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POTENTIAL OF RHIZOBIA AND FREE LIVING RHIZOBACTERIA CONTAINING ACC-DEAMINASE FOR IMPROVING NODULATION AND YIELD OF CHICKPEA (Cicer arietinum L.)

NO DULATION AND YIELD OF CHICKPEA ( Cicer arietinum L.) SHER MUHAMMAD SHA HZAD M.Sc. (Hons.)

SHER MUHAMMAD SHA HZAD

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 :

MEMBER

MEMBER

:

:

(DR. AZEEM KHALID)

(DR. MUHAMMAD ARSHAD)

(DR. KHALIL-UR-REHMAN)

DEDICATED TO MY BELOVED SISTER [TASLEEM KAUSAR] TO THE ONE I ADORE AND TELL MY
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 2 ND 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

LIST OF CONTENTS Chapter Ti tle Page 1. Introduction 1 2. Review of Literature 7 2.1.
LIST OF CONTENTS Chapter Ti tle Page 1. Introduction 1 2. Review of Literature 7 2.1.

Chapter

Title

Page

LIST OF CONTENTS Chapter Ti tle Page 1. Introduction 1 2. Review of Literature 7 2.1.

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

Chapter Ti tle Page 3.5.2. Field experiments 53 3.6. Characterization of rhizobact erial isolates for
Chapter Ti tle Page 3.5.2. Field experiments 53 3.6. Characterization of rhizobact erial isolates for

Chapter

Title

Page

Chapter Ti tle Page 3.5.2. Field experiments 53 3.6. Characterization of rhizobact erial isolates for

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

4.3.3.

Nodulation

72

4.3.

Growth pouch experiment

74

4.3.1.

Root growth

74

4.3.2.

Shoot growth

77

4.3.3.

Nodulation

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 4.4.1.1. Root growth 81 4.4.1.2. Shoot growth 81
Chapter Title Page 4.4.1. Jar experiment 80 4.4.1.1. Root growth 81 4.4.1.2. Shoot growth 81

Chapter

Title

Page

Chapter Title Page 4.4.1. Jar experiment 80 4.4.1.1. Root growth 81 4.4.1.2. Shoot growth 81

4.4.1.

Jar experiment

80

4.4.1.1.

Root growth

81

4.4.1.2.

Shoot growth

81

4.4.2.3.

Nodulation

83

4.4.2.

Growth pouch experiment

83

4.4.2.1.

Root growth

85

4.4.2.2.

Shoot growth

85

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 (2 nd 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

Chapter Ti tle Page 4.7.1.1.3. Number of pods per plant 111 4.7.1.1.4. Grain yield 113
Chapter Ti tle Page 4.7.1.1.3. Number of pods per plant 111 4.7.1.1.4. Grain yield 113

Chapter

Title

Page

Chapter Ti tle 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.

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

4.7.1.2.1.

Plant height

120

4.7.1.2.2.

Fresh biomass

120

4.7.1.2.3.

Number of pods per plant

120

4.7.1.2.4.

Grain yield

122

4.7.1.2.5.

100-grain weight

122

4.7.1.2.6.

Number of nodules per plant

123

4.7.1.2.7.

Nodule dry weight per plant

123

4.7.1.2.8.

Root length

125

4.7.1.2.9.

Root dry weight

125

4.7.1.2.10.

Nitrogen content in grain

125

4.7.1.2.11.

Nitrogen content in straw

126

4.7.1.2.12.

Phosphorous content in grain

126

4.7.1.2.13.

Phosphorous content in straw

126

4.7.2.

Second year trials

128

4.7.2.1.

First trial: site-I (UAF)

128

4.7.2.1.1.

Plant height

128

4.7.2.1.2.

Fresh biomass

128

4.7.2.1.3.

Number of pods per plant

129

4.7.2.1.4.

Grain yield

129

4.7.2.1.5.

100-grain weight

131

4.7.2.1.6.

Number of nodules per plant

131

4.7.2.1.7.

Nodule dry weight per plant

131

4.7.2.1.8.

Root length

132

4.7.2.1.9.

Root of dry weight

132

4.7.2.1.10.

Nitrogen content in grain

134

4.7.2.1.11.

Nitrogen content in straw

134

4.7.2.1.12.

Phosphorous content in grain

134

4.7.2.1.13.

Phosphorous content in straw

136

4.7.2.2.

Second trial: site-II (Nawaz Farm, Jhang)

136

4.7.2.2.1.

Plant height

136

4.7.2.2.2.

