobacteria with ACC-deaminase activity lower induced stress ethylene levels and may
be useful to promote plant growth under salt stress conditions. For this purpose, 20 strains
of rhizobacteria isolated from salt affected soils and were screened for their growth
promoting activity and ACC-deaminase activity under axenic conditions at 6 dS m-1.
Three rhizobacterial strains S5, S15 and S20 (out of twenty) promoted maximum plant
growth under axenic conditions were selected for pot study at 0, 5, 10 dS m-1. Results of
pot trial revealed that with increase in salinity levels reduced the growth of maize
seedlings. While, inoculation seeds with these rhizobacterial strains showed better at all
salinity levels and the rhizobacterial strains S20 at 5 dS m-1 significantly promoted root
and shoot length, root fresh and dry weights, and shoot fresh and dry weights up to 56
and 62, 51 and 71, 52 and 61%, respectively, over untreated control. At 10 dS m-1
increase was up to 120 and 63, 52 and 71, 59 and 118%, respectively, over control.
Similarly, due to inoculation with S20 strain maximum increase in chlorophyll a, b and
carotenoid contents of fresh leaves was recorded up to 86% at 5 dS m-1 and 84% at 10 dS
m-1 over its respective control. Results revealed negative effects of stress induced
19
ethylene could be partially reduced through inoculation with rhizobacteria containing
ACC-deaminase.
Dodd et al. (2004) conducted a study to evaluate the effect of inoculation with
Variovorax paradoxus 5C-2 having ACC-deaminase activity on growth of peas at two
different levels of soil moisture. They documented that the bacteria promoted biomass of
root from 20 to 25% irrespective of soil moisture levels, and total plant biomass was also
improved up to 25% in plants grown in drying soil. They also measured the effect of
inoculation with AVG (inhibitor of ethylene). While both treatments were compared with
control (irrigated with distilled water). However, both bacterial and AVG treatments
promoted root growth at both soil moisture regimes while, shoot and total biomass was
only improved as compared to control plants in case of bacterial inoculation under dry
soil conditions. Plant responses to bacteria were qualitatively similar to AVG treatment,
recommending that bacterial ACC-deaminase was concerned in plant-bacterium
interaction and observed the effect of Variovorax paradoxus 5C-2 on pea plants were
mediate by ethylene.
2.3.1.3. Metals and organic contaminants
Most plants synthesize growth inhibitory levels of stress ethylene in the presence
of high level of metals and also become severely iron depleted. This is readily remediate
in laboratory by adding siderophores and ACC-deaminase producing rhizobacteria which
can also help the plant to overcome many of the effects of high levels of metal (Burd et
al., 2000; Reed and Glick, 2005). Similarly, transgenic plants that express a bacterial
ACC-deaminase gene under the control of a root specific promoter are more resistant to
the toxic effects of metals than non-transformed plants (Nie et al., 2002; Stearns et al.,
20
2005; Li et al., 2006).
Field experiments aimed to facilitate the growth of plant in metal contaminated
soils so that plant can take up and concentrate the metal (i.e. metal phytoremediation/or
phytoaccumulation) are more complex than laboratory experiment. In the field, both
ACC-deaminase producing plant growth promoting bacteria and transgenic plants that
express a bacterial ACC-deaminase gene under the control of a root specific promoter
grow better than non-transformed and uninoculated plants. Although many metal
contaminants are present at high levels in the field, in this environment they are generally
not especially bio-available so that only a small fraction of the metals are taken up by the
plants (Farwell et al., 2007).
Considerable success has been attained in the phytoremediation of organic
contaminants such as oil spills, polycyclic aromatic hydrocarbons and polycyclic
biphenyls such that this technology is organized for commercialization. Many varieties of
plants and trees can take up and degrade some organic compounds; but, large molecules
are less water soluble and more difficult to degrade, often requiring degradative bacteria
as well as plant roots for their breakdown. While the degradative bacterial population in
the bulk soil is insufficient to efficiently breakdown complex organic molecules, the
bacterial in the rhizosphere is typically 100-1000 times greater than in bulk soil so that
most of the degradation of environmental organic contaminants occurs in the rhizosphere
of plant.
Despite the fact that many plants, together with rhizospheric degradative bacteria,
can readily degrade many organic pollutants; most of these compounds are exhibited
inhibitory effect on plant growth. Not astonishingly, significant part of this growth
21
inhibition is a consequence of the production of stress ethylene by plant. Therefore,
treatment of plant seeds or roots with ACC-deaminase producing growth promoting
bacteria relieves much of this growth inhibition, allowing the plant to grow to near
normal size and degradation of contaminants to proceed at a much faster rate (Huang et
al., 2005; Greenberg et al., 2006a, b).
2.3.1.4. Soil compaction
It increases both soil strength and reduces availability of nitrogen and both these
factors can increase ethylene production (Lege et al., 1997; Hussain et al., 1999). A
number of studies have shown that ethylene might be involved in the inhibitory effects of
compacted soil on root growth and development (Simojoki et al., 2001). In case of soil
compaction only roots face the stress conditions. In response to mechanical stress, it is
known that roots showed increase of ACC and consequently ethylene, and that ethylene
is a cause of root growth inhibition (Sarquis et al., 1991).
2.3.1.5. Excessive moisture
Intensive rainfall for long period often causes inadequate soil oxygen supply in
the rhizosphere for microbial and root respiration. This condition is more encouraging the
ethylene production due to increase stability and entrapment of ethylene in water (140
ppm, 25 oC, in water) leading to great persistence and less degradation (Drew, 1992).
Higher ethylene levels results in certain morphological adaptation in plant root
architecture, such as inhibition of more root elongation and adventitious rooting (Visser
et al., 1996). In an experiment, chickpea plants were exposed to excessive moisture
stress. As a result, its lower leaves became chlorotic, floral development ceased and wide
spread leaf abscission occurred. Similar, physiological responses were found when
22
chickpea plants were exposed to exogenous application of ethylene as Ethephon (Cowie
et al., 1996). Similarly, high concentration of ethylene was observed in a soil amended
with its precursor CaC2 in the presence of high moisture levels (Kashif, 2008).
2.3.1.6. Pathogen infection
Synthesis of plant ethylene is often drastically increased during infection by
pathogens (Frankenberger and Arshad, 1995). Ethylene acts as a messenger in plant-
microbe interaction (Ton et al., 2002). This increased stress ethylene during pathogenesis
may play a role in disease symptoms development and in weakening of endogenous
resistance (Yang and Hoffman et al., 1984; Abeles et al., 1992; Lund et al., 1989; Gupta
et al., 2001). All known type of disease promoted ethylene synthesis caused by bacteria,
fungi, nematodes and viruses. Increase ethylene synthesis is generally associated with
starting of tissues necrosis. Fungal infected tissues of plant exhibit peak production of
ethylene. The rate of this increased ethylene synthesis is mostly correlated with the
amount of tissue damage (Herbad and Shain, 1988; Spanu and Boller, 1989).
Infection increased ethylene stress was observed in caster bean leaves when
infected by Fusarium oxysporum (Vander Molen et al., 1983). Ethylene production
accelerated during pathogenesis is also an effect of ethylene production by fungal and
bacterial attacks, which have been reported as synthesis of ethylene in vitro (Makovitzki
et al., 2007). Strain of Fusarium oxysporum increased production of ethylene in liquid
media (Swart and Kamerbeek, 1977; Hottoger and Boler, 1991). Studies on ethylene
synthesis by nineteen various species of Fusarium indicate that ethylene synthesis is a
constant trait of the isolates of Fusarium oxysporum from tulip (Swart and Kamerbeek,
1976). Abnormally high levels of ethylene synthesis by F. oxysporum has also been
23
documented in vitro (de Munk and de Rooy, 1971), 4,000 times higher as compared to
other fungi tested.
2.3.2. Ethylene as an inhibitor of nodulation
Since ethylene affects several aspects of root development and nodule formation
(Goodlass and Smith, 1979; Ligero et al., 1991; Hirsch and Fang, 1994; Arshad and
Frankenberger, 2002; Shaharoona et al., 2006), it also acts as an inhibitor of nodulation in
various legumes (Heidstra et al., 1997; Nukui et al., 2000). Ethylene has negative effects
on tip growth and cell division in roots of dicotyledonous plants. It blocks cortical cell
division (Goodlass and Smith, 1979; Lee and LaRue, 1992a, b) and inhibits infection in
legumes (Spaink, 1997). In addition, ethylene blocks the formation of nodule primodia,
whereas the numbers of infarctions do notdecrease (Lee and LaRue, 1992).
It has been reported that application of exogenous ethylene or ACC (the
immediate precursor of ethylene in higher plants) inhibits nodule formation in Psisum
sativum, Medicago truncatula, Medicago sativa, Macroptilium atropurpureum, and Lotus
japonicus (Nukui et al., 2000; Peters and Crist-Estes, 2001). Moreover, ethylene reduced
nodule formation in Phaseolus bean (Grobbelar et al., 1971), pea, clover (Goodlass and
Smith, 1979; Lee and LaRue, 1992a, b), and Vicia (Zaat et al., 1989). Several authors
reported that ethylene release was stimulated after inoculation in alfalfa (Ligero et al.,
1987), Vicia (van Workum et al., 1995), and soybean (Hunter, 1993; Suganuma et al.,
1995), and after nitrogenous fertilizer application in alfalfa (Ligero et al., 1991).
Nodulation can be promoted by using inhibitors of ethylene production (Yuhashi
et al., 2000; Nukui et al., 2000). Inhibitors of ethylene synthesis (AVG and Cobalt) and
action (Silver) promoted nodulation, as in Vicia (Zaat et al., 1989), pea (Guinel and
24
LaRue, 1992), and alfalfa (Peters and Crist-Estes, 1989; Ligero et al., 1991).
Aminoethoxyvinylglycine (AVG) reduced the generation of ethylene induced by nitrate
and restored nodulation (Ligero et al., 1991). Like chemical inhibitors, PGPR containing
ACC-deaminase promotes nodulation in leguminous plants through inhibition of ethylene
production and consequently, they promote symbiosis and N-fixation in plants (Nukui et
al., 2000; Oldroyd et al., 2001; Okazaki et al., 2004)
Production of ethylene in most of the plant tissues is normally low; however, its
accelerated production can be induced by a wide range of developmental and
investigational indications including seed germination, fruit ripening, leaf and fruit
senescence and a number of biotic and abiotic stresses (Yang and Hoffman, 1984;
Theologies, 1992; Saleem et al., 2007). However, any factor which induces a change in
the endogenous levels of ethylene in a plant results in modified growth and development
(Arshad and Frankenberger, 2002; Jennifer et al., 2006; Munusamy et al., 2006).
2.4. Plant growth promoting rhizobacteria
In recent years, the use of microorganisms to improve plant growth has been
increased in various parts of the world (Khalid et al., 2009). Plant growth promoting
rhizobacteria (PGPR) affect growth and development of plants by direct or indirect
mechanisms. The direct mechanisms include N2-fixation, mobilization of nutrients via
production of phosphatases, siderophores or organic acids, and production of
phytohormones and enzymes (Lucy et al., 2004; Khalid et al. 2004a; Gray and Smith,
2005; Burgmann et al., 2005; Tsavkelova et al., 2005; Cakmakci et al., 2006; Cheng and
Zhao, 2007; Golovatskaya and Karnachuk, 2007; Hayat and Ahmad, 2007; Beneduzi et
al., 2008; Hathway, 2008). Indirectly, the bacteria may exert positive influence on plant
25
growth by lessening certain deleterious effects of a pathogenic organism by inducing host
resistance to the pathogen or by knocking out the pathogen from root surfaces or
producing chitinases or other pathogen suppressing substances (Raj et al. 2003; Guo et al.
2004; Van Loo and Glick, 2004; Van Loo, 2007). Although scientists have reported both
direct and indirect ways of growth stimulation by PGPR, but there is no clear separation
between these two mechanisms. Some bacteria possess multiple traits through which
plant growth where one trait may dominate the other one (Hafeez et al., 2004;
Shaharoona et al. 2006a, 2008). A bacterium influencing plant growth by regulating
synthesis of plant hormones can also play a role in controlling plant pathogens and
diseases and, vice versa.
2.4.1. Effect of plant growth promoting rhizobacteria on plant growth
Plant growth promoting bacteria exert influence on plant growth through various
mechanisms of action and several authors have reported positive effect of inoculation on
various crops (Arshad and Frankenberger, 2002; Nadeem et al., 2006; Tanimoto, 2005;
Kausar and Shahzad, 2006; Watt et al., 2006; Glick et al., 2007; Hardoim et al., 2008;
Egamberdieva, 2008; Hameeda et al., 2008; Hathway, 2008; Figueiredo et al., 2008;
Yang et al., 2009; Khalid et al., 2009). Here, the main focus is to review the effects of
plant growth promoting bacteria containing ACC-deaminase on growth and yield of
various crops which is presented in the following section.
2.4.2. Plant growth promoting bacteria containing ACC-deaminase
Plant growth promoting bacteria can modulate plant growth through changes in
the levels of plant hormone ethylene. Production of ethylene in plants has been found
directly related to the amount of ACC (Machackova et al., 1997). Certain bacteria contain
26
an enzyme ACC-deaminase that hydrolyses ACC into NH3 and α-ketobutyrate (Glick et
al., 1998; Mayak et al., 1999; Shaharoona et al., 2006a, b; Saleem et al., 2007; Contesto
et al., 2008). The uptake and cleavage of ACC by plant growth promoting bacteria
containing ACC-deaminase reduce the amount of ACC, as well as ethylene, outside the
germinating seeds, in that way acting as a sink for ACC. The biological activity of
bacteria controls the relative production of ACC-deaminase and ACC-oxidase in the
system to lower ethylene levels in plant (Glick et al., 1998; Arshad and Frankenberger,
2002). Reduction in the levels of ACC results in lower levels of endogenous ethylene,
which reduces the inhibitory effects of higher ethylene levels (Glick et al., 1998; Geraats
et al., 2003; Dodd et al., 2004; Andrea et al., 2007). Numerous bacteria have been found
to stimulate plant growth through their ACC-deaminase activity (Glick et al., 1998;
Mayak et al., 1999; Contesto et al., 2008). Furthermore, plants that are inoculated with
PGPR containing ACC-deaminase are resistant to the harmful effects of stress ethylene
that is generated due to stress conditions (Grichk and Glick, 2001; Wang et al., 2004;
Mayak et al.,2004a; Kausar and Shahzad, 2006; Saleem et al., 2007; Egamberdieva, 2008).
2.4.2.1. Effect of bacteria containing ACC-deaminase on leguminous
crops
A number of factors affect the nodulation on the roots of legume together with
physicochemical and host microsymbiont compatibility conditions of the soil and
presence of known and unknown bio-molecules such as flavonoides, hormones and
polysaccharides (Antoun et al., 1978; Tisdale et al., 1990; Glick et al., 1998; Mayak et
al., 1999). Besides other aspects, phytohormones have major role in developing
nodulation in leguminous plant (Hirsch and Fang, 1994). Now days, it is well established
27
that ethylene plays a regulatory role in almost all the plant developmental processes
under any stress condition. While it is produced in all the organs of higher plants, but
mostly in ripening fruits and senescing tissues, its higher levels have been found
(Frankenberger and Arshad, 1995; Holguin and Glick, 2001; Arshad and Frankenberger,
2002). These days, it is well-known that ethylene possesses a main place between the
factors that affecting the development of the rhizobia-legume association. But it may
function as an auto regulator to control nodulation (Nukui et al., 2000; Arshad and
Frankenberger, 2002). Ethylene applied directly as a gas and/or indirectly as ACC, acted
as an inhibitor of nodulation on the root of legumes (Jadhave et al., 1994; Yuhashi et al.,
2000). Endogenous ethylene production negatively affects the nodulation; while
inhibitors of ethylene production improve nodulation (Delgado et al., 1994; Ligero et al.,
1999; Penrose et al., 2001; Grichko and Glick, 2001a; Dodd et al., 2004).
Any approach that can reduce stress ethylene evolution might alleviate its negative
effects on root growth and nodulation. PGPR containing ACC-deaminase could promote
plant growth though ACC-deaminase activity. Belimov et al. (2009) evaluated the effect
root associated bacterium Variovorax paradoxus 5C-2 containing ACC-deaminase on pea
(Pisum sativum) plants grown in dry soil. Inoculation with V. paradoxus 5C-2 with ACC-
deaminase activity improved growth, yield, nodulation and water use efficiency of
droughty peas. Systemic effects of V. paradoxus 5C-2 included an amplified soil drying
induced increase of xylem abscisic acid (ABA) concentration, but an attenuated soil
drying-induced increase in xylem ACC concentration. Dey et al. (2007) isolated 233
isolates of rhizobacteria from the rhizosphere of peanut and nine isolates were selected on
the basis of ACC-deaminase activity. These selected isolates significantly increased the
28
seed germinating potential and root length of the seedling peanut as compared with
untreated control. All identified isolates were belonged to Pseudomonas spp. The
effectiveness of the selected PGPR strains was repetitively tested in pot and field
conditions for 3 years. Seed inoculation of these three isolates, viz. PGPR1, PGPR2 and
PGPR4, resulted in a significantly higher pod yield than the control, in pots, during rainy
and post-rainy seasons. The contents of nitrogen and phosphorus in soil, shoot and kernel
were also enhanced significantly in treatments inoculated with these rhizobacterial
isolates in pots during both the seasons. In the field trials, however, there was wide
dissimilarity in the efficacy of the PGPR isolates in enhancing the growth and yield of
peanut in different years. Plant growth promoting fluorescent pseudomonad isolates, viz.
PGPR1, PGPR2 and PGPR4, significantly enhanced pod yield (23 to 26%, 24 to 28% and
18 to 24%, respectively), haulm yield and nodule dry weight over the control in three
years. Other traits like root length, pod number, 100-kernel mass, shelling out-turn and
nodule number were also enhanced. Similarly, Zafar-ul-Hye et al. (2007) isolated twenty
seven isolates of PGPR containing ACC-deaminase from the lentil rhizosphere and
screened on the based of their growth promotion of lentil seedlings under axenic
conditions. Mostly it was observed that all the PGPR isolates had the potential to promote
growth of lentil seedlings under axenic conditions. Results of jar experiment revealed that
inoculation increased the root and shoot length, fresh root and shoot weights, dry root and
shoot weights of lentil seedlings up to 2.4 and 2.3, 2.6 and 2.7, 2.6 and 2.2 folds,
respectively over control. It is recommended that ACC-deaminase trait could be a useful
technique for screening efficient PGPR for promoting plant growth before testing their
potential under field conditions.
29
Lucas Garcia et al. (2004) investigated the effects of inoculation with three
different strains of PGPR containing ACC-deaminase activity, Aur 6, Aur 9 and Cell 4,
belonging to P. fluorescens, C. balustinum and S. fonticola, respectively; on biological
nitrogen fixation, nodulation and growth promotion by soybean (Glycine max) var.
Osumi plants. Inoculation modes for the PGPR and Sinorhizobium fredii (carried out
through irrigation), were examined. In the first mode, PGPR and S. fredii were co-
inoculated. In the second mode, we first inoculated S. fredii and after the PGPR, which
were added 5 or 10 days later. In the third mode, the PGPR were inoculated first, and the
S. fredii was inoculated 5 days later. Plants were maintained in a green house under
controlled conditions, and were sampled three months after sowing. The results obtained
exhibited the effects of the inoculation sequence. The most significant effects on growth
parameters (stem plus leaf weight and fresh root weight) were found when inoculations
with PGPR and S. fredii were at different times or when we inoculated only with PGPR
and the plants were watered with nitrogen. Co-inoculation had no positive effects on any
parameter, probably due to competition between the PGPR and S. fredii. The results
indicated that the inoculation modes with PGPR and rhizobia played a very important
role in the effects produced. Thus, plant growth promoting rhizobacteria may interact
synergistically with root-nodulating rhizobia, PGPR selected for one crop should be
assessed for potentially hazardous effects on other crops before being used as inoculants.
Shaharoona et al. (2006a) evaluated the effectiveness of bacterial strains
possessing ACC-deaminase activity on nodulation in legumes upon co-inoculation with
rhizobia. Results of pot trial showed that co-inoculation with Bradyrhizobium and PGPR
isolates enhanced the nodulation in mung bean compared with inoculation with
30
Bradyrhizobium alone. It is highly expected that inoculation with rhizobacteria
containing ACC-deaminase hydrolysed endogenous ACC into ammonia and alpha-
ketobutyrate instead of ethylene. Consequently, root and shoot growth as well as
nodulation were promoted. Similarly, Remans et al. (2007) examined the potential of
PGPR to enhance nodulation of common bean (Phaseolus vulgaris). It was observed that
the effect on nodulation of three (out of four) PGPR was strongly dependent on P
nutrition. Further, the use of specific PGPR mutant strains indicated that bacterial indole-
3-acetic-acid production and 1-aminocyclopropane-1-carboxylate deaminase activity play
an important role in the host nodulation response, particularly under low P conditions.
Moreover, it was shown that the differential response to PGPR under low versus high P
conditions was associated with changes in the host hormone sensitivity for nodulation
induced under P deficiency.
Tittabutr et al. (2008) conducted a study to evaluate effect of ACC-deaminase on
nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC-
deaminase were cloned from bacterial strain TAL1145 and bacterial BL3 in multicopy
plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC-
deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants
of TAL1145 containing the native or BL3 acdS gene could grow in minimal media
containing 1.5 mM ACC, whereas BL3 could tolerate up to 3 mM ACC. The TAL1145
acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was
highly inducible by ACC and not by mimosine. The transconjugants of TAL1145
containing the native and BL3-acdS genes formed nodules with greater number and sizes,
and produced higher root mass on L. leucocephala than by TAL1145. This study showed
31
that the acdS gene increased ACC-deaminase activities of TAL1145 and also enhanced
its symbiotic efficiency on L. leucocephala. Similarly, Ma et al. (2003) reported that
ethylene inhibits nodulation in various legumes. In order to examine the way by which
rhizobacteria regulate nodulation, the ACC-deaminase gene was isolated. Insertion
mutants with mutations in the bacterial ACC-deaminase gene (acdS) and its regulatory
gene, a leucine-responsive regulatory protein like gene (lrpL), were constructed and
assessed their abilities to nodulate Pisumsativum L. cv. Sparkle (pea). Both mutants,
neither of which synthesized ACC-deaminase, showed decreased nodulation efficiency in
comparison with parental strain. The study demonstared that the presence of ACC-
deaminase activity in bacteria enhanced the nodulation of P. sativum L. cv. Sparkle, by
modulating ethylene levels in the plant roots during the early stages of nodule
development. However, Nukui et al. (2006) examined the regulation of the acdS gene
encoding ACC-deaminase in bacteria during symbiosis with the host legume Lotus
japonicus; they introduced the glucuronidase (GUS) gene into acdS so that GUS was
showed under control of the acdS promoter, and also generated disruption mutants with
mutations in a nitrogen fixation regulator gene, nifA. While, the histochemical GUS assay
exhibited that there was exclusive expression of acdS in mature root nodules. Two
homologous nifA genes, mll5857 and mll5837, were observed in the symbiosis island of
M. loti and were designated nifA1 and nifA2, respectively. Quantitative reverse
transcription-PCR explained that nifA2 disruption resulted in considerably reduced
expression of acdS, nifH, and nifA1 in bacteroid cells. In contrast, nifA1 disruption
slightly promoted expression of the acdS transcripts and suppressed nifH to some degree.
These results indicated that the acdS gene and other symbiotic genes were positively
32
regulated by the NifA2 protein, but not by the NifA1 protein, in M. loti. The mode of
gene expression suggested that M. loti acdS participates in the establishment and/or
maintenance of mature nodules by interfering with the production of ethylene, which
induces negative regulation of nodulation.
The efficacy of PGPR for improving growth and yield of plants was also tested
under different abiotic stresses i.e. water shortage, high and low temerature, salinity and
under deficiency nutreints. The effect of inoculation with PGPR containing ACC-
deaminase on growth and water use efficiency under water stress conditions was
confirmed using peas as test plant which is very sensitve to ethylene changes. It was
found that inoculation with PGPR containing ACC-deaminase was highly effective in
removing the effects of water stress on growth, yield and ripening of peas (Arshad et al.,
2008). Moreover, inoculation of peas with PGPR containing ACC-deaminase
significantly decreased the effects of water stress on growth and yield of peas. Results
showed that inoculation increased the grain yield up to 62% over the untreated water
stressed control while water use efficiency was also significantly increased because of
inoculation. It was concluded that reduction in the severity of water stress imposed
effects might be due to reduction in drought induced ethylene levels.
2.4.2.2. Effect of bacteria containing ACC-deaminase on other crops
Several authors have reported that PGPR containing ACC-deaminase enhance
growth of crops. Ghosh et al. (2003) isolated three strains of PGPR from the soil of
southeastern Wisconsin, on based of their efficacy to use ACC as sole nitrogen source.
These bacterial isolates were identified as Bacillus circulans DUC1, Bacillus firmus
DUC2, and Bacillus globisporus DUC3. Each isolated strain of bacteria gave similar
33
results of ACC-deaminase activity and improved root elongation in canola seedlings
under axenic conditions. Soil inoculations with these rhizobacterial strains increased root
length, shoot length fresh biomass and dry weight of potted canola plants. Likewise, a
soil inoculation with B. globisporus DUC3 positively promoted the roots and shoot
growth of plants. Similarly, Hynes and Nelson (2001) isolated a bacterial strain
Pseudomonas putida that was named as 6-8 from the rhizosphere of pea obtained from a
field. It was one of the bacterial strains selected from the culture collection based on its
ability to use ACC as sole source of nitrogen for growth, to produce siderophores and
improve root elongation of canola in growth pouches. Glick et al. (1994b) studied that
mutants’ strains of Pseudomonas putida GR12-2 that was deficient in ACC-deaminase
enzyme activity did not have the ability to promote canola root elongation under
gnotobiotic conditions. Similarly, an ACC-deaminase activity minus mutant of E.
cloacae UW4 strain was improved root growth of canola seedlings (Li et al., 2000).
Several strains of PGPR increase plant growth by regulating the endogenous
ethylene production in the roots through their ACC-deaminase activity. However, nitrate
in the surrounding area of the roots may decrease the efficiency of PGPR by increasing
the activity of ACC-oxidase enzyme resulting in greater ethylene synthesis by the roots.
Shaharoona et al (2006) evaluated the efficacy of PGPR containing ACC-deaminase for
improving growth and yield of maize grown in N-amended soil. A number of strains of
rhizobacteria containing ACC-deaminase were screened on the based of growth
promoting activity in maize roots under axenic conditions. Significant increases in plant
height, root weight and total biomass were recorded due to effect of inoculation. Results
of the field experiment showed that the inoculation performed comparatively better in the
34
absence of N-fertilizer. Bacterial strain Pseudomonas fluorescens biotype G (N3) was the
most successful strain both in the presence and absence of N fertilizer. Similarly,
Shaharoona et al. (2008) conducted a study to test effects of two PGPR strains containing
ACC)-deaminase, Pseudomonas fluorescens and P. fluorescens biotype F on growth,
yield and nutrient use efficiency of wheat under different levels of three major nutrients
N, P, and K (at 0, 25, 50, 75, and 100% of recommended levels). Results of pot and field
trials showed that the efficacy of strains reduced with the increasing levels of NPK in the
soil for improving growth and yield of wheat. In another study, they observed significant
effects in case of inoculation with PGPR on root elongation, root weight, tillers per pot,
and grain and straw yields in the pot trials (Shaharoona et al., 2007). Inoculation with
these PGPR was also shown to promote wheat growth and yield significantly under field
conditions. Similarly, Ahmed et al. (2004) isolated six PGPR strains Q50, N3, Q7, N7,
Q14 and Y showing ACC-deaminase activity from rhizosphere soil of wheat and tested
their efficacy at recommended levels of mineral fertilizers NPK @ 150-120-70 kg ha-1,
respectively. Results of pot revealed that all isolates showed significant increase in root
elongation, root weight, straw yield and grain yield up to 31, 83, 69 and 60%,
respectively, over untreated control. These findings demonstrate that ACC-deaminase
containing PGPR could be efficiently used for growth promotion of various crops.
2.5. Rhizobia
The gram-negative soil bacteria such as rhizobia have ability to cause infection in
root tissues of the compatible host plant of legume and start the formation of nitrogen
fixing nodules (Stougaard, 2000; Prell and Poole, 2006). The biochemistry, physiology,
genetics and ecology of the bacteria forming symbiotic relationship with legumes have
35
been well-studied. These bacteria are known as the rhizobia. But they are taxonomically
different members of the “a” and “b” sub-classes of the Proteobacteria. Sahgal and Johri
(2003) expressed the current situation of rhizobial taxonomy and enlisted thirty six
species from seven genera (Azorhizobium, Allorhizobium, Bradyrhizobium,
Methylobacterium, Mesorhizobium, Sinorhizobium and Rhizobium). Unlike other plant
growth promoting bacteria, Rhizobium sp. plays an important role in increasing growth
and yield of legumes through symbiotic nitrogen fixation (Young and Haukkam, 1996).
2.5.1. Nodulation
It is the site for symbiotic nitrogen fixation formed as a result of series of
interactions between rhizobia and leguminous plants. Most rhizobium isolates can
nodulate more than one host plant species, while several different bacterial species are
often isolated from a single legume host plant (Cooper, 2007). Rhizobia have four main
types of surface polysaccharides which play their role at different stages of symbiotic
development as well as including root colonization, host recognition, infection thread
formation and nodule invasion. They are also found to be implicated in biofilm
formation on root hair surfaces (Fujishige et al., 2006). A variety of different
compounds has been found to be found in root exudates of plant (Bertin et al., 2003).
The plants generally can exude as much as 21% of the fixed photosynthate (Walker et
al., 2003), and in seedlings, even up to 40% (Bertin et al., 2003) into the rhizosphere.
However, different sugars such as mannose, rhamnose and galacturonic acids are also in
exudates and in the root mucilage of leguminous plants (Knee et al., 2001). Rhizobia
can also grow on several unusual compounds found in the rhizosphere e.g. mimosine,
stachydrine, calystegines, homoserine, rhizopines and trigonelline (Soedarjo and
36
Borthakur, 1998). In the legume plant rhizosphere, the rhizobia become affected by
growth promoting compounds and chemo-tactic while combined effect results in root
colonization. The rhizobia are certainly chemo-tactic to unfractionated legume
epidermal exudates containing flavonoids as well (Cooper, 2004). Simple sugars, amino
acids, hydroxyaromatic and dicarboxylic acids in root exudates can also cause to come
out a strong chemo-tactic reaction.
Upon root colonization, bacteria start to generate signal molecules that are
transduced within the cells of the plant, resulting in increased growth and development of
the plant. The relation between rhizobia and legumes initiates with attachment of the
bacteria to the root hairs during lectin binding (Gray et al., 1992). Thus, any approach
which could be helpful to increase root system would facilitate rhizobia to form more
nodules in legumes.
In addition to other factors, plant hormones have very imperative role in the
growth and development of nodulation (Frankenberger and Arshad, 1995). Plant growth
regulators produced by microbes could have major role in symbiosis, especially in the
establishment of nodulation during legume and rhizobium interaction (Hirsch et al.,
1997), or in direct plant growth promotion (Kobayashi et al., 1993). But ethylene is also
found to be involved in an inhibiting nodulation in legumes by rhizobium (Glick, 2005).
2.5.2. Rhizobium inoculation of legumes
A successful symbiosis and nitrogen fixation may be attained, if the conditions of
rhizobium inoculants remain optimized (Zahran, 2001). The isolation and screening of
highly effective and competitive strains from native rhizobial population to be used as
inocula, could be much beneficial under field conditions (Chatel and Greenwood, 1973).
37
Huang and Erickson (2007) tested the effectiveness of R. leguminosarum for
improving growth and yield of pea and lentil. They found improved seedling growth,
nodule biomass and shoot and root biomass in peas. Grain yield of pea was also
increased. In case of lentil, seed treatment with R. leguminosarum exhibited increase in
seedling height shoot and nodule biomass. Similarly, improvement in nodulation was
observed in peanut by inoculation with Rhizobium species (Dey et al., 2004). Miller et al.
(2007) reported that the strains of R. leguminosarum bv. trifolii formed effective nodules
on T. ambiguum i.e., Caucasian clover and ineffective nodules on T. repensi i.e., white
clover. Similarly, Ahmad et al. (2008) reported the effect of different methods of
rhizobial inoculation on yield, root nodulation, and seed protein contents of two lentil
varieties (Masoor-93 and Massor-2002). The variables such as seed protein contents, root
nodulation, N-contents of root and shoot of lentil, N-contents of soil, seed yield and yield
components were significantly affected by inoculation with rhizobium. Similarly, several
researchers have reported significant effect of rhizobial inculcation on growth, yield and
nodulation of various legume crops (Asghar et al., 1988; Yanni et al., 1992; Hoque and
Haq, 1994; van Rhijn et al., 2001; Huang et al., 2007).
2.6. Co-inoculation with rhizobia and plant growth promoting bacteria
There are several reports which reveal that effectiveness of rhizobia could be
enhanced by co-inoculation with plant growth promoting bacteria. Co-inoculation with
symbiotic and rhizosphere bacteria may improve nodulation by a number of mechanisms.
For example, they can generate phytoalexin, antibiotics against pathogens, siderophores
chelating insoluble cations and colonize root surfaces by out-competing pathogenic
organisms, and thus increase nodulation and growth (Parmer and Dadarwal, 1999). It has
38
been confirmed with Fahraeus slides that A. brasilense causes an increase in the number
of root hairs and root diameter in alfalfa (Itzigsohn et al., 1993). Inoculation with A.
brasilense also increase root hair formation in seedling roots of common bean (Burdman
et al., 1996). Fluorescent pseudomonads have been observed to increase nodulation in
chickpea and thus promoting biological nitrogen fixation (Parmar and Dadarwal, 2000).
Co-inoculation of Bradyrhizobium with P. striata has also been observed to enhance
biological nitrogen fixation in soybean. The interactions between PGPB and rhizobia
could prove to be synergistic or antagonistic. These interactions may be used for
improving the biological nitrogen fixation and crop yield (Dubey, 1996). There has also
been indication for the presence of plant growth promoting Bacillus strains in the root
nodules of soybean (Bai et al., 2002).
Many scientists have reported that ACC-deaminase activity of PGPB strains
coupled with rhizobial activity play a very imperative role in growth, yield and nutrients
uptake of peanut. There was significant promotion in the number and nodule dry weight
of peanut by inoculation with PGPB containing ACC-deaminase (Jeffries et al., 2003).
The promotion in the number of nodules might be due to increase in root length and
growth, consequently, providing more number of active sites and contact to rhizobial
strains for nodulation. Shaharoona et al. (2006a) observed that co-inoculation with
Pseudomonas and Bradyrhizobium species significantly improved root length, total
biomass and nodulation in mung bean. Co-inoculation showed the most promising
increases in case of number of nodules and fresh and dry nodule weight. Co-inoculation
with P. putida biotype A and Bradyrhizobium produced significant increase in number of
nodules per plant in comparison with untreated control and 48% increase, over sole
39
inoculation with Bradyrhizobium. They suggested that increase in nodulation in mung
bean might be due to bacterial ACC-deaminase activity. Babar et al. (2007) carried out
study to assess the effectiveness of co-inoculation of chickpea with rhizobium and
Enterobacter strains. They isolated Enterobacter and rhizobial strains from nodules and
roots of chickpea and applied in combinations to improve nodulation and plant growth.
They stated that co-inoculation improved nodulation and growth of chickpea in most of
the treatments and the increase was dependent on co-inoculation and the host cultivar.
Rosas et al. (2006) studied the promising action of two phosphate solubilizing
Pseudomonas strains on the symbiosis of rhizobial strains (S. meliloti and B. japonicum)
with alfalfa and soybean. A greater number of nodules and dry weight was recorded in
soybean when the co-inoculation with B. Japonicum and Pseudomonas was done. They
found that Pseudomonas supplied phosphorous when growing with B. Japonicum.
Likewise, Anandham et al. (2007) carried out a study to evaluate the potential of co-
inoculation of the sulfur-oxidizing bacteria with a rhizobium strain that had no sulfur-
oxidizing potential in groundnut. Clay like pellet formulations (2.5×107 cfu g-1 pellet) of
the Thiobacillus strains were developed and their efficacy to stimulate plant growth were
tested in groundnut under pot and field conditions with sulfur-deficit soil. Studies in pot
conditions gave more promising results on groundnut increasing the fresh biomass,
nodule number and dry weight, and pod per plant. Co-inoculation of Thiobacillus strain
with rhizobium under field conditions observed significantly higher nodule number,
nodule dry weight and plant biomass 136.9 per plant, 740 mg and 15 g per plant,
respectively, on eighty days after sowing and increased the pod yield up to 18% in
comparison with untreated control. Also inoculation of sulfur-oxidizing bacteria
40
improved the soil available sulfur 7.4 to 8.43 kg ha-1. It is concluded from these results
that inoculation of sulfur-oxidizing bacteria along with rhizobia results in synergistic
effects promoting the yield and oil contents of groundnut, in sulfur-deficit soils. Effect of
co-inoculation with B. japonicum A1017 and a gusA-marked strain of P. fluorescens
2137, P. fluorescens WCS365, A. agilis 125 and A. lipoferum 137 was assessed on
soybean (Chebotar et al., 2001). The gusA-marked rhizobacteria successfully colonized
the root tips and near the surfaces on the roots tips. P. fluorescence 2137 had the
maximum colonization activity on soybean roots either inoculated alone or inoculated
with B. japonicum A1017. Co-inoculation of P. flouresens 2137 and B. japonicum A1017
improved the colonization of B. japonicum A1017 on soybean roots, nodule numbers and
acetylene reduction activity at 10 and 20 days after inoculation. The effect of
rhizobacteria P. flourescens, A. chroococcum and A. brasilense, sole and in co-
inoculation with Rhizobium species and G. mosseae was assessed by Siddiqui et al.
(2001) on the growth of chickpea. In case of sole inoculation, G. mosseae was better in
improving plant growth and reducing nematode reproduction than any other tested
organism. Use of A. brasilense induced similar increase in the plant growth to that caused
by Rhizobium species, while application of A. chroococcum was better than A. brasilense
in improving growth of nematode infected plants. But combined application of P.
flourescens and G. mosseae was better in improving plant growth and reducing nematode
reproduction than any other treatment. Likewise, Sindhu et al. (2002) studied that co-
inoculation with four Bacillus strains MRS12, MRSA18, MRS 31 and MRS27 increased
shoot dry mass of green gram more than sole inoculation with Bradyrhizobium. Similarly,
Yuming et al. (2003) co-inoculated three strains of Bacillus species with B. japonicum
41
under green house and natural field conditions. These strains improved nodulation,
nodule weight, root weight, total nitrogen and grain yield. The strain B. thuringiensis
NEB 17 was the most effective in improving soybean production. Effects of the co-
inoculation dose of two PGPR strains, S. proteamaculans 1-102 and S. liquefaciens 2-68,
with B. japonicum on soybean growth, nodulation and nitrogen fixation were investigated
under axenic conditions. The co-inoculation of PGPR significantly increased nodule
number, plant dry weight and fixed nitrogen (Bai et al., 2002). Madhaiyan et al. (2006b)
recorded that the dual inoculation of methyl-bacterium with rhizobium significantly
improved plant growth, nodulation and yield contributing traits in groundnut in
comparison with sole application of rhizobium.
Figueiredo et al. (2007) conducted a study in a greenhouse to assess the potential
of PGPR on nodulation, biological nitrogen fixation and growth of Phaseolus vulgaris L.
cv. Tenderlake. The disinfected seeds werre inoculated with Rhizobium tropici alone and
in combination with PGPR strain. It was observed that beans, co-inoculated with R.
tropici and Paenibacillus polymyxa had higher concentrations of leghemoglobin,
nitrogenase activity and N2-fixing efficacy and thus formed associations of greater
symbiotic efficiency. Inoculation with Rhizobium and P. polymyxa strain stimulated
nodulation as well as nitrogen fixation.
2.7. Composted organic material and biological activity
Use of compost in the rhizosphere of plant can provide a better environment for
bacteria to increase their growth and activities in the rhizosphere. This would have
positive effect on plant root growth by supporting bacterial activities (Bajpai et al., 2002;
Cheuk et al., 2003). Biologically active high quality composts can play an important role
42
in sustainable crop production. The high quality compost has been found to improve soil
structure, humus contents of soil and supply of macro and micro nutrients (Cheuk et al.,
2003; Ahmad et al., 2008a, b). The presence of such composts activates and stabilizes the
soil microflora. Soil organic matter boosts up the physical, chemical and biological
properties of the soil, and consequently crop production (Karlen et al., 1997). The
interaction of soil physical, chemical and biological properties define a particular soil’s
“quality” and determine how effectively the soil performs the ecosystem functions
(Larson and Pierce, 1991). The benefits of increased SOM contents in terms of crop yield
and nutrients uptake have also been observed by the long term trials (Johnston, 1986).
With continued use of composts, levels of P in soil are increased (Sharpley and
Rekolainen, 1997). Furthermore, the use of composts and manures makes available
nutrients other than NPK.
All studies clearly demonstrated that the integrated use of rhizobia and PGPR
containing ACC-deaminase along with organic fertilizers could be highly effective for
improving growth, yield and nodulation of chickpea crop.
43
Co-inoculation of Chickpea Materials and Methods
MATERIALS AND METHODS

