Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web...

357
In the name of Allah, the Most Beneficent, the Most Merciful

Transcript of Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web...

Page 1: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

In the name of Allah, the Most Beneficent, the Most Merciful

Page 2: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Role of endophytic bacteria containing carbonic anhydrase in improving the photosynthesis and plant biomass of cereals at

different moisture regimes

By

ANA ASLAM

M.Sc. (Hons.) Soil Science 2005-ag-1704

A thesis submitted in partial fulfilment ofthe requirements for the degree

of

DOCTER OF PHILOSOPHY

In

SOIL SCIENCE

Institute of Soil & Environmental SciencesFaculty of Agriculture

University of Agriculture, Faisalabad, Pakistan 2019

Page 3: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency
Page 4: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency
Page 5: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

DEDICATED

To

My Beloved Parents, Husbandand

Respected Supervisor

Whose encouragement, spiritual inspiration and sincere prayers

motivated me to achieve my academic goals

Page 6: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

ACKNOWLEDGEMENTS All praises and humblest thanks are to Almighty ALLAH, the most Beneficent and the most Merciful, whose blessings flourished my thoughts to finally shape up the cherished fruit of my humble proceedings to this study. I pay my homage to Holly Prophet Hazrat Muhammad (P.B.U.H), the most perfect and exalted among us, who is forever a source of wisdom and knowledge for humanity as a whole.

I feel highly priviledge to express my heartiest gratitude to my honorable supervisor Dr. Zahir Ahmad Zahir, Professor, Institute of Soil & Environmental Sciences, University of Agriculture, Faisalabad, for his dynamic supervision, constant guidance, valuable suggestions, constructive and thoughtful criticism during the study. I am also thankful to Dr. Hafiz Naeem Asghar, Assistant Professor, Institute of Soil & Environmental Sciences Dr. Muhammad Shahid Associate Professor, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, for their intellectual suggestion and cooperative guidance.

A deep sense of appreciation is owed to Dr. Muhammad Arshad (Late) (T.I.), (D.N.P), Professor, Institute of Soil & Environmental Sciences, University of Agriculture, Faisalabad, for his cooperative attitude, constructive criticism and valuable suggestion during the research study.

My sincere thanks are extended to Dr. Muhammad Naveed, Assistant professor, Institute of Soil & Environmental Sciences, University of Agriculture, Faisalabad, for his constant help, guidance and valuable discussion.

I feel utmost pleasure in expressing my gratitude to Dr. Peer Schenk, Professor, Dr. Lilia Costa Carvalhais, Plant microbe interaction Laboratory, The University of Queensland, Australia for their supportive attitude and kind guidance during my stay in Australia for opening a new era of research in plant microbe interaction

I am highly thankful to Higher Education Commission, Islamabad, Pakistan (HEC) for providing financial assistance and IRSIP.

Special and particular thanks are extended to my Fellows in the Soil Microbiology and Biochemistry Lab., for their help and cooperation to accomplish this script.

No acknowledgements could ever adequately express my obligations to my beloved Parents for their encouragement, love and support throughout my career. I can only say I am here just due to prayers of family specially my father. I am also grateful to my husband Muhammad Adeel for his supportive and motivative attitude. I can’t ignore my dear brother M. Usman and my sweet sisters Sana and Safina who have always inspired and encouraged me. Their prayers will be always with me for my success.

Cordial thanks to my friend Sakeena tul Ain Haider, and all other well-wishers for their encouragement and consistent support during my studies.

Ana Aslam

Page 7: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency
Page 8: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Chapter Title Page3.10. Plant analysis 353.10.1. Chemical analysis 353.10.2. Digestion 353.10.3. Nitrogen determination 353.10.4. Phosphorus determination 363.10.5. Potassium determination 363.10.6. Leaf relative water contents (RWC) 363.10.7. Electrolyte leakage 373.10.8 Chlorophyll content 373.10.9. Gaseous exchange parameter 373.10.10 Carbonic anhydrase activity 373.10.11. Proline content 383.10.12. Total protein content 383.10.13. Malondialdehyde content 383.10.14. Catalase in leaves 383.10.15. Glutathione reductase in leaves 393.10.16. Ascorbate peroxidase in leaves 393.10.17. Total phenolics in leaves 393.10.18. Total soluble sugars in leaves 393.10.19 Colonization of plant tissues 403.11. Characterization and identification of selected endophytic

bacteria40

3.11.1. Indole 3-acetic acid production under normal and stressed environment

40

3.11.2. Phosphate solubilization 413.11.2.1. Phosphate solubilization (plate assay) 413.11.2.2 Phosphate solubilization under normal and stressed conditions 413.11.3. Siderophore production 433.11.4. Exopolysaachride (EPS) production 433.11.5. Chitinase activity 433.11.6. Catalase activity 433.11.7. Oxidase activity 433.11.8 Organic acid production 453.11.9 Microbial aggregation ability 453.11.10 Survival under starved condition 453.11.11 Survival of bacterial inocula in soil 453.11.12 Cellulase activity 473.11.13 Xylanase activity 473.11.14 Protease activity 473.11.15 Identification of selected isolate 473.12. Influence of endophytic bacteria on gene expression in

Arabidopsis thaliana under drought stress47

3.12.1. Sample collection and isolation of endophytic bacteria from Arabidopsis

47

3.12.2. Screening of endophytic bacteria for stress tolerance and carbonic anhydrase activity

48

Page 9: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Chapter Title Page3.12.3. Screening of selected bacterial isolates for plant growth

promotion under PEG-induced water deficit stress48

3.12.4. Effect of selected isolates on gene expression in Arabidopsis thaliana under PEG-induced water deficit stress

50

3.12.5. RNA extraction 503.12.6. Preparation of cDNA and primers sequence 503.12.7. Expression profiling through Real Time PCR 513.13. Statistical analysis 51Chapter 4 Results 544.1. Drought tolerance ability of endophytic bacteria 544.1.1. Drought tolerance ability of endophytic bacterial isolates from

wheat54

4.1.2. Drought tolerance enhancing ability of endophytic bacterial isolates of maize

57

4.2. Carbonic anhydrase activity of drought tolerant isolates 574.2.1. Carbonic anhydrase activity of drought tolerant wheat isolates 574.2.2. Carbonic anhydrase activity of drought tolerant maize isolates 574.3. Screening of selected drought tolerant CA containing

endophytic bacteria for plant growth promotion under axenic conditions

61

4.3.1. Screening of wheat isolates for growth promotion 614.3.1.1. Root length 614.3.1.2. Shoot length 614.3.1.3. Root fresh weight 654.3.1.4. Shoot fresh weight 654.3.1.5. Root dry weight 674.3.1.6. Shoot dry weight 674.3.1.7. Chlorophyll contents 694.3.1.8. Carbonic anhydrase activity 694.3.1.9. Photosynthetic rate 714.3.1.10. Transpiration rate 714.3.1.11. Stomatal conductance 734.3.1.12. Substomatal conductance 734.3.1.13. Relationship between photosynthetic rate and CA activity

exhibited by drought tolerant endophytic bacterial isolates75

4.3.2. Screening of maize isolates for growth promotion 754.3.2.1. Root length 754.3.2.2. Shoot length 754.3.2.3. Root fresh weight 804.3.2.4. Shoot fresh weight 804.3.2.5. Root dry weight 824.3.2.6. Shoot dry weight 824.3.2.7. Chlorophyll contents 844.3.2.8. Carbonic anhydrase activity in leaves 844.3.2.9. Photosynthetic rate 864.3.2.10. Transpiration rate 864.3.2.11. Stomatal conductance 88