Fresh biomass

137

Chapter Title Page 4.7.2.2.4. Grain yield 139 4.7.2.2.5. 100-grain weight 139 4.7.2.2.6. Number of
Chapter Title Page 4.7.2.2.4. Grain yield 139 4.7.2.2.5. 100-grain weight 139 4.7.2.2.6. Number of

Chapter

Title

Page

Chapter Title Page 4.7.2.2.4. Grain yield 139 4.7.2.2.5. 100-grain weight 139 4.7.2.2.6. Number of

4.7.2.2.4.

Grain yield

139

4.7.2.2.5.

100-grain weight

139

4.7.2.2.6.

Number of nodules per plant

139

4.7.2.2.7.

Nodule dry weight per plant

140

4.7.2.2.8.

Root length

140

4.7.2.2.9.

Root of dry weight

140

4.7.2.2.10.

Nitrogen content in grain

143

4.7.2.2.11.

Nitrogen content in straw

143

4.7.2.2.12.

Phosphorous content in grain

143

4.7.2.2.13.

Phosphorous content in straw

145

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

LIST OF TABLES Table Title Page 3.1. Bacterial isolates collected from different locations 46 3.2.
LIST OF TABLES Table Title Page 3.1. Bacterial isolates collected from different locations 46 3.2.

Table

Title

Page

LIST OF TABLES Table Title Page 3.1. Bacterial isolates collected from different locations 46 3.2.

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 nodulation of chickpea seedlings under axenic conditions (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

(1 st 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 (1 st year pot trial) 95

4.15.

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 (1 st year pot trial) 99

Table Title Page 4.16. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deam
Table Title Page 4.16. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deam

Table

Title

Page

Table Title Page 4.16. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deam inase

4.16. 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 (2 nd year pot trial)

102

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 (2 nd year pot trial)

106

4.18. Effect of inoculation and 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 (2 nd year pot trial)

109

4.19. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on growth and yield of chickpea in absence and presence of P-enriched compost (1 st year field trial)

112

4.20. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on nodulation and root growth of chickpea in absence and presence of P-enriched compost (1 st year field trial)

115

4.21. Effect of inoculation and 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 (1 st year field trial)

119

4.22. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on growth and yield of chickpea in absence and presence of P-enriched compost (1 st year field trial)

121

4.23. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on nodulation and root growth of chickpea in absence and presence of P-enriched compost (1 st year field trial)

124

4.24. Effect of inoculation and 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 (1 st year field trial)

127

4.25. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on growth and yield of chickpea in absence and presence of P-enriched compost (2 nd year field trial)

130

4.26. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on nodulation and root growth of chickpea in absence and presence of P-enriched compost (2 nd year field trial)

133

Table Title Page 4.27. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-de
Table Title Page 4.27. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-de

Table

Title

Page

Table Title Page 4.27. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-de aminase

4.27. Effect of inoculation and 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 (2 nd year field trial)

135

4.28. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on growth and yield of chickpea in absence and presence of P-enriched compost (2 nd year field trial)

138

4.29. Effect of inoculation and co-inoculation with rhizobia and rhizobacteria containing ACC-deaminase on nodulation and root growth of chickpea in absence and presence of P-enriched compost (2 nd year field trial)

141

4.30. Effect of inoculation and 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 (2 nd 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 (CO 2+ ) 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

LIST OF FIGURES Figure Titl e Page 2.1. Yang cycle and ethylene biosynthe sis pathway in
LIST OF FIGURES Figure Titl e Page 2.1. Yang cycle and ethylene biosynthe sis pathway in

Figure

Title

Page

LIST OF FIGURES Figure Titl e Page 2.1. Yang cycle and ethylene biosynthe sis pathway in

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 (Co 2+ ) on etiolated chickpea seedlings in the presence of 10 mM ACC concentration

150

Co-inoculation of Chickpea

Abstract

Co-inoculation of Chickpea Abstract POTENTIAL OF RHIZOBIA AND FREE LI VING RHIZOBACTERIA CONTAINING ACC-DEAMINASE FOR
Co-inoculation of Chickpea Abstract POTENTIAL OF RHIZOBIA AND FREE LI VING RHIZOBACTERIA CONTAINING ACC-DEAMINASE FOR

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

Co-inoculation of Chickpea Introduction The plant rhizosphere is a remarkable INTRODUCTION ecological environment where
Co-inoculation of Chickpea Introduction The plant rhizosphere is a remarkable INTRODUCTION ecological environment where