A series of laboratory, net house and field studies were conducted to test the efficacy of
inoculation with rhizobia and rhizobacteria containing ACC-deaminase separately as well
as in combination (co-inoculation) for improving growth, yield, and nodulation of
chickpea (Cicer arietinum L.). Effectiveness of single and co-inoculation was also
evaluated in the soil amended with the compost enriched with phosphorous (P), both
under pot and field conditions.
3.1. Isolation of rhizobacteria containing ACC-deaminase and rhizobia
Rhizobacterial isolates were isolated from rhizosphere soil of the chickpea plants,
while rhizobial isolates were isolated from nodules of chickpea plants. Plants were
collected from different locations of Faisalabad region (Faisalabad and Jhang districts)
situated at longitude 72o0’ and 73o45’ east, and 30o30’ and 32o0’ north and Rawalpindi
region (Attock and Chakwal districts) situated at longitude 72 o0’ and 74 o0’ east, and 33
o
0’ and 34 o0’ north of Pakistan.
Plants of chickpea (45-60 days old) were uprooted and taken to the laboratory in
polythene bags. Non-rhizosphere soil from chickpea plants was removed by gentle
agitation of the roots, and the soil strictly adhering to the roots (rhizosphere soil) was
separated. The rhizobacteria were isolated by using minimal salt medium (MSM)
containing ACC as sole nitrogen source (Glick et al., 1994). Dilution plate technique
(Wollum II, 1982) was employed for isolation under aseptic conditions. The soil
suspension was plated onto agar medium, and incubated at 30 oC. Twenty nine bacterial
44
colonies with different growth characteristics (shape, size, colour, and growth rate) were
isolated and purified by further streaking on fresh prepared MSM plates, and the pure
cultures were stored at -40 °C in eppendrof containing 20% glycerol to be used them for
further experiments.
For the isolation of rhizobia, the roots of chickpea were washed gently with tap
water, and nodules were separated from the roots. The collected nodules were surface
disinfected for a moment (<10 s) by dipping in 95% ethanol solution, followed by
dipping in 0.2% HgCl2 solution for 3 min (Russel et al., 1982). Nodules were then
washed several times with sterilized (autoclaved) water to remove surface disinfectant.
The surface sterilized nodules with the help of a sterilized glass rod were crushed in 5 mL
of sterilized water to obtain a milky suspension. A loopful of the suspension was streaked
on yeast extract mannitol (YEM) agar medium (Holt et al., 1994) plates, and incubated at
30 oC. The isolated single colonies were picked, and re-streaked on agar plates. This
process was repeated 3-4 times on fresh prepared agar plates to obtain pure cultures of
rhizobia. In this way, 35 rhizobial isolates were collected and they were designated as S1,
S2,…………., S35. These isolates were stored in 20% glycerol at -40 oC for subsequence
use. The bacteria isolated from different locations are summarized in Table 1.
3.2. Testing ability of rhizobacteria to use ACC as sole source of
nitrogen
Five milliliter of ½ Tryptic soy broth (TSB) were inoculated with rhizobacterial
isolates. The cultures were incubated for 48 h at 30 °C under shaking conditions (100
rpm). Cultures were diluted 10 times in sterilized 0.1 M MgSO4 solution. In 96-well
plate, 120 µL MSM were added to each well. In first 4 lanes, 15 µL 0.1 M MgSO4, and in
45
Table 3.1: Bacterial isolates collected from different locations
Location Rhizobial isolates
Faisalabad S1, S2, S3, S4, S5, S6, S7, S8 and S9
Jhang S10, S11, S12, S13, S14, S15, S16, S17 and S18
Chakwal S19, S20, S21, S22, S23, S24, S25, S26 and S27
Attock S28, S29, S30, S31, S32, S33, S34 and S35
Rhizobacterial isolates
Faisalabad J27, J28, J41, J105, J107, J108, J109 and J112
Jhang J114, J115, J117, J118, J119, J120, J122 and J127
Chakwal J16, J17, J18, J19, J24 and J26
Attock J4, J5, J6, J7, J10, J14 and J15
46
second 4 lanes, 15 µL 0.1 M (NH4)2SO4 were added. The 3 mM ACC were filter
sterilized with 0.2 µm filter membrane and was stored at -20 °C before the assay. This
was allowed to thaw before use; 15 µL of thawed ACC were filled in the rest of the 4
lanes. For inoculation of each well, 15 µL of bacterial culture were used. In untreated
control wells, 15 µL 0.1 M MgSO4 were used instead of inocula. Optical density (OD)
was measured after 48 h at 600 nm using Biolog® identification system. The OD value of
ACC and (NH4)2SO4 wells were compared along with MgSO4 wells to determine the
ability of bacteria to utilize ACC for their growth as described by Jacobson et al. (1994).
The rhizobacterial isolates were grown on two N sources [ACC and (NH4)2SO4],
and one mineral source (MgSO4), to observe the growth rate of each isolate, for ACC
substrate in parallel to (NH4)2SO4. The rhizobacterial isolates were categorized into three
groups, as isolates with higher (Group A), medium (Group B), and lower (Group C) ACC
utilizing rate depending upon their OD values at 600 nm for ACC substrate as compared
to (NH4)2SO4. The isolates with higher ACC-utilization rate possessed OD value for the
wells of ACC substrate similar/near to the OD value for the wells of (NH4)2SO4 in the
initial 48 h of growth. Similarly, isolates with medium ACC-utilization rate possessed
lower OD value for ACC wells as compared to those for (NH4)2SO4 in the initial 48 h of
growth. Isolates with lower ACC-metabolic rate possessed the lowest OD value for ACC
wells (near to the OD value of wells for MgSO4) in the same time.
3.3. Screening rhizobacteria and rhizobia for their abilities to promote
growth and nodulation of chickpea
The inocula of selected rhizobacteria and rhizobia were prepared by using MSM
and YEM, respectively. For this purpose, MSM and YEM broths were prepared in 250
47
mL flasks separately and autoclaved at 121 oC for 20 min. Each broth after cooling was
inoculated with respective bacterial isolates. All the flasks were incubated at 30 oC for 48
h in the orbital shaking incubator at 100 rpm. Optical density was measured at 600 nm by
spectrophotometer and uniform cell density containing 108-109 CFU mL-1 was achieved.
For all experiments, the inocula containing 108-109 CFU mL-1 were used; however, fresh
inoculum was prepared for each experiment.
3.3.1. Screening of rhizobacteria
3.3.1.1. Germination assay
Germination tests were carried out to determine the effect of inoculation with
rhizobacteria having different ACC-utilization rates on seed germination. For this,
chickpea (Cicer arietinum L.) cv. Bittal-98 seeds were surface-disinfected by dipping in
95% ethanol and 0.2% (w/v) HgCl2 solutions as described by Russel et al. (1982) and
Khalid et al. (2004). The treated seeds were rinsed with sterilized water thoroughly.
These seeds were inoculated by dipping them in the rhizobacterial culture for 20 min. Six
inoculated seeds were placed in each petri-plate containing soaked (with distilled water)
filter papers. Uninoculated control was used for comparison. The petri-plates were
incubated at 25±2 oC for 10 days. Each treatment was replicated three times. Percent
germination was calculated.
3.3.1.2. Jar experiment
Jar experiments were conducted in the growth room under axenic conditions for
screening rhizobacteria containing ACC-deaminase for promoting root and shoot growth
of chickpea seedlings. Sterilized glass jars were used for the experiment.
48
The surface disinfected seeds of chickpea were placed in petri-plate containing
soaked (with distilled water) filter paper for germination. The petri-plates were incubated
at 25±2 oC for 4 days. Slightly germinated seeds were inoculated with relevant bacterial
cell suspension (OD at 600 nm, 108-109 CFU mL-1) by dipping them for 10 min. Two
sterilized filter paper sheets were saturated with the sterilized distilled water and then
three inoculated slightly germinated seeds were placed in between the filter papers, which
were rolled and put in sterilized glass jars. Uninoculated control was included for
comparison. Jars were incubated at 20±2 oC in the growth room using completely
randomized design (CRD) with three replications for each treatment. Nutrients were
supplied by adding 20 mL of sterilized Hoagland solution (½ strength) (Hoagland and
Arnon, 1950). Light and dark period was adjusted to 10 and 14 h, respectively. After 15
days of germination the data regarding root and shoot growth were recorded. On the basis
of screening experiment, six (J107, J108, J112, J118, J119 and J120) rhizobacterial
isolates were selected based on their potential to promote root growth (root length, lateral
root length and numbers, and dry root biomass) for further studies.
3.3.2. Screening of rhizobia
Jar and pouch experiments were conducted to screen the most effective rhizobial
isolates for improving growth and nodulation of chickpea under controlled conditions.
3.3.2.1. Jar experiment
For this study, each jar was filled with 500 g sand after sieving through 2 mm
sieve. Forty milliliter of distilled water were added to each jar before autoclaving. All the
jars after wrapping in papers were autoclaved three times at 121 oC for 30 min. After this,
all the jars were placed in laminar flow hood for cooling.
49
Slightly germinated chickpea seeds were inoculated as described earlier in the
section 3.3.1. Three inoculated seeds were sown in each jar. In case of control, seeds
were dipped in sterilized YEM broth. Nitrogen free sterilized Hoagland solution (½
strength) was to provide nutrition (Fahraeus, 1957). Jars were placed in the growth room
at 20±2 oC using CRD with three replications. The light period was adjusted to 10 hours
(day) and 14 hours dark (night) period. After 50 days of sowing, the data regarding root
growth and nodulation were recorded.
3.3.2.2. Pouch experiment
Pouch experiment was conducted in the growth room for further confirmation of the
results of jar experiment under axenic conditions. In this study, the same procedure as
described in the jar experiment was followed for inoculation of chickpea seedlings.
Three inoculated slightly germinated seeds were placed in each sterilized growth
pouch. To fulfill nutrient deficiency, 20 mL of N free Hoagland solution (½ strength) was
applied to each pouch once a week. Each treatment was replicated thrice. Growth
pouches were placed in growth room at 20±2 oC adjusted to 10 h light and 14 h dark
period. The data regarding root and shoot growth and nodulation were recorded after 50
days. Based on jar and pouch experiments, six rhizobial isolates (S4, S9, S14, S21, S27
and S32) exhibiting highest growth and nodulation were selected for carrying out further
tests.
3.4. Screening effective isolates of rhizobacteria and rhizobia for co-
inoculation (Jar and Pouch experiments)
The selected bacterial isolates (rhizobacteria and rhizobia) were tested in
combination by conducting jar and pouch experiments to screen the most effective
50
isolates of rhizobacteria containing ACC-deaminase and rhizobia for co-inoculation for
improving growth and nodulation of chickpea. Inocula of rhizobacteria and rhizobia were
prepared separately in MSM and YEM, respectively and uniform cell densities (108-109
CFU mL-1) were maintained. For co-inoculation, broth cultures of rhizobacteria and
rhizobia were used in 1:1 ratio. Experimental procedure was the same as described in the
above section. The data on growth and nodulation parameters were recorded after 50
days.
To confirm the results of jar experiment, the same above experiment was repeated
in pouch for evaluating co-inoculation effects on growth and nodulation of chickpea.
Like jar experiment, this trial was also conducted in the growth room with three
replications using CRD. The data regarding growth and nodulation parameters were
observed after 50 days. Based on screening experiments, four the most effective isolates
(two each from rhizobacteria and rhizobia) were selected to evaluate their potential under
natural conditions.
3.5. Effect of single and co-inoculation on growth, yield and nodulation
of chickpea under natural conditions
Both pot and field experiments were conducted to test the potential of selected
rhizobacterial (J119 and J120) and rhizobial (S4 and S14) isolates alone as well as in
combination for promoting growth, nodulation and yield of chickpea under natural
conditions.
3.5.1. Pot experiments
Pot trials were conducted in the net house of the Institute of Soil and
Environmental Sciences, University of Agriculture, Faisalabad. The inocula for the pot
51
trials were prepared by culturing the selected isolates of rhizobacteria on MSM and
rhizobia on YEM broths.
The inocula (108-109 CFU mL-1) of selected rhizobacteria and rhizobia were
injected into sterilized peat (100 mL kg-1 peat, seed to peat ratio 10:1 w/w) containing 50
mL of 10% sterilized sugar solution. For inoculation, seeds of chickpea were mixed with
the inoculated peat. In case of control, the seeds were coated with the same slurry but
without inocula. The experiments were conducted with different set of treatments i.e.
effectiveness of inoculation with rhizobacteria or rhizobia either alone or in combination
was tested in the presence of recommended levels (@ 25: 60: 25 kg ha-1) of NPK
fertilizers respectively. These isolates were also tested in the soil amended with P-
enriched compost. For this, 50% recommended level of chemical fertilizer P was used for
the enrichment of composted organic material and remaining 50% recommended level of
chemical fertilizer P was applied at the time of sowing. The P-enriched compost was
applied at 300 kg ha-1. Five seeds were sown in each pot containing 12 kg soil per pot
which were thinned to one plant after 15 days of germination. The uprooted plants were
incorporated into the soil of same pot. Pots were placed in the net house under ambient
light and temperature using CRD with six replications. Canal water was used for
irrigation of pots when needed. At flowering stage, data about nodulation were noted
from three (out of six) replications. Remaining three replications were harvested at
maturity stage and data regarding yield and yield contributing parameters were collected
and analyzed statistically. Grain and straw samples were analyzed to determine the N and
P concentrations.
52
3.5.2. Field experiments
Field experiments were conducted at three different sites i.e. at Research Farm of
the Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad
and on Sajawal Farm at Chak No. 72/GB, Tehsil Jaranwala, District Faisalabad and
Nawaz Farm, Mouza Wasava, Tehsil & District Jhang. The experiments were conducted
for the consecutive two years.
The selected rhizobacterial and rhizobial isolates were tested alone as well as in
combination under field conditions. All the agronomic practices were same as used in pot
experiments. Effectiveness of these isolates was also evaluated in the soil amended with
compost enriched with P. For this, 50% of recommended dose of chemical fertilizer P
was used for the enrichment of composted organic material while remaining 50% of
recommended dose of P was applied at the time of sowing. The P-enriched compost was
applied @ 300 kg ha-1. The inoculated/ co-inoculated and uninoculated seeds were sown
in the well-prepared field @ 80 kg ha-1. Treatments were replicated thrice, using
randomized complete block design (RCBD). Canal water was used for irrigation when
needed. At flowering stage, data regarding nodulation were collected. At maturity, the
crop was harvested and data about growth and yield parameters were recorded. Grain and
straw samples were analyzed for N and P concentrations.
3.6. Characterization of rhizobacterial isolates for ACC-deaminase
activity
ACC-deaminase activity was determined by monitoring the amount of ammonia
generated due to hydrolysis of ACC by the rhizobacterial isolates containing ACC-
deaminase. In Erlenmeyer flasks, 500 mL broth of each isolate was prepared in MSM
53
-1
containing 2 g L ammonium sulfate as N source and incubated at 30 ºC in a shaker at
100 rpm for 48 h. Then the culture was centrifuged at 8300 × g for 10 min. and cell
pellets were collected in phosphate buffer (pH 7) and re-centrifuged. Cell pellets were
-1
transferred to MSM containing ACC substrate (5 mmol L ) and incubated under shaking
for 1 h at 30 ºC. After 1 h, the culture was centrifuged at 8300 × g for 10 min. and
ammonia was determined in supernatant solution by the protocol described by Nagatsu
and Yagi (1966).
3.7. Classical triple response bioassay
Etiolated pea seedlings are considered sensitive to ethylene and show a highly
specific classical “triple” response upon exposure to plant hormone ethylene (Shaharoona
et al., 2007). This response includes reduction in shoot and root length, increase in stem
diameter and more horizontal growth (Neljubow, 1901). Therefore, changes in ethylene
level through ethylene decreasing bacteria were investigated by using classical “triple”
response of etiolated chickpea seedlings. Pea seedlings were grown in the presence of
ACC and then Co2+ and two selected rhizobacterial isolates (J119 and J120) containing
ACC-deaminase were applied. There was a control treatment in which neither ACC nor
Co2+ was applied. Three surface disinfected seeds were sandwiched in the folds of
sterilized filter paper placed in 100 mL beaker. The beakers were placed in air tight 1L
mason jars, wrapped in green foil to provide “safe” green light. All the treatments were
replicated thrice. Incubation was carried out in complete darkness throughout the
experiment at 20±2 °C. After seven days, the data regarding root length, shoot length and
shoot diameter were observed.
54
3.8. Characterization of rhizobacteria and rhizobia for other traits
The selected isolates were characterized for some other biochemical
characteristics which are described below:
3.8.1. Phosphate solubilization assay
The bacterial isolates were evaluated for their ability to solubilize inorganic
phosphate. Agar medium containing tri-calcium phosphate as an inorganic form of
phosphate was utilized in this assay (Goldstein, 1986). The bacterial isolates were
cultured and a loop full of each strain was placed on the plates; five per plate, and the
plates were incubated at 30 °C for seven days. A zone of clearing around the colonies
after 6 days was scored as positive for phosphate solubilization. The experiment was
performed twice with four replications for each bacterial isolate.
3.8.2. Chitinase activity
Chitinase activity was examined by the method described by Chernin et al. (1998)
with some modification. Colloidal chitin [0.2% (w/v)] was added to flask containing
MSM. The autoclaved medium was cooled and poured into sterilized petri plates. After
solidification, a loopful of each culture was placed on the plates at five equidistant places.
The plates were incubated at 30 °C for 7 days until clearing zones of the chitin were seen
around the colonies.
3.8.3. Siderophores production assay
Fifty milliliter of C chrome azurol S (CAS) agar medium was prepared as
described by Schwyn and Neilands (1987). Thirty milliliter of CAS agar medium were
taken in petri-plate. Three wells were made on media in petri-plate by using No. 1
55
sterilized cork borer and 30 µL of the respective cultures of rhizobacteria and rhizobia
were added into the wells. Untreated control was used for comparison. The plates were
incubated at 30 oC for 72 h after which change in colour in media was observed and
characterized as positive for siderophores production
3.8.4. Root colonization assay
Root colonization ability of different isolates in the chickpea was studied under
axenic conditions as described by Simon et al. (1996). Sterilized glass jars were filled
will sterilized sand. The sand was moistened with Hoagland solution (½ strength) and
surface-sterilized chickpea seeds were sown after inoculation with respective
rhizobacteria and rhizobial isolates. Jars were placed in growth room at 25±1 oC. After 7
days, 0.2 g root tips were removed and shaken vigorously in 5mL sterilized water on
orbital shaking incubator at 100 rpm. The bacterial suspension was diluted from 10-1 to
10-4. Sterilized YEM and MSM were poured into petri plates along with 1 mL of each
dilution and were incubated at 30 oC. The colonies were counted and CFU mL-1 was
calculated using a colony counter.
3.9. Identification of selected isolates
The rhizobacteria and rhizobia exhibiting the highest growth promoting activity
under axenic conditions were identified using Biolog ® identification system