Page 10: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.3.2.12. Substomatal conductance 88Chapter Title Page4.3.2.13. Relationship between photosynthetic rate and CA activity

exhibiting by drought tolerant endophytic bacteria90

4.5. Evaluation of selected GUS labeled endophytic bacterial isolates in pot trial

90

4.5.1. Effect of selected Gus labelled endophytic bacterial isolates on wheat

90

4.5.1.1. Plant height 904.5.1.2. Root dry weight 934.5.1.3. Soot dry weight 934.5.1.4. Carbonic anhydrase activity 934.5.1.5. Photosynthetic rate 954.5.1.6. Transpiration rate 954.5.1.7. Stomatal conductance 954.5.1.8. Relative water content (RWC) 974.5.1.9. Electrolyte leakage (EEL) 974.5.1.10. Proline Content 974.5.1.11 Melanaldehyde content 994.5.1.12. Grain yield 994.5.1.13. Colonization of plant tissues 994.5.1.14. Characterization of selected bacterial isolates for IAA

production under normal and stressed conditions101

4.5.1.15. Characterization of selected bacterial isolates for P solubilization under normal and stressed conditions

101

4.5.2. Study the selected Gus labeled endophytic bacterial isolates on maize

104

4.5.2.1. Plant height 1044.5.2.2. Root dry weight 1044.5.2.3. Shoot dry weight 1044.5.2.4. Carbonic anhydrase activity in leaves 1064.5.2.5. Photosynthetic rate 1064.5.2.6. Transpiration rate 1064.5.2.7. Stomatal conductance 1084.5.2.8. Relative water content (RWC) 1084.5.2.9. Electrolyte leakage (EEL) 1084.5.2.10. Proline content 1104.5.2.11. Melanaldehyde content (MDA) 1104.5.2.12. Grain yield 1104.5.2.13. Colonization of plant tissues 1124.5.2.14 Characterization of selected bacterial isolates for IAA

production under normal and stressed conditions112

4.5.2.14 Characterization of selected bacterial isolates for P solubilization under normal and stressed conditions

112

4.6. Evaluation of selected endophytic bacterial isolates in field trials

116

4.6.1. Evaluation of selected endophytic bacterial isolates for wheat 1164.6.1.1. Number of tillers 1164.6.1.2. Carbonic anhydrase activity 116

Page 11: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.6.1.3. Photosynthetic rate 116Chapter Title Page4.6.1.4. Transpiration rate 1184.6.1.5. Water use efficiency (WUE) 1184.6.1.6. Grain Yield 1184.6.1.7. Catalase contents 1204.6.1.8. Ascorbate peroxidase (APX) contents 1204.6.1.9. Glutathione reductase (GR) contents 1204.6.1.10. Total protein contents 1224.6.1.11. Total soluble sugars 1224.6.1.12. Total phenolic contents 1224.6.1.13. Grain nitrogen (%) 1244.6.1.14. Grain phosphorus (%) 1244.6.1.15. Grain potassium (%) 1244.6.2. Evaluation of selected endophytic bacterial isolates for maize 1264.6.2.1. Number of grains per cob 1264.6.2.2. Carbonic anhydrase activity 1264.6.2.3. Photosynthetic rate 1264.6.2.4. Transpiration rate 1284.6.2.5. Water use efficiency (WUE) 1284.6.2.6. Grain Yield 1284.6.2.7. Catalase contents 1304.6.2.8. Ascorbate peroxidase (APX) contents 1304.6.2.9. Glutathione reductase (GR) contents 1304.6.2.10. Total protein contents 1324.6.2.11. Total soluble sugars 1324.6.2.12. Total phenolic contents 1324.6.2.13. Grain nitrogen (%) 1344.6.2.14. Grain phosphorus (%) 1344.6.2.15. Grain potassium (%) 1344.7. Evaluation of potential endophytic bacterial isolates for gene

expression in Arabidopsis thaliana under PEG-induced water deficit conditions

136

4.7.1. Screening of endophytic bacterial isolates based on drought tolerance ability, CA activity and plant growth promotion

136

4.7.2. Effect of endophytic bacterial isolates on plant growth of Arabidopsis thaliana

136

4.7.2.1. Root length 1364.7.2.2 Number of lateral roots 1364.7.2.3. Root fresh weight 1364.7.2.3. Shoot fresh weight 1394.7.3. Effect of selected isolates on gene expression and

transcriptional response of Arabidopsis thaliana139

4.7.3.1. Expression pattern of dehydration responsive protein (RD22) 1394.7.3.2. Expression pattern of dehydration responsive element

(RD29B)139

4.7.3.3. Expression pattern of late embryogenesis (LEA) 1394.7.3.4. Expression pattern of dehydrin (RAB18) 141

Page 12: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.7.3.5. Expression of dehydration-response element binding protein 2A (DREB2A)

141

Chapter Title Page4.7.3.6. Expression of defense related gene (PR1.2.) 1414.7.3.7. Expression of WRKY57 transcription factors 1414.7.3.8. Expression of WRKY8 transcription factors 1414.7.3.9. Expression pattern of C2H2–Zinc finger protein (Zat 10) 1434.7.3.10. Expression pattern of dehydrins (COR47) 1434.7.3.11. Expression of ethylene responsive transcription factor 7

(AtERF 7)143

4.7.3.12. Expression pattern of dehydrins (LTI78) 1434.7.3.13. Expression pattern of MYB domain protein 15 (MYB 15) 1454.7.3.14. Expression pattern of abscisic acid dependent dehydrins

(ERD10)145

4.7.3.15. Expression of ethylene responsive factor (ERF 13) 1454.8. Characterization and identification of endophytic bacterial

isolates147

Chapter Discussion 1505.1. Drought tolerance ability of bacterial endophytes 1505.2. Carbonic anhydrase activity of drought tolerant isolates 1515.3. Screening of CA producing drought tolerant endophytic

bacteria for growth promotion in wheat and maize seedlings under PEG-imposed water deficit stress in axenic conditions

151

5.4. Evaluation of selected endophytic bacterial isolates for wheat and maize in pot trials

153

5.5. Interaction between endophytic bacterial population and plant tissues

155

5.6. Evaluation of selected endophytic bacterial isolates in field trial

156

5.7 Influence of drought tolerant CA containing endophytic bacteria on plant growth promotion and gene expression

159

Summary 161Future directions 164References 165

Page 13: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

List of TablesTable Title Page3.1. Phyico-chemical characteristics of the soils used for wheat and

maize trials34

3.2. Primer sequence for arabidopsis used in RT-PCR 524.1. Selected drought tolerant endophytic bacterial isolates from

wheat56

4.2. Selected drought tolerant endophytic bacteria isolates from maize

59

4.3. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot length of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

64

4.4. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot fresh weight of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

66

4.5. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot dry weight of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

68

4.6. Effect of drought tolerant CA containing endophytic bacterial isolates on chlorophyll content and CA activity in leaves of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

70

4.7. Effect of drought tolerant CA containing endophytic bacterial isolates on photosynthetic and transpiration rate of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

72

4.8. Effect of drought tolerant CA containing endophytic bacterial isolates on stomatal and substomatal conductance of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

74

4.9. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot length of drought tolerant(H1) and sensitive (H2) maize hybrids under non-stressed and PEG-induced water deficit conditions

79

4.10. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot fresh weight of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG-induced water deficit conditions

81

4.11. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot dry weight of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG induced water deficit conditions

83

Page 14: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table Title Page4.12. Effect of drought tolerant CA containing endophytic bacterial

isolates on chlorophyll contents and carbonic activity of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG-induced water deficit conditions

85

4.13. Effect of drought tolerant CA containing endophytic bacterial isolates on photosynthetic and transpiration rate of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG-induced water deficit conditions

87

4.14. Effect of drought tolerant CA containing endophytic bacterial isolates on stomatal substomatal conductance of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG-induced water deficit conditions

89

4.15. Characterization of selected drought tolerant endophytic bacterial isolates

148

Page 15: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

List of FiguresFig. Title Page4.1. Principal component analysis of optical density of endophytic

bacteria isolates from wheat at different PEG-6000 induced water deficit stress levels

55

4.2. Principal component analysis of optical density of endophytic bacteria isolates from maize at different PEG-6000 induced water deficit stress levels

58

4.3. Drought tolerant endophytic bacterial isolates from wheat with high carbonic anhydrase activity

60

4.4. Drought tolerant endophytic bacteria isolates from maize with their high carbonic anhydrase activity

60

4.5. Relationship between photosynthetic rate and carbonic anhydrase activity exhibited by drought tolerant endophytic bacterial isolates in wheat cv. Fsd-2008

76

4.6. Relationship between photosynthetic rate and carbonic anhydrase activity exhibited by drought tolerant endophytic bacterial isolates in wheat cv. Uqab-2000

76

4.7. Relationship between photosynthetic rate and carbonic anhydrase activity exhibited by drought tolerant endophytic bacterial isolates in maize hybrid (H1)

91

4.8. Relationship between photosynthetic rate and carbonic anhydrase activity exhibited by drought tolerant endophytic bacterial isolates in maize hybrid (H2)

91

4.9. Effect of drought tolerant CA containing endophytic bacteria on plant height (A), root dry weight (B) and shoot dry weight (C) in both wheat cultivars at different field capacity levels

92

4.10. Effect of drought tolerant CA containing endophytic bacteria on carbonic anhydrase activity (A), photosynthetic rate (B) and transpiration rate (C) in both wheat cultivars at different field capacity levels

94

4.11. Effect of drought tolerant CA containing endophytic bacteria on stomatal conductance (A), relative water content (B) and electrolyte leakage (C) in both wheat cultivars at different field capacity levels