The

plant

rhizosphere

is

a

remarkable

INTRODUCTION

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

Co-inoculation of Chickpea

Introduction

Co-inoculation of Chickpea Introduction facilitate plant growth by va rious mechanisms (Lugtenberg et al., 2002; Raj
Co-inoculation of Chickpea Introduction facilitate plant growth by va rious mechanisms (Lugtenberg et al., 2002; Raj

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 N 2 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

N 2 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

Co-inoculation of Chickpea

Introduction

Co-inoculation of Chickpea Introduction Recently, some PGPR have shown great potential to enhance plant growth by
Co-inoculation of Chickpea Introduction Recently, some PGPR have shown great potential to enhance plant growth by

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

(C 2 H 4 )

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

Frankenberger, 2002).

determinate

nodules

(Nukui

et

al.,

2000;

Arshad

and

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

Co-inoculation of Chickpea

Introduction

Co-inoculation of Chickpea Introduction the rhizosphere by the ACC-deaminase of a bacterium. Furthermore, the ACC from
Co-inoculation of Chickpea Introduction the rhizosphere by the ACC-deaminase of a bacterium. Furthermore, the ACC from

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 C 2 H 4 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.

4

(2007a)

reported

that

inoculation

with

Co-inoculation of Chickpea

Introduction

Co-inoculation of Chickpea Introduction Psedumonas putida containing ACC-deaminase diluted the ACC-imposed effect on
Co-inoculation of Chickpea Introduction Psedumonas putida containing ACC-deaminase diluted the ACC-imposed effect on

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 Co 2+ , 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

Co-inoculation of Chickpea

Introduction

Co-inoculation of Chickpea Introduction in roots, inoculation with PGPR containing ACC-deaminase could be very effective
Co-inoculation of Chickpea Introduction in roots, inoculation with PGPR containing ACC-deaminase could be very effective

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

Co-inoculation of Chickpea Review of Literature REVIEW OF LITERATURE Crops which belong to family Leguminosae are
Co-inoculation of Chickpea Review of Literature REVIEW OF LITERATURE Crops which belong to family Leguminosae are

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature Chickpea can thrive under good moisture conditions with daytime
Co-inoculation of Chickpea Review of Literature Chickpea can thrive under good moisture conditions with daytime

Chickpea can thrive under good moisture conditions with daytime temperature

between 21 to 29 o C and night time temperature near 20 o C (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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature produces ethylene for normal development of plant instead functions to
Co-inoculation of Chickpea Review of Literature produces ethylene for normal development of plant instead functions to

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature Ethylene is generated by most of the plant tissues. Biosynthesis
Co-inoculation of Chickpea Review of Literature Ethylene is generated by most of the plant tissues. Biosynthesis

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature HPO 2- 4 HCOOH - +NH3 MTR – 1- P
Co-inoculation of Chickpea Review of Literature HPO 2- 4 HCOOH - +NH3 MTR – 1- P
HPO 2- 4 HCOOH - +NH3 MTR – 1- P KMBA | RCHOO - O
HPO 2- 4
HCOOH -
+NH3
MTR – 1- P
KMBA
|
RCHOO -
O
MTR-kinase
RCOO -
YANG CYCLE
L-methionine
PPi + Pi ATP
PPi + Pi
ATP
MTR SAM -synthase ATP Adenine MTA-nuclosidase SAM MTA PPi + Pi ACC-Synthase ACC N-malonyl transferase
MTR
SAM -synthase
ATP
Adenine
MTA-nuclosidase
SAM
MTA
PPi + Pi
ACC-Synthase
ACC N-malonyl transferase
γ- glutmyl transpeptidase
ACC N-malonyl transferase γ- glutmyl transpeptidase MACC GACC ACC ETHYLENE Malonyl Co A γ- glutmyl moiety
ACC N-malonyl transferase γ- glutmyl transpeptidase MACC GACC ACC ETHYLENE Malonyl Co A γ- glutmyl moiety
MACC
MACC
GACC ACC
GACC
ACC
transferase γ- glutmyl transpeptidase MACC GACC ACC ETHYLENE Malonyl Co A γ- glutmyl moiety of glutathione
ETHYLENE
ETHYLENE
Malonyl Co A γ- glutmyl moiety of glutathione ACC O 2 oxidase HCNN CO 2
Malonyl Co A
γ- glutmyl moiety of glutathione
ACC
O 2
oxidase
HCNN
CO 2
glutmyl moiety of glutathione ACC O 2 oxidase HCNN CO 2 Figure 2.1: Yang cycle and
glutmyl moiety of glutathione ACC O 2 oxidase HCNN CO 2 Figure 2.1: Yang cycle and