TM
(Microlog System Release 4.2, Hayward, CA, USA). For identification, the isolates
were grown on Biolog universal growth medium. Cells from the agar plate were removed
with a sterile swab in a way that no nutrients were carried out from the agar medium into
suspension. The swab was swirled and pressed against the inside surface of the tube on
the dry glass above the fluid line to break up clumps and release cells. The swab was
56
moved up and down the wall of the tube until the cells mixed with the fluid and became a
homogenous, clump free suspension. The turbidity was adjusted to appropriate value for
each isolate. This suspension was poured into the multi-channel pipette reservoir. Eight
sterile tips were fastened securely onto the 8-channel repeating pipette. Tips were primed
and 150 µL of inoculum was poured into each well. Micro-plate were covered with its lid
and incubated at 30 oC for 6 h. After incubation, micro-plate was loaded to micro plate
reader and identified putatively by Microlog Software.
3.10. Soil analyses
The soil used for pot and field trials was analyzed by standard procedures as
described below.
3.10.1. Soil textural class (Mechanical analysis)
For determining soil textural class, 50 g of each soil sample was taken and 40 mL
of 1% sodium hexametaphosphate solution and 250 mL of distilled water were poured
and kept it for over night. Soil was stirred for 10 min with a mechanical stirrer and
transferred in 1 L graduated cylinder and the volume was made up to the mark. After
mixing the suspensions reading was noted by bouyoucos hydrometer (Moodie et al.,
1959). Soil textural class was determined by using the International Textural Triangle.
3.10.2. Saturation percentage (SP)
Saturated paste was prepared and transferred to a tarred china dish and weighed.
China dish (containing soil paste) was put in an oven and dried to a constant weight at
105 °C and weighed again. Saturation percentage was calculated by using method
(Method 27a, U.S. Salinity Laboratory Staff, 1954).
57
Mass of wet soil – Mass of dry soil

SP = x 100
Mass of oven dry soil
3.10.3. pH of saturated soil paste (pHs)
The pH of saturated soil paste was determined after preparing soil paste. For this,
250 g soil was saturated with distilled water. The paste was allowed to stand for 1 h and
pH was recorded (Method 21a, U.S. Salinity Laboratory Staff, 1954) by using pH meter
(Kent-Eil 7015).
3.10.4. Electrical conductivity (ECe)
For determining ECe, extract of each soil paste was obtained by vacuum pump.
ECe was noted with digital Jenway conductivity meter model 4070 (Method 3a and 4b).
3.10.5. Organic matter (OM)
Soil organic matter content was determined according to the standard method
described by Moodie et al. (1959). For this, 1 g of soil sample was mixed thoroughly with
10 mL of 1N K2Cr2O7 solution and 20 mL of concentrated H2SO4. Then 150 mL of
distilled water and 25 mL of 0.5 N FeSO4 solutions was added and the excess was treated
with 0.1 N KMnO4 solutions to pink end point.
3.10.6. Total nitrogen
Nitrogen was determined by Ginning and Hibbard’s method of H2SO4 digestion
and distillation was made with macro Kjeldhal’s apparatus (Jackson, 1962).
58
3.10.7. Available phosphorus
Five gram soil was extracted with 0.5 M NaHCO3 solution adjusted to pH 8.5.
Five milliliter of filtrate was taken in 100 mL volumetric flask and then added 5 mL color
developing reagent (ascorbic acid) and volume was made up to the mark. Reading was
taken on spectrophotometer at 410 nm and available P was calculated with standard curve
(Watanabe and Olsen, 1965).
3.10.8. Extractable potassium
Extraction was done with 1 N ammonium acetate (pH 7) and potassium was
determined by Flame Photometer (Jenway PFP-7) (Method 1la, Salinity Laboratory Staff,
1954).
3.11. Plant analysis
At maturity, plants were harvested and the data about growth and yield
parameters were recorded. Straw and grains samples were analyzed for N and P
concentrations.
3.11.1. NP analysis
N and P were determined after digestion. The plant samples were digested as
described by Wolf (1982). For this, the dried and ground plant sample of 0.1 g was
placed in digestion tubes; 2 mL of conc. H2SO4 was added and incubated over night at
room temperature. Then 1 mL of H2O2 (35% pure) was added into digestion tubes and
was rotated. Tubes were put in the digestion block and heated up to 350 oC until fumes
were generated and continued heating for 20 min. Then digestion tubes were removed
and 1 mL of H2O2 was added slowly and again the digestion tubes were placed back in
the digestion block until fumes were produced for 20 min above step was repeated until
59
Table 3.2: Physico-chemical characteristics of soils used for field
trials
Soil characteristics Site-I Site-II Site-III
Textural class Sandy clay loam Sandy clay loam Sandy loam
ECe 3.3 3.1 3.4
pH (dS m-1) 7.7 7.8 7.4
Organic matter (%) 0.64 0.61 0.34
Total nitrogen (%) 0.059 0.056 0.031
Available phosphorus (mg kg-1) 8.9 7.9 7.7
Extractable potassium (mg kg-1) 112 108 103
Site I: Institute of Soil & Environmental Sciences, University of Agriculture, Faisalabad

Site II: Sajawal Farm, Tehsil Jaranwala, District Faisalabad
Site III: Nawaz farm, Mouza Wasava, Tehsil and District Jhang
60
the material became colorless. The volume of extract was made up to 50 mL with
distilled water. After that, it was filtered and used for determination of N and P as under.
3.11.1.1. Nitrogen
Five milliliter of aliquot was taken in Kjeldhal flask and it was placed on the
Kjeldhal ammonium distillation unit. After adding 10 mL of 40% NaOH the flask was
connected to the distillation apparatus. A receiver was prepared in 100 mL flask by
adding 5 mL of 2% H3BO3 and few drops of indicator (bromocresol green and methylene
red). When the distillate was become approximately 50 mL, then conical flask was
removed and cooled for few min. Then the content of the flask were titreated with 0.01N
standard H2SO4 up to pink end point.
3.11.1.2. Phosphorus
Five milliliter of aliquot was mixed in 10 mL of Barton reagents and total volume
was made as 50 mL. The samples were kept for half an hour and phosphorus was
determined by spectrophotometer using standard curve.
a). Barton reagent
The Barton reagent was prepared by mixing A and B solutions as described by
Ashraf et al. (1992).
I). Solution A
Ammonium molybdate (25 g) was dissolved in 300 mL of distilled water.
II). Solution B
Ammonium metavenadate (1.25 g) was dissolved in boiling water (300 mL), and
then cooled and 250 mL of concentrated HNO3 was added and cooled at room
temperature. Both solutions (A and B) were mixed and volume was made up to 1 L.
61
3.12. Statistical analysis
In all the studies, statistical procedures were applied to analyze the data (Steel et
al., 1997) using appropriate designs and means were compared by Duncan’s Multiple
Range Tests (Duncan, 1955).
62
Co-inoculation of Chickpea Results
RESULTS
Several isolates of rhizobacteria and rhizobia were isolated from the rhizosphere soil and
nodule of chickpea, respectively. The most efficient plant growth promoting rhizobacteria
(PGPR) were screened on the basis of their effectiveness for improving growth and
nodulation of chickpea under axenic conditions. The selected rhizobacteria and rhizobia
were further evaluated under pot and field conditions. Field experiments were conducted
at different locations for consective two years, at the Research Farm of the University and
farmers’ fields. The results of these trials are discussed below.
4.1. Screening rhizobacteria under controlled conditions
4.1.1. Utilization of ACC as source of N by rhizobacteria
The ability of rhizobacterial isolates to utilize ACC as a source of N was assessed
on the basis of bacterial growth. Test isolates utilized ACC as N source (i.e., positive for
ACC-deaminase enzyme activity) but with different degrees of efficacy. On the basis of
their growth measured in terms of cell density (OD600), these isolates were divided into
three groups (Table 4.1). Rhizobacterial isolates (J16, J18, J107, J108, J112, J118, J119
and J120) showing highest growth (OD > 0.75) by utilizing ACC as sole N source, were
categorized as Group-H. Similarly, eleven isolates showing medium growth (OD 0.75-
0.50), were placed in Group-M while ten isolates exhibiting least growth (OD < 0.50)
were shown in Group-L.
63
Table 4.1: Rhizobacterial isolates showing variable growth (measured as OD)
on the media containing ACC as sole N source
(Average of seven replications)

Grouping of isolates based on their ACC-utilization rate
Rhizobacterial
isolates Group-H Group-M Group-L
O.D>0.75 O.D=0.75-0.50 O.D<0.50
J4
J5
J6
J7
J10
J14
J15
J16
J17
J18
J19
J24
J26
J27
J28
J41
J105
J107
J108
J109
J112
J114
J115
J117
J118
J119
J120
J122
J127
64
4.1.2. Effect of rhizobacteria on germination of chickpea seeds
Inoculation with rhizobacterial isolates did not affect the germination percentage
of chickpea seeds significantly; however; there was a strong effect of inoculation with
some rhizobacterial isolates on the root growth of germinated seedlings (Table 4.2). Up
to 75.2% increase in root length was observed in case of inoculation as compared to
uninoculated control.
4.1.3. Effect of rhizobacteria on growth of chickpea seedlings under
axenic conditions (Jar experiment)
4.1.3.1. Root growth
The increase in root length in response to rhizobacterial inoculation was up to
107.5% as compared to uninoculated control (Table 4.3). Six the most effective
rhizobacterial isolates (J107, J108, J112, J118, J119 and J120), increased root length
ranging from 63.8 to 107.5% over uninoculated control. Three isolates had negative
effect on root length (up to 8.1%) compared to uninoculated control while the rest of the
test isolates increased root length up to 59.6% compared with uninoculated control.
The maximum increase (86.7% greater than control) in root dry weight of
chickpea seedlings was observed in response to inoculation with J119 (Table 4.3).
Overall, 25 isolates had positive effect on root dry weight that increase ranged from 12.2
to 86.7% over uninoculated control. Four isolates (J10, J17, J109 and J122) had negative
effect on root dry weight and a decrease in root dry weight up to 8.8% was observed upon
inoculation compared with uninoculated control.
65
Table 4.2: Germination percentage and root enlongation of chickpea as
affected by inoculation with rhizobacterial isolates
(Average of six replications)

Root length (cm)
Bacterial isolates Germination (%) Means ± S.E.
Range
Control 88.0 ***** 1.69 ± 0.06
High 82.3 1.34 - 4.43 2.96 ± 0.08
Medium 85.0 1.32 - 2.98 2.14 ± 0.11
Low 82.1 1.35 - 3.90 2.58 ± 0.05
Standarad error of means (± S.E.)
66
Table 4.3: Effect of rhizobacterial inoculation on root growth of chickpea
seedlings under axenic conditions
(Jar experiment)
(Means of three replications)
Root dry Number of Lateral Lateral root
Rhizobacterial Root length
weight lateral root length dry weight
Isolate (cm)
(g plant-1) roots (cm) (g)
Control 13.64 ± 1.07 0.178 ± 0.05 6 ± 1.02 2.31 ± 0.10 0.019 ± 0.02
J4 14.78 ± 1.10 0.200 ± 0.03 8 ± 1.05 3.52 ± 0.13 0.025 ± 0.03
J5 19.04 ± 1.31 0.208 ± 0.05 9 ± 1.01 4.12 ± 0.09 0.024 ± 0.02
J6 21.14 ± 1.43 0.280 ± 0.08 12 ± 1.41 5.02 ± 0.02 0.035 ± 0.04
J7 22.47 ± 1.29 0.215 ± 0.06 9 ± 1.32 2.62 ± 0.11 0.027 ± 0.03
J10 20.04 ± 1.23 0.167 ± 0.03 10 ± 1.40 3.12 ± 0.10 0.022± 0.01
J14 19.54 ± 1.18 0.287 ± 0.05 13 ± 1.01 5.79 ± 0.13 0.033 ± 0.05
J15 17.04 ± 1.13 0.217 ± 0.03 11 ± 1.56 4.29 ± 0.14 0.021 ± 0.03
J16 20.84 ± 1.37 0.256 ± 0.07 12 ± 1.61 3.85 ± 0.09 0.032 ± 0.04
J17 21.54 ± 1.53 0.162 ± 0.04 10 ± 1.06 3.45 ± 0.11 0.020 ± 0.01
J18 22.84 ± 1.83 0.273 ± 0.05 13 ± 1.23 4.56 ± 0.15 0.030 ± 0.05
J19 19.74 ± 1.61 0.281 ± 0.04 12 ± 1.11 5.12 ± 0.08 0.029 ± 0.03
J24 17.44 ± 0.97 0.281 ± 0.03 11 ± 1.08 3.28 ± 0.10 0.035 ± 0.06
J26 21.04 ± 1.08 0.295 ± 0.02 13 ± 1.32 4.09 ± 0.07 0.034 ± 0.04
J27 18.84 ± 1.31 0.291 ± 0.02 11 ± 1.41 4.15 ± 0.11 0.028 ± 0.03
J28 15.34 ± 1.12 0.285 ± 0.01 12 ± 1.54 4.39 ± 0.09 0.036 ± 0.05
J41 15.94 ± 1.21 0.251 ± 0.06 10 ± 1.29 2.91 ± 0.10 0.030 ± 0.02
J105 13.30 ± 0.86 0.258 ± 0.09 9 ± 1.02 1.94 ± 0.07 0.032 ± 0.03
J107 28.30 ± 2.31 0.314 ± 0.05 14 ± 1.65 6.89 ± 0.13 0.039 ± 0.06
J108 21.34 ± 2.14 0.321 ± 0.06 16 ± 2.01 7.12 ± 0.11 0.038 ± 0.04
J109 12.90 ± 1.03 0.167 ± 0.07 3 ± 0.62 1.02 ± 0.05 0.014 ± 0.03
J112 22.54 ± 2.37 0.297 ± 0.09 16 ± 1.68 7.63 ± 0.09 0.037 ± 0.02
J114 20.34 ± 2.01 0.288 ± 0.04 12 ± 0.96 2.89 ± 0.11 0.033 ± 0.02
J115 17.64 ± 1.10 0.268 ± 0.03 10 ± 1.12 3.16 ± 0.10 0.029 ± 0.03
J117 14.34 ± 1.13 0.257 ± 0.03 11 ± 1.05 3.06 ± 0.09 0.032 ± 0.04
J118 23.63 ± 2.59 0.300 ± 0.02 17 ± 2.13 7.23 ± 0.14 0.038 ± 0.05
J119 27.80 ± 2.68 0.332 ± 0.08 22 ± 1.89 8.93 ± 0.11 0.040 ± 0.04
J120 27.38 ± 1.97 0.318 ± 0.04 20 ± 1.64 8.51 ± 0.13 0.042 ± 0.03
J122 12.54 ± 0.96 0.167 ± 0.05 4 ± 0.72 1.89 ± 0.07 0.016 ± 0.02
J127 15.12 ± 1.10 0.243 ± 0.03 10 ± 0.89 3.08 ± 0.10 0.023 ± 0.04
SED (Standard error of difference between two means) = Root length: 2.163; Root dry weight:
0.0319; Number of lateral roots: 2.371; Lateral root length: 0.0431; Lateral root dry weight: 0.0053;
P value at 5%
67
Results regarding number of later roots are presented in table 4.3. Overall, it was
observed that inoculation with rhizobacteria containing ACC-deaminase increased the
number of lateral roots of chickpea seedlings up to 266.7% more than uninoculated
control. Rhizobacteria isolates J107, J108, J112, J118, J119 and J120 showed the most
prolific increases in number of lateral roots that were 133.3 to 266.7% higher in
comparison to uninoculated control. Two isolates (J109 and J122) decreased the number
of later roots up to 50% as compared to control. Rest of the isolates caused maximum
increase in number of later roots that ranged from up to 33.3 to 116.7% over control.
The increase in lateral root length caused by inoculation with rhizobacteria
containing ACC-deaminase was recorded up to 286.6% as compared to uninoculated
control (Table 4.3). The most prominent increase was found in response to inoculation
with the rhizobacteria such as J107, J108, J112, J118, J119 and J120 that ranged from
198.3 to 286.6% over uninoculated control. Isolate J119 was the most effective isolate of
rhizobacteria among the tested 29 isolates. However, three isolates (J105, J109 and J122)
showed negative effect and decreased the lateral root length up to 55.8% as compared to
The maximum increase (121.1% higher than control) in lateral root dry weight of
chickpea seedlings was recorded through inoculation with J119 (Table 4.3). In general,
27 isolates had positive effect on lateral root dry weight that increase ranged from 5.3 to
121.1% more than uninoculated control. Two isolates (J109 and J122) had negative effect
on lateral root dry weight and a decrease in lateral root dry weight up to 26.3% was found
upon inoculation compared to uninoculated control.
68
Picture of a jar trial: Effect of inoculation with a rhizobacterium containing ACC-

deaminase on root and shoot growth of chichkpea seedlings under axenic conditions
69
4.1.3.2. Shoot growth
Inoculation with rhizobacteria containing ACC-deaminase increased the shoot
length of chickpea seedlings up to 57.4% over uninoculated control (Table 4.4). The
isolates J107, J108, J112, J118, J119 and J120 showed substantial increases in shoot
length that were 20.9 to 57.4% higher than uninoculated control. Four isolates (J4, J105,
J109 and J122) decreased the shoot length up to 8.3% as compared to control. The
remaining isolates caused maximum increase in shoot length up to 23.9% over
Increase in shoot dry weight in response to inoculation with rhizobacteria
containing ACC-deaminase was found up to 83.5% as compared to uninoculated control
(Table 4.4). The most promising increase was observed in case of inoculation with the
rhizobacteria such as J107, J108, J112, J118, J119 and J120 that ranged from 67.1 to
83.5% over control. Isolate J119 was the most effective isolate among the tested 29
isolates. However, three isolates (J10, J109 and J122) gave negative effect and decreased
the shoot dry weight up to 4.5% as compared to uninoculated control.
4.2. Effect of rhizobia on growth and nodulation of chickpea under
axenic conditions (Jar experiment)
The rhizobial isolates were tested in jars (sand culture) to evaluate their
effectiveness on growth and nodulation of chickpea seedling under axenic conditions.
4.2.1. Root growth
Rhizobial isolates were effective for promoting root and shoot growth, and
nodulation of chickpea in sand culture. The maximum increase in root length (72% more
than control) was observed where inoculation was done with isolate S9 (Table 4.5). The
70
Table 4.4: Effect of rhizobacterial inoculation on shoot growth of chickpea
(Jar experiment)
Rhizobacterial Shoot length Shoot dry weight
Isolate (cm) (g plant-1)
Control 15.60 ± 1.19 0.167 ± 0.04
J4 15.57 ± 1.06 0.177 ± 0.02
J5 17.33 ± 1.12 0.219 ± 0.07
J6 19.07 ± 1.21 0.268 ± 0.04
J7 18.50 ± 1.10 0.199 ± 0.06
J10 19.03 ± 2.01 0.163 ± 0.07
J14 19.33 ± 1.16 0.267 ± 0.02
J15 17.67 ± 0.89 0.172 ± 0.03
J16 18.53 ± 1.03 0.200 ± 0.04
J17 19.32 ± 1.24 0.252 ± 0.06
J18 18.47 ± 1.13 0.258 ± 0.07
J19 18.77 ± 0.96 0.266 ± 0.08
J24 17.93 ± 1.39 0.269 ± 0.05
J26 19.47 ± 1.41 0.236 ± 0.03
J27 18.22 ± 1.31 0.255 ± 0.04
J28 19.83 ± 1.51 0.271 ± 0.06
J41 18.10 ± 0.93 0.231 ± 0.01
J105 15.57 ± 1.03 0.238 ± 0.03
J107 18.87 ± 1.43 0.289 ± 0.04
J108 19.67 ± 1.63 0.292 ± 0.06
J109 14.30 ± 1.01 0.159 ± 0.03
J112 19.60 ± 2.11 0.274 ± 0.05
J114 18.93 ± 2.03 0.247 ± 0.06
J115 16.17 ± 1.16 0.231 ± 0.02
J117 16.31 ± 1.13 0.211 ± 0.04
J118 19.42 ± 1.89 0.280 ± 0.07
J119 24.55 ± 1.96 0.306 ± 0.08
J120 21.97 ± 1.51 0.293 ± 0.03
J122 15.30 ± 1.31 0.160 ± 0.04
J127 18.81 ± 1.41 0.221 ± 0.06
SED (Standard error of difference between two means) = Shoot length: 1.137; Shoot dry weight:
0.0293; P value at 5%
71
six top most isolates of rhizobia S4, S9, S14, S21, S27 and S32, caused increase in root
length of chickpea seedlings ranging from 55.2 to 72.2% over uninoculated control.
Three rhizobial isolates (S2, S17, and S23) had negative effect and decreased the root
length up to 8%, in comparison with uninoculated control.
Almost the same trend was observed in case of root dry weight of chickpea in
response to inoculation with rhizobia (Table 4.5). The highest root weight (0.36 g) was
recorded when plants were inoculated with S14. Next to it, isolates S9 and S21 were the
most effective, and both had similar effect on root dry weight. All other tested isolates
had positive effect on root dry weight except five isolates (S3, S17, S23, S26 and S31) as
inoculation with these isolates resulted in decreased root dry weight compared with
4.2.2. Shoot growth
The increase in shoot length was up to 77% in case of inoculation with rhizobial
isolate S14. Three isolates (S3, S17 and S23) decreased root length of chickpea seedlings
as compared to uninoculated control (Table 4.5). Rhizobial isolates S4, S9, S14, S21, S27
and S32, were the most efficient isolates which enhanced root length ranging from 49 to
77% over uninoculated control while remaining isolates also showed an increase in root
length up to 48% as compared to uninoculated control.
Results regarding shoot dry weight showed that the six most effective rhizobial
isolates (S4, S9, S14, S21, S27 and S32), gave an increase in shoot dry weight up to
78.3% as compared to uninoculated control (Table 4.5). Eight isolates gave similar
results as that of control while two isolates (S26 and S28) showed decrease in shoot dry
72
Table 4.5: Effect of rhizobial inoculation on root and shoot growth of
chickpea under axenic conditions
(Jar experiment)
Rhizobial Root length Root dry weight Shoot length Root dry weight
-1
Isolate (cm) (g plant ) (cm) (g plant-1)
Control 12.92 ± 1.06 0.21 ± 0.01 13.92 ± 1.21 0.23 ± 0.02
S1 16.00 ± 1.13 0.24 ± 0.03 17.75 ± 1.14 0.28 ± 0.05
S2 12.42 ± 1.31 0.21 ± 0.02 14.58 ± 1.03 0.23 ± 0.01
S3 13.75 ± 0.94 0.20 ± 0.02 13.83 ± 0.98 0.25 ± 0.03
S4 19.75 ± 1.59 0.31 ± 0.03 21.83 ± 1.69 0.35 ± 0.01
S5 16.33 ± 1.09 0.27 ± 0.04 19.17 ± 1.56 0.25 ± 0.02
S6 17.50 ± 1.22 0.29 ± 0.02 20.58 ± 1.23 0.26 ± 0.01
S7 15.33 ± 0.84 0.24 ± 0.03 17.25 ± 0.95 0.30 ± 0.03
S8 14.83 ± 1.03 0.26 ± 0.01 17.75 ± 0.79 0.28 ± 0.02
S9 22.25 ± 2.03 0.34 ± 0.04 22.67 ± 2.01 0.36 ± 0.03
S10 14.08 ± 1.01 0.25 ± 0.01 17.42 ± 0.93 0.23 ± 0.05
S11 13.08 ± 0.79 0.21 ± 0.02 15.92 ± 1.31 0.28 ± 0.02
S12 14.67 ± 1.11 0.28 ± 0.04 19.00 ± 1.05 0.23 ± 0.03
S13 13.67 ± 1.14 0.21 ± 0.01 16.17 ± 0.86 0.23 ± 0.01
S14 21.08 ± 1.39 0.36 ± 0.03 24.63 ± 2.36 0.39 ± 0.06
S15 14.75 ± 1.06 0.23 ± 0.02 17.92 ± 1.07 0.24 ± 0.02
S16 15.33 ± 1.31 0.27 ± 0.02 19.17 ± 1.11 0.28 ± 0.02
S17 11.92 ± 1.02 0.20 ± 0.01 12.67 ± 0.89 0.23 ± 0.02
S18 15.50 ± 0.91 0.26 ± 0.02 19.92 ± 1.21 0.25 ± 0.04
S19 14.50 ± 0.88 0.25 ± 0.03 17.75 ± 1.01 0.23 ± 0.01
S20 14.33 ± 0.79 0.22 ± 0.03 16.00 ± 1.31 0.23 ± 0.03
S21 20.75 ± 2.09 0.34 ± 0.03 20.75 ± 2.14 0.36 ± 0.05
S22 15.17 ± 1.56 0.27 ± 0.04 18.83 ± 2.01 0.24 ± 0.02
S23 12.58 ± 0.81 0.20 ± 0.01 13.25 ± 1.01 0.23 ± 0.01
S24 14.50 ± 1.15 0.22 ± 0.02 16.50 ± 0.86 0.31 ± 0.04
S25 15.50 ± 1.07 0.27 ± 0.04 18.92 ± 0.99 0.28 ± 0.03
S26 13.08 ± 0.90 0.21 ± 0.01 14.42 ± 1.02 0.22 ± 0.01
S27 20.42 ± 1.94 0.32 ± 0.03 23.00 ± 2.01 0.35 ± 0.04
S28 13.42 ± 1.02 0.24 ± 0.02 14.75 ± 1.01 0.22 ± 0.02
S29 18.42 ± 1.56 0.29 ± 0.01 21.75 ± 1.63 0.34 ± 0.03
S30 17.50 ± 1.08 0.27 ± 0.03 19.50 ± 1.29 0.26 ± 0.02
S31 13.58 ± 0.87 0.20 ± 0.01 14.33 ± 1.08 0.27 ± 0.03
S32 19.67 ± 2.04 0.31 ± 0.03 23.33 ± 1.37 0.35 ± 0.04
S33 13.83 ± 1.13 0.21 ± 0.01 14.00 ± 1.21 0.28 ± 0.01
S34 15.08 ± 1.01 0.24 ± 0.02 16.42 ± 0.96 0.25 ± 0.03
S35 14.00 ± 1.14 0.21 ± 0.03 14.67 ± 1.30 0.25 ± 0.02
0.033; Shoot length: 1.60; Shoot dry weight: 0.026; P value at 5%
73
weight (up to 11.8%) as compared to uninoculated control. Remaining rhizobial isolates
increased the shoot dry weight from 4.6 to 55.2% over uninoculated control.
4.3.3. Nodulation
The data regarding nodules per plant and nodule weight are summarized in Table
4.5. Inoculation with rhizobial isolates was highly effective in increasing number of
nodules per plant (Table 4.6). The most effective isolate was S14 among the tested
isolates, and maximum (21) nodules per plant were observed by inoculation with this
isolate. The other isolates produced up to 16 nodules per plant ranging from 3 to 16 while
no nodulation was noted in 11 isolates as well as in uninoculated control.
The results revealed that maximum nodule dry weight (0.118 g) was resulted from
inoculation with isolate S14 (Table 4.6). Other tested rhizobial isolates produced nodule
dry weight ranging from 0.004 to 0.09 g per plant except 11 isolates, which showed
similar results as observed in case of control.
4.3. Growth pouch experiment
Rhizobial isolates were also tested in growth pouches to further confirm their effect
on growth and nodulation in chickpea seedlings under axenic conditions.
4.3.1. Root growth
The results showed that inoculation with rhizobial isolates was effective in
increasing root growth of chickpea seedlings compared to uninoculated control (Table
4.7). Six isolates (S4, S9, S14, S21, S27 and S32) caused more than 50% increase in root
length as compared with uninoculated control. The other isolates showed maximum
increase up to 50% compared with control except S6, S8, S13, S16, S22 and S31. The
later six isolates had negative effect on root length as compared to uninoculated control.
74
Table 4.6: Effect of rhizobial inoculation on nodulation of chickpea under

axenic conditions
(Jar experiment)
Number of nodules Nodule dry weight
Rhizobial isolate
plant-1 (g plant-1)
Control 0.0 ± 0.00 0.000 ± 0.000
S1 5.0 ± 1.31 0.029 ± 0.004
S2 0.0 ± 0.00 0.000 ± 0.000
S3 6.0 ± 1.06 0.041 ± 0.003
S4 9.0 ± 1.56 0.056 ± 0.005
S5 0.0 ± 0.00 0.000 ± 0.000
S6 6.0 ± 1.43 0.047 ± 0.008
S7 0.0 ± 0.00 0.000 ± 0.000
S8 0.0 ± 0.00 0.000 ± 0.000
S9 12.0 ± 2.29 0.066 ± 0.009
S10 0.0 ± 0.00 0.000 ± 0.000
S11 0.0 ± 0.00 0.000 ± 0.000
S12 8.0 ± 1.09 0.049 ± 0.006
S13 0.0 ± 0.00 0.003 ± 0.001
S14 21.0 ± 3.52 0.118 ± 0.008
S15 5.0 ± 1.08 0.031 ± 0.002
S16 0.0 ± 0.00 0.000 ± 0.000
S17 0.0 ± 0.00 0.000 ± 0.000
S18 5.0 ± 101 0.029 ± 0.003
S19 5.0 ± 1.13 0.037 ± 0.002
S20 5.0 ± 0.98 0.035 ± 0.004
S21 16.0 ± 2.34 0.092 ± 0.005
S22 0.0 ± 0.00 0.000 ± 0.000
S23 3.0 ± 0.72 0.014 ± 0.002
S24 5.0 ± 0.78 0.030 ± 0.002
S25 6.0 ± 1.06 0.043 ± 0.004
S26 7.0 ± 1.21 0.038 ± 0.005
S27 15.0 ± 2.16 0.076 ± 0.009
S28 0.0 ± 0.00 0.000 ± 0.000
S29 4.0 ± 0.86 0.035 ± 0.003
S30 0.0 ± 0.00 0.000 ± 0.000
S31 7.0 ± 1.03 0.052 ± 0.004
S32 10.0 ± 1.56 0.073 ± 0.006
S33 4.0 ± 0.72 0.033 ± 0.002
S34 3.0 ± 0.72 0.026 ± 0.003
S35 5.0 ± 1.03 0.037 ± 0.004
SED (Standard error of difference between two means) = Nodules per plant: 0.865; Nodule dry
weight: 0.013; P value at 5%
75
Table 4.7: Effect of rhizobial inoculation on root and shoot growth of
chickpea under axenic conditions
(Growth pouch experiment)