96

4.12. Effect of drought tolerant CA containing endophytic bacteria on proline content (A), melanaldehyde (B) and grain yield (C) in both wheat cultivars at different field capacity levels

98

4.13. Colonization of root (A), shoot (B) and leaf (C) tissues with drought tolerant CA containing endophytic bacteria in both wheat cultivars at different field capacity levels

100

4.14. IAA production of drought tolerant CA containing endophytic bacterial isolates with reference to time

102

4.15. P-solubilization of drought tolerant CA containing endophytic bacterial isolates with reference to time

103

Page 16: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Fig. Title Page4.16. Effect of drought tolerant CA containing endophytic bacteria

on plant height (A), root dry weight (B) and shoot dry weight (C) in both maize hybrids at different field capacity levels

105

4.17. Effect of drought tolerant CA containing endophytic bacteria on carbonic anhydrase activity (A), photosynthetic rate (B) and transpiration rate (C) in both maize hybrids at different field capacity levels

107

4.18. Effect of drought tolerant CA containing endophytic bacteria on stomatal conductance (A), relative water content (B) and electrolyte leakage (C) in both maize hybrids at different field capacity levels

109

4.19. Effect of drought tolerant CA containing endophytic bacteria on proline content (A), melanaldehyde content (B) and grain yield (C) in both maize hybrids at different field capacity levels

111

4.20. Colonization of root (A), shoot (B) and leaf (C) tissues with drought tolerant CA containing endophytic bacteria in both maize hybrids at different field capacity levels

113

4.21. IAA production of drought tolerant CA containing endophytic bacterial isolates with reference to time

114

4.22. P-solubilization of drought tolerant CA containing endophytic bacterial isolates with reference to time

115

4.23. Effect of drought tolerant CA containing endophytic bacteria on number of tillers (A), carbonic anhydrase activity (B) and photosynthetic rate (C)of wheat under water deficit stress

117

4.24. Effect of drought tolerant CA containing endophytic bacteria on transpiration rate (A), water use efficiency (B) and grain yield (C) of wheat under water deficit stress

119

4.25. Effect of drought tolerant CA containing endophytic bacteria on catalase (A), ascorbate peroxidase (B) and glutathione reductase (C) of wheat under water deficit stress

121

4.26 Effect of drought tolerant CA containing endophytic bacteria on total protein (A), total soluble sugars (B) and total phenolic content (C) of wheat under water deficit stress

123

4.27. Effect of drought tolerant CA containing endophytic bacteria on grain nitrogen (A), phosphorus (B) and potassium (C) of wheat under water deficit stress

125

4.28. Effect of drought tolerant CA containing endophytic bacteria on no. of grains per cob (A), carbonic anhydrase activity (B) and photosynthetic rate (C) of maize under water deficit stress

127

4.29 Effect of drought tolerant CA containing endophytic bacteria on transpiration rate (A), water use efficiency (B) and grain yield (C) of maize under water deficit stress

129

4.30 Effect of drought tolerant CA containing endophytic bacteria on catalase (A), ascorbate peroxidase (B) and glutathione reductase (C) content of maize under water deficit stress

131

Page 17: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Fig. Title Page4.31 Effect of drought tolerant CA containing endophytic bacteria

total protein contents (A), total soluble sugars (B) and total phenolic contents (C) of maize under water deficit stress

133

4.32 Effect of drought tolerant CA containing endophytic bacteria on grain nitrogen (A), phosphorus (B) and potassium (C) of maize under water deficit stress

135

4.33. Effect of drought tolerant CA containing endophytic bacterial isolates (AR4, AR14) on root length (A) number of lateral roots (B), root fresh weight (C) and shoot fresh weight (D) in Arabidopsis thaliana under normal (0%) as well as PEG-mediated water deficit conditions (3%)

138

4.34 Effect of drought tolerant CA containing endophytic bacterial isolates (AR4, AR14) on stress responsive genes RD22 (A), RD29B (B), LEA (C) and RAB18 (D) in Arabidopsis thaliana under normal (0%) as well as PEG-mediated water deficit conditions (3%)

140

4.35. Effect of drought tolerant CA containing endophytic bacterial isolates (AR4, AR14) on DREB2A (A) PR1.2 (B) WRKY57 (C) and WRKY 8 (D) in Arabidopsis thaliana under normal (0%) as well as PEG-mediated water deficit conditions (3%)

142

4.36 Effect of drought tolerant CA containing endophytic bacterial isolates (AR4, AR14) on transcription factors and gene Zat 10 (A), COR47 (B), AtERF7 (C) and LTI78 (D) in Arabidopsis thaliana under normal (0%) as well as PEG- mediated water deficit conditions (3%).

144

4.37 Effect of drought tolerant CA containing endophytic bacterial isolates (AR4, AR14) on transcription factors and gene MYB15 (A), ERD10 (B), ERF13 (C) in Arabidopsis thaliana under normal (0%) as well as PEG-mediated water deficit conditions (3%)

146

4.38 Identification of selected endophytic bacterial isolates on the basis of 16S rRNA sequence similarities

149

Page 18: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

List of PicturesPicture Title Page1 Phosphorus solubilization by endophytic bacterial isolates 422 Exopolysacchride production by endophytic bacterial isolates 443 Catalase production by endophytic bacteria 464 Oxidase production by endophytic bacteria 465 Screening of drought tolerant CA containing endophytic

bacterial isolates for plant growth promotion in Arabidopsis thaliana under axenic conditions

49

6 Effect of drought tolerant CA containing endophytic bacteria on root length under normal conditions

62

7 Effect of drought tolerant CA containing endophytic bacteria on root length under PEG-induced water deficit conditions

62

8 Effect of drought tolerant CA containing endophytic bacterial isolates on shoot length under normal conditions

63

9 Effect of drought tolerant CA containing endophytic bacteria on shoot length under PEG-induced water deficit conditions

63

10 Effect of drought tolerant CA containing endophytic bacteria on root length under normal conditions

77

11 Effect of drought tolerant CA containing endophytic bacteria on root length under water deficit conditions

77

12 Effect of drought tolerant CA containing endophytic bacteria on shoot length under normal conditions

78

13 Effect of drought tolerant CA containing endophytic bacteria on shoot length under PEG-induced water deficit conditions

78

14 Effect of drought tolerant CA containing endophytic bacteria on Arabidopsis thaliana growth under normal conditions

137

15 Effect of drought tolerant CA containing endophytic bacteria on Arabidopsis thaliana growth under PEG-induced water deficit conditions

137

Page 19: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

AbstractClimate change is one of the serious threats to food security throughout the world

especially in arid and semi-arid regions, affecting agricultural productivity. Rapid

changes in global climate such as alteration in rain fall pattern and increase in temperature

lead to severe drought stress that limits the crop production by reducing photosynthetic

rate and suppressing plant growth. Endophytic bacteria containing carbonic anhydrase

may improve plant growth and elicit tolerance under water deficit stress by enhancing the

photosynthesis in plants. Carbonic anhydrase (CA) catalyzes the reversible conversion of

atmospheric carbon dioxide into bicarbonate, first substrate of photosynthesis in C3 and

C4 plants. Therefore, present study was conducted to assess the potential of drought

tolerant CA containing endophytic bacteria for improving photosynthesis and plant

biomass of cereals under different moisture regimes. One hundred and fifty bacterial

isolates were isolated form two cereals (wheat and maize) and tested for their ability to

tolerate PEG-induced water deficit conditions. Fifty isolates exhibiting higher drought

tolerance from each crop were further analyzed for CA activity. Ten drought tolerant

isolates with higher CA activity were further assessed for growth promotion in wheat

(C3) and maize (C4) plants. Isolates WR2, WS11 and WL19 showed higher

photosynthetic rate and plant growth in both wheat cultivars; however, increase was more

for Uqab-2000 than Fsd-2008 under PEG- induced water deficit conditions. Moreover,

isolates MR17, MS1 and MG9 gave significant increase in photosynthesis and plant

growth in both maize hybrids, especially for H2 hybrid under PEG-mediated water stress.

Selected isolates from both crops were labeled with Gus and tested for plant growth

promotion as well as colonization efficiency in wheat and maize under water deficit

stress. Inoculation of selected isolates showed significant results for photosynthesis,

growth and colonization efficiency of wheat and maize under well watered (100% FC)

and stressed (70 and 40%) conditions especially for Uqab-2000 and H2. In the same

ways, isolates WR2, WS11 and WL19 gave significant results for growth, physiology and

yield of wheat under field condition where water deficit stress was induced by skipping

irrigation at tillering, flowering and grain filling stage. On the other hand, inoculation

with isolates MR17, MS1 and MG9 improved growth under normal and stressed

conditions which were induced by withholding irrigation at vegetative and reproductive

stage of maize. Selected isolates also proved to be efficient auxin producer and p-

solubilizer under normal and stressed conditions. These isolates were identified as

Page 20: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Bacillus sp. In separate study, it was observed that endophytic bacterial isolates carrying

CA activity AR4 and AR14 (belonged to Microbacterium sp. and Psychrobacter sp.,

respectively) also stimulated the expression of various genes and transcription factors in

Arabidopsis thaliana under normal as well as PEG-induced water deficit conditions.