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature It is well known that ethylene action in plants resulting
Co-inoculation of Chickpea Review of Literature It is well known that ethylene action in plants resulting

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature Polyaminase Spd/Spm Inhibited by : DFMO ODC DFMA Orinthine Putrescine
Co-inoculation of Chickpea Review of Literature Polyaminase Spd/Spm Inhibited by : DFMO ODC DFMA Orinthine Putrescine

Polyaminase

Spd/Spm

of Chickpea Review of Literature Polyaminase Spd/Spm Inhibited by : DFMO ODC DFMA Orinthine Putrescine A

Inhibited by : DFMO

Review of Literature Polyaminase Spd/Spm Inhibited by : DFMO ODC DFMA Orinthine Putrescine A r g

ODC

DFMA

Orinthine

Polyaminase Spd/Spm Inhibited by : DFMO ODC DFMA Orinthine Putrescine A r g i n i

Putrescine

Arginine

dSAM

ADC

Lignification/Betaine L-Methionine SAMS SA M D C
Lignification/Betaine
L-Methionine
SAMS
SA M D C
STRESS Cell damage
STRESS
Cell damage
L-Methionine SAMS SA M D C STRESS Cell damage E t h y l e n
L-Methionine SAMS SA M D C STRESS Cell damage E t h y l e n

Ethylene

ACCS ACCO SAMS Inhibited by Induced by Fruit ripening IAA; Ethylene Ca-cytokinin Physical wounding Chilling
ACCS
ACCO
SAMS
Inhibited by
Induced by Fruit ripening
IAA; Ethylene
Ca-cytokinin
Physical wounding
Chilling injury, Drought stress,
Anaerobiosis, Flooding
AVG, AOA,
Rhizobitoxine
Induced by
Ripening
wounding
Inhibited by
Anaerobiosis,
Uncouplers cobalt/S
Temperature 35 0 C
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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature Contradictory effects of ethylene on plan ts have also been
Co-inoculation of Chickpea Review of Literature Contradictory effects of ethylene on plan ts have also been

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

14

ethylene

Ethylene

Co-inoculation of Chickpea

Review of Literature

Ethylene Co-inoculation of Chickpea Review of Literature A Deleterious peak Beneficial peak Ethylene Time B
Ethylene Co-inoculation of Chickpea Review of Literature A Deleterious peak Beneficial peak Ethylene Time B
A Deleterious peak Beneficial peak Ethylene
A
Deleterious peak
Beneficial peak
Ethylene

Time

B

Deleterious peak Beneficial peak
Deleterious peak
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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature production in plants on exposure to various types of stresses
Co-inoculation of Chickpea Review of Literature production in plants on exposure to various types of stresses

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 o C than 55 o C. Ethylene production was 2.3 to 3.8 fold greater at 50 o C

than at ambient (22 o C) conditions (Frankenberger and Phelen, 1985). Similarly,

increased ethylene evolution was observed in two soils when heated up to 80 o C (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

o C

resulted

in

substantially lower rate of ethylene evolution and higher temperature (30 to 35 o C)

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature increases in ethylene production of Pinus sylvestris needles. Fl-abd et
Co-inoculation of Chickpea Review of Literature increases in ethylene production of Pinus sylvestris needles. Fl-abd et

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature the roots 0, 50, 100 and 200 mM NaCl. Epinasty
Co-inoculation of Chickpea Review of Literature the roots 0, 50, 100 and 200 mM NaCl. Epinasty

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature dry weight of tomato seedlings grown up to 172 mM
Co-inoculation of Chickpea Review of Literature dry weight of tomato seedlings grown up to 172 mM

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature ethylene could be partially re duced through inoculation with
Co-inoculation of Chickpea Review of Literature ethylene could be partially re duced through inoculation with

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature 2005; Li et al., 2006). Field experiments aimed to facilitate
Co-inoculation of Chickpea Review of Literature 2005; Li et al., 2006). Field experiments aimed to facilitate

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

Co-inoculation of Chickpea

Review of Literature

Co-inoculation of Chickpea Review of Literature inhibition is a consequence of the production of stress ethyl
Co-inoculation of Chickpea Review of Literature inhibition is a consequence of the production of stress ethyl

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 o C, in water) leading to great persistence and less degradation (Drew, 1992).

Higher

ethylene

levels

results

in

certain