Rhizobial Root length Root dry weight Shoot length Shoot dry weight
-1
Isolate (cm) (g plant ) (cm) (g plant-1)
Control 12.08 ± 1.02 0.23 ± 0.01 12.50 ± 1.30 0.26 ± 0.02
S1 13.75 ± 1.12 0.25 ± 0.03 13.92 ± 1.14 0.27 ± 0.01
S2 15.17 ± 1.06 0.26 ± 0.02 16.08 ± 1.48 0.33 ± 0.04
S3 14.33 ± 0.87 0.25 ± 0.01 16.00 ± 0.89 0.29 ± 0.03
S4 19.92 ± 1.48 0.38 ± 0.03 20.42 ± 2.08 0.43 ± 0.05
S5 14.25 ± 0.97 0.25 ± 0.02 12.75 ± 0.94 0.29 ± 0.03
S6 11.58 ± 0.69 0.24 ± 0.03 11.58 ± 1.03 0.27 ± 0.01
S7 12.17 ± 1.03 0.22 ± 0.01 12.67 ± 1.54 0.27 ± 0.03
S8 11.33 ± 1.23 0.22 ± 0.02 12.25 ± 1.20 0.26 ± 0.04
S9 22.83 ± 1.74 0.35 ± 0.03 22.50 ± 1.83 0.38 ± 0.04
S10 14.25 ± 0.79 0.27 ± 0.01 14.08 ± 1.01 0.34 ± 0.03
S11 12.14 ± 1.01 0.23 ± 0.01 16.67 ± 1.48 0.27 ± 0.04
S12 15.58 ± 1.13 0.25 ± 0.02 15.42 ± 1.13 0.29 ± 0.03
S13 11.67 ± 0.93 0.22 ± 0.02 12.83 ± 1.54 0.26 ± 0.01
S14 20.92 ± 1.32 0.43 ± 0.04 21.67 ± 1.95 0.44 ± 0.04
S15 16.08 ± 0.88 0.28 ± 0.01 14.50 ± 0.92 0.34 ± 0.03
S16 11.58 ± 1.01 0.24 ± 0.02 13.25 ± 1.23 0.28 ± 0.01
S17 10.58 ± 0.87 0.23 ± 0.01 10.75 ± 1.12 0.25 ± 0.02
S18 16.50 ± 1.05 0.26 ± 0.03 16.92 ± 1.03 0.31 ± 0.03
S19 13.92 ± 1.06 0.22 ± 0.01 13.50 ± 1.31 0.26 ± 0.02
S20 14.58 ± 0.94 0.23 ± 0.03 16.33 ± 1.24 0.28 ± 0.04
S21 19.17 ± 1.23 0.35 ± 0.02 21.17 ± 1.69 0.41 ± 0.05
S22 11.58 ± 0.79 0.23 ± 0.01 16.58 ± 0.89 0.25 ± 0.01
S23 13.33 ± 1.01 0.20 ± 0.02 11.58 ± 1.02 0.24 ± 0.01
S24 14.25 ± 1.11 0.25 ± 0.01 14.75 ± 1.14 0.30 ± 0.03
S25 16.25 ± 1.03 0.27 ± 0.01 16.25 ± 1.01 0.34 ± 0.02
S26 13.08 ± 1.07 0.23 ± 0.02 13.92 ± 1.07 0.28 ± 0.03
S27 19.50 ± 1.56 0.38 ± 0.03 19.67 ± 1.34 0.40 ± 0.05
S28 12.50 ± 1.13 0.24 ± 0.02 13.25 ± 1.43 0.29 ± 0.02
S29 18.08 ± 1.48 0.33 ± 0.04 18.33 ± 1.39 0.38 ± 0.03
S30 13.75 ± 1.00 0.23 ± 0.01 13.17 ± 0.92 0.26 ± 0.02
S31 11.08 ± 0.98 0.21 ± 0.01 10.92 ± 0.89 0.25 ± 0.01
S32 18.33 ± 1.38 0.36 ± 0.04 19.00 ± 1.70 0.40 ± 0.04
S33 13.50 ± 0.92 0.25 ± 0.01 14.83 ± 1.12 0.31 ± 0.03
S34 13.92 ± 0.89 0.23 ± 0.02 15.42 ± 1.62 0.27 ± 0.01
S35 14.25 ± 1.03 0.24 ± 0.01 14.75 ± 131 0.32 ±0.02
SED (Standard error of difference between two means) = Shoot length: 1.58; Root length: 1.71;
Shoot dry weight: 0.029; Root dry weight 0.031; P value at 5%
76
Like root length, six rhizobial isolates (S4, S9, S14, S21, S27 and S32), caused
increase in root dry weight ranging from 51.5 to 84.9% as compared with uninoculated
control (Table 4.7). Five isolates (S7, S13, S19, S23 and S31) decreased the root dry
weight up to 13.5%, as compared to uninoculated control. Rest of the isolates had neutral
(no effect) to positive effect (up to 42.8% increase in root dry weight) compared to
control.
4.3.2. Shoot growth
In case of shoot length, almost the same trend was observed and once again six
isolates of rhizobia (S4, S9, S14, S21, S27 and S32), were able to increase shoot length
more than 50% (52 to 80%) as compared to control (Table 4.7). Five isolates (S6, S8,
S17, S23 and S31) had negative effect and their inoculation resulted in up to 14%
decrease in shoot length as compared to uninoculated control. Up to 46.7% increase in
shoot length was recorded in response to inoculation with rest of the isolates.
The maximum increase in shoot dry weight in to response rhizobial inoculation
was up to 67.1% as compared to uninoculated control (Table 4.7). The S14 isolate was
most effective in increasing shoot dry weight. Four isolates (S8, S13, S19 and S30) gave
the same effect on shoot dry weight as that of control and four isolates (S17, S22, S23
and S31) decreased the shoot dry weight up to 6.9%, as compared to uninoculated
control.
4.3.3. Nodulation
The data regarding number of nodules showed that most of the rhizobial isolates
were able to produce nodules in chickpea plants (Table 4.8). Out of 35 isolates, 22
isolates produced nodules ranging from 3 to 19 per plant, as compared to no nodulation in
77
Table 4.8: Effect of rhizobial inoculation on nodulation of chickpea under
axenic conditions

(Means of three replicates)
Rhizobial isolate
plant-1 (g plant-1)
Control 0.0 ± 0.00 0.000 ± 0.000
S1 3.0 ± 0.62 0.021 ± 0.001
S2 0.0 ± 0.00 0.000 ± 0.000
S3 5.0 ± 0.72 0.032 ± 0.002
S4 8.0 ± 1.44 0.051 ± 0.003
S5 0.0 ± 0.00 0.000 ± 0.000
S6 6.0 ± 0.72 0.041 ± 0.005
S7 0.0 ± 0.00 0.000 ± 0.000
S8 0.0 ± 0.00 0.000 ± 0.000
S9 9.0 ± 1.02 0.062 ± 0.007
S10 0.0 ± 0.00 0.000 ± 0.000
S11 0.0 ± 0.00 0.000 ± 0.000
S12 7.0 ± 0.89 0.042 ± 0.002
S13 0.0 ± 0.00 0.000 ± 0.000
S14 19.0 ± 2.31 0.111 ± 0.008
S15 4.0 ± 0.51 0.021 ± 0.001
S16 0.0 ± 0.00 0.000 ± 0.000
S17 0.0 ± 0.00 0.000 ± 0.000
S18 3.0 ± 0.39 0.024 ± 0.003
S19 5.0 ± 0.62 0.035 ± 0.004
S20 4.0 ± 0.37 0.031 ± 0.002
S21 13.0 ± 1.86 0.082 ± 0.003
S22 0.0 ± 0.00 0.000 ± 0.000
S23 0.0 ± 0.00 0.000 ± 0.000
S24 3.0 ± 0.31 0.023 ± 0.003
S25 6.0 ± 0.61 0.030 ± 0.004
S26 6.0 ± 0.53 0.032 ± 0.005
S27 11.0 ± 1.05 0.071± 0.006
S28 0.0 ± 0.00 0.000 ± 0.000
S29 4.0 ± 0.43 0.031 ± 0.004
S30 0.0 ± 0.00 0.000 ± 0.000
S31 7.0 ± 1.01 0.041 ± 0.003
S32 12.0 ± 1.46 0.063 ± 0.008
S33 4.0 ± 0.19 0.022 ± 0.001
S34 3.0 ± 0.54 0.021 ± 0.003
S35 5.0 ± 0.98 0.033 ± 0.002
78
Control S14
[Uninoculated]
Picture of a pouch trial: Effect of inoculation with rhizobia on nodulation of chichkpea

79
uninoculated control while, no nodulation was recorded in case of inoculation with 13
rhizobial isolates.
Like nodule number, there was also a variation in nodule dry weight of chickpea
in response to inoculation with different rhizobial isolates and most of the isolates were
highly effective in producing nodule dry weight and up to 0.111 g nodule dry weight was
observed in case of inoculation and S14 was the most effective among the test isolates.
4.4. Screening effective isolates of rhizobia and rhizobacteria for co-
inoculation of chickpea
80
Based upon inoculation effects on growth and nodulation under axenic conditions,
the six most effective isolates each from rhizobia (S4, S9, S14, S21, S27 and S32) and
rhizobacteria (J107, J108, J112, J118, J119 and J120) were selected to further evaluate
their potential in combination (co-inoculation) for promoting growth and nodulation of
chickpea. Rhizobial isolates were selected preferably on the basis of their efficiency to
improve nodulation while rhizobacterial isolates containing ACC-deaminase were
selected on the basis of root growth promotion and changes in root architecture induced
by these rhizobacteria. Selected isolates of rhizobia and rhizobacteria were evaluated in
all possible combinations for promting growth and nodulation of chickpea by conducting
growth pouch and jar experiments.
4.4.1. Jar experiment
All possible combinations of selected rhizobia and rhizobacteria were tested in
jars (sand culture) to assess their efficacy on growth of seedlings and nodulation in
chickpea under axenic conditions. The results are given below.
The increase in root length in response to co-inoculation was up to 85% as
compared to uninoculated control (Table 4.9). Two bacterial combinations (J119 x S14
and J120 x S4), were the most effective and increase in root length ranged from 80.8 to
85% over uninoculated control. Three combinations (J107 x S9, J108 x S21 and J120 x
S21) had negative effect on root length (up to 6.6%), in comparision with uninoculated
control while, rest of the combinations also increased root length up to 64.5% compared
with uninoculated control.
81
The maximum increase (78.8% higher than control) in root dry weight of
chickpea seedlings was observed in response to co-inoculation with J120 x S4 (Table
4.9). Overall, 33 combinations had positive effect on root dry weight that caused increase
ranging from 2.9 to 78.8% over uninoculated control. Three combinations (J107 x S9,
J108 x S21 and J120 x S21) had negative effect on root dry weight and decrease in root
dry weight up to 12% was observed upon co-inoculation as compared to uninoculated
control.
Co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase
increased the shoot length of chickpea seedlings up to 92.7% compared to uninoculated
control (Table 4.9). The bacterial combinations (J119 x S14 and J120 x S4) showed the
most promising increase in shoot lengths that was 82.9 to 92.7% higher than uninoculated
control. Rest of the test combinations also increased shoot length up to 70.6% as
compared to uninoculated control while bacterial combination (J108 x S21) exhibited the
least increase in shoot length over uninoculated control.
Table 4.9: Effect of co-inoculation with rhizobacteria and rhizobia on root
and shoot growth of chickpea seedlings under axenic conditions
(Jar experiment)
Root length Root weight Shoot length Shoot dry
Combinations
(cm) dry (g) (cm) weight (g)
Control 13.83 ± 1.12 0.22 ± 0.01 13.58 ± 1.01 0.23 ± 0.02
J107 x S4 18.25 ± 0.89 0.23 ± 0.01 16.67 ± 1.33 0.24 ± 0.02
J107 x S9 12.92 ± 0.96 0.21 ± 0.02 14.33 ± 0.96 0.23 ± 0.04
J107 x S14 16.58 ± 1.24 0.25 ± 0.01 17.50 ± 1.18 0.26 ± 0.03
J107 x S21 18.33 ± 1.03 0.24 ± 0.03 18.25 ± 1.53 0.27 ± 0.02
J107 x S27 17.33 ± 1.52 0.23 ± 0.01 15.67 ± 1.03 0.25 ± 0.01
J107 x S32 17.67 ± 1.06 0.24 ± 0.01 15.17 ± 1.21 0.26 ± 0.06
J108 x S4 17.17 ± 0.83 0.25 ± 0.03 16.58 ± 0.76 0.27 ± 0.01
82
J108 x S9 19.83 ± 1.57 0.25 ± 0.04 18.92 ± 0.94 0.26 ± 0.05

J108 x S14 19.50 ± 0.86 0.23 ± 0.01 19.33 ± 1.21 0.24 ± 0.03
J108 x S21 13.58 ± 0.98 0.20 ± 0.02 13.92 ± 1.26 0.23 ± 0.02
J108 x S27 17.67 ± 2.05 0.25 ± 0.03 18.42 ± 1.07 0.27 ± 0.04
J108 x S32 21.25 ± 1.69 0.33 ± 0.03 22.25 ± 1.74 0.38 ± 0.05
J112 x S4 22.75 ± 1.63 0.36 ± 0.04 23.17 ± 0.95 0.37 ± 0.02
J112 x S9 18.75 ± 1.13 0.30 ± 0.03 18.67 ± 1.23 0.35 ± 0.05
J112 x S14 19.08 ± 0.96 0.25 ± 0.02 20.92 ± 1.27 0.27 ± 0.01
J112 x S21 17.75 ± 1.21 0.29 ± 0.02 17.58 ± 1.06 0.31 ± 0.02
J112 x S27 16.83 ± 1.08 0.26 ± 0.01 15.83 ± 1.34 0.28 ± 0.03
J112 x S32 17.50 ± 1.12 0.28 ± 0.03 18.17 ± 0.83 0.31 ± 0.02
J118 x S4 16.25 ± 0.89 0.27 ± 0.01 17.33 ± 1.19 0.29 ± 0.03
J118 x S9 18.25 ± 1.02 0.26 ± 0.02 20.08 ± 1.16 0.27 ± 0.04
J118 x S14 17.75 ± 0.79 0.25 ± 0.04 19.33 ± 1.06 0.27 ± 0.01
J118 x S21 16.58 ± 0.88 0.26 ± 0.02 17.17 ± 0.85 0.28 ± 0.04
J118 x S27 17.75 ± 0.78 0.25 ± 0.03 16.17 ± 1.20 0.28 ± 0.02
J118 x S32 16.58 ± 1.65 0.27 ± 0.01 15.75 ± 1.32 0.29 ± 0.03
J119 x S4 19.08 ± 1.06 0.30 ± 0.03 19.08 ± 1.53 0.34 ± 0.04
J119 x S9 21.42 ± 1.09 0.32 ± 0.01 20.42 ± 1.14 0.36 ± 0.03
J119 x S14 25.58 ± 1.68 0.39 ± 0.03 24.83 ± 1.19 0.39 ± 0.04
J119 x S21 18.33 ± 1.59 0.29 ± 0.02 19.00 ± 1.35 0.31 ± 0.03
J119 x S27 18.75 ± 1.04 0.30 ± 0.01 20.75 ± 1.12 0.32 ± 0.03
J119 x S32 17.58 ± 0.89 0.26 ± 0.01 18.00 ± 0.94 0.28 ± 0.01
J120 x S4 25.00 ± 1.83 0.37 ± 0.04 26.17 ± 1.49 0.44 ± 0.06
J120 x S9 19.17 ± 1.29 0.27 ± 0.02 18.33 ± 1.29 0.29 ± 0.05
J120 x S14 19.42 ± 1.17 0.28 ± 0.02 19.83 ± 1.06 0.30 ± 0.03
J120 x S21 13.33 ± 0.78 0.19 ± 0.01 14.83 ± 0.72 0.24 ± 0.04
J120 x S27 19.00 ± 1.21 0.27 ± 0.03 19.33 ± 1.09 0.29 ± 0.02
J120 x S32 18.33 ± 1.34 0.25 ± 0.02 17.58 ± 1.26 0.28 ± 0.03
*SED (Standard error of difference between two means) = Shoot length: 1.53; Root length: 1.19;
Shoot dry weight: 0.029; Root dry weight: 0.021; P value at 5%
The maximum increase in shoot dry weight in response to co-inoculation with
rhizobial and rhizobacterial isolates was up to 89.9% as compared to uninoculated control
(Table 4.8). The bacterial combinations J119 x S14 and J120 x S4 were most effective in
increasing shoot dry weight which ranged from 70.7 to 89.9% over uninoculated control.
Two combinations J107 x S9 and J108 x S21 decreased the shoot dry weight up to 4.8%,
as compared to uninoculated control. While remaining combinations also increased shoot
dry weight up to 58.6% as compared to uninoculated control.
4.4.2.3. Nodulation
83
Co-inoculation with bacterial combinations was highly effective in increasing
number of nodules per plant (Table 4.10). The most effective combinations among the
tested combinations were J119 x S14 and J120 x S4, and maximum nodules per plant
(29) were observed by co-inoculation with these combinations. The other combinations
produced maximum up to 24 nodules per plant while, no nodulation was noted in seven
combinations (J107 x S9, J107 x S32, J108 x S21, J112 x S32, J119 x S14, J120 x S21
and J120 x S27), as well as in uninoculated control.
The data regarding nodule dry weight revealed that maximum nodule dry weight
(0.151 g) was recorded in response to co-inoculation with bacterial combinations J120 x
S14. Other tested combinations produced nodule dry weight ranging 0.023 to 0.113 g per
plant, while 7 combinations gave similar results as that of uninoculated control.
4.4.2. Growth pouch experiment
The selected bacterial isolates (rhizobia and rhizobacteria) were also tested in
growth pouches to further confirm their effect on growth and nodulation in chickpea
under axenic conditions.
Table 4.10: Effect of co-inoculation with rhizobacteria and rhizobia on

(Jar experiment)
Combinations Number of nodules per Nodule dry weight
plant (g plant-1)
Control 0.0 ± 0.00 0.000 ± 0.000
J107 x S4 6.3 ± 1.41 0.033 ± 0.004
J107 x S9 0.0 ± 0.00 0.000 ± 0.000
J107 x S14 5.0 ± 0.89 0.025 ± 0.003
J107 x S21 7.0 ± 2.00 0.032 ± 0.007
J107 x S27 7.3 ± 1.29 0.042 ± 0.005
J107 x S32 0.0 ± 0.00 0.000 ± 0.000
J108 x S4 6.3 ± 0.59 0.031 ± 0.006
84
J108 x S9 6.0 ± 1.03 0.029 ± 0.007

J108 x S14 8.3 ± 1.48 0.039 ± 0.005
J108 x S21 0.0 ± 0.00 0.000 ± 0.000
J108 x S27 6.0 ± 0.49 0.029 ± 0.004
J108 x S32 17.8 ± 2.12 0.113 ± 0.006
J112 x S4 20.3 ± 2.59 0.107 ± 0.009
J112 x S9 9.3 ± 0.51 0.045 ± 0.006
J112 x S14 6.7 ± 1.01 0.027 ± 0.005
J112 x S21 16 ± 0.98 0.040 ± 0.004
J112 x S27 9.0 ± 1.05 0.041 ± 0.003
J112 x S32 0.0 ± 0.00 0.000 ± 0.000
J118 x S4 11.7 ± 1.67 0.056 ± 0.006
J118 x S9 13 ± 1.43 0.031 ± 0.002
J118 x S14 7.7 ± 1.11 0.032 ± 0.006
J118 x S21 9.3 ± 1.29 0.044 ± 0.003
J118 x S27 6.0 ± 1.09 0.027 ± 0.002
J118 x S32 5.3 ± 0.85 0.026 ± 0.005
J119 x S4 0.0 ± 0.00 0.000 ± 0.000
J119 x S9 9.3 ± 1.27 0.043 ± 0.003
J119 x S14 29.0 ± 3.78 0.149 ± 0.006
J119 x S21 9.3 ± 1.21 0.042 ± 0.004
J119 x S27 7.0 ± 1.01 0.032 ± 0.003
J119 x S32 9.0 ± 1.12 0.045 ± 0.006
J120 x S4 27.0 ± 1.04 0.151 ± 0.013
J120 x S9 24.0 ± 2.79 0.078 ± 0.005
J120 x S14 8.0 ± 1.08 0.041 ± 0.003
J120 x S21 0.0 ± 0.00 0.000 ± 0.000
J120 x S27 0.0 ± 0.00 0.000 ± 0.000
J120 x S32 5.3 ± 1.40 0.023 ± 0.002
weight: 0.019; P value at 5%.
resulted in tremendous increase in root length of chickpea seedlings as compared to
uninoculated control (Table 4.11). The increase in root lenght of chickpea seedlings in
response to inoculation with various combinations ranged from 15.5 to 80% except four
combinations (J107 x S9, J108 x S21, J118 x S32 and J120 x S21) which showed
decrease in root length up to 10.4%, when compared with uninoculated control.
85
Combinations J119 x S14 and J120 x S4 were the most effective bacterial co-inoculant
isolates.
The maximum root dry weight was recorded in response to co-inoculation with
J119 x S14 followed by J120 x S4 which was 91% greater than uninoculated control
(Table 4.11). All other rhizobial plus rhizobacterial isolates also increased root dry
weight except J107 x S9, J108 x S21, J108 x S14 and J120 x S21 which had negative
effect on root dry weight compared to control.
Maximum increase in shoot length was recorded up to 89.2% in response to co-
inoculation with different bacterial combinations while two combinations (J108 x S21
and J120 x S21) decreased root length of chickpea seedlings up to 5.4%, as compared to
uninoculated control (Table 4.11). Microbial combinations J119 x S14 and J120 x S4
were the most effective combinations which promoted shoot length from 83.7 to 89.2%
over uninoculated control. Rest of the test combinations also increased the shoot length
up to 75.5% as compared to uninoculated control.
Table 4.11: Effect of co-inoculation with rhizobacteria and rhizobia on root
and shoot growth of chickpea seedlings under axenic conditions

Root length Root dry weight Shoot length Shoot dry weight
Combinations -1
(cm) (g plant ) (cm) (g plant-1)
Control 12.92 ± 1.33 0.18 ± 0.02 12.25 ± 1.47 0.19 ± 0.03
J107 x S4 16.92 ± 0.96 0.20 ± 0.02 15.33 ± 1.89 0.21 ± 0.05
J107 x S9 11.58 ± 1.09 0.17 ± 0.01 12.50 ± 0.54 0.18 ± 0.03
J107 x S14 15.25 ± 1.24 0.22 ± 0.02 16.17 ± 0.92 0.22 ± 0.02
J107 x S21 17.00 ± 0.87 0.21 ± 0.04 16.92 ± 1.88 0.23 ± 0.01
J107 x S27 16.00 ± 1.45 0.19 ± 0.03 14.33 ± 1.37 0.23 ± 0.03
86
J107 x S32 16.33 ± 1.02 0.21 ± 0.02 13.83 ± 1.54 0.23 ± 0.02
J108 x S4 19.42 ± 1.27 0.22 ± 0.04 15.25 ± 0.53 0.27 ± 0.01
J108 x S9 18.50 ± 1.59 0.22 ± 0.01 17.58 ± 0.59 0.23 ± 0.05
J108 x S14 18.17 ± 0.92 0.18 ± 0.02 18.00 ± 1.62 0.21 ± 0.02
J108 x S21 12.25 ± 1.07 0.17 ± 0.01 11.58 ± 1.36 0.17 ± 0.01
J108 x S27 17.67 ± 1.99 0.22 ± 0.02 17.08 ± 0.78 0.23 ± 0.06
J108 x S32 20.08 ± 1.56 0.30 ± 0.05 20.92 ± 2.03 0.32 ± 0.04
J112 x S4 21.00 ± 1.16 0.31 ± 0.04 21.50 ± 1.61 0.33 ± 0.03
J112 x S9 17.42 ± 0.93 0.27 ± 0.02 17.33 ± 0.07 0.27 ± 0.04
J112 x S14 17.75 ± 1.31 0.22 ± 0.03 19.58 ± 1.06 0.23 ± 0.05
J112 x S21 16.42 ± 0.73 0.26 ± 0.01 16.25 ±1.35 0.27 ± 0.02
J112 x S27 16.92 ± 0.19 0.23 ± 0.02 14.50 ± 1.42 0.24 ± 0.03
J112 x S32 16.17 ± 1.02 0.25 ± 0.03 16.83 ± 0.98 0.26 ± 0.02
J118 x S4 14.92 ± 1.15 0.24 ± 0.04 16.00 ± 1.01 0.25 ± 0.03
J118 x S9 16.92 ± 0.95 0.23 ± 0.02 18.75 ± 1.66 0.24 ± 0.04
J118 x S14 16.42 ± 1.03 0.22 ± 0.01 18.00 ± 0.83 0.23 ± 0.01
J118 x S21 15.25 ± 0.94 0.23 ± 0.02 15.83 ± 0.81 0.24 ± 0.01
J118 x S27 16.42 ± 0.72 0.22 ± 0.01 14.83 ± 1.42 0.24 ± 0.04
J118 x S32 12.58 ± 1.12 0.24 ± 0.03 14.42 ± 0.72 0.25 ± 0.03
J119 x S4 17.75 ± 0.94 0.27 ± 0.04 17.75 ± 1.43 0.29 ± 0.02
J119 x S9 19.33 ± 1.83 0.29 ± 0.03 19.08 ± 1.65 0.30 ± 0.04
J119 x S14 23.25 ± 1.30 0.34 ± 0.05 22.50 ± 1.14 0.35 ± 0.03
J119 x S21 17.00 ± 1.12 0.26 ± 0.02 17.67 ± 1.63 0.29 ± 0.01
J119 x S27 17.42 ± 0.98 0.27 ± 0.03 19.42 ± 1.49 0.25 ± 0.01
J119 x S32 16.25 ± 1.36 0.23 ± 0.01 16.67 ± 1.07 0.25 ± 0.02
J120 x S4 22.08 ± 1.33 0.32 ± 0.03 23.17 ± 1.24 0.37 ± 0.03
J120 x S9 17.83 ± 0.89 0.24 ± 0.02 17.00 ± 1.59 0.33 ± 0.04
J120 x S14 18.08 ± 1.44 0.25 ± 0.03 18.50 ± 1.13 0.18 ± 0.02
J120 x S21 12.00 ± 1.19 0.16 ± 0.01 11.67 ± 0.69 0.25 ± 0.04
J120 x S27 17.67 ± 1.02 0.24 ± 0.03 18.00 ± 0.78 0.27 ± 0.04
J120 x S32 18.75 ± 2.13 0.22 ± 0.02 16.25 ± 1.43 0.35 ± 0.05
0.019; Shoot length: 1.69; Shoot dry weight: 0.026; P value at 5%
increased the shoot dry weight from 8.12 to 92.6% but three combinations (J107 x S9,
J108 x S21 and J120 x S21) decreased shoot dry weight up to 9.7%, as compared to
uninoculated control (Table 4.11). Maximum increase in shoot dry weight was obtained
in response to co-inoculation with combinations J119 x S14 which was 92.6% more than
uninoculated control. While remaining test combinations also increased shoot dry weight
up to 73.8% over control.
87
4.4.2.3. Nodulation
The results about number of nodules revealed that most of the bacterial
combinations were capable to produce nodules in chickpea plants (Table 4.12). Out of 36
combinations, 29 produced nodules rangings from 4 to 26 per plant, as compared to no
nodulation in uninoculated control while, no nodulation was recorded in case of
inoculation with 7 combinations.
Like number of nodules, there was also diversity in nodule dry weight of chickpea
in response to inoculation with different combinations of rhizobial and rhizobacterial
isolates and most of the combinations were highly effective in producing nodule dry
weight. Up to 0.129 g nodule dry weight was observed in case of inoculation with
bacterial combination J119 x S14.
4.5. Effect of inoculation and co-inoculation on nodulation, growth and
yield of chickpea under natural conditions
Based on the ability of inocula to promote growth and nodulation under axenic
conditions, two isolates each from from rhizobia and rhizobacteria were selected and
Table 4.12: Effect of co-inoculation with rhizobacteria and rhizobia on