Therefore, it can be suggested that inoculation of endophytic bacterial isolates (WR2,

WL19 for wheat and MR17, MG9 for maize) is good for enhancing photosynthesis and

plant biomass under water deficit conditions. Moreover, multi-site field experiments for

these isolates are suggested for evaluating the successful performance in field. However,

molecular studies are required to confirm role of bacterially synthesized CA in

photosynthesis.

Page 21: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Chapter I

INTRODUCTION

The current and predicted changes in global climate are major concerns for the

productivity of agriculture sector (Lepetz et al., 2009) and food security (Misra, 2014).

The rapid increase in carbon dioxide (CO2) concentration due to insatiable demand of

burgeoning human population for energy from combustion of fossil is projected to result

in significant changes in climate. These climatic changes lead to increase in temperature

and alteration in rainfall patterns (Solomon et al., 2007) coupled with intensified events of

rain and drought. The global average temperature is expected to exceed from 1.4 - 5.8 ˚C

by the 21st century (year 2100) and would cause pronounced reduction in crop yield.

Furthermore, precipitation is expected to decrease 20% or more by the next century in

arid and semi-arid regions (Misra, 2014). Several uncertainties are also associated with

future climate pattern (Parry et al., 2007; Randall et al., 2007). Some of the climate

change impacts are growing desert area and increase in frequency and severity of floods

and drought.

Drought is one of the major constraints to agricultural productivity throughout the

world, suppressing plant growth and crop productivity. About, 25% of world arable land

is primarily affected by drought stress (Jajarmi, 2009). However, overall effects of

drought are likely to accelerate with increasing climatic changes (Walter et al., 2011) and

growing water crisis. Drought stress accounts for 50% reduction in crop yield (Wood,

2005). The degree of destruction caused by water deficit stress in plants depends on plant

species, genotypes, duration of exposure, severity of stress, age of plant and

developmental stage (Safarnejad, 2004). Drought stress decreases cell division and

expansion, root proliferation, stem elongation, leaf size and disturbs plant nutrient

relationship, thereby impairs water use efficiency and crop productivity (Li et al., 2009).

Leaf area also reduces under drought stress due to lower number of leaves and loss of

turgidity (Farooq et al., 2010). Number of physiological and biochemical changes appear

under drought stress at cellular level including variation in membrane fluidity, loss of

turgidity, variation in osmolyte concentration, protein-lipid and protein to protein

interaction (Chaves et al., 2009). Closure of stomata is the earlier response of plant under

water limited conditions (Schroeder et al., 2001). Stomatal closure due to change in leaf

water status under limited supply of water limits the photosynthesis (Nogueira et al.,

1

Page 22: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

2001, Silva et al., 2003). Reduction in photosynthesis is primarily explained by limited

availability of intercellular CO2 that affects photosynthesis at acceptor site (Cornic et al.,

1992) and inhibits the activity of photosynthetic enzyme such as Rubisco (Haupt-Herting

and Fock, 2000) or ATP synthase (Nogues and Baker, 2000). Drought stress also impairs

photosynthetic machinery and its components, thus affects biochemical processes

associated with photosynthesis and reduces agriculture productivity (Dreesen et al.,

2012). Therefore, increasing productivity per drop of water is becoming important for

many regions (Luquet et al., 2005).

Several efforts have been made to lower drought induced yield reduction and

improve crop productivity including production of drought tolerant genotypes (Stikic et

al., 2014). Prerequisite to develop drought resistant plants include phenotypic

improvement to elucidate plant response and adaptability to drought stress, selection of

gene that directly contribute in drought stress and evaluate impact of drought resistance

on crop production and quality of produce but these tasks are very difficult because plant

drought tolerance is complex phenomenon (Chaves et al., 2003). Moreover, lack of

proficient selection process and lower genetic variability for yield component are major

problem for their limited success (Gosal et al., 2009). Approaches including molecular

breeding and genetic improvement have been followed to enhance adaptability against

drought stress but have certain limitations (Ashraf and Akram, 2009). Genetically

modified (GM) plants are also not well adopted in many parts around the globe (Wahid et

al., 2007) owing to various reasons.

Therefore, another eco-friendly alternative approach is required. One of such

feasible aproach could be use of bacteria such as plant growth facilitating bacteria that are

living freely either in soil, rhizolpane, rhizophere or phyllosphere having potential role in

growth enhancement under stressed environment (Bashan and de-Bashan, 2005). Some

beneficial bacterial strains mitigate biotic stress also protect plant against abiotic stresses

where they colonize rhizophere and improve plant growth by maintaining proper soil

moisture, improving soil structure and increasing absorption of plant mineral nutrition

(de-Bashan et al., 2012; Kim et al., 2012). Different species of plant growth facilitating

rhizobacteria such as Pseudomonas, Bacillus, Azospirillum, Acetobacter, Burkholderia

etc., produce various phytohormones expecially auxins, cytokinins, gibberellins and may

attribute to growth improvement and development in stressed conditions compared to

plant gown under normal growth environment (Bashan and de-Bashan, 2005).

2

Page 23: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Inoculation of crop plant with beneficial bacteria leads to formation of extensive

root, root hairs and lateral roots that may increase plant ability to survive under different

stress environments (Hayat et al., 2010). Regardless of these positive impacts, bacterial

products can show variability among the experiments (Montesinos, 2003). Furthermore,

there are certain disadvantage associated with the use of plant growth facilitating bacteria

(PGPB) as they are more receptive to the environmental constraints particularly drought

and soil temperature etc., inconsistent efficacy under field conditions (Labuschagne et al.,

2010) and short persistence in soil environment and rhizosphere.

Possible way to solve this delimma is use of endophytic bacteria under drought

stress, since these bacterial strains colonize the interior of plant that is stable and

protected niche. These bacteria colonize the plant without showing any external infection

or substantive detrimental effect on plant (Schulz and Boyle, 2006). Endophytic bacteria

may enter inside the plant through root hair cell by penetration (Huang, 1986) or with the

help of cell wall degenerating enzymes (Quadt- Hallmann et al., 1997) where they

colonize the intercellular spaces as well as vascular bundle and then systematically

colonize the tissues of plant (Compant et al., 2005). The capability to colonize interior

tissues of host has invented them priceless for agriculture to facilitatecrop productivity.

The role of endophytes in growth facilitation has gained attention as their

inoculation provides effective and consistent improvement in plant productivity

(Morrissey et al., 2004; Shi et al., 2010). Endophytic bacteria are known to contribute

plant growth facilitation by number of mechanisms either through production of various

phytohormones particularly auxin, gibberellins and cytokinins (Madhaiyan et al., 2006),

breakdown of endogenously produced plant ethylene by an enzyme 1-

aminocyclopropane-1-carboxylate (ACC) deaminase (Long et al., 2008; Ryan et al.,

2008), better plant nutrient acquisition (Malinowski et al., 2000), nitrogen fixation (Doty

et al., 2009; Jha and Kumar, 2007), phosphorus solubilization (Vessey, 2003; Kuklinsky-

Sobral et al., 2004; Puente et al., 2009a), siderophore production (Ramesh et al., 2009),

iron chelation or pathogen infection (Compant et al., 2005; Forchetti et al., 2007) through

antifungal (Compant et al., 2005; Zachow et al., 2008) or antibacterial agent and by

inducing systemic resistance (Gomez-Lema Cabanas et al., 2014). These bacteria improve

root length and growth of secondary root, thus enhance plant growth (Amaresan et al.,

2012). Endophytic bacteria significantly enhance shoot biomass compared to non-

inoculated control plant (Montanez et al., 2012). Inoculation with these beneficial

3

Page 24: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

bacteria increase the total soluble sugars and starch which in turn could compensate the

drought effects and results in better uptake of water and minerals from the soil (Gagne-

Bourque et al., 2015). Endophytic bacteria also confer drought tolerance by facilitating

the induction of several stress associated genes in leaves of plant (Sherameti et al., 2008).

Inoculation with endophytic bacteria enhance drought tolerance by over expressing the

gene associated with drought stress (LEA-14- and DHN3-like) and also transcription

factor regulating the dehydration responsive element binding gene (DREB2B) under

limited supply of water (Gagne-Bourque et al., 2015). These bacteria also improve the

photosynthetic activity (Chi et al., 2005) seedling emergence and establishment under

unfavorable conditions (Puente et al., 2009b; Forchetti et al., 2010).

Moreover, endophytic bacteria may possess carbonic anhydrase that enhances the

efficiency of carbon fixation. This enzyme is involved in several physiological processes

such as photosynthesis and CO2 transport (Supuran, 2008; 2011). Carbonic anhydrase

(CA) is an enzyme that catalyzes the conversion of carbon dioxide (CO2) into bicarbonate

(HCO3-) (Badger and Price, 1994; Sly and Hu, 1995; Tripp et al., 2001; Hisar et al., 2005)

with high catalytic rate up to 106 s-1 (Raven, 1995). Carbonic anhydrase facilitates the

supply of CO2 to ribulose-1,5-bisphosphate carboxylase (RuBisco) by converting HCO3-

to CO2 in C3 plants and also enhances the supply of HCO3- to phosphoenolpyruvate

carboxylase (PEPC) by converting the CO2 to HCO3 in C4 plants for photosynthesis.

Carbonic anhydrase may also regulate the stomatal conductance by maintaining

equilibrium CO2 and HCO3 (Tiwari et al., 2005). Furthermore, CA required for CO2

regulated stomatal opening and closing can be alternative approach to provide protection

against unfavorable conditions (Wei-Hong et al., 2014). Drought tolerant CA containing

endophytic bacteria improves CA activity, photosynthetic rate and plant biomass under

non-stressed as well as PEG induced water deficit stressed conditions (Aslam et al.,

2018). Therefore, it may be an important subject to stimulate the CO2 assimilation by

artificially regulating the CA expression. Increase in photosynthesis is important for

better crop productivity.

Although, an enormous number of studies reported that endophytic bacteria

facilitate the plant growth but little is known about the use of drought tolerant CA

containing endophytic bacteria for enhancing the plant growth by stimulating the

photosynthesis. Thus, monitoring the role of CA containing endophytic bacteria in

enhancing the photosynthesis and facilitating cereal biomass is novel idea.

4

Page 25: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Wheat (C3 plant) and maize (C4 plant) are major food grain crops in Pakistan and

occupy the large area. They are cultivated on 9180 thousand hectare with annual

production 25,478 thousand hectare and 1,130 thousand hectare with annual production

4695 thousand hectare, respectively (Pakistan Economic Survey, 2014-2015). The

decreased production may be associated with several factor including, environmental

stresses. Keeping in view the above discussion, it can be hypothesized that inoculation

with carbonic anhydrase containing endophytic bacteria may enhance the photosynthesis

and improve the biomass of cereals (wheat C3 and maize C4) under water deficit

conditions.

For this purpose, series of experiments were conducted to pursue the following

objectives:

Isolation and screening of CA containing endophytic bacteria for plant growth

promotion under axenic conditions at different moisture levels

Characterization and identification of selected CA containing endophytic bacteria

Studying the potential of CA containing endophytic bacteria on photosynthesis

and plant growth promotion under pot and field conditions under drought stress

Studying the potential of CA containing endophytic bacteria on gene expression in

Arabidopsis thaliana under normal as well as stressed conditions

5

Page 26: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Chapter II

REVIEW OF LITERATURE

Water scarcity and food security are the biggest challenges under changing

climatic pattern as both are highly vulnerable to future climatic change. Continuous

severity in the process of climate change shows that our planet will soon be hotter and

drier and global food supply may become inadequate to meet the demands of rapidly

expanding world’s population. To provide food for all of the people, it is absolutely

necessary that cost effective and eco-friendly strategies should be used to ameliorate the

problem of drought in dry areas and increase the agricultural productivity within next few

years. Application of endophytic bacteria can be potential means for facilitating growth

and yield under drought stress in sustainable manner. In this review, role of carbonic

anhydrase containing bacterial endophytes for enhancing photosynthesis and plant

biomass in cereals under limited water supply has been discussed. Moreover, effect of

these bacteria on drought responsive genes and transcription factors of Arabidopsis

thaliana has also been reviewed.

2.1. The problem of drought

Drought is one of the devastating threats to food productivity and livelihoods of

more than two billion people who live on drylands which contribute to 41% of world land

surface. It refers to creeping phenomena as it develops slowly and its impacts remain for

longer period of time after initiation of event (Mazhar et al., 2015). Arid and semi-arid

zones around the globe are most prone to drought (Deng et al., 2004). Drought is climatic

condition of an area where moisture supply is below the average value over continuous

period of time (Anjum et al., 2012). Frequency of droughts is common in developed and

developing world but it leaves long run impacts on the economy of developing world

because most of agriculture in this world is rainfed (Anjum et al., 2010). Drought events

may be short and intense as well as these can persist for many years and constitute

significant loss to local economy. Water deficit in soil can be chronic in the regions

facing low water availability or unpredictable due to changing weather conditions during

the plant growth period. The drought stress is expected to increase with growing water

scarcity and climate change (Harb et al., 2010). Climatic factors such as low humidity,

high temperature and wind are associated with drought stress in many territories of the

world and can significantly enhance its severity (Edwards et al., 1997). Continuous

6

Page 27: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

decrease in precipitation combined with high evapotranspiration yields agricultural

drought (Mishra and Cherkauer, 2010). Agricultural drought occurs due to lack of

adequate moisture necessary for normal growth and expansionto complete plant life cycle

(Manivannan et al., 2008). Drought stress is not only limited to desert regions but also

dramatic reduction in agriculture productivity occurred in temperate regions due to global

warming (Ciais et al., 2003).

In, Pakistan, around 15 million hectares of cultivated land is suffered by water

deficit stress (Mujtaba and Alam, 2002). Out of 79.6 million hectares area, 88% area

encompasses arid to semiarid climate. According to Anjum et al. (2010) about 9% area

receives rain fall above the 508 mm, whereas 22% receives rain between 254-508 mm

and about 69% area receives rain below the 254 mm. The intensity of drought is more

severe in Baluchistan and Sindh because they lie in hyperarid regions; however, Punjab

and Khyber Pakhtunkhwa (KPK) are also affected by drought stress (Mazhar et al., 2015).

2.2. Drought and its impact on photosynthesis and plant growth

Plants are primarily sessile organisms in agriculture environment and are

constantly exposed to plethora of biotic and abiotic constraints including pathogen, heat,

cold, salinity, floods and drought that severely impair crop productivity throughout the

world. Among the stress inducing abiotic factors, limiyed supply of water is a major

constraint that reduces agricultural crop production in tropical world (Kim et al., 2012). It

causes 50% or more decline in crop yield (Wang et al., 2003). It impairs seed germination

and causes poor strengthening (Harris et al., 2002) due to less water uptake in imbibition

phase during germination and disturbed enzyme activity (Taiz and Zeiger, 2010). It also

decreases leaf size, stems elongation, proliferation of root as well as hampers plant water

relations (Anjum et al., 2011). Limited supply of water reduces plant height and growth

by impairing cell expansion, elongation and mitosis (Kaya et al., 2006; Hussain et al.,

2008). Cell elongation is usually inhibited in drought stress due to disturbance in flow of

water from xylem to elongating cell (Nonami, 1998). Drought stress affects water

relations by decreasing total water, water content and turgor. It also limits the gaseous

exchange and transpiration through stomatal closure. Plant cells lower their water

potential and turgor in response to water stress that enhances concentration of solute in

cytosol and other extracellular matrices, resulting in cell enlargement which leads to

growth inhibition and reproductive impairment (Lisar et al., 2012). Moreover, reactive

oxygen species (ROS) increase dramatically under drought stress and induce oxidative

7

Page 28: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

damage (Farooq et al., 2009) to DNA, protein and Lipid (Apel and Hirt, 2004). These

ROS (O2−, H2O2 and OH radicals) can attack membrane lipid and markedly increase their

peroxidation (Mittler, 2002). Loss of membrane stability also reflects lipid peroxidation

induced by ROS. Overproduction of ROS stimulates melonaldehydes content under

drought stress. The increased level of melonaldehyde is signal of oxidative stress (Moller

et al., 2007). The ROS are extremely reactive and impose severe impairment in plant by

enhancing protein degradation, lipid peroxidation and DNA fragmentation which

ultimately causes cell death.