Combinations
plant-1 (g plant-1)
Control 0.00 ± 0.00 0.000 ± 0.000
J107 x S4 5.67 ± 1.36 0.029 ± 0.007
J107 x S9 0.00 ± 0.00 0.000 ± 0.000
J107 x S14 4.0 ± 0.72 0.014 ± 0.002
88
J107 x S21 6.33 ± 1.66 0.030 ± 0.006

J107 x S27 6.67 ± 0.89 0.035 ± 0.014
J107 x S32 0.00 ± 0.00 0.000 ± 0.000
J108 x S4 0.00 ± 0.00 0.000 ± 0.000
J108 x S9 6.00 ± 1.02 0.028 ± 0.009
J108 x S14 7.33 ± 1.10 0.023 ± 0.005
J108 x S21 0.00 ± 0.00 0.000 ± 0.000
J108 x S27 5.67 ± 0.89 0.034 ± 0.004
J108 x S32 16.78 ± 1.49 0.110 ± 0.012
J112 x S4 19.33 ± 2.13 0.108 ± 0.016
J112 x S9 8.33 ± 1.19 0.034 ± 0.005
J112 x S14 0.00 ± 0.00 0.000 ± 0.000
J112 x S21 7.67 ± 0.98 0.041 ± 0.007
J112 x S27 8.00 ± 1.70 0.044 ± 0.004
J112 x S32 0.00 ± 0.00 0.000 ± 0.000
J118 x S4 10.33 ± 1.29 0.051 ± 0.005
J118 x S9 5.67 ± 1.44 0.025 ± 0.007
J118 x S14 0.00 ± 0.00 0.000 ± 0.000
J118 x S21 8.67 ± 1.26 0.051 ± 0.009
J118 x S27 5.33 ± 0.94 0.021 ± 0.003
J118 x S32 5.00 ± 1.19 0.027 ± 0.008
J119 x S4 0.00 ± 0.00 0.000 ± 0.000
J119 x S9 9.33 ± 1.31 0.048 ± 0.081
J119 x S14 26.0 ± 303 0.129 ± 0.089
J119 x S21 8.67 ± 1.19 0.040 ± 0.002
J119 x S27 7.00 ± 0.96 0.042 ± 0.006
J119 x S32 8.00 ± 1.43 0.063 ± 0.012
J120 x S4 24.67 ± 2.19 0.121 ± 0.049
J120 x S9 13.00 ± 1.41 0.050 ± 0.001
J120 x S14 7.33 ± 0.76 0.036 ± 0.005
J120 x S21 0.00 ± 0.00 0.000 ± 0.000
J120 x S27 0.00 ± 0.00 0.000 ± 0.000
J120 x S32 4.67 ± 0.72 0.022 ± 0.006
Co-inoculation picture [nodulation]
89
further evaluated for their potential to promote growth, yield and nodulation of chickpea
under natural conditions by conducting pot and field experiments.
4.5.1. Pot experiments
Pot experiments were conducted in the net house to assess the effectiveness of
selected rhizobacteria (J119 and J120) as well as rhizobia (S4 and S14) for improving
growth, yield and nodulation of chickpea in the presence and absence compost enriched
with P. The results are described in the following section.
4.5.1.1. Plant height
Inoculation had significant effect on plant height of chickpea over uninoculated
control (Table 4.13). Although single inoculation with the four isolates caused a
significant increases in the plant height over uninoculated control both in the presence
and absence of P-enriched compost; they varied significantly with each other. Up to
24.2% increase in plant height observed in case of single inoculation as compared with
control; however, effect was more pronounced (45.7% higher than control) when
inoculated plants were grown in the soil amended with P-enriched. Co-inoculation of
chickpea with rhizobial isolate S14 plus rhizobacteria containing ACC-deaminase J120
resulted in highly significant positive effect as 73.4% increase in plant height was
recorded compared to uninoculated control. In case of P-enriched compost, inoculation
with all the four combinations of rhizobia and rhizobacteria resulted in significant
increase (73.4%) in plant height as compared to uninoculated control. Three
combinations J119 x S4, J119 x S14 and J120 x S14 also differed significantly from the
effects of single inoculation.
90
Table 4.13: Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on
growth and yield of chickpea in presence and absence of P-enriched compost
(1st year pot trial)

(Average of three replications)
Plant height Fresh biomass Number of pods Grain yield 100-grain weight
Treatments (cm) (g pot-1) plant-1 (g pot-1) (g)
Without With Without With Without With Without With Without With
compost compost compost compost compost compost compost compost compost compost
Control 29.1 h 31.0 h 41.2 g 44.9 g 50 i 56 h 7.9 m 8.8 l 31.2 h 32.6 g
J119 37.9 f-g 44.3 b-d 49.7f 56.1 c-e 64 g 74 bd 10.0 jk 11.6 gh 34.9 f 35.5 c-f
J120 36.5 g 43.5 c-e 50.8ef 56.6 cd 62 g 76 bd 9.8 k 12.3 d-f 35.3 ef 35.7 b-f
S4 36.6 g 41.4 d-g 50.4 f 54.3 d-f 67 f-g 73 ce 9.6 k 12.5 ce 35.2 ef 36.0 b-f
S14 37.2 g 43.0 d-f 51.9 d-f 57.0 cd 66 f-g 79 a-c 10.1 jk 12.1 e-g 34.7 f 36.6 a-e
J119 x S4 38.8 e-g 49.1 ab 54.7 d-f 60.3 bc 68 e-g 80 ab 10.4 j 12.8 b-d 35.4 d-f 37.2 ab
J119 x S14 38.3 e-g 51.8 a 53.9 d-f 65.1 ab 66 f-g 84 a 11.0 i 13.4 a 36.9 a-c 36.9 a-c
J120 x S4 38.5 e-g 48.4 a-c 54.6 d-f 66.6 a 77 bc 78 bc 11.3 hi 13.1 ab 35.2 e-f 36.8 a-d
J120 x S14 43.3 d-f 50.3 a 55.1 d-f 62.0 ab 70 d-f 79 ac 11.8 f-g 12.9 ac 35.3 e-f 37.6 a
LSD Value 4.730 4.728 5.705 0.494 1.283
Values sharing similar letter (s) in a column are non-significant at P< 0.05, according to Duncan’s multiple range test
J: Rhizobacterial isolate
S: Rhizobia isolate
91
4.5.1.2. Fresh biomass
All inoculation/co-inoculation treatments significantly increased fresh biomass
both in the presence and absence of P-enriched compost (Table 4.13). The maximum
increase of 61.9% in fresh biomass was observed in case of co-inoculation with J120 x
S4, followed by J119 x S4, J119 x S14, and J120 x S 14 along with P-enriched compost
which increased fresh biomass ranging from 46.6 to 58.1% compared to uninoculated
control. In the absence of P-enriched compost, increase in fresh biomass was recorded up
to 33.8% due to inoculation of J120 x S 14 compared to control. In case of single
inoculation, rhizobial isolate S14 was the most effective among rhizobia and
rhizobacteria both in the compost amended and unamended soils.
4.5.1.3. Number of pods per plant
Maximum number of pods per plant were recorded when rhizobial isolate S4 was
applied in co-inoculation with rhizobacteria J120, there was 54% increase in number of
pods over uninoculated control (Table 4.12). It was followed by co-inoculation with J120
x S4 which showed an increase of 40% in number of pods per plant over control. Other
treatments where bacteria were applied either alone or in combinations increased number
of pods per plant up to 34% over uninoculated control. Inoculation/co-inoculation with P-
enriched compost yielded highest increase in number of pods per plant which was 68%
greater than control. Overall, inoculation/co-inoculation treatments showed better
performance in the presence of P-enriched compost than the soil not amended with P-
enriched compost.
92
Picture: Effect of inoculation plus P-enriched compost on growth and number of pods of
chickpea under pot conditions
93
4.5.1.4. Grain yield
Both single and co-inoculation treatments significantly affected grain yield
compared to control (Table 4.13). The co-inoculation with J119 x S14 caused 38.5%
more grain yield over control, however, it was raised up to 68.4% when inoculation was
tested in the presence of P-enriched compost. Increase in grain yield over respective
control ranged from 21.4 to 56.8% in response to all other inoculation/co-inoculation
treatments.
4.5.1.5. 100-grain weight
In case of 100-grain weight of chickpea, all inoculation/co-inoculation treatments
showed significant increases in 100-grain weight over control (Table 4.13).
Inoculation/co-inoculation caused a maximum increase in 100-grain weight of 18.3%
over uninoculated control. But inoculation/co-inoculation along with P-enriched compost
yielded highest increase of 20.4% in 100-grain weight, followed by 19% increase with
co-inoculation J119 x S4 in the presence of P-enriched compost over uninoculated
control. Rest of the treatments also showed significant increase in 100-grain weight
ranged from 13.7 to 18.3% compared to uninoculated control.
4.5.1.6. Number of nodules per plant
All the inoculation/co-inoculation treatments showed an increase in number of
nodules per plant significantly over uninoculated control both in absence and presence of
P-enriched compost (Table 4.14). The maximum increase (up to 72.7%) in number of
nodules per plant was observed in response to co-inoculation with J120 x S4 and J120 x
S14 over control but this increase was reached up to 93.9% when the same inoculants
94
Table 4.14: Effect of co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on nodulation and
root growth of chickpea in presence and absence of P-enriched compost

Number of nodules Nodule dry weight Root length Root dry weight
Treatments plant-1 plant-1 (cm) (g plant-1)
Without With Without With Without With Without With
compost compost compost compost compost compost compost compost
Control 33 h 36 h 0.22 j 0.25 i 18.4 g 19.8 f-g 1.8j 2.0 i
J119 49 ef 51 ef 0.35 g-h 0.41 cd 24.8 c-e 26.2 bc 2.1 gh 2.2 g
J120 45 g 52 d-f 0.34 h 0.38 e 25.3 cd 25.4 cd 2.2 gh 2.5 d
S4 50 ef 50 ef 0.35 f-g 0.39 d 22.6 d-f 24.6 c-e 2.1 gh 2.4 de
S14 48 f-g 53 de 0.37 e-g 0.42 c 20.5 f-g 24.8 c-e 2.1 gh 2.4 de
J119 x S4 56 bc 59 bc 0.36 e-g 0.44 ab 29.1 ab 29.9 a 2.4 de 2.7 bc
J119 x S14 55 cd 64 a 0.35 g-h 0.46 a 28.8 ab 31.3 a 2.4 de 2.8 b
J120 x S4 57 bc 59 b 0.38 e-f 0.44 ab 20.1 f-g 30.2 a 2.3 ef 2.9 b
J120 x S14 57 bc 64 a 0.40 cd 0.43 b 22.0 ef 30.4 a 2.6 c 3.0 a
LSD Value 3.417 0.0165 2.848 0.0741
Values sharing similar letter (s) in a column are non-significant at P< 0.05, according to Duncan’s multiple range test.
S: Rhizobia isolate
95
were applied in soil amended with P-enriched compost. In case of sole application,
highest increase in number of nodules was recorded up to 51.5% due to inoculation with
S4 over control. The increase in number of nodules per plant was up to 60.6% when
isolate S14 was used with P-enriched compost, in comparison with uninoculated control.
4.5.1.7. Nodule dry weight
The data regarding nodule dry weight revealed highest increase was up to 70.7%
where seeds were inoculated with S14 while in the presence of P-enriched compost; it
was 90.7% greater than uninoculated control (Table 4.14). Next effective isolate was
J119 that produced nodule dry weight up to 88.4% over control when tested in
combination with P-enriched. But in case of co-inoculation, maximum increase in nodule
dry weight was 84.7% with J120 x S14 over control and it was followed by treatments
J120 x S4 (72.6%). Treatments J119 x S4 and J119 x S14 also showed significant
increase in nodule dry weight (69.3 and 62.8% respectively) over uninoculated control.
When these treatments were tested with P-enriched compost, the maximum increase in
nodule dry weight was recorded up to 113% in response to co-inoculation with J119 x
S14 plus P-enriched compost, as compared with control.
4.5.1.8. Root length
The highest increase of 69.7% in root length was recorded in response to co-
inoculation with J119 x S14 in the presence of enriched compost compared with
respective uninoculated control (Table 4.14). Co-inoculation treatments such as J119 x
S4, J120 x S4, and J120 x S14 caused up to 65.1% increases in root length over
uninoculated control. In case of inoculation treatments, highest increase in root growth
was recorded up to 42.1% over uninoculated control in response to inoculation with J119
96
Un-inoculated control vs J119 x S14 + P-enriched compost
Picture: Effect of co-inoculation with J119 x S14 on nodulation of chickpea under pot
conditions
97
plus P-enriched compost. But rest of the inoculation treatments also showed significant
increasing effect over control and that increase in root length ranged from 11.3 to 38%
over control.
4.5.1.9. Root dry weight
A significant increase in the root dry weight over uninoculated control was
observed in response to inoculation (Table 4.14). In case of single inoculation, maximum
increase in root dry weight was recorded up to 20.7% due to inoculation with J120 which
was increased up to 34.1% when inoculation was tested in the presence of P-enriched
compost. In case of co-inoculation, maximum increase (34.6%) in root dry weight was
recorded up to 34.63% in case of co-inoculation with J120 x S14, over control and this
increase was further enhanced up to 67% over control when applied along with P-
enriched compost. Remaining co-inoculation treatments J119 x S4, J119 x S14 and J120
x S4, also showed significant increases in the root dry weight in the presence of P-
enriched compost that ranged from 47.5 to 59.8%, over uninoculated control.
4.5.1.10. Nitrogen content in grain
In case of sole inoculation, maximum nitrogen content in grain was observed in
case of inoculation with S14 (8.9% increase over control), followed by J120 which
showed an increase of 6.5% over control (Table 4.15). When inoculation was tested in
the presence of P-enriched compost then a significant (29.6%) in N content was observed
when S4 was applied. While the remaining inoculation treatments plus P-enriched
compost also resulted in an increase of up to 25.8% in N content uninoculated control.
In case of co-inoculation, maximum increase (30.8%) in N content in grain was
observed in response to co-inoculation of J120 x S14, over control. The same co-
98
Table 4.15: Interactive effect of co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on N and
P content in grain and straw of chickpea in absence and presence of P-enriched compost

N content in grain N content in straw P content in grain P content in straw
Treatments (%) (%) (%) (%)
Control 2.60 j 2.64 ij 0.56 i 0.59 hi 0.32 h 0.34 g 0.17 j 0.19 i
J119 2.74 hi 2.94 ef 0.62 gh 0.66 e-g 0.39 ef 0.40 c-e 0.20 hi 0.23 d-f
J120 2.77 gh 3.31 d 0.63 f-h 0.71 e 0.38 f 0.39 d-f 0.21 gh 0.23 d-f
S4 2.76 g-i 3.37 cd 0.62 g-i 0.77 d 0.38 f 0.42 a-c 0.21 gh 0.24 cd
S14 2.83 f-h 3.27 d 0.65 f-g 0.78 d 0.38 f 0.41 b-d 0.22 e-h 0.24 cd
J119 x S4 2.88 f-g 3.46 bc 0.67 e-g 0.92 a 0.39 ef 0.43 ab 0.23 d-f 0.25 bc
J119 x S14 2.85 f-h 3.59 a 0.67 e-g 0.85 b 0.38 f 0.42 a-c 0.21 gh 0.27 ab
J120 x S4 2.87 f-h 3.54 ab 0.66 e-g 0.84 bc 0.39 ef 0.42 a-c 0.22 e-h 0.28 a
J120 x S14 3.04 e 3.38 cd 0.69 ef 0.80 cd 0.38 f 0.43 a 0.23 d-f 0.27 ab
LSD Value 0.1173 0.0524 0.0166 0.0165
S: Rhizobia isolate
99
Inoculation strains caused an increase of 38.1% in N content when applied in the soil
amended with P-enriched compost. Similarly, all other co-inoculation treatments resulted
in 50% increase in N content over control.
4.5.1.11. Nitrogen content in straw
Similar to N content in grain, inoculation/co-inoculation had significant effect on
N content in straw compared with control (Table 4.15). Maximum increase in N content
in straw in response to sole inoculation was 16.1% due to inoculation with S14 that was
further increase of 39.3% when applied in soil amended with P-enriched compost. While
S14 in co-inoculation with J120 caused up to 23.2% increase in N content compared
woth uninoculated control. The effectiveness of inoculation/co-inoculation was increased
when chickpea seeds were inoculated in the presence of P-enriched compost. Up to
51.8% increase in N content was observed as compared to uninoculated control.
4.5.1.12. Phosphorous content in grain
Like N content, inoculation/co-inoculation was also effective in improving P
content in grain of chickpea (Table 4.15). Co-inoculation caused maximum improvement
in P content up to 21.9% over control. In soil amended with P-enriched compost,
maximum increase in P content in grain were recorded 34.1% in response to co-
inoculation with J120 x S14, over uninoculated control.
4.5.1.13. Phosphorous content in straw
Phosphorous nutrition was also improved in straw in response to inoculation with
rhizobia and rhizobacteria either applied alone or in combination with each other (Table
4.15). Single inoculation exhibited maximum increase in P content in straw up to 21.3%
due to inoculation with S14, over uninoculated control. Combined use of inoculation
100
treatments plus P-enriched compost further enhanced the P content in straw and an
increase of 42.9% over uninoculated control. But in co-inoculation treatments, highest
increase in P contents in straw was 37.1% in response to co-inoculation with J120 x S14,
over control. It was followed by J119 x S4, J119 x S14 and J120 x S4. On the other hand,
when co-inoculation was tested in presence of P-enriched compost, the highest increase
in P content in straw was 64.7% with the application of J120 x S4, as compared with
control. The rest of the co-inoculation treatments J119 x S4, J119 x S14 and J120 x S14,
also caused significant increases (up to 58.8%) in P content in straw when these
treatments were tested in the presence of P-enriched compost.
4.6. Pot experiment-II (2nd year)
In second year, the rhizobacterial isolates containing ACC-deaminase (J119 and
J120) and rhizobial isolates (S4 and S14) were tested again for evaluating their potential
to improve nodulation and growth of chickpea crop, applied alone or in combinations in
the presence and absence of P-enriched compost.
4.6.1. Plant height
Inoculation/co-inoculation significantly promoted the plant height of chickpea
plant compared to control (Table 4.16). The highest increase (32.4%) was observed with
J120 compared with uninoculated control and followed by J119, S4 and S14. When
inoculation treatments were tested with P-enriched compost, then maximum increase
observed in palnt height was up to 54.3% over control. In case of co-inoculation
treatments, maximum increase in plant height was 50% due to co-inoculation with J120 x
S14, over control and followed by J119 x S4, J119 x S14 and J120 x S4 treatments which
increased the plant height up to 35.3%. When co-inoculation was tested with P-enriched
101
growth and yield of chickpea in presence and absence of P-enriched compost
(2nd year pot trial)

Treatments (cm) (g plant-1) plant-1 (g plant-1) (g)
Control 28.1 h 29.1 h 39.1 i 41.8 hi 46 i 52 h 6.9 k 7.8 j 29.1 f 31.1 e
J119 36.7 g 43.3 b-d 45.3 gh 51.8 d-f 57 gh 68 b-d 8.8 hi 10.5 f 32.4 b-e 32.3 c-e
J120 37.2 f-g 41.8 c-e 46.4 f-h 51.6 d-f 57 gh 71 a-c 8.6 i 11.1 de 32.1 c-e 33.4 a-d
S4 36.3 g 42.4 cd 46.0 f-h 50.0 e-g 61 fg 67 c-e 8. 5 i 10.9 ef 32.5 b-e 32.5 b-e
S14 36.2 g 41.4 d-f 47.0 f-h 53.0 c-e 60 fg 72 a-c 8.9 hi 11.3 c-e 32.3 c-e 32.8 b-d
J119 x S4 38.0 e-g 45.7 a-d 49.4 e-g 56.1 b-d 62 e-g 73 ab 9.2 h 11.7 a-c 32.2 c-e 33.7 a-c
J119 x S14 36.8 g 48.4 a 49.4 e-g 62.6 a 60 fg 74 ab 9.7 g 12.2 a 33.4 a-d 34.4 a
J120 x S4 37.5 f-g 47.0 ab 50.0 e-g 57.6 a-c 70 b-d 76 a 9.9 g 11.6 b-d 32.0 d-e 33.6 a-c
J120 x S14 42.1 c-e 46.3 a-c 51.6 d-f 60.4 ab 64 d-f 72 a-c 10.5 f 11.9 ab 32.5 b-e 34.0 ab
LSD Value 3.937 5.061 5.474 0.483 1.376
S: Rhizobial isolate
102
compost then the maximum increase in plant height was up to 72.4% as a result of co-
inoculation with (J119 x S14). Rest of co-inoculation treatments J119 x S4, J120 x S4
and J120 x S14 along with P-enriched compost, also caused significant increase in plant
height compared to uninoculated control.
4.6.2. Fresh biomass
In case of inoculation, highest increase in fresh biomass was 20.2% with S14 and
inoculation of which isolate along with P-enriched compost, further promoted it over
uninoculated control (Table 4.16). The other isolates in single inoculation plus P-enriched
compost caused increases in fresh biomass up to 32.5% over control. In case of co-
inoculation, maximum increase in fresh biomass was noted up to 32% with J120 x S14,
over control which was followed by 26.4, 26.5 and 28% increase with inoculation of J119
x S4, J119 x S14 and J120 x S4 respectively, and their effect wase non-significant with
each other. When co-inoculation was examined in the soil amended with P-enriched
compost, it resulted in an increase of 60.1% in fresh biomass due to co-inoculation with
J119 x S14, compared with uninoculated control. Other co-inoculation treatments J119 x
S4, J120 x S4 and J120 x S14, also produced significantly higher fresh biomass when
tested with P-enriched compost.
4.6.3. Number of pods per plant
Inoculation with rhizobia and rhizobacteria either alone or in co-inoculation had
highly significant positive effect on number of pods per plant. In case of inoculation, the
highest increase (32.6%) in number of pods per plant was recorded as a result of
inoculation with S4 while S14 showed highest increase (56.6%) in the presence of P-
enriched compost (Table 4.16). In the case of co-inoculation, the most promising results
103
were obtained with J120 x S4 resulting in 52.2% increase over control and this increase
was further enhanced up to 65.2% when tested in combination with P-enriched compost.
4.6.4. Grain yield
In case of grain yield, a significant enhancement (27%) in grain yield resulted in
case of S14 as compared to uninoculated control (Table 4.16). This increase was further
rasised as 62.7% more increase was observed over control when P-enriched compost was
applied @ 300 kg ha-1. Rest of the isolates also had significant positive effect on grain
yield, both in the absence and presence of enriched compost. While in case of co-
inoculation, J120 x S14 combination was the most effective and significantly increased
grain yield (52%) compared to uninoculated control in the absence of P-enriched compost
while J119 x S14 was more effective (76% increase over control) compared to compost
unamended soil. Other co-inoculant combinations had also significant positive effect on
grain yield.
4.6.5. 100-grain weight
Like other parameters, increase in 100-grain weight was also observed in response
to inoculation/co-inoculation (Table 4.16). Overall, co-inoculant strains J119 x S14 were
the most effective in producing maximum weight of 100-grains as compared to
uninoculated control, in the absence and presence of P-enriched compost.
4.6.6. Number of nodules per plant
Number of nodules per plant was enhanced significantly by inoculation with
rhizobia and rhizobacteria as compared to control (Table 4.17). An increase in number of
nodules per plant ranged from 34.5 to 48.3% due to single inoculation over control. The
integrated use of inoculation plus P-enriched compost was further increased number of
104
nodules per plant and it reached up to 65.5% compared with control. While in case of co-
inoculation treatments, maximum increase in number of nodules per plant were produced
up to 58.6% and 96.6% in absence and presence of P-enriched compost compared with
uninoculated/untreated control respectively. The most effective combination was J119 x
S14 followed by J119 x S4, J120 x S4 and J120 x S14.
4.6.7. Nodule dry weight
All the treatments promoted the nodule dry weight per plant over uninoculated
control (Table 4.17). In case of sole inoculation S14 was most efficient strain to increase
nodule dry weight per plant which was 57.8% more than uninoculated control. It was
followed by J119, J120 and S4. In soil amended with enriched compost, the increase in
nodule dry weight per plant was up to 78.4% over uninoculated control. The most
promising increase in nodule dry weight was recorded in response to co-inoculation plus
P-enriched compost as 98.7% increase in weight was recorded as result of inoculation
with J120 x S14 as compared to uninoculated control. However, in case of untreated soil,
the observed increased in nodule dry weight by the same above combination was 72.5%
compared to uninoculated control.
4.6.8. Root length
The results regarding root length showed significant increase in root length of
chickpea (Table 4.17). A maximum increase of 44.6% in root length was recorded due to
inoculation with J120 followed by the treatments J119, S4, and S14, which increased the
root length ranging from 17.4 to 29.4% as compared to control. When inoculation was
tested with P-enriched compost, then maximum increase in root length was 49.6% over
105
Table 4.17: Effect of co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on nodulation and
root growth of chickpea in absence and presence of P-enriched compost