Under drought stress, plants modulate their physiological responses such as

stimulation of stomatal closure by increasing ABA contents, accumulation of

osmoprotectant/compatible solutes and increase in expression of vacuolar pyrophosphates

and aquaporins through osmotic adjustment (Bartels and Sunkat, 2005). Closure of

stomata contributes to less CO2 assimilation. Water stress affects photosynthesis through

stomatal and non-stomatal reductions (Chaves et al., 2003). Stomatal limitation increases

under drought stress along with decrease in photosynthetic parameters (Zlative and

Yordanov, 2004). The decrease in photosynthetic CO2 assimilation is resulted from

stomatal closure that reduces the diffusion of CO2 into leaves and ultimately decreases

intracellular concentration of CO2 (Cornic, 2000). Reduction in leaf mesophyll and

stomatal conductance restricts the diffusion of CO2 at the carboxylation site from air

under mild drought stress (Flexas and Medrano, 2002). Net photosynthetic carbon

assimilation as well as relative water content is reduced in wheat leaves due to stomatal

closure (Dulai et al., 2006). Decrease in photosynthesis has also been observed as severity

of water stress progresses either due to disruption in photosystems (Havaux et al., 1986)

resulted from light which is absorbed more than its reduction capability (Navari-Izzo and

Rascio, 1999), or deterioration of photophosphorylation. It reduces gaseous exchange and

disrupts the photosynthetic pigments leading to reduced growth and plant productivity

(Anjum et al., 2011). Chlorophyll content, relative water content and cell membrane

stability also decrease under drought stress, although, decrease is more pronounced in

drought sensitive compared to tolerant wheat varieties (Almeselmani et al., 2011).

Drought stress also induces substantial reduction in crop yield. Decline in crop

yield occurs probably due to disturbance in gaseous exchange parameters which not only

decreases source and sink size but also hampers phloem loading, transfer of assimilate

and partitioning of dry matter under deficit water conditions (Farooq et al., 2009).

8

Page 29: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Substantial reduction in maize growth and its yield components including cobs, kernel per

cob, 100 kernal weight, grain yield and biological yield occurred under drought stress

(Anjum et al., 2011). Yield reduction in drought stress also occurs due to stomatal closure

in response to reduced water content in soil that decreases the diffusion of CO2 and results

in decline in photosynthesis (Flexas et al., 2004). Stomatal conductance, mesophyll

conductance, transpiration rate, photosynthetic rate, pigment content, relative water

content and dry weight also reduced under drought stress in wheat. Photosynthetic

contents such as chlorophyll b to carotenoid also decrease and lead to yield reduction in

wheat. Furthermore, spike weight, number of grains and grains per spike also decreased

in wheat in water deficit conditions (Allahverdiyev et al., 2015). Drought stress also

influences crop performance such as decreases grain filling period and rate, weight of

grains, grain yield and water use efficiency by affecting all the growth stages but its

severity is more at booting and heading stage compared to anthesis and grain filling stage

in wheat (Nawaz et al., 2013) where more yield reduction at early growth stages may be

due to hampered pollination and seed setting (Farooq et al., 2009) that reduced the grain

count per spike in wheat (Mary et al., 2001). Transport and availability of nutrient also

becomes limited in rhizosphere under water deficit condition that ultimately produces

wilting symptoms in plants. Therefore, plant nutritional status is considered as water

stress indicator (Raza et al., 2012). Drought affects mineral nutrition and metabolism that

reduces the leaf area and alters the partitioning of photassimilates among the different

organs (Lisar et al., 2012).

Stress condition also stimulates and represses genes involved in various functions

(Shinozaki et al., 2003; Yamaguchi-Shinozaki and Shinozaki, 2005). Stress induced genes

have been observed in various plant species such as Arabidopsis and rice (Chinnusamy et

al., 2007; Shinozaki and Yamaguchi-Shinozaki, 2007). Molecular analysis showed that

several droughts induced genes with different functions as well as transcription factors

that regulate the expression of stress related genes are found in rice, arabidopsis and many

other plants (Shinozaki and Yamaguchi-Shinozaki, 2007). Transcription factor

dehydration responsive element binding protein, 2A (DREB2A), is activated under limited

supply of water (Sakuma et al., 2006). Futhermore, expression pattern of genes belonging

to dehydrins (DHNs; Td11, Td16) and transcription factors (DREBS), cell wall

polysaccharides (xylanase inhibitor and endo beta 1.4 glucanase) and cellular metabolism

(ALDH7) was upregulated, however, regulation of ethylene-responsive element binding

9

Page 30: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

factor genes (ERF) and actin binding protein were slightly not significantly down

regulated in leaves of Triticum durum under water deficit stress (Melloul et al., 2014).

2.3. Adaptation measures to mitigate the drought stress

Several adaptation measures are practiced throughout the world to alleviate yield

reduction elicited by drought stress coupled with increase in crop productivity. Control of

irrigation models such as furrow, drip, sprinkler, recognition of resistant resource through

development of different screening methods, use of crop residues and rotation of crop are

different agronomic ways to mitigate the drought stress (Nezhadahmadi et al., 2013).

Breeding of drought tolerant plant has also become high priority under changing climatic

condition. Different traditional selection methods, genomic and genetic tools are being

used to induce tolerance to water stress (Fleury et al., 2010). Traditional breeding requires

identification of genetic variability between plant genotypes and introduction of tolerant

lines with suitable agronomic traits. Hence, traditional breeding seems to be valuable

under drought stress but it is a slow strategy and limited due to selection of suitable gene

for the breeding. Moreover, these strategies are labor intensive that requires a lot of

efforts to segregate the desirable traits from the undesirable traits (Nezhadahmadi et al.,

2013). Tolerance to drought is complex trait, however, variety of genes associated with

stress tolerance is reported but still large gaps remain (Price et al., 2002; Wang et al.,

2003; Fleury et al., 2010). Transgenic approaches have also been used for understanding

molecular process associated to drought tolerance. Majority of these approaches are not

successful to obtain drought tolerant crop with high yield because overexpressed

promoters are used for expression of drought tolerant genes instead of tissue specific and

drought regulated promoters (Reguera et al., 2012; Cominelli et al., 2013). However,

approaches with tissue specific and drought inducible promoter may be useful in future to

improve tolerance in crop against drought stress (Cominelli et al., 2013). Moreover, under

changing stressed environment and enormous number of crops with variety of cultivars,

key genes need to be engineered into plant but seems unclear that genetic engineering will

be fast enough to cope the burgeoning demand of food in near future (Timmusk and

Behers, 2012). Studies showed that certain microbial strains enhanced tolerance against

environmental constraints such as nutrient and drought stress (Yang et al., 2009).

Specifically, PGPB and mycorrhizal fungi have ability to modulate plant physiological

responses and promote their survival under unfavorable environmental situations

(Marasco et al., 2012; Milosevic et al., 2012).

10

Page 31: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

2.4. Plant growth promoting bacteria under drought stress

Being, relatively simple and cost effective alternative strategy, use of PGPB has

emerged as a promising tool with broad spectrum benefits for improving the plant growth

(Timmusk and Wagner, 1999; Timmusk, 2003; Mayak et al., 2004). Timmusk and

Wagner (1999) firstly reported drought tolerance induced by PGPB. (Timmusk and

Wagner, 1999; Mayak et al., 2004). Plant growth facilitating rhizobacteria (PGPR) and

endophytes exhibit a variety of plant growth facilitating properties that support the plant

growth under limited water supply (Marasco et al., 2013). The root length and biomass in

pepper plants increased up to 40% by the PGPB that mediated larger root system being

responsible for water uptake in dry soil (Marasco et al., 2013). Bacteria having capability

to colonize the plant root and facilitate plant growth have been considered as PGPR

(Kloepper and Schroth, 1978). Rhizospheric bacteria harbor different adaptive traits and

improve plant vigour under both biotic and abiotic stressed environments. The exact

mechanism of drought tolerance by rhizospheric bacteria is speculative but possible ways

include production of phytohormones, production of ACC-deaminase enzyme to reduce

the ethylene level, induced systemic resistance and formation of biofilm (Yang et al.,

2009; Timmusk and Nevo, 2011; Kim et al., 2012). Bacterial priming of wheat enhances

drought tolerance by improving the plant biomass upto 78% and its survival up to five

fold greater under drought stress through production of stress related hormones (Timmusk

et al., 2014). Studies also showed the role of these PGPR in eliciting the drought

resistance in peppers and tomatoes (Mayak et al., 2004). Despite the beneficial effect,

application of these microbes is often hampered under field condition due to inconsistent

performance (Thomashow et al., 1996). Ability of inoculum to colonize root is primary

factor that determines its efficacy for yield enhancement (Weller, 1988). This may lead to

selection of beneficial bacteria that can colonize the root system effectively (Raaijmakers

and Weller, 2001). Endophytic bacteria colonize the internal tissues of plant without

inducing any harm (Schulz and Boyle, 2006). Plant pre-exposed to these microbes or

primed plant can respond more rapidly and efficiently compared to non-primed plants as

they are pre-acclimated to host cell metabolism and can survive under challenging

environment betterly (Sturz and Nowak, 2000).