Control 29 i 33 h 0.22 k 0.24 j 16.8 i 18.0 hi 1.5 j 1.6 ij
J119 43 ef 47 cd 0.31 i 0.38 cd 23.8 ef 25.2 c-e 1.7 hi 1.8 e-h
J120 39 g 48 c 0.33 gh 0.35 ef 24.3 e 24.8 de 1.8 f-h 2.0 d
S4 42 fg 48 c 0.33 gh 0.39 c 21.8 fg 23.8 ef 1.7 hi 2.0 d
S14 43 ef 47 cd 0.34 f-h 0.37 de 19.8 gh 23.5 ef 1.7 hi 2.0 d
J119 x S4 45 c-f 52 b 0.34 f-h 0.42 ab 25.8 a-e 27.7 ab 1.9 d-g 2.2 c
J119 x S14 44 d-f 57 a 0.33 gh 0.43 a 25.4 b-e 27.3 a-c 1.8 e-h 2.6 a
J120 x S4 46 c-e 52 b 0.36 ef 0.41 b 19.7 gh 28.0 a 1.9 d-f 2.5 ab
J120 x S14 45 c-f 55 ab 0.38 cd 0.42 ab 21.4 fg 27.0 a-d 2.0 d 2.4 b
LSD Value 3.476 0.0156 2.199 0.148
S: Rhizobia isolate
106
control. Inoculation with other test isolate plus P-enriched compost also significantly
increased root length ranging from 39.6 to 47.5% than uninoculated control. But in case
of co-inoculation, the highest increase (53.5%) in root length was observed due to co-
inoculation with J119 x S4, over control. When co-inoculation treatments were tested
with P-enriched compost, the maximum increase was 66.4% in root length due to co-
inoculation with J120 x S4. Rest of the co-inoculation treatments J119 x S4, J119 x S14
and J120 x S14 in the presence of P-enriched compost, also promoted the root length up
to 64.4%, over control.
4.6.9. Root dry weight
Root dry weight per plant was significantly improved by inoculation by with
different isolates and a maximum increase of 19.9% over uninoculated control was
observed as result of inoculation with J120 (Table 4.17). However, the effectiveness of
the inoculation was further enhanced in the presence of P-enriched compost. Likewise,
co-inoculation with J120 plus S14 showed greatest effect as 32.2% more root dry weight
was observed compared to control. In compost amended soil, J119 plus S14 combination
was most effective and significantly increased root dry weight by 76.7% compared with
4.6.10. Nitrogen content in grain
The maximum increase in N content in garin was 24.6% when inoculated with
J119 x S4, over respective control (Table 4.18). While all other inoculation/co-
inoculation treatments J119, J120, S4, S14, J119 x S14, J120 x S4 and J120 x S14
increased N content ranging from 16.5 to 23.5% compared to uninoculated control. In
compost amended soil, co-inoculation with rhizobia and rhizobacteria was more effective
107
as compared to inoculation/co-inoculation in the unamended soil. Maximum increase
(32.3%) in N content in garin was observed in case of co-inoculation with J119 x S14 as
4.6.11. Nitrogen content in straw
A highest increase (15.4%) in N content in straw was observed in case of
inoculation with J119 x S14 compared to uninoculated control (Table 4.18).
Inoculation/co-inoculation plus P-enriched compost was more effective in increasing N
content as compared to inoculation/co-inoculation only. The highest N content was found
with J119 x S14 which was 35.4% more than uninoculated control.
4.6.12. Phosphorous content in grain
There was a significaltly higher P content (23.7% more over control) in response
to inoculation with J120 x S4 (Table 4.18). It was followed descending order by J119,
J120, S4, S14, J119 x S4, J119 x S14, and J120 x S14. Inoculation/co-inoculation plus P-
enriched compost was more effective than alone inoculation/co-inoculation treatments.
Maximum increase in P content in garin was 38.9% with J120 x S4 compared with
uninoculated control. While other inoculation/co-inoculation treatments in the presence
of P-enriched compost also promoted P content in garin that ranged from 24.7 to 36.8%
as compared to uninoculated control
4.6.13. Phosphorous content in straw
The maximum increase (55.9%) in P content was observed in reponse to co-
inoculation with J119 x S14, over control (Table 4.18). But other inoculation/co-
inoculation treatments also gave significant increase (up to 49%) in P content compared
108
N and P content in grain and straw of chickpea in absence and presence of P-enriched compost

Treatments (%) (%) (%) (%)
Control 2.60 j 2.64 j 0.65 h 0.70 g 0.28 j 0.31 i 0.14 k 0.16 j
J119 3.03 hi 3.18 ef 0.72 fg 0.76 e 0.35 f-h 0.36 d-g 0.19 hi 0.22 d-g
J120 3.03 i 3.20 ef 0.72 fg 0.79 cd 0.34 h 0.35 e-h 0.18 i 0.23 d-f
S4 3.06 g-i 3.24 c-e 0.73 fg 0.82 b 0.34 h 0.36 cf 0.18 i 0.23 d-f
S14 3.13 f-h 3.30 b-d 0.72 fg 0.77 de 0.34 h 0.37 b-e 0.19 hi 0.24 b-e
J119 x S4 3.24 c-e 3.32 bc 0.74 f 0.81 bc 0.34 f-h 0.38 a-c 0.22 d-g 0.25 ab
J119 x S14 3.18 ef 3.44 a 0.75 ef 0.88 a 0.34 h 0.39 ab 0.22 d-g 0.24 b-e
J120 x S4 3.15 e-g 3.40 ab 0.73 f 0.81 bc 0.35 f-h 0.39 a 0.21 g-h 0.25 a-c
J120 x S14 3.21 d-f 3.34 b 0.74 f 0.86 a 0.34 gh 0.38 a-d 0.21 g-h 0.26 a
LSD Value 0.0907 0.0234 0.0166 0.0165
S: Rhizobia isolate
109
to uninoculated control. In soil amended with P-enriched compost, inoculation/co-
inoculation significantly promoted P content as compared to inoculation/co-inoculation
only. The most promising increase in P content in straw was 76.9% due to inoculation
with J120 x S14, over control. While other inoculation/co-inoculation in the presence of
P-enriched compost increased P content significantly in straw compared to uninoculated
control.
4.7. Field trials
Effectiveness of selected isolates of PGPR containing ACC-deaminase (J119 and
J120) and rhizobia (S4 and S14) alone as well as in combinations were tested for
promoting growth, nodulation and yield of chickpea under field conditions. Field trials
were conducted at different locations for two consecutive years.
4.7.1. First year trials
During the first year, field trials were conducted at two different sites i.e.
Research Farm of the University of Agriculture, Faisalabad and Farmer’s field at Chak
No. 72/GB, Faisalabad.
4.7.1.1. First trial: Site-I (UAF)
Inoculation and co-inoculation effects of selected PGPR containing ACC-
deaminase (J119 and J120) and rhizobial isolates (S4 and S14) were evaluated in the
absence and presence of P-enriched compost. Recommended dose of chemical fertilizers
was applied in case of soil not amended with P-enriched compost. While in case of soil
amended with P-enriched compost, the remaining nutrients (equivalent to chemical
fertilizer) were supplied through chemical fertilizers. The results of field trials are
summarized below.
110
4.7.1.1.1. Plant height
The data regarding plant height are presented in Table 4.19. Single inoculation
had significant effect on plant height and up to 22.8% increase in plant height was
observed compared to uninoculated control. Effects of all isolates were non-significant
with each other. While up to 32.8% increase in plant height was noted in response to
inoculation plus P-enriched compost, as compared with uninoculated control. The co-
inoculation with J120 x S14 showed maximum plant height up to 29% as compared with
uninoculated control, followed by J119 x S4, J120 x S4 and J11x S14. Up to 42%
improvement in plant height was recorded as a result of co-inoculation plus P-enriched
compost, as compared with uninoculated control but all the co-inoculation plus P-
enriched compost treatments were statistically non-significant with each other.
4.7.1.1.2. Fresh biomass
The maximum increase in fresh biomass was noted up to 27% in case of single
inoculation with S14 in the absence of P-enriched compost while in the presence of P-
enriched compost; J120 caused 41% increase in fresh biomass as compared to control
(Table 4.19). It was noted that co-inoculation with J120 x S14 produced maximum fresh
biomass that was 37.6% more than uninoculated control. Combined use of co-inoculation
with P-enriched compost gave maximum increase in fresh biomass as compared with
control. The combination J119 x S4 plus P-enriched compost was the most effective and
increased 61% fresh biomass compared with control.
4.7.1.1.3. Number of pods per plant
It is clear from the results that co-inoculation plus P-enriched compost was much
better for improving number of pods per plant than uninoculated control (Table 4.19). In
111
growth and yield of chickpea in absence and presence of P-enriched compost
(1st year field trial)

Treatments (cm) (t ha-1) plant-1 (t ha-1) (g)
Control 43.7 j 45.5 j 7.5 l 8.1 k 39 j 43 i 1.9 j 2.1 ij 31.9 f 33.3 e
J119 50.7 i 56.4 c-g 8.7 j 11.1 c 55 h 62 fg 2.3 hi 3.0 bc 35.6 d 35.8 cd
J120 51.7 hi 57.0 b-f 9.1 ij 10.6 d 56 h 68 c-e 2.4 h 2.9 cd 35.9 cd 35.8 cd
S4 53.6 f-i 58.0 b-e 9.2 h-j 10.5 d 54 h 67 d-f 2.5 gh 2.6 e-g 35.3 d 37.3 ab
S14 53.1 g-i 56.0 d-g 9.5 g-i 9.9 e-g 58 gh 66 d-f 2.4 f-h 2.7 d-g 35.6 d 37.3 ab
J119 x S4 55.0 e-h 58.7 a-d 9.6 gh 12.1 a 63 ef 74 b 2.7 d-f 3.3 a 35.8 cd 37.2 ab
J119 x S14 56.1 c-g 60.3 ab 9.7 f-g 11.8 ab 63 fg 80 a 2.7 d-g 3.3 a 36.18 cd 37.9 ab
J120 x S4 55.3 d-g 62.0 a 10.2 d-f 11.5 bc 57 h 72 bc 2.7 d-g 3.1 a-c 36.0 cd 38.6 a
J120 x S14 56.4 cg 59.6 a-c 10.4 de 11.3 bc 63 e-g 69 b-d 2.8 de 3.2 ab 35.8 cd 36.9 bc
LSD Value 3.096 0.454 4.323 0.228 1.098
112
the absence of P-enriched compost, inoculation with J120 x S4 resulted in maximum
number of pods per plant which was 64.1% more than control. While, co-inoculation
(J119 x S14) along with P-enriched compost gave maximum increase (105%) in number
of pods per plant as compared to uninoculated control.
4.7.1.1.4. Grain yield
Maximum grain yield in compost unamended soil was resulted from co-
inoculation with J120 x S14 which was 41% greater than uninoculated control (Table
4.19). When inoculation/co-inoculation was tested along with P-enriched compost then
further increase in grain yield was recorded. Through, grain yield was improved from
35.4 to 69.7% by inoculation/co-inoculation plus P-enriched compost but the highest
increase (69.7%) in grain yield was obtained with co-inoculation with J119 x S14
4.7.1.1.5. 100-grain weight
It is evident from Table 4.19 that both inoculation and co-inoculation had
significant positive effect on 100-grain weight of chickpea and maximum increase of
20.4% in weight was recorded in response to co-inoculation with J120 x S4 in the
presence of P-enriched compost. The increase in 100-grain weight varied from 10.5 to
18.9% over uninoculated control in case of the inoculation/co-inoculation treatments in
the presence or absence of P-enriched compost.
4.7.1.1.6. Number of nodules per plant
The number of nodules per plant were highest (106% increase over control) when
the plants were co-inoculated with J120 x S14 and sown in compost amended soil (Table
4.20). The maximum numbers of nodules per plant were up to 62.5% upon inoculation
113
with J120 x S14 compared with untreated control in the soil not amended with compost.
The composted treated soil without inoculation also enhanced number of nodules per
plant over uninoculated control.
4.7.1.1.7. Nodule dry weight
Co-inoculation produced 50 to 53.9% more dry nodule weight per plant, while
simple inoculation showed up to 37.6% increase compared to uninoculated control (Table
4.20). The integrated use of co-inoculation and P-enriched compost further increased
nodule dry weight (up to 69.4% more over control) and maximum effect was observed in
case of inoculation with J119 x S14. It was followed by J120 x S4, J120 x S14 and J119 x
S4. While inoculation plus P-enriched compost also significantly increased nodule dry
weight which was 55.2% higher than control.
4.7.1.1.8. Root length
Application of inoculation/co-inoculation with and without P-enriched compost
was significantly better than sole application of P-enriched compost and/or uninoculated
control (Table 4.20). An increase of up to 24.5% in root length was shown in case of
single inoculation; while upon co-inoculation it was 29.1% greater than uninoculated
control. But in case of inoculation plus P-enriched compost, maximum increase (37.9%)
in root length was observed due to inoculation with S14, followed by J119, J120 and S4
which also showed an improvement in root length ranging from 29.5 to 36.8% over
control. When co-inoculation treatments were tested in presence of P-enriched compost
then the highest increase in root length was recorded up 41.4% compared to control.
114
nodulation and root growth of chickpea in absence and presence of P-enriched compost

Control 32 j 36 j 0.51 j 0.57 i 21.7 g 23.2 fg 1.40 i 1.48 h
J119 43 i 54 c-e 0.67 h 0.73 e-g 27.1 b-e 28.4 a-e 1.74 ef 1.78 e
J120 46 g-i 50 e-g 0.70 f-h 0.74 d-g 26.3 e 29.8 a-d 1.63 g 1.86 d
S4 45 hi 51 d-f 0.69 gh 0.74 d-g 26.8 c-e 28.2 a-e 1.62 g 1.96 c
S14 48 f-h 55 b-d 0.71 f-h 0.80 bc 26.0 ef 30.0 a-c 1.64 fg 1.90 cd
J119 x S4 50 e-g 59 b 0.77 ce 0.78 c-e 27.7 a-e 30.0 a-c 1.78 e 2.37 b
J119 x S14 49 f-h 64 a 0.75 c-f 0.87 a 26.6 de 30.3 ab 1.67 e-g 2.43 b
J120 x S4 49 f-h 66 a 0.72 e-h 0.84 ab 27.1 b-e 30.8 a 1.72 e-g 2.52 a
J120 x S14 52 d-f 58 bc 0.79 b-d 0.81 bc 28.1 a-e 30.5 a 1.76 ef 2.35 b
LSD Value 4.106 0.0524 2.837 0.091
115
4.7.1.1.9. Root dry weight
It is evident from the Table 4.20 that integrated use of co-inoculation and P-
enriched compost showed significantly better performance for enhancing root dry weight
in comparison with simple inoculation /co-inoculation as well as the uninoculated
control. There is a little variation within single inoculation and co-inoculation treatments
in case of root dry weight in the absence of P-enriched compost. Maximum increase (up
to 23.5%) in root dry weight was observed as a result of inoculation with J119 compared
with control. Non-significant effect was found among the inoculation treatments. Co-
inoculation showed further increase in root dry weight up to 28.7% as result of J119 x S4
inoculation, and other remaining treatments of co-inoculation also exhibited an increase
in root dry weight up 27.9% over uninoculated control. When these treatments were
tested in the presence of P-enriched compost, the effects on root dry weight were more
pronounced when compared with control. In case of inoculation/co-inoculation plus P-
enriched compost, maximum increase was 85.3% due to inoculation with J120 x S4 plus
enriched compost.
4.7.1.1.10. Nitrogen content in grain
Simple inoculation with S14 resulted in 11.2% increase in N content in grain and
this increase was further improved up to 18.1%, as compared to control when inoculation
was tested along with P-enriched compost (Table 4.21). While, co-inoculation (J119 x
S14) along with P-enriched compost gave maximum increase of 23% over control,
followed by combinations J120 x S14, J120 x S4 and J119 x S4.
116
Un-inoculated control vs J119 x S14 plus P-enriched compost
Picture: Effect of co-inoculation plus P-enriched compost on nodulation of chickpea

under field conditions
117
4.7.1.1.11. Nitrogen contents in straw
As far as N content in straw is concerned, co-inoculation along with P-enriched
compost significantly inproved N conetent compared to simple inoculation or
uninoculated control (Table 4.21). Only simple inoculation/co-inoculation resulted in 9.6
to 15.1% increase in N content in straw and J119 x S14 combination was highly ranked
among all the treatments.
4.7.1.1.12. Phosphorous content in grain
The data reveal that simple inoculation significantly increased P content in grain
which was up to 14.3% greater than control (Table 4.21). While co-inoculation caused up
to 21.6% increase as compared to uninoculated control. In case of integrated use of
inoculation/co-inoculation and P-enriched compost showed maximum effect on P content
was 27% when co-inoculants J120 x S14 were applied along with enriched compost.
4.7.1.1.13. Phosphorous content in straw
The combined use of co-inoculation and P-enriched compost gave better
performance for improving P content in straw in comparison with simple inoculation/co-
inoculation as well as uninoculated control (Table 4.21). In case of single inoculation,
maximum increase in P content in straw was observed by inoculation with J119 strain
that was up to 38.9% more than uninoculated control. Co-inoculation with J119 x S14
resulted in 55.6% increase in P content as compared to control. When inoculation and co-
inoculation were testes with P-enriched compost, a further increase in P content was
observed, as up to 83.3% more P content were found with co-inoculant J120 x S4.
118

Treatments (%) (%) (%) (%)
Control 2.87 i 2.97 h 0.73 i 0.78 hi 0.37 h 0.40 g 0.18 j 0.20 ij
J119 3.03 h 3.24 e-g 0.82 f-h 0.86 c-f 0.42 d-f 0.43 c-f 0.25 fg 0.28 de
J120 3.01 h 3.30 d-f 0.81 f-h 0.89 c-e 0.41 fg 0.42 e-g 0.23 gh 0.28 de
S4 3.05 h 3.20 fg 0.80 gh 0.92 bc 0.41 fg 0.43 c-f 0.22 hi 0.29 b-d
S14 3.19 g 3.39 b-d 0.82 f-h 0.85 d-g 0.41 fg 0.42 d-f 0.23 gh 0.28 c-e
J119 x S4 3.33 c-e 3.39 b-d 0.82 f-h 0.90 b-d 0.44 b-d 0.45 b 0.27 de 0.31 ab
J119 x S14 3.22 fg 3.53 a 0.84 e-g 0.95 ab 0.43 b-e 0.44 b-e 0.28 de 0.31 ab
J120 x S4 3.21 fg 3.40 bc 0.83 f-h 0.97 a 0.43 b-e 0.46 a 0.26 ef 0.31 a-c
J120 x S14 3.27 e-g 3.48 ab 0.83 e-h 0.91 bc 0.45 b 0.47 a 0.27 d-f 0.33 a
LSD Value 0.091 0.0524 0.0166 0.0235
119
4.7.1.2. Field trial-II: Site-II (Sajawal Farm)
The second field trial during the first year was conducted at Sajawal Farm,
72/GB, Faisalabad.
All the selected rhizobacteria and rhizobia significantly affected plant height of
chickpea (Table 4.22). In case of single inoculation, all the isolates were statistically at
par with each other but differed significantly from control. The isolate S4 was the most
effective isolate when applied along with P-enriched compost. However, the same isolate
on co-inoculation with J119 in compost amended soil showed maximum plant height,
resulting in 47% increase compared with control.
4.7.1.2.1.2. Fresh biomass
The maximum fresh biomass was observed in case of co-inoculation where the
two combinations i.e., J119 x S4 and J120 x S14 along with enriched compost produced
almost the same fresh biomass (up to 62.7% increase over uninoculated control). While,
co-inoculation of S4 with J120 also statistically at par with above two combinations
(Table 4.22). All other inoculation treatments also had significantly increasing effect on
fresh biomass both in the absence and presence of P-enriched compost as compared to
over uninoculated control
4.7.1.2.1.3. Number of pods per plant
Numbers of pods per plant were also significantly affected by inoculation/co-
inoculation both in the presence and absence of P-enriched compost (Table 4.22). The
highest number of pods (90.2% more than control) were recorded when co-inoculant
J119 x S14 were applied with P-enriched compost. Other inoculation/co-inoculation
120

Control 41.0 h 43.5 gh 7.7 i 8.0 i 41 j 46 i 1.8 i 1.9 h 32.7 g 34.1 f
J119 45.6 e-g 47.6 d-f 9.1 h 9.6 f-h 53 h 67 b-d 2.3 g 2.5 f 35.5 de 35.8 c-e
J120 45.2 fg 48.8 d 9.3 gh 10.3 d 52 h 66 cd 2.3 g 2.8 bc 35.1 ef 36.4 b-e
S4 45.3 fg 56.3 bc 9.2 gh 10.5 d 55 gh 70 bc 2.3 g 2.7 cd 35.9 c-e 36.1 c-e
S14 46.2 d-g 46.7 d-f 9.2 gh 11.2 c 56 f-h 69 bc 2.3 g 2.8 bc 35.8 c-e 37.7 ab
J119 x S4 48.3 d-f 60.2 a 10.0 d-f 12.5 a 67 b-d 69 bc 2.6 ef 3.2 a 36.3 b-e 36.6 a-e
J119 x S14 48.6 de 55.3 c 9.9 d-f 11.8 b 64 de 78 a 2.7 cd 3.1 a 35.7 c-e 37.8 ab
J120 x S4 48.0 d-f 58.7 ab 9.7 e-g 12.2 ab 58 fg 71 b 2.6 ef 2.8 bc 36.3 b-e 38.0 a
J120 x S14 47.8 d-f 54.2 c 10.3 de 12.5 a 60 ef 70 bc 2.7 cd 2.9 b 36.9 a-d 37.2 a-c
LSD Value 2.703 0.538 4.441 0.117 1.318
121
treatments along with P-enriched compost resulted in significant more pods per plant
which ranged from 61 to 73.2% over uninoculated control. When inoculation/co-
inoculation was tested without P-enriched compost, J119 x S4 showed the most
promising results and caused up to 63.4% increase over uninoculated control. Rest of all
the inoculation/co-inoculation treatments had significant effect on number of pods per
plant as compared to uninoculated control.
4.7.1.2.1.4. Grain yield
As observed in the first trial, co-inoculation along with P-enriched compost
resulted in highly significant improvement in the grain yield as compared to simple
inoculation as well as uninoculated control (Table 4.22). In response to simple co-
inoculation, maximum increase in grain yield was recorded up to 50.8%, while remaining
treatments of simple inoculation/co-inoculation also showed increase in grain yield
ranging from 26.3 to 48.6% higher over uninoculated control. Increase in grain yield
ranged from 38 to 77.7% in case of inoculation/co-inoculation with P-enriched compost
while maximum improvement in grain yield (77.7%) was achieved with the application
of J119 x S4. It was statistically similar with effect of co-inoculant strains J119 x S14.
Combined application of J120 x S14 without enriched compost resulted in maximum
grain yield compared to uninoculated control.
4.7.1.2.1.5. 100-grain weight
Maximum increase in 100-grain weight was 13.2% due to co-inoculation with
J120 x S14. Integrated use of co-inoculation with J120 x S4 plus P-enriched compost
resulted in maximum increase in 100-grain weight up to 16.2% over uninoculated control
(Table 4.22). Next to it, J119 x S14 was the effective co-inoculant strains compared with
122
uninoculated control. In the compost unamended soil, J120 x S14 was the most effective
isolates in enhancing 100-grain weight as compared to uninoculated control.
4.7.1.2.1.6. Number of nodules per plant
Inoculation/ co-inoculation resulted in significant increase in number of nodules
per plant compared with uninoculated control when tested with P-enriched compost
(Table 4.23). The maximum increase in number of nodules was found with J120 x S4
along with P-enriched compost which was 85.7% greater than control. This treatment had
non-significant effect on number of nodules per plant with combined application of J119
x S14. In compost unamended soil, J119 x S4 was top ranked combination, causing an
increase of 54.3% compared to control. Overall, all inoculation/ co-inoculation treatments
had significant effect on number of nodules per plant as compared to uninoculated
control.
4.7.1.2.1.7. Nodule dry weight
Single inoculation caused up to 33.2% increase in dry nodule weight per plant as
compared to uninoculated control while up to 47.5% increase was noted as a result of co-
inoculation (Table 4.23). In case of inoculation treatments, the isolates J119, J120, and S4
were statistically at par to each other. But in case of co-inoculation, maximum increase in
nodule dry was attained up to 57.9% due to J120 strain, while rest of the single strains
also promoted the nodule dry weight that increase ranged from 31.7 to 48.6% over
uninoculated control. Maximum increase in nodule dry weight was obtained up to 82.3%,
when J119 x S14 was applied in combination with P-enriched compost in comparison
with uninoculated control.
123

Control 35 h 39 gh 0.36 l 0.39 k 21.8 f 23.9 e 1.50 j 1.64 i
J119 46 ef 52 cd 0.46 ij 0.47 j 27.7 d 30.3 a-c 1.85 gh 1.89 f-h
J120 46 d-f 53 cd 0.45 j 0.56 d 28.2 d 28.9 b-d 1.75 hi 1.96 e-g
S4 45 ef 54 c 0.45 j 0.53 e 27.3 d 29.2 b-d 1.77 h 2.14 b-d
S14 43 fg 56 bc 0.47 hi 0.52 e 27.7 d 28.9 b-d 1.80 h 2.00 d-f
J119 x S4 54 c 60 ab 0.50 f 0.59 bc 29.3 b-d 30.8 ab 1.89 f-h 2.26 b
J119 x S14 53 c 63 a 0.53 e 0.65 a 28.4 cd 31.6 a 1.85 gh 2.53 a
J120 x S4 52 c-e 65 a 0.49 fg 0.57 cd 29.2 b-d 30.3 a-c 1.87 f-h 2.16 b-c
J120 x S14 53 c 61 ab 0.48 gh 0.61 b 28.9 b-d 30.4 a-c 1.89 f-h 2.09 c-e
LSD Value 5.951 0.0165 1.765 0.128
124
4.7.1.2.1.8. Root length
Simple inoculation/co-inoculation significantly increased root length over
uninoculated control (Table 4.23). Single inoculation caused up to 29% increase in root
length while this increase was further promoted up to 38.6% due to inoculation plus
enriched P-compost compared to uninoculated control. All the co-inoculation treatments
showed significant increase in root length over control. Co-inoculation with J119 x S14
in the presence of P-enriched compost gave highest increase 44.7% in root length
compared with control, followed by J119 x S4, J120 x S4 and J120 x S14.
4.7.1.2.1.9. Root dry weight
All inoculation/co-inoculation treatments gave significant increase in root dry
weight as compared to control (Table 4.23). Co-inoculation increased root dry weight up
to 26% in response to J120 x S14 in the absence P-enriched compost, compared to
uninoculated control. Maximum root dry weight per plant (68.7% increase over control)
was achieved when co-inoculation with J119 x S14 was tested in compost amended soil.
While, rest of the treatments of inoculation/co-inoculation plus enriched compost also
significantly increased root dry weight as compared with uninoculated control
4.7.1.2.1.10. Nitrogen content in grain
The maximum increase in N content in grain was recorded up to 34.5% due to co-
inoculation with J119 x S14 along with P-enriched compost compared to uninoculated
control (Table 4.24). While other remaining inoculation/co-inoculation treatments also
showed significant increase in N content in grain ranging from 5.4 to 31% over
uninoculated control when tested in the absence and presence of P-enriched compost.
Effect of single inoculation treatments was non-significant with each other.
125
4.7.1.2.1.11. Nitrogen content in straw
All inoculation/co-inoculation treatments increased significantly N content in
straw (Table 4.24). In case of compost unamended soil, the maximum increase in N
content was 18.3% due to inoculation with combination J120 x S14, over control. The
highest increase in N content in straw was noted up to 23.3% with same combination
when tested in the presence of P-enriched compost, and this increase was statistically at
par with S4, J119 x S4, J119 x S14 and J120 x S4 treatments.
4.7.1.2.1.12. Phosphorous content in grain
Simple inoculation/co-inoculation significantly increased P content in grain
compared to control (Table 4.24). Sole inoculation showed 19% increase in P content in
grain, while in case of co-inoculation this increase was promoted up to 21.5% over
uninoculated control. While combined use of co-inoculation and P-enriched compost
gave highest increase in P content in grain was 29.8% due to co-inoculation with J120 x
S4, followed by J119 x S14, J120 x S14 and S4 treatments. Non-significant differences
were observed only between these treatments J119, J120, S4, S14, and J120 x S4.
4.7.1.2.1.13. Phosphorous content in straw
Results about P content in straw are presented in Table 4.24. Maximum increase
in P content in straw was 45.6 % when co-inoculation with J120 x S4 was done in
compost amended soil compared with uninoculated control, followed by J119 x S14,
J120 x S14 and J119 x S4. In the presence of compost amended soil, single inoculation
also increased P content in straw up to 21.3% compared to control while their effect was
at par with each other.
126

Treatments (%) (%) (%) (%)
Control 2.58 k 2.63 jk 0.60 h 0.65 g 0.35 h 0.39 g 0.16 g 0.17 fg
J119 2.79 hi 3.00 de 0.68 fg 0.71 b-d 0.41 ef 0.42 c-e 0.18 ef 0.19 d-f
J120 2.75 i 3.06 d 0.69 d-f 0.70 b-e 0.41 ef 0.43 b-d 0.18 ef 0.20 c-e
S4 2.72 ij 2.96 d-f 0.68 ef 0.73 ab 0.40 f-g 0.44 a-c 0.18 ef 0.19 d-f
S14 2.82 g-i 3.00 de 0.69 c-f 0.71 b-d 0.42 c-e 0.42 c-e 0.18 ef 0.19 d-f
J119 x S4 2.89 e-h 3.31 b 0.70 c-f 0.72 a-c 0.43 b-d 0.43 b-d 0.19 de 0.21 bc
J119 x S14 2.88 f-h 3.47 a 0.69 d-f 0.73 ab 0.42 c-e 0.45 ab 0.20 c-e 0.22 ab
J120 x S4 2.93 e-g 3.38 ab 0.69 d-f 0.73 ab 0.42 d-f 0.46 a 0.18 ef 0.23 a
J120 x S14 2.96 d-f 3.18 c 0.71 b-d 0.74 a 0.43 b-d 0.46 a 0.18 ef 0.21 bc
LSD Value 0.1049 0.02347 0.0235 0.0165
127
4.7.2. Second year trials
In the second year, field trials were conducted at two different locations i.e.
Research Farm of the University of Agriculture, Faisalabad and Farmer’s field at Mouza
Wasva, Jhang.
4.7.2.1. First trial: Site-I (UAF)
During second year, once again inoculation and co-inoculation with selected
PGPR containing ACC-deaminase (J119 and J120) and rhizobial isolates (S4 and S14)
had almost similar type of effects as observed in the first year, which are summarized
below.
Single inoculation had significant effect on plant height and up to 15.5% increase
in plant height was recorded compared to uninoculated control (Table 4.25). While up to
26% increase in plant height was achieved in response to inoculation plus P-enriched
compost, as compared with uninoculated control. The co-inoculation with J120 x S4 gave
highest increase in plant height up to 18.9% as compared with uninoculated control,
followed by J120 x S14, J119 x S14 and J120 x S4. Up to 32.4% enhancement in plant
height was observed as a result of co-inoculation plus P-enriched compost, as compared
with uninoculated control but all the co-inoculation plus P-enriched compost treatments
were statistically non-significant with each other.
Highest increase (27.7%) in fresh biomass was observed as a result of simple
inoculation with S14 in case compost unamended soil, while in the presence of compost,
J120 caused 37.8% increase in fresh biomass as compared to control (Table 4.25). Other
128
remaining inoculation treatments had also significant effect on fresh biomass when tested
in the presence of enriched compost. It was recorded that co-inoculation with J120 x S14
produced maximum fresh biomass that was up to 32.1% higher than uninoculated control,
followed by J119 x S14, J120 x S4 and J119 x S4. Integrated use of co-inoculation with
P-enriched compost gave maximum increase in fresh biomass as compared with control.
The combination J120 x S14 plus P-enriched compost was the most effective and
increased 48.8% fresh biomass compared with uninoculated control.
In the absence of P-enriched compost, single inoculation with J119 produced
maximum number of pods per plant that was up to 47.6% compared to uninoculated
control and effects of all isolates were at par with each other (Table 4.25). While up to
66.7% increase in number of pods per plant was observed in case of to inoculation plus
P-enriched compost, compared to over uninoculated control control. Co-inoculation with
J120 x S14 gave maximum increase in number of pods up to 61.9% as compared to
uninoculated control, followed by J119 x S4, J119 x S14 and J120 x S4. Up to 92.9%
increase in number of pods per plant was recorded as a result of co-inoculation plus P-
enriched compost, as compared with uninoculated control.
In case of simple inoculation, maximum increase in grain yield was up to 18.1%
compared with uninoculated control and effects of all treatments were non-significant to
each other (Table 4.25). Up to 46.3% increase in grain yield was achieved as a result of
inoculation with J120 plus P-enriched compost, followed by S14, S4 and J119. While
129
(2nd year field trial)

Control 38.8 g 40.4 g 8.5 i 8.9 h 42 j 48 i 1.7 j 1.8 j 32.3 g 33.2 fg
J119 44.0 ef 44.8 ef 10.7 fg 10.9 f 62 f-h 69 cd 2.1 hi 2.2 gh 34.6 ef 35.1 de
J120 44.5 ef 48.6 bc 10.5 g 11.8 c 58 h 70 cd 2.1 hi 2.6 d 35.0 de 36.6 b-d
S4 43.9 f 47.4 cd 10.8 fg 11.6 c-d 58 h 68 c-f 2.0 i 2.4 ef 35.1 de 35.4 de
S14 44.8 ef 48.9 bc 10.9 f 11.5 de 61 gh 67 c-f 2.1 hi 2.4 e 35.6 de 36.0 c-e
J119 x S4 46.2 de 50.3 ab 10.5 g 12.3 b 63 e-h 76 ab 2.1 g-i 2.7 c 35.7 de 36.6 b-d
J119 x S14 45.0 ef 51.0 a 10.8 f 12.5 ab 65 d-g 77 ab 2.3 f 2.9 b 36.1 c-e 37.8 ab
J120 x S4 45.4 d-f 51.4 a 10.9 f 12.3 b 67 c-g 81 a 2.2 g 2.6 d 35.7 de 38.4 a
J120 x S14 44.3ef 50.3 ab 11.4 e 12.8 a 68 c-e 72 bc 2.3 ef 3.1 a 35.0 de 37.5 a-c
LSD Value 1.907 0.283 5.117 0.091 1.450
130
simple co-inoculation gave an increase in grain yield up to 30.51% than uninoculated
control. Co-inoculation with J120 x S14 in the presence of P-enriched compost gave
highest increase in grain yield that was up to 75.1% over uninoculated control.
It is clear from the results that both inoculation and co-inoculation had significant
positive effect on 100-grain weight of chickpea and maximum increase 18.7% in weight
was observed in response to co-inoculation with J120 x S4 in the presence of P-enriched
compost (Table 4.25). The increase in 100-grain weight varied from 7 to 18.7%
compared to uninoculated control in case of the inoculation/co-inoculation treatments in
the presence or absence of P-enriched compost.
In response to single inoculation with S14, maximum number of nudules per plant
was found up to 47% more than uninoculated control, following by J120, J119 and S4
(Table 4.26). While up to 73.3% increase in number of nudules per plant was noted when
inoculation with S14 was tested in the presence of enriched compost. However, simple
co-inoculation with J120 x S14 gave 61.8% increase in number of nodules per plant
compared with control, followed by J120 x S4, J119 x S4 and J119 x S14. Combined use
of co-inoculation plus enriched compost produced maximum number of nodules per plant
that was 88.2% more than uninoculated control.
4.7.2.1.7. Nodule dry weight
Co-inoculation produced maximum 41.9% more dry nodule weight per plant,
while simple inoculation showed up to 35.1% increase compared to uninoculated control
(Table 4.26). While integrated use of co-inoculation and P-enriched compost further
131
increased in nodule dry weight (up to 70.1% more over control) and maximum effect was
observed in case of inoculation J119 x S14. It was followed by J119 x S4, J120 x S4 and
J120 x S14. Whilereas, inoculation plus P-enriched compost also significantly increased
nodule dry weight which was 46.8% higher than control.
Maximum increase in root length was observed up to 22.6% as a result of
inoculation/co-inoculation, when compared with control (Table 4.26). While up to 37.3%
increase in root dry weight was obtained due to inoculation with S14 plus P-enriched
compost, followed by the J119, J120 and S4. When co-inoculation with J119 x S14 was
tested in the presence of P-enriched compost, the highest increase in root length was 48%
over uninoculated control but this increase in root length was at par with each other.
4.7.2.1.9. Root of dry weight
Maximum increase (20.8% more over control) in root dry weight per plant was
observed in response to inoculation with J120 that was at par with other strains, however,
increase in root dry weight varied from 16.3 to 20.8% compared to control (Table 4.26).
Likewise, co-inoculation of S4 and S14 with J119 strain improved the root dry weight
significantly as compared to other treatments with percent increase of 29.7 to 33.2%
respectively, as compared to uninoculated control. Co-inoculation with J120 x S4 plus P-
enriched compost showed maximum (up to 56.4% more than control) increase in root dry
weight per plant compared to control and this effect was non-significant with J119 x S4
and J120 x S14.
132

Control 34 j 37 j 0.41 h 0.44 gh 21.0 g 22.8 fg 2.0 k 2.2 j
J119 46 hi 50 f-h 0.53 d-f 0.56 c-e 24.7 ef 26.4 c-e 2.4 hi 2.6 ef
J120 44 i 59 a-d 0.48 fg 0.59 b-d 25.5 de 28.4 bc 2.4 hi 2.8 d
S4 47 hi 56 c-e 0.52 ef 0.60 bc 25.3 e 27.9 b-d 2.3 i 2.6 fg
S14 50 f-h 57 b-e 0.55 c-e 0.55 c-e 25.3 e 28.8 a-c 2.4 hi 2.7 de
J119 x S4 50 f-h 61 a-c 0.58 c-e 0.64 ab 25.6 de 30.3 ab 2.6 ef 2.9 c
J119 x S14 53 e-g 64 a 0.55 c-e 0.69 a 25.8 de 31.1 a 2.7 de 3.1 ab
J120 x S4 49 g-i 60 a-d 0.55 c-e 0.66 a 25.2 e 29.6 ab 2.5 gh 3.2 a
J120 x S14 55 d-f 62 ab 0.56 c-e 0.67 a 25.4 de 29.8 ab 2.5 fg 3.0 b
LSD Value 4.717 0.0524 2.228 0.0908
133
Single inoculation with S14 resulted in 20.9% increase in N content in grain and
this increase was further enhanced up to 23.2% when inoculation was tested in the
presence of P-enriched compost compared to control, followed by J119, J120 and S4
strain (Table 4.27). Simple co-inoculation increased N content in grain up to 30.9%
caused by J119 x S14 combination, as compared with control. While up to 36.3%
increase in N content in grain by co-inoculation with J119 x S14 plus P-enriched compost
was recorded compared with uninoculated control, followed by J120 x S4, J119 x S4 and
J120 x S14.
4.7.2.1.11. Nitrogen content in straw
Simple inoculation/co-inoculation resulted in 16.4 to 24.7% increase in N content
in straw, while J120 x S14 showed maximum 24.7% increase in N content compared with
control (Table 4.27). Further increase in N content in straw was noted, when
inoculation/co-inoculation was tested with compost amended soil. Highest increase in N
content in straw was 38.6%, caused by co-inoculation with J120 x S14 plus P-enriched
compost, followed by J119 x S14, J119 x S4 and J120 x S4.
Simple inoculation/co-inoculation significantly increased P content in grain as
compared to uninoculated control (Table 4.27). Sole inoculation caused by 19.2%
increase in P content in grain, while in case of co-inoculation this increase was promoted
up to 21.4% over uninoculated control. While integrated use of co-inoculation with J120
x S and P-enriched compost gave maximum increase in P content in grain was 32%
compared with uninoculated control. In case of simple inoculation/co-inoculation, non-
134

Treatments N content in grain N content in straw P content in grain P content in straw
(%) (%) (%) (%)
Control 2.59 i 2.74 h 0.73 m 0.78 l 0.31 h 0.33 g 0.23 i 0.24 h-i
J119 3.07 fg 3.17 d-f 0.86 jk 0.93 ef 0.36 ef 0.38 b-e 0.26 f-h 0.27 fg
J120 3.04 g 3.16 d-f 0.85 k 0.95 de 0.37 c-f 0.39 b 0.27 fg 0.30 a-d
S4 3.11 e-g 3.18 c-e 0.88 ij 0.90 gh 0.36 f 0.38 b-e 0.27 fg 0.30 b-e
S14 3.13 e-g 3.19 c-e 0.87 i-k 0.93 e-g 0.37 d-f 0.39 b-d 0.26 g-h 0.29 c-f
J119 x S4 3.21 c-e 3.40 b 0.89 h-i 0.96 cd 0.36 f 0.41 a 0.27 fg 0.32 ab
J119 x S14 3.39 b 3.53 a 0.88 ij 1.00 ab 0.37 d-f 0.39 bc 0.28 d-g 0.31 a-c
J120 x S4 3.25 cd 3.44 ab 0.88 h-j 0.98 bc 0.38 b-e 0.41 a 0.27 fg 0.31 a-c
J120 x S14 3.28 c 3.39 b 0.91 fg 1.01 a 0.37 d-f 0.39 b 0.27 fg 0.33 a
LSD Value 0.0908 0.0235 0.0165 0.0235
135
significant effects were observed in these treatments J119, J120, S4, S14, and J119 x S4,
J119 x S14 and J120 x S14.
The combined use of co-inoculation in the presence of P-enriched compost
showed better performance for improving P content in straw compared to uninoculated
control (Table 4.27). In case of single inoculation, maximum increase in P content in
straw was observed by inoculation with J120 strain that was up to 15% more than
uninoculated control. While co-inoculation with J119 x S14 resulted in 20.2% increase in
P content as compared to control. When inoculation and co-inoculation were testes with
P-enriched compost, a further increase in P content was observed, as up to 39.1% higher
P content were observed in case of co-inoculation with J120 x S14.
4.7.2.2. Second trial: Site-II (Nawaz Farm, Jhang)
The second field trial during the second year was carried out at Nawaz Farm,
Mouza-Wasava, Jhang to confirm the results of previous trials.
All the selected rhizobacteria containing ACC-deaminase and rhizobia significantly
promoted plant height of chickpea (Table 4.28). In case of single inoculation, all the
treatments were non-significant with each other but differed significantly from control.
Simple inoculation resulted in 15.9% increase in plant height, while co-inoculation
increased the plant height up to 19.4% compared to unoculated control and co-
inoculation treatments were also statistically similar to each other. While up to 36.7%
increase in plant height was achieved as a results of co-inoculation with J119 x S4 plus P-
136
enriched compost, followed by J119 x S14, J120 x S14 and J120 x S4. Effect of these
treatments J119 x S4, J119 x S14 and J120 x S14 was at par to each other.
Maximum increase (29.1% more over control) in fresh biomass was obtained in
case of inoculation with S14, followed by J119, J120 and S4 (Table 4.28). While up to
44.2% increase in fresh biomass was observed due to inoculation with S4 plus P-enriched
compost compared with control. Simple co-inoculation with J120 x S14 increased the
fresh biomass up to 33.4%, followed by J119 x S4, J119 x S14 and J120 x S4. While
combined use of co-inoculation with J119 x S4 plus P-enriched compost resulted in
maximum increase (63.7% more than uninoculated control) in fresh biomass and
statistically this treatment was top ranked as compared with all other treatments.
It is obvious from the results that co-inoculation plus P-enriched compost was
more effective in improving number of pods per plant than uninoculated control (Table
4.28). Maximum increase (97.4% over uninoculated control) in number of pods per plant
was observed as result of co-inoculation with J119 x S14 in the presence of enriched
compost, while other co-inoculation treatments in the presence of compost amended soil,
also showed significant increase in number of pods per plant ranged from 73.7 to 94.7%
more than control. Up to 52.3% increase in number of pos per plant was achieved by
single inoculation with S14 compared with uninoculated control. While in compost
amended soil, inoculation with J120 produced maximum (76.3% more over control)
number of pods per plant, followed by S14, S4 and J119.
137

Control 35.6 i 38.3 hi 6.9 j 7.6 i 38 j 44 i 1.6 l 1.7 kl 32.0 f 32.5 ef
J119 40.4 gh 42.1 d-g 8.4 h 8.9 ef 54 gh 63 c-e 1.8 jk 2.2 fg 33.8 de 34.6 cd
J120 41.3 f-h 43.7 d-f 8.5 gh 9.9 d 53 h 67 bc 1.8 jk 2.4 de 34.5 cd 34.9 cd
S4 40.6 f-h 44.7 c-e 8.8 f-g 10.0 d 54 gh 63 c-e 1.8 jk 2.3 e 35.0 cd 34.9 cd
S14 40.9 f-h 45.18 b-d 8.9 ef 9.7 d 59 e-g 62 d-f 1.9 jk 2.3 e 34.6 cd 35.5 a-c
J119 x S4 42.6 d-g 48.8 a 9.0 ef 11.4 a 57 f-h 75 a 2.1 hi 2.7 a 34.8 cd 35.9 a-c
J119 x S14 41.8 e-g 47.8 ab 8.9 ef 10.9b 60 d-f 70 ab 2.1 gh 2.6 bc 35.6 a-c 36.7 ab
J120 x S4 41.3 f-h 45.2 b-d 9.1 ef 10.4 c 64 c-e 66 b-d 2.0 hi 2.5 cd 35.2 bc 36.0 a-c
J120 x S14 42.3 d-g 46.8 a-c 9.2 e 10.8 b 61 d-f 74 a 1.9 ij 2.7 a 34.7 cd 37.0 a
LSD Value 2.738 0.327 4.992 0.117 1.405
138
Simple inoculation showed statistically similar effect with each other, and
maximum increase in grain yield through inoculation was recorded up to 13.3% over
uninoculated control (Table 4.28). While up to 43% increase in grain yield was achieved
due to inoculation plus enriched P-compost, over uninoculated control. But performance
of co-inoculation was statistically better than simple inoculation. In compost amended
soil, co-inoculation J119 x S4 increased grain yield which was up to 65.5% compared to
uninoculated control, followed by J120 x S4, J119x S14, and J120 x S14.
The results about 100-grain weight are summarized in Table 4.28 that both
inoculation and co-inoculation had significant positive affect on 100-grain weight of
chickpea and maximum increase 15.3% in 100-grain weight was recorded in response to
co-inoculation with J120 x S14 in the presence of compost amended soil, followed by S4,
J119 x S4, J119 x S14, J120 x S4 and J120 x S14 treatments. The increase in 100-grain
weight differed from 5.6 to 15.3% over uninoculated control in case of the
inoculation/co-inoculation treatments in the presence or absence of P-enriched compost.
In the presence of P-enriched compost, the maximum number of nodules per plant
were (89.7% more than uninoculated control) when the plants were co-inoculated with
J120 x S14 while other co-inoculation treatments increasd number of nodules per plant
up to 82.8% more than control (Table 4.29). The maximum numbers of nodules per plant
were up to 75.9% upon inoculation with J120 x S4 compared to control in the soil not
amended with compost. Up to 48.3% increase in number of nodules per plant was
139
observed due to inoculation with S14, while this was further improved up to 62.1% when
inoculation was tested in compost amended soil.
4.7.2.2.7. Nodule dry weight per plant
In the absence of P-enriched compost, co-inoculation produced up to 48.61%
more dry nodule weight per plant, while simple inoculation showed up to 30.7% increase
compared to uninoculated control (Table 4.29). While combined use of co-inoculation
and enriched compost further increased nodule dry weight (up to 87% more over control)
and maximum effect was observed in case of inoculation J119 x S14. It was followed by
J120 x S4, J120 x S14 and J119 x S4. Whereas, inoculation plus P-enriched compost also
significantly increased nodule dry weight which was 72.1% more than uninoculated
control.
Simple inoculation/co-inoculation showed statistically non-significant effects on
root length compared to uninoculated control (Table 4.29). Maximum increase in root
length was observed up to 28.4% as a result of inoculation/co-inoculation in the absence
of enriched compost over uninoculated control. While inoculation/co-inoculation plus
enriched P-compost increased root length ranged from 34.3 to 55% and the most effective
combination was J119 x S14 when tested in the presence of enriched compost compared
to uninoculated control.
4.7.2.2.9. Root of dry weight
Maximum root dry weight (20.4% more over control) was noted in response to
inoculation with S14, followed by treatments J119, J120 and S4. Co-inoculation resulted
140

Control 29 k 32 jk 0.32 l 0.36 k 22.6 g 24.9 f-g 2.3 i 2.5 j
J119 38 hi 42 f-i 0.40 j 0.47 f-g 27.8 ef 30.3 be 2.6 ef 2.7 e
J120 37 ij 43 e-h 0.40 j 0.55 e 28.0 e 32.1 bc 2.6 fg 2.8 cd
S4 41 g-i 45 d-g 0.41 ij 0.47 de 28.3 e 30.4 b-e 2.6 g 2.7 de
S14 43 e-h 47 c-g 0.42 i 0.56 de 27.7 ef 31.3 b-d 2.7 ef 2.8 cd
J119 x S4 45 d-g 49 b-d 0.46 gh 0.57 cd 29.3 c-e 32.8 ab 2.8 de 3.2 b
J119 x S14 48 b-e 53 ab 0.46 gh 0.61 a 28.4 de 35.0 a 2.9 c 3.4 a
J120 x S4 51 a-c 55 a 0.45 h 0.58 bc 27.7 ef 32.1 bc 2.7 ef 3.3 ab
J120 x S14 47 c-f 50 a-d 0.48 f 0.59 ab 29.0 de 33.3 ab 2.9 c 3.2 b
LSD Value 5.005 0.0165 2.627 0.105
141
Co-inoculation with J120 x S14 vs J120 x S14 + P-enriched compost
Picture: Effect of co-inoculation on nodulation in the absence and presence of P-

enriched compost under field conditions
142
in maximum increase of root dry weight (up to 28.4% higher than control) by co-
inoculation with J119 x S14 followed by J120 x S14, J119 x S4 and J120 x S4 (Table
4.29). While up to 50.2% increase in root dry weight was obtained as a result of co-
inoculation with J119 x S14 in compost amended soil. Inoculation/co-inoculation
significantly increased root dry weight both in the absence and presence of P-enriched
compost.
Both inoculation/co-inoculation significantly improved N content in grain in the
compost amended and unamended soil (Table 4.30). Simple inoculation with S14
resulted in 17.8% increase in N content in grain and this increase was further improved
4.7.2.2.11. Nitrogen content in straw
Maximum increase in N content in straw was 29% which caused by co-
inoculation with J120 x S4, while other treatments of inoculation/co-inoculation also
increased N content in straw that ranged from 18.8 to 27.5% more than control (Table
4.30). The heighest increase (40.6%) in N content in straw was observed through co-
inoculation with J120 x S14 plus enriched compos, followed by J120, J119 x S4, J119 x
S14 and J120 x S4 in the presence of enriched compost.
The results revealed that simple inoculation significantly increased P content in
grain which was up to 18.7% more than uninoculated control (Table 4.30). While co-
inoculation caused up to 19.7% increase in P content in grain as compared to
uninoculated control. In case of integrated use of inoculation/ co-inoculation and P-
143

Treatments (%) (%) (%) (%)
Control 2.64 l 2.69 k 0.69 k 0.73 j 0.32 k 0.34 j 0.23 h 0.24 gh
J119 3.03 i 3.09 f-h 0.86 g-i 0.91 e-i 0.36 g-i 0.37 e-i 0.25 f-h 0.26 d-f
J120 3.00 j 3.35 b 0.82 c-h 0.92 a-d 0.38 c-h 0.39 a-d 0.25 f-h 0.28 c-e
S4 3.03 ij 3.18 d 0.87 i 0.88 d-i 0.36 i 0.38 d-i 0.25 fg 0.28 c-e
S14 3.11 e-g 3.20 d 0.83 h-i 0.91 b-f 0.36 h-i 0.39 b-f 0.25 fg 0.27 c-e
J119 x S4 3.08 gh 3.32 c 0.87 f-i 0.95 ab 0.37 f-i 0.40 ab 0.25 fg 0.31 a
J119 x S14 3.13 e 3.45 a 0.83 c-g 0.96 a-c 0.38 c-g 0.40 ab 0.26 ef 0.30 b
J120 x S4 3.07 h 3.36 b 0.89 e-i 0.92 a 0.37 f-i 0.41 a 0.25 ef 0.29 b-c
J120 x S14 3.11 ef 3.31c 0.88 f-i 0.97 b-e 0.370 f-i 0.39 b-e 0.26 d-f 0.28 b-d
LSD Value 0.0287 0.0525 0.0166 0.0164
144
enriched compost exhibited maximum effect on P content was 28.3% when co-inoculants
J120 x S4 were applied along with enriched compost.
Single inoculation/co-inoculation increased P content in straw up to 13.4%
compared with uninoculated control and effect of all inoculation/co-inoculation
treatmrents were at par to each other (Table 4.30). Maximum increase in P content in
straw was recorded up to 35.3% by co-inoculation with J119 x S4 in the presence of
enriched compost, followed by J119 x S14, J120 x S4 and J120 x S14. While, simple
inoculation also showed further increase in P content in straw when tested in the presence
of enriched compost.
4.9. Classical “triple” response
Classical “triple” response is a bioassay to test the effect of ethylene biosynthesis
on plant. In this study, the potential of selected rhizobacterial isolates containing ACC-
deaminase to reduce the effects of ethylene produced from ACC (i.e. precursor of
ethylene) was examined. It is clear from the results that selected isolates containing ACC-
deaminase significantly diluted the effect of exogeneously applied ACC (Table 4.31).
The seedlings length and stem diameter of etiolated pea seedlings was affected by
exogenous use of ACC. The seedling diameter increased with the increase in ACC
concentration and exhibited significant positive correlation with ACC (r = 0.98**) (Fig.
4.1). Maximum increase in diameter was recorded at 10 mM concentration of ACC.
Length of pea seedlings decreased as the concentration of ACC increased. Maximum
reduction in seedling length was observed at concentration of 10 mM ACC that reduced
the length of pea seedling up to 63.6% as compared with control (without ACC). There
145
Table 4.31: Classical “triple” response of etiolated pea seedlings at different concentrations of ACC (mM)

Parameter Control 2 mM ACC 4 mM ACC 6 mM ACC 8 mM ACC 10 mM ACC LSD Value
Shoot diameter (mm) 0.83 f 0.90 e 1.197d 1.34 c 1.45 b 1.59 a 0.056
Shoot length (cm) 14.3 a 13.1 a 10.3 b 9.8 c 7.3 d 5.2 e 1.017
Root length (cm) 10.1 a 8.8 b 7.6 c 6.9 cd 5.9 d 4.0 e 1.058
Fresh biomass (g) 2.5 a 2.3 b 2.2 bc 2.1 c 1.9 d 1.6 e 0.138
Shoot dry weight (g) 0.83 a 0.90 b 1.20 c 1.34 d 1.45 e 1.59 f 0.056
Root dry weight (g) 0.37 a 0.32 b 0.31 bc 0.29 c 0.26 d 0.18 e 0.018
Mean values sharing similar letter (s) in a column are non-significant at P < 0.05, according to Duncan’s multiple range test.
146

Figure 4.1: Classical “triple” response of etiolated pea seedlings at different

concentrations of ACC (mM)
147
was a linear decline in seedling length with increasing ACC concentration and significant
positive correlation (r = -0.99**) was observed between them.
Apploication of cobalt to ACC treated plants significantly reduced the stem
diameter and increased shoot length (Table 4.32). Similarly, the study demonstrated that
inoculation with rhizobacterial isolates (J119 and J120) containing ACC-deaminase
activity reduced the effect of ACC and significantly increased shoot length and decreased
stem diameter (Table 4.32), confirming that bacteria with ACC-deaminase activity can
eliminate inhibitory effect of ethylene on plant growth by lowering its endogenous levels.
148
Table 4.32: Comparative effect of inoculation with rhizobacteria containing ACC-deaminase activity and cobalt
(CO2+) on etiolated pea seedlings in the presence of 10 mM ACC
Shoot diameter Shoot length Root length Fresh biomass Shoot dry Root dry
Treatments
(mm) (cm) (cm) (g) weight (g) weight (g)
10 mM ACC 1.58 a 5.2 c 4.0 c 1.6 c 0.163 c 0.17 c
CO2+ + 10 mM ACC 0.83 b 12.8 b 9.7 b 2.2 b 0.413 b 0.31 b
J119 + 10 mM ACC 0.77 d 14.1 a 10.7 a 2.5 a 0.446 a 0.33 a
J1120 + 10 mM ACC 0.85 c 13.6 ab 9.9 b 2.4 a 0.433 a 0.32 ab
LSD Value 0.033 1.023 0.65 0.12 0.019 0.018
Mean values sharing similar letter (s) in a column are non-significant at P < 0.05, according to Duncan’s multiple range test
149

Figure 4.2: Comparative efficacy of inoculation with rhizobacteria containing ACC-

deaminase activity and cobalt (Co2+) on etiolated chickpea seedlings
150
4.10. Identification and characterization of selected rhizobacteria and
rhizobia
The selected bacteria were identified and characterized for various charctiristics
(Table 4.33). The rhizobacteria identified as Serratia proteamaculans (J119) and
Citrobactor koseri (J120) were positive for in vitro ACC-deaminase activity, P
solubilization and chitinase activity, and siderophores production, in addition to showing
high root colonization ability. Similarly, both rhizbial isolates S4 and S14 identified as
Mesorhizobium ciceri were able to solubilize P and form siderophores. Chitinase activity
was observed in case of S14 which also showed high root colonization rate.
151

Table 4.33: Identification and characterization of selected strains of plant growth promoting rhizobacteria and
rhizobia isolated from chickpea

Colony ACC-deaminase Chitinase Siderophore Phosphate Root
Bacterial strains color activity activity production solubilization colonization
(α-ketobutyrate (qualitative) (cfu g-1)
nmol g-1 biomass h-1)
Serratia proteamaculans (J119) Pink 451 ± 10.3 ++ve ++ve +ve 5.63 × 106
Citrobacter koseri (J120) White 429 ± 8.5 +ve +ve ++ve 4.79 × 105
Mesorhizobium ciceri (S4) White ND -ve +ve +ve 7.59 × 104
Mesorhizobium ciceri (S14) Pink ND +ve ++ve +ve 6.93 × 105
±: Standard error of means; ND: Not determine
152