2.5. Comparison of other plant growth promoting rhizobacteria and endophytic

bacteria

Endophytic bacteria can be differentiated from non-endophytic bacteria due to

11

Page 32: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

their unique behavior with the host plant. These bacteria efficiently colonize the tissues

(above and below ground) of host plant and form long term association without causing

harm to host. They develop lifelong associations usually latent or dormant infections

which are distinguished from associations of other transient bacteria that will not sustain

longer. These bacteria are further differentiated from others by regulating the growth of

other nematodes, bacteria and fungi (Hallmann et al., 1997; Bacon and Hinton, 2006).

Endophytic bacteria reside in the interior of plant as it is continuous source of nutrients

and protected niche. Intercellular spaces are very rich source of organic and inorganic

nutrients essential for supporting the growth of complicated mixture of bacterial

endophytes (Bacon and Hinton, 2006). Being inside the protected niche, endophytic

bacteria are less exposed to different biotic stresses and face less competition with other

microbes due to frequent source of plant nutrients (Bacon and Hinton, 2006). These

bacteria are considered better than the rhizospheric and their rhizoplanic counterparts

because they can fix nitrogen directly into their host plant, provide benefit to plant by

nitrogen fixation (Cocking, 2003) and face less competition being sheltered in the plant

interior (Gupta et al., 2012). Bacterial endophytes are better sheltered from high biotic

and abiotic constraints and competitive environment of soil (Sturz et al., 2000). In

comparison to rhizobacteria, these bacteria may be less exposed to the stress inducing

biotic and abiotic factors (Hallmann et al., 1997). Being habituate to their host and

present at rhizosphere initiation and seedling development, these bacterial endophytes

offer twins of advantage, thereby provide ecological benefits compared to other wild type

resident soil microflora that are often associated with failure of seed treatments (Sturz and

Nowak, 2000). Moreover, growth enhancement by bacterial endophytes are more

compared to rhizosphere localized bacteria or bacteria restricted to root surface (Chanway

et al., 2000). Beneficial effects of endophytic bacteria are more than rhizosphere

colonizing bacteria (Pillay and Nowak, 1997). Therefore, colonization efficiency of

endophytic bacteria and survival ability of inoculum both in plant and rhizosphere is

prerequisite for efficient delivery and management of inocula (Compant et al., 2005)

2.6. Endophytic bacteria and their relationship with host plant

Plants harbor an abundant and variety of microflora mostly bacteria and fungi

inside the xylematic vessels which carry out various operation for nutrition of host plant

are called endophytes (Hallmann et al., 1997; Rosenblueth and Martinez-Romero, 2006).

Bacterial endophytes are referred to those bacteria which colonize the interior tissues of

12

Page 33: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

plant without showing any sign of infection and causing deleterious effects in host plant

(Holliday, 1989; Schulz and Boyle, 2006). These bacteria are virtually found in every

plant studied, nearly 300,000 species of plant reported to exist in our planet and each of

them is host of one or several bacterial endophytes (Strobel et al., 2004). These bacteria

do not generate impairment to host plant and humans (Di Fiore et al., 1995; Chiarini et

al., 2006; Schmid et al., 2006), on contrary, they promote the growth of plant (Castro-

Sowinski et al., 2007). In contrast to other endosymbionts, they do not make membrane

bound structures/compartments. They are spread all over the plant organs including roots,

shoots, leaves, flowers, fruits, seeds. Their association with the host plant may be obligate

and facultative and they cause no impairment to host plant. Obligate endophytes are

closely bound to host for their survival and growth except for transportation to alternate

plants either vertically or with vectors whereas facultative endophytes can live either

inside the plant or other habitats. Some endophytes are opportunistic (enter occasionally)

and some are passenger endophytes (enter inside the plant accidently). However, a newly

emerged plant bacteria association is competent endophytes. Competent endophytic

bacteria possess genetic machinery necessary for colonizing of endosphere and persist in

it. These bacteria actively enter inside the plant, colonize host plant tissue, regulate plant

physiology and maintain beneficial plant microbe relationship (Hardoim et al., 2008).

Large numbers of endophytic bacteria form symbiotic association but some establish

mutualistic association (Bacon and Hinton, 2006). Plants restrict the growth of

endophytes but endophytes use variety of processes to acclimate to their living

environment (Dudeja et al., 2012). Endophytes produce stable compounds to maintain the

symbiosis that help their survival in new environment (Lee et al., 2004; Das and Varma,

2009). Within the plant, endophytic bacteria do not induce morphological changes as root

nodule and do not cause disease as phytopathogen. Transient intercellular dwellers are

excluded here that can become intracellular, sometime invade xylem tissue and become

problematic to plant. Xylem endophytes are regarded not good for bicontrol because they

are viewed as problematic since infected and weakened vessel become impaired and

hazardous to host plant (McCully, 2001). Soil or rhizospheric bacteria can turn to good

endosphere colonizers if these bacteria have ability to cope with unexpected events of

changing environments from exosphere to endosphere that requires different bacterial

responses in different tissues (Hardoim et al., 2008).

13

Page 34: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

2.7. Origin of endophytic bacteria and mode of entry in plants

Bacterial endophytes may originate from seed (Adams and Kloepper, 1996),

rhizosphere soil (Mahafee and Kloepper, 1997), vegetative organs (Dong et al., 1994) and

phylloplane (Beattie and Lindow, 1995). Several observations revealed that rhizosphere is

a primary source for bacterial endophytes (Hallmann et al., 1997; Mahafee and Kloepper,

1997) but other site of transmission can not be ignored. Bacteria can also enter in plant

tissue through lenticels, stomata, site of emergence of lateral root and germinating

radicals (Huang, 1986). Gluconobacter diazotrophicus enters through stomata in

sugarcane (James et al., 2001). Streptomyces galbus facilitates leaf surface colonization

and lateral establishment (Suzuki et al., 2005). Endophytic bacteria mostly originate from

soil and infect host plant by colonizing the cracks made at the site of lateral root junction

and spread fastly into intercellular spaces of root (Chi et al., 2005). Cracks in root are the

major hot spot for the colonization of endophytic bacteria (Sorensen and Sessitsch, 2006).

Colonization by bacteria at secondary root emergence site has also been discussed by

various authors (Reinhold and Hureck, 1998; Mahaffee et al., 1997). After entering inside

the plant tissues, these bacteria remain either confine to specific tissue including root

cortex or colonize consistently by transporting through xylem vessel (Quadt-Hallmann et

al., 1997). These bacteria were observed in xylem vessels of internode, leaf and

substomatal chambers but were not found on stem and leaf surfaces (Compant et al.,

2005). Compant et al. (2011) also reported that bacteria get access into plant tissue from

soil through cracks developed by lateral roots and moved from roots to leaves, then

flower and fruit through vascular system (Hardoim et al., 2008; Compant et al., 2011).

2.8. Colonization by endophytic bacteria

There is variety of ways through which endophytes get enter to the interior of host

plant.

2.8.1. Rhizoplane colonization

Bacterial endophytes attach the rhizoplane (solid root surface) to enter into roots

such as root tip, site of lateral root emergence (Hardoim et al., 2008). A momentous

amount of studies have revealed that attachment to root is key step for the establishment

of endophytic colonization. Attachment of bacteria and their subsequent entry into host

root occurs through apical root including zone of active penetration (root hair and thin

walled surface root layer) as well as zone of passive penetration (basal root where cracks

14

Page 35: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

are formed due to lateral root formation). Variety of bacterial components is also involved

in this process. The association of Herbaspirillum seropedicae, diazotrophic endophyte

bacteria, to surfaces of maize roots depends on lipopolysacchrides (LPS) (Balsanelli et

al., 2010). Another study showed that colonization of rice rhizoplane and endosphere by

Gluconacetobacter diazotrophicus required exopolysaccharides (EPS) (Meneses et al.,

2011). Some bacterial strains lost their adhesion capability used bacterial structural

component for colonization (Malfanova et al., 2013). Recently, diazotrophic Azoarcus sp.

BH72, a pilT mutant has been reported to impair its twitching motility, resulting in

defective root colonization (Bohm et al., 2007) while the pilT locus plays important role

in motility of attached bacteria on the surface of plant. Any destruction to pilA as well as

pilT that are essential for the formation of pilus decreased its movement, indicating the

role of these genes in colonization potential of Azoarcus sp. (Bohm et al., 2007).

Similarly, rice endophyte Azoarcus sp. BH72 required type IV pili for the attachment of

root (Dorr et al., 1998). These bacteria increase in numbers after attaching to plant root

through cell divisions and result in formation of microcolonies (Compant et al., 2008).