Co-inoculation of Chickpea Discussion
DISCUSSION
Ethylene is a potent plant growth regulator that is involved in the regulation of many
physiological processes occurring in the plant. It is effective in evoking physiological
response in plant even when present in extremely low concentrations. Although ethylene
is crucial for many physiological processes, its higher concentrations are often inhibitory
for plant growth. Elevated levels of ethylene are produced in plants in response to biotic
and abiotic stresses, and thus suppress root and shoot growth. Certain plant growth
promoting bacteria are capable of lowering ethylene levels in plant through their ACC-
deaminase activity. They convert ACC in to NH3 and -ketobutyrate instead of ethylene.
So reduction in the levels of ethylene under suboptimal growth conditions results in
stimulation of root growth and consequently shoot growth. In the present studies,
rhizobacteria containing ACC-deaminase and rhizobia were isolated from the rhizosphere
soil and nodules of chickpea plants, respectively. The rhizobacteria along with rhizobia
were screened for their potential to promote nodulation and growth of chickpea under
axenic conditions. The most prolific co-inoculation strains were selected (two each from
rhizobia and rhizobacteria) for inoculation studies under natural soil conditions.
5.1. Screening rhizobacteria and rhizobia for their potential to promote
growth and nodulation of chickpea under axenic conditions
5.1.1. Screening rhizobacteria containing ACC-deaminase for growth promoting
activity
A series of laboratory studies were conducted under controlled (axenic)

153
conditions to screen effective strains of rhizobacteria with ACC-deaminase activity for
promoting growth and nodulation of chickpea. First of all, ability of rhizobacterial
isolates to utilize ACC as sole source of N was confirmed by determining their cell
densities (OD). All the rhizobacterial isolates, originated from the rhizosphere soil of
chickpea, were capable of utilizing ACC and variable growth rates were shown on MSM
containing ACC as sole N source. This may imply that ACC-deaminase enzyme in
different rhizobacteria might have variable potential to hydrolyze ACC and, thus could
have differential affect on growth of inoculated plants. Several bacterial species
belonging to different genera such as Azospirillum, Agrobacterium, Achromobacter,
Burkholderia, Enterobacter, Pseudomonas and Ralstonia have been reported to possess
variable ACC-deaminase activity (Nukui et al., 2000; Guinel and Sloetjes, 2000; Yuhashi
et al., 2000; Wang et al., 2001; Duan et al., 2006; Shaharoona et al., 2006 b; Nadeem et
al., 2007; Arshad et al., 2008).
Plate (germination assay) and jar (root and shoot growth) experiments were
conducted for evaluating growth promoting activities of rhizobacteria containing ACC-
deaminase under axenic conditions. Inoculation with rhizobacteria containing ACC-
deaminase did not affect germination of chickpea significantly. Since ethylene is required
for germination of chickpea seeds (Gallardo et al., 1994, 1995; Matilla and Matilla-
Vazquez, 2008), so germination was slightly decreased in response to inoculation with
rhizobacteria containing ACC-deaminase compared to uninoculated control. However, in
this study, a significant increase in root length and weight was observed in the case of
inoculated plants compared with uninoculated control. It is highly likely that
rhizobacteria promoted root growth by lowering ethylene levels in plant and/or in the
154
vicinity of roots because of their ACC-deaminase activity. This premise is further
supported by the results obtained from jar experiment (Table 4.3) in which observed root
growth was much better in the plants inoculated with the rhizobacteria containing ACC-
deaminase than the uninoculated control. These results are in agreement with the findings
of many researchers who reported better root growth in plants inoculated with bacteria
containing ACC-deaminase (Glick et al., 1995; Xie et al., 1996; Hall et al., 1996; Shah et
al., 1998; Matilla, 2000; Penrose et al., 2001; Mayak et al., 2004a, b; Shaharoona et al.,
2006a, b; Soares et al., 2006). A direct correction has been found between in vitro
bacterial ACC-deaminase activity and root growth (Mayak et al., 2004b; Shaharoona et
al., 2006a).
In this study, it was also observed that rhizobacteria containing ACC-deaminase
were highly effective in promoting number of lateral roots, later root length and root dry
weight of chickpea seedlings, in addition to improving roots length. These changes in
root architecture of the inoculated plants could be attributed to bacterial ACC-deaminase
activity as described earlier. Furthermore, a tremendous positive effect on shoot growth
by rhizobacteria containing ACC-deaminase was also observed. A significant direct
relationship has been observed between root and shoot growth (Shaharoona et al., 2006a).
Ghosh et al. (2003) reported that rhizobacteria containing ACC-deaminase promoted
plant growth, while root length was significantly increased under axenic conditions.
Similar kind of findings have been documented by other researchers (Shah et al., 1998;
Penrose et al., 2001; Belimov et al., 2002; Dodd et al., 2004; Cakmakci et al., 2005;
Sergeeva et al., 2006). These findings may imply that rhizobacteria with ACC-deaminase
activity could prove to be effective inoculants for improving growth of chickpea plants.
155
5.1.2. Screening rhizobia for their ability to promote nodulation and growth of
chickpea
Pouch and jar trials were conducted in the growth room to screen effective
isolates of rhizobia for promoting nodulation and growth of chickpea under
controlled (axenic) conditions. The results illustrated that all the test rhizobial isolates
exhibited growth promoting activity in chickpea but with different degrees of efficacy.
Some of the isolates were highly effective in increasing nodulation in chickpea in both
pouch and jar (sand) experiments. Similarly, several researchers reported significant
increases in nodulation of various legumes upon inoculation with different rhizobial
strains (Roy et al., 1995; Belimov et al., 2002; Ghosh et al., 2003; Dodd et al., 2004;
Mayak et al., 2004; Sergeeva et al., 2006; Shaw et al., 2006; Miller et al., 2007;
Figueiredo et al., 2008). Huang and Erickson (2007) studied the effect of inoculation with
R. leguminosarum on lentil and pea. They observed a significant improvement in seedling
growth, nodule fresh weight, root and shoot biomass in peas. Furthermore, a significant
increase in seed yield of pea was recorded. Similarly, inoculation of chickpea seeds with
Mesorhizobium ciceri, promoted seedling height, root nodule mass and shoot biomass.
Likewise, nodulation was enhanced in peanut by Rhizobium species (Dey et al., 2004).
The study also demonstrated that some of the rhizobial isolates did not induce
nodulation in chickpea under axenic conditions. Failure in nodulation might be due to
some antagonism that prohibited Rhizobium to colonize the root surface (Jadhave et al.,
1994). Likewise, the van Rhijn et al. (2001) reported no nodulation in pea (Pisum
sativum) and alfalfa (Medicago sativa L.) plants in response to Rhizobium inoculants.
Rhizobia, in certain cases, become deficient in exopolysaccharides due to mutation or
156
some unknown reasons. However, screening of effective rhizobial strains under
gnotobiotic conditions could be useful strategy for the selection of efficient strains to
improve nodulation process under field conditions. Furthermore, nodulation may be
enhanced by co-inoculating the Rhizobium sp. with plant growth promoting rhizobacteria
containing ACC-deaminase which may eliminate inhibitory effect of ethylene on
nodulation by lowering its levels in plants and/or in the vicinity of roots through ACC-
deaminase activity.
5.1.3. Effect of co-inoculation on nodulation and growth of chickpea under axenic
conditions
Twelve isolates, six each from rhizobacteria and rhizobia, were selected to find
out the most effective co-inoculant strains for the improvement of nodules and growth of
chickpea. Co-inoculation of rhizobia with certain isolates of rhizobacteria containing
ACC-deaminase activity was more effective in improving nodulation and growth of
chickpea than the sole inoculation of rhizobia. It is very likely that PGPR containing
ACC-deaminase activity promoted growth and nodulation by lowering ethylene levels in
roots (Glick et al., 1998; Paul and Verma, 1999; Belimove et al., 2002; Ma et al., 2004;
Shaharoona et al., 2006a, b; Duan et al., 2006; Glick et al., 2007; Arshad et al., 2008).
Such rhizobacteria might also have stimulated nodulation indirectly through increased
root growth that provides rhizobia more infection sites for nodulation. This promise is
supported by the data presented in Table 4.9, which illustrated that some rhizobacteria
containing ACC-deaminase significantly promoted root length, and lateral root number
and length of chickpea seedlings under axenic conditions. Similarly, Parmar and
Dadarwal (2000) reported that increase in root growth provides more number of active
157
sites and access to nodulation for rhizobia. They documented enhanced nodulation in
chickpea plant by rhizobacteria. Similarly, Dashti et al. (1998) demonstrated an increase
in nodule weight per plant in two soybean cultivars because of co-inoculation of B.
japonicum with plant growth promoting bacteria at early stages in comparison to sole
inoculation of the B. japonicum. Co-inoculation of Bradyrhizobium and P. striata also
promoted biological nitrogen fixation in soybean (Dubey, 1996). Similarly, dual
inoculation with Bradyrhizobium sp. and the PGPR promoted nodulation, growth of
plants and N2-fixation in Vigna radiata (Gulati et al., 2001). Improvement in nodule
occupancy of Bradyrhizobium in soybean has been reported due to co-inoculation with P.
fluorescens (Bai et al., 2002; Nishijima et al., 1988; Fuhrmann and Wollum, 1989; Chen
et al., 2000; Matilla, 2000; Jeffries et al., 2003; Dechassa and Schenk, 2004; Hafeez et
al., 2004; Vikram et al., 2007; Mishra et al., 2009).
Some combinations of rhizobial and rhizobacterial isolates could not improve
growth and nodulation compared to uninoculated control, which might be due to certain
compounds (i.e., toxic for plants to some extent) produced by the bacteria. Production of
antibiotics and competition for attachment sites on root surfaces could be one of the
reasons for negative effects of co-inoculation of PGPR with Rhizobium (Chebotar et al.,
2001; Valverde et al., 2006; Mirza et al., 2007). Raverkar and Konde (1988) also reported
the adverse effects of co-inoculation with Rhizobium and Azospirillum lipoferum on
nodulation, nitrogen contents and yield of two peanut cultivars.
5.2. Effect of inoculation/ co-inoculation on chickpea under natural conditions
Based upon laboratory and growth room trials conducted under axenic conditions
for screening plant growth promoting bacteria, two isolates from each of the rhizobia (S4
158
and S14) and rhizobacteria (J119 and J120) containing ACC-deaminase were selected to
use them as inocula for pot and field experiments. Pot and field trials were conducted
under natural conditions to evaluate the potential of selected plant growth promoting
bacterial isolates for improving nodulation, growth and yield of chickpea. Seeds of
chickpea were treated with peat based inocula of rhizobia and rhizobacteria before
sowing in the pots and into the fields. Field experiments were conducted at three different
locations i.e. at research area of the University as well as at farmers’ fields for
consecutive two years. The results of both pot and field experiments indicated that
inoculation with selected isolates of rhizobia and rhizobacteria containing ACC-
deaminase significantly promoted nodulation, growth and yield of chickpea, when the
inocula were applied either alone or in combination (co-inoculation) with each other. In
general, co-inoculation exhibited more pronounced effect on growth, yield and
nodulation of chickpea than single inoculation. Here, rhizobacteria with ACC-deaminase
were shown to influence positively on growth and nodulation, most probably through
lowering of ethylene in plant at early stage of development as a results of ACC-
deaminase activity. Reduction in ethylene by the selected rhizobacteria with ACC-
deaminase activity and its influence on plant growth was further confirmed by conducting
classical “triple” response bioassay on etiolated chickpea seedlings under controlled
(axenic) conditions. It is obvious from the results (Table 4.31) that application of ACC
caused classical “triple” response in etiolated pea seedlings while inoculation with
Serratia proteamaculans (J119) and Citrobacter koseri (J120) containing ACC-
deaminase diluted the ACC-imposed effect similar to that of Co2+. It is very likely that
intensity of classical “triple” response decreased in response to reduction in endogenous
159
ethylene levels in etiolated pea seedlings due to cleavage of ACC into HN3 and α-
ketobutyrate by the enzyme ACC-deaminase of the rhizobacteria. Several authors have
reported the effect of PGPR on legume crops. Shaharoona et al. (2006a) reported a
significant positive effect of PGPR containing ACC-deaminase on nodule fresh and dry
weight and number of nodules of mung bean. Remans et al. (2007) viewed that bacterial
ACC-deaminase activity can play an important role in the host nodulation response,
mainly under low P conditions.
Field experiments were conducted at different sites (i.e. at Research Farm and
farmers’ fields) to evaluate the potential of inoculation/co-inoculation with rhizobia plus
rhizobacteria to promote growth, nodulation and yield of chickpea PGPR isolate J119
performed better under field conditions at all sites, for improving growth and yield of
chickpea; however, a site variation in their performance was observed at different
locations. This variation could be attributed to different factors including ability of
bacteria to colonize and compete, climatic factors, soil characteristics and indigenous
micro-flora present at different places (Khalid et al., 2004; Kannan et al., 2005;
Lalfakzual et al., 2008).
The presence of PGPR in the root vicinity may also improve ability of rhizobia to
compete with indigenous populations for nodulation. This was demonstrated by Gupta et
al. (1998) by conducting experiment on green gram (Vigna radiata) grown in a non-
sterile soil, in which two strains of Enterobacter were co-inoculated with two strains of
Bradyrhizobium sp. (Vigna). PGPR improved the nodule occupancy of the two rhizobial
strains. Bradyrhizobium sp. (strain S24) occupied 60% of nodules in sole inoculation
which was reached up to 81% in the presence of Enterobacter (strain EG-ER-1) while
160
other Enterobacter (KG-ER-1) improved nodule occupancy of Bradyrhizobium (strain
Cog15) from 77 to 88% (Gupta et al., 1998). However, it appears that PGPR strains have
no effect on the in vitro growth of Bradyrhizobium, as described by the same researchers
through using 10 Bradyrhizobium strains co-inoculated with 14 strains of PGPR,
including Enterobacter (Gupta et al., 2003). In a study with B. japonicum, 18 bacteria
belonging to the genera Pseudomonas and Aeromonas spp. did not interfere with the
nodulation capacity of soybean, but three of these strains increased nodule numbers and
others enhanced plant growth (Polonenko et al., 1987). Similarly, strain dependent effects
have also been reported in a study where co-inoculation with P. fluorescens 2137
increased the colonization of B. japonicum on soybean roots and nodule numbers while
co-inoculation with P. fluorescens WCS365 had the opposite effects (Chebotar et al.,
2001). The same study also suggested that the high root colonization of P. fluorescens
2137 could enhance nodulation by the release of growth promoting substances that
stimulate B. japonicum. Dey et al. (2007) tested efficacy of PGPR belonging to
Pseudomonas sp. under pot and field conditions for three consecutive years. Seed
inoculation with three isolates PGPR1, PGPR2 and PGPR4, significantly increased pod
yield than the control, in pots. In the field trials, however, there was wide dissimilarity in
the efficacies of the PGPR isolates in improving growth and yield of peanut in different
years. Plant growth promoting fluorescent pseudomonads, PGPR1, PGPR2 and PGPR4,
significantly enhanced pod yield (23 to 26%, 24 to 28% and 18 to 24%, respectively),
haulm yield and nodule dry weight over the control in three consecutive years. Lian et al.
(2001) observed that Bacillus circulans produces a chemical compound analog to the nod
factor of B. japonicum. This compound causes root hair deformation activity on soybean.
161
It was observed that co-inoculation of rhizobia with rhizobacteria containing
ACC-deaminase along with P-enriched compost showed highly positive effect on growth,
nodulation and yield of chickpea as compared to uninoculated/ untreated control.
Composted organic materials not only serve as a source of plant macro- and micro-
nutrients but also supports the activity of inocula (Badr EL-Din et al., 2000; El-Din et al.,
2000; Pooran et al., 2002; Biswas and Narayanasamy, 2002; Bajpai et al., 2002; Cheuk et
al., 2003; Tengerdy and Szakacs, 2003; Indira-Sarangthem et al., 2004; Zayed et al.,
2005; Ayaga et al., 2006; Ahmad et al., 2008a, b, Abd El-Gawad, 2008). So application
of P-enriched compost affected the growth and nodulation of chickpea by enhancing
nutrient mobilization and uptake, in addition to enhancing bacterial activities. Several
reports have suggested that PGPR can stimulate plant growth through their P-solubilizing
activity (Indira-Sarangthem et al., 2004; Zayed et al., 2005; Ayaga et al., 2006; Khan et
al., 2007; Wani et al., 2007d, e; Afzal and Bano, 2008). PGPR are known to affect the
nutrient availability to the plant through mineralization, acidification, redox changes or
by producing iron cheaters and siderophores (Burd et al., 2000; Romkens et al., 2002;
Abou-Shanab et al., 2003; Raghothama, 2005). Since selected PGPR also exhibited P-
solubilization and chitinase activity, and siderophores production, so promotion in growth
and nodulation might also have been facilitated through these useful traits.
Synergism among Rhizobium and PGPR strains is also beneficial for legume
crops, as observed previously with chickpea plants (Alagawadi and Gaur, 1988). Co-
inoculation of Rhizobium and P. striata or B. polymyxa (PSB) increased plant growth,
nodulation, nitrogenase activity, and uptake of N and P. PGPR also improved available P
content of the soil. Similarly, positive effect of PGPR (Pseudomonas KB-133), a PSB (B.
162
megatherium) and Rhizobium sp. strain on growth, nodulation and yield of black gram,
has been documented (Gunasekaran et al., 2004; Kumar and Chandra, 2008). Nitrogenase
enzyme also requires a lot of iron (Fe), and so siderophores production could be useful
for improving nodulation (Leong, 1986; Catellan et al., 1999; Vivekananthan et al.,
2004).
PGPR Serratia proteamaculans J119 exhibited highest ACC-deaminase activity
(Table 4.32), production of siderophores, chitinase activity and colonization to the
surfaces of plant roots. In general, Serratia proteamaculans J119 performed better alone
as well as in co-inoculation with rhizobia for improving growth and yield of chickpea. In
case of rhizobia, Mesorhizobium ciceri (S14) was the most effective strain and was
capable of producing siderophores. Hence, ACC-deaminase activity of the rhizobacteria
coupled with other useful traits was, most likely, responsible for improving nodulation,
growth and yield of chickpea when co-inoculated with rhizobial strains.
In conclusion, the use of co-inoculants such as rhizobia and rhizobacteria
containing ACC-deaminase along with P-enriched compost could be the most effective
and novel approach for achieving better root growth, nodulation and yield of chickpea.
163
Co-inoculation of Chickpea Summary
SUMMARY
Chickpea (Cicer arietinum L.) is an important legume crop, usually grown in water
deficit areas of Pakistan. Among several factors which contribute to low productivity of
chickpea, elevated levels of ethylene in plant could be one of the major growth limiting
factors under sub-optimal growth conditions. It is established fact that accelerated levels
of ethylene are produced in plant in response to biotic and abiotic stresses, which inhibit
root growth and nodulation. Some PGPR are capable of lowering ethylene levels through
their ACC-deaminase activity and thus enhance root growth and nodulation and,
consequently shoot growth and yield. It is very likely that the use of PGPR containing
ACC-deaminase along with rhizobia could be effective for promoting growth, nodulation
and yield of chickpea. In the present study, PGPR containing ACC-deaminase and
rhizobia were isolated from the rhizosphere soil and nodules of chickpea plants (collected
from different locations) on MSM containing ACC-deaminase as sole N source and YEM
media, respectively. The ability of rhizobacteria to utilize ACC as N source was further
confirmed by examining their growth and cell densities (in terms of OD) in MSM broth.
Effective strains of rhizobacteria along with rhizobia were screened for their potential to
produce nodulation and growth of chickpea by conducting experiments under axenic
(controlled) conditions.
Some isolates of rhizobacteria were very effective in promoting root and shoot
growth (up to 107 and 57.4% respectively, over uninoculated control) of chickpea in jar
experiment. Similarly, most of the rhizobial isolates (from a total of 35) significantly
164
improved growth and nodulation as compared to uninoculated control. Six isolates of
rhizobacteria were selected on the basis of morphological changes in root growth (root
length, lateral root number and length, and root dry weight) under axenic conditions.
Likewise, six rhizobial isolates were selected on the basis of improvement in root growth
and nodulation of chickpea.
The selected rhizobacteria and rhizobia were tested in all possible
combinations under axenic conditions to screen effective co-inoculant strains for the
improvement of growth and nodulation of chickpea. In most cases, co-inoculation with
rhizobacteria and rhizobia was highly effective and significantly promoted root growth
and nodulation compared to single inoculation with either of the bacteria or uninoculated
control.
Two most effective combinations (J119 x S14 and J120 x S4) of rhizobia and
rhizobacteria containing ACC-deaminase were selected to further examine their potential
by conducting pot and field trials under natural conditions.
Pot experiments were conducted in the net house to evaluate the potential of
selected isolates of rhizobacteria containing ACC-deaminase (J119 and J120) and
rhizobia (S4 and S14) alone as well as in combination for improving growth, nodulation
and yield of chickpea. The results revealed that inoculation with these isolates
significantly increased yield and yield contributing parameters as compared to
uninoculated control. The maximum increase in grain yield (up to 76%), nodule number
(up to 94%) and nodule dry weight (up to 96.6%) was observed in response to co-
inoculation of rhizobial strain S14 with rhizobacteria J119 or J120 compared to
165
Like pot trials, the selected bacterial isolates were tested under field conditions at
different locations for consecutive two years. The results show that both single and co-
inoculation with selected bacteria significantly promoted growth, nodulation, and yield of
chickpea. In case of single inoculation, the isolate S14 was the most effective and caused
up to 27.2% increase in grain yield. Nodulation was also improved significantly by
inoculation with this bacterium. In case of co-inoculation, strains J119 and S14 were the
most effective in improving grain yield and nodulation.
The integrated use of co-inoculation of rhizobia and rhizobacteria containing
ACC-deaminase along with P-enriched compost was the most effective approach to
improve growth, nodulation and yield of chickpea under both pot and field conditions.
The results of classical “triple” response bioassay illustrated that application of
ACC had significant effect on etiolated pea seedlings and caused reduction in seedling
length and increase in stem diameter while inoculation with the selected rhizobacteria
diluted the ACC-imposed effect on etiolated pea seedlings, most likely by reducing the
production of ethylene from ACC as a result of their ACC-deaminase activities.
Similarly, application of cobalt (inhibitor of ethylene) eliminated the effect of ACC on
etiolated pea seedlings.
The selected rhizobacteria and rhizobia were identified and then characterized for
different traits. In addition to in vitro ACC-deaminase activity, the rhizobacteria Serratia
proteamaculans (J119) and Citrobacter koseri (J120) exhibited other useful traits such as
production of siderophores, chitinase and phosphate solubilization activity. Both strains
(S4 and S14) of rhizobium Mesorhizobium ciceri had ability to produce sideriophores and
solubilize phosphates while chitinase activity was only observed in case of S14. The
166
maximum root colonization was recorded in the case of Serratia proteamaculans (J119)
while Mesorhizobium ciceri (S14) was next the most active root colonizing bacterium.
In general, Serratia proteamaculans (J119) and Mesorhizobium ciceri
(S14) were the most effective bacteria in improving growth, nodulation and yield of
chickpea.
167
Co-inoculation of Chickpea Conclusions
CONCLUSIONS
In this study, two approaches were simultaneously employed to select effective PGPR
and rhizobia for chickpea to be used them as inocula in pot and field trials. The
approaches include screening of rhizobacteria for in vitro ACC-deaminase activity and
for their growth-promoting activity (particularly root growth) under gnotobiotic (axenic)
conditions. The rhizobia were selected based on their ability to produce high number of
nodules and nodule dry weight under axenic conditions. The results demonstrated that
bacterial ACC-deaminase-induced changes in root architecture could be crucial for the
selection of efficient strains of PGPR. Such bacteria could be very effective as co-
inoculants to improve growth, nodulation and yield of chickpea. However, the degree to
which these inoculants impart benefits to plant growth can vary with the conditions and
PGPR strains. A PGPR strain with multifarious traits could be more useful under diverse
conditions compared to a strain containing single trait. Application of composted organic
material along with inocula may further enhance the effectiveness of the PGPR. This
premise is supported from the results that inoculation/ co-inoculation had promising
positive effects on growth, nodulation and yield of chickpea grown in the soil amended
with P-enriched compost. Thus, it can be concluded from my study that the use of co-
inoculation (rhizobia and rhizobacteria containing ACC-deaminase) in the presence of P-
enriched compost could be the most effective and novel approach for promoting
nodulation and yield of chickpea grown under natural conditions.
168
Co-inoculation of Chickpea Conclusions
Future research should focus on managing plant-microbe and microbe-microbe (rhizobia-
rhizobacteria containing ACC-deaminase) interactions, mainly with respect to their mode
of actions and adaptability to extreme environmental conditions for the betterment of
plants. Screening efficient strains of Rhizobium sp. with ACC-deaminase activity
compatible with the environment and plant sp. could be very useful approach to enhance
the nodulation in legumes. In addition, researchers should explore how nutrition and root
exudation could affect the activity of useful bacteria and promote nodulation.
169

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POTENTIAL OF RHIZOBIA AND FREE LIVING RHIZOBACTERIA

CONTAINING ACC-DEAMINASE FOR IMPROVING NODULATION AND

A thesis submitted in partial fulfillment of the requirements for the degree of

INSTITUTE OF SOIL AND ENVIRONMENTAL SCIENCES

FOR ALWAYS BEING THERE WHEN

YOU MAKE A SAD SONG SEEM NOT SO SAD.

IF I COULD BUY YOU THE WORLD,

TO GO FORWARD WITH PURPOSE AND DETERMINATION

SHE DIED ON 2ND OF JULY, 2007 (MAY SHE REST IN JANNATULFIRDOUS)

ALLAH BLESS YOU IN HEAVEN

SHER MUHAMMAD SHAHZAD

2.1. Yang cycle and ethylene biosynthesis pathway in plant 11

POTENTIAL OF RHIZOBIA AND FREE LIVING RHIZOBACTERIA CONTAINING

The plant rhizosphere is a remarkable ecological environment where numerous

either by releasing plant growth regulators (PGRs) or other biologically active

substances, altering endogenous levels of PGRs, enhancing availability and uptake of

nutrients through fixation and mobilization, reducing harmful effects of pathogenic

microorganisms on plants and/or by employing multiple mechanisms of action for plant

Rhizobium, Bradyrhizobium, Pseudomonas, Azospirillum, Azotobacter, Bacillus,

Biological N2 fixation by rhizobia is one of the widely studied mechanisms by

releasing carbon as root exudates. A range of bacteria participates in interactions with

altering the synthesis of endogenous phytohormones through the production of specific

enzymes. Among these enzymes, bacterial 1-aminocyclopropane-1-carboxylate (ACC)

1998). Ethylene in higher concentration has also been known as an inhibitor of

Any factor/stimulus which causes a change in ethylene levels of a plant either by

2007a). Seed or root inoculation with PGPR containing ACC-deaminase decreases

2003; Shaharoona et al., 2006a, b, 2007a, b, 2008). Lowering of endogenous levels of

ethylene occurs because of hydrolyses of ACC (an immediate precursor of ethylene) in

endogenous production of ethylene reduces in response to decreased ACC levels within

partially eliminate the stress-induced ethylene-mediated negative impact on plants by

al., 2006a, b, 2007a, b, 2008; Shahzad et al., 2008).

growth by promoting either proliferation of primary roots or lateral roots or by increasing

chemical inhibitors of ethylene synthesis (aminoethoxyvinylglycine and cobalt) and

Psedumonas putida containing ACC-deaminase diluted the ACC-imposed effect on

while the inoculation with Acinetobacter calcoaceticus or Pseudomonas fluorescens in

Contesto et al., 2008).

of PGPR is measured. Stimulation in root growth and biomass production of different

establishment of roots, whether by elongation of primary roots, is advantageous for

nutrients from the surrounded environment.

in roots, inoculation with PGPR containing ACC-deaminase could be very effective in

improving growth and nodulation of chickpea.

The use of rhizobacteria containing ACC-demainase in co-inoculation with

growth enhancing mechanisms. Furthermore inoculation benfitscan be enhanced by

 Isolation and screening of effective rhizobia and PGPR with ACC-deaminase

 Testing potential of selected rhizobia and PGPR strains alone as well as in

combination (co-inoculation) for improving growth, yield and nodulation of

chickpea under pot and field conditions

 Evaluating effectiveness of selected rhizobia and PGPR inoculants for further

 Identification and characterization of selected rhizobia and PGPR containing

Crops which belong to family Leguminosae are generally referred to as legumes or

causing major yield losses.

of Asia was 7.36 million tons (89.4%) (http://faostat.fao.org/). However, Australia,

Oceania together share only 6.2% of the world chickpea production.

between 21 to 29 oC and night time temperature near 20 oC (Siddique et al., 1999;

2.2. Causes of low yield of chickpea in Pakistan

ground water, buildup of pathogenic fungal diseases, temperature stress during

reproductive stage, a bad soil structure, excessive moisture, ineffectiveness of Rhizobium

site of stress. Production of excessive amounts of ethylene in plants causes inhibition of

Norastehnia et al., 2007).

exposed to environmental and biological stresses could be helpful in minimizing the

by lowering ethylene in plant under suboptimal growth conditions.

2.3. Ethylene biosynthesis and plant growth

Ethylene is a simplest unsaturated hydrocarbon which regulates many metabolic

Isolation and screening of effective rhizobia and PGPR with ACC-deaminase

Testing potential of selected rhizobia and PGPR strains alone as well as in

Evaluating effectiveness of selected rhizobia and PGPR inoculants for further

Identification and characterization of selected rhizobia and PGPR containing