Bacteria form colonies at these sites (Zachow et al., 2010). Infection of root tissues may

occur through these microcolonies for instance at the junction of lateral roots. These

bacteria produce cellulytic enzymes that can hydrolyze cell wall. Enzymatic activity is

important for the degeneraion of plant cell envelop in this invasion process. Many of the

endophytic bacteria produce these enzymes under in vitro conditions (Reinhold-Hurek et

al., 2006). Bacterial gene endoglucanase is considered essential for colonization of

Burkholderia sp. PsJN and Azoarcus sp. BH72 (Compant et al., 2005; Reinhold-Hurek et

al., 2006). In addition, expression of an enzyme, endoglucanase that hydrolyze the β

(1→4) linkage in cellulose was observed at the entry of Azoarcus sp. (Reinhold-Hurek et

al., 2006). Root colonizing bacteria produce low level of cell wall degrading enzymes that

differentiates these bacterial endophytes from the other bacterial pathogens which

produce deleteriously high level of enzymes (Elbeltagy et al., 2000). Bacterial cell wall

degenerating enzymes are involved in induction of plant defense system as many defense

related protein are present in cell wall of bacteria. Elicitation of plant defense response

reduces the induction of plant pathogens (Iniguez et al., 2005). On the other hand,

endophytes may enter inside the plant root and other tissues without producing cell wall

degrading enzymes possibly through the automatically formed cracks in displaced

epidermal cell or through spaces constituted by soil herbivores. These bacteria enter

inside the plant via cracks at the site of lateral root emergence and last invisible to defense

15

Page 36: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

system of plant. This is the case that endophytes may escape from immune system

(Malfanova et al., 2013). After entering the roots, these competent endophytes penetrated

through the casparian strips present in endoderm to transmit systematically to the other

above ground plant parts. Highest bacterial population was observed in root, stem and

leaves, respectively (Lamb et al., 1996).

2.8.2. Endophytic colonization of different plant tissues

When, these competent endophytic bacteria are present inside the host plant, they

react with plant signals for inducing cellular processes that are essential for their entry

into endophytic stage and further translocate to cortex intercellular tissues of roots and

beyond. For this process, generation of endoglucanases (Compant et al., 2005) and

endopolygalacturonidases (Reinhold-Hurek et al., 2006) seem indispensable. Some

bacteria penetrate endodermal cell and colonize the xylem vessels. Once bacteria crossed

the endodermal cell wall either last at the spot of entry (Timmusk et al., 2005) or transfer

deeper intercellular spaces in cortex (Gasser et al., 2011). These endophytes multiply

inside the plants (Zakria et al., 2007) and attain high cell density (e.g. 108 cells g-1 dry

weight root tissue) (Barraquio et al., 1997). Inoculation of rice with Sinorhizobium

meliloti labeled with green florescent protein under gnotobiotic conditions showed that

endophytic population dynamics inside plant is greater (i.e. 9 x 1010 cells cm-3 of root and

leaf tissue) (Chi et al., 2005). It is well explained that population of bacterial endophytes

decreases as the area from root increases and only a few bacteria reach the upper part of

shoot, leaf and reproductive organ, such as flower fruit and seed (Compant et al., 2010;

Furnkranz et al., 2011). Chi et al. (2005) found that dynamic infection process began with

colonization of rhizoplane, then endophytic colonization of roots followed by

transmission into stem, leaves and leaf sheath. It is observed that amount of nutrients in

xylem decreased along the axis of plant. The presence of these bacteria in reproductive

tissues was assured by colonization (Okunishi et al., 2005; Furnkranz et al., 2011) and

through microscopic visualization (Coombs and Franco, 2003; Compant et al., 2011). If

one reproductive cell such as male gametes and egg cell possess microbe, the endosperm

and embryo developed from them may be colonized. These results explain the

translocation of endophytes from plant to seed (Malfanova et al., 2013). So far, infection

of reproductive tissues has been observed only with viruses (Agarwal and Sinclair, 1996).

However, the exact procedure for the translocation of bacteria from vascular bundle to

reproductive organ and latterly to other plant needs to be explored.

16

Page 37: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

2.9. Bacterial endophytes and their potential role in growth promotion

Endophytic bacteria can facilitate plant growth and induce resistance to different

environmental stresses (Ryan et al., 2008). Interaction between plant and endophytes has

been reported to elicit integrity, proper functioning and feasibility of agro-ecosystem

(Nagarajkumar et al., 2004). These bacteria stimulate seedling emergence, enhance plant

survival under stressful environmental conditions and improve plant growth and

development (Bent and Chenway, 1998). Mechanism of plant growth facilitation by

endophytic bacteria has been postulated to be similar with plant growth promoting

rhizobacteria (Madhaiyan et al., 2006). Bacterial endophytes elicit growth promotion

either directly i) by producing the 1-aminocyclopropane- 1-carboxylate (ACC)

deaminase, an enzyme that lowers ethylene level in plants, ii) by producing the plant

hormones e.g. auxin (Prasad and Dagar et al., 2014) and cytokinin or indirectly i) by

helping the plant in nutrient acquisition (Sessitsch et al. 2002) such as phosphorus

solubilization, nitrogen fixation (Iniguez et al., 2004; Sevilla et al., 2001 ) and chelation

of iron (Costa and Loper, 1994) ii), by preventing the infection caused by pathogen, iii)

by antibacterial (Hardoim et al., 2015) or antifungal agent, iv) by competing the pathogen

for plant nutrient either though siderophore production or inducing systemic resistance in

plant. Plant provides protective niche for endophytes and these endophytes produce useful

signals and metabolites (Rosenblueth and Martinez- Romero, 2006) that stimulate uptake

of nutrients (Ramos et al., 2011), modify plant growth and biomass (Hardoim et al., 2008)

and induce tolerance against osmotic stress (Sziderics et al., 2007) and other abiotic

factors. Endophytic bacteria have been reported to facilitate growth of tomato plants

where 61% of isolated bacteria enhanced tomato growth and 50 to 64% improved

biomass accumulation (Majeed et al., 2014). Jasim et al. (2014) also found that

Pseudomonas sp. affected the growth of ginger by producing IAA, siderophores and

ACC-deaminase activity. Similarly, improvement in root number and root and shoot

length was observed by the inoculation of endophytic bacterial isolates BETL9, BETL13,

BECL8, BECS1 and BECS7 in tomato and chilli seedlings (Amaresan et al., 2012).

2.9.1. Endophytic bacteria as stimulator of plant nutrients

Plant growth facilitation has been recorded by several bacterial endophytes

(Zachow et al., 2010; Gasser et al., 2011; Malfanova et al., 2011) directly mediated

through nutrient availability. The ability to fix N2 in plant by endophytic bacteria was

demonstrated in many studies. It has been investigated that endophytic diazotrophs help

17

Page 38: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

plant in acquiring the 70% of required N2 through BNF (James, 2001). Studies also

showed that Acetobacter diazotrophicus (Gluconoacetobacter diazotrophicus) has ability

to fix 150 kg N ha −1 year −1 and act as main contributor to N in sugarcane

(Muthukumarasamy et al., 2005). It has been anticipated that use of endophytes can

produce 200 kg N ha-1 year-1 in rice (Ladha and Reddy, 1995). This fact was well

explained by isotope analysis (Elbeltagy et al., 2001) and by observing the nitrogenase

genes expression in nitrogen-fixing cells (Egener et al., 1999; Roncato-Maccari et al.,

2003; You et al., 2005). 15N2 incorporation studies showed that inoculation of sugarcane

with G. diazotrophicus Pal5 fixed 0.6% of N through BNF (Sevilla et al., 2001) and this

value was 0.14% for rice plant harboring Herbaspirillum sp. (Elbeltagy et al., 2001).

Secondly, N2 fixation is greatly influenced by nitrogen availability and oxygen

concentration. Nitrogenase expression was suppressed in free air (21%) and improved

under microoxic (having low oygen) conditions i.e. 2% O2 in Herbaspirillum sp. B501

(You et al., 2005) indicating that interior of plant is suitable place for nitrogen fixation.

Endophytic bacteria fixed nitrogen with nitrogen fixing ability have an edge over their

rhizospheric counterparts because they make available fixed nitrogen to host plant.

Secondly, low partial pressure of oxygen is required for activity of nitrogenase, an

oxygen sensitive enzyme, however, endosphere of root makes them more acquiescent for

nitrogen fixation reaction. Bacterial endophytes with nitrogen fixing capacity have been

reported to derive 47% nitrogen from the air and promote plant growth (Gupta et al.,

2012). Moreover, nitrogen starvation also depresses the biosynthesis of IAA. Brandi et al.

(1996) revealed that supernatant obtained from Erwinia herbicola culture showed 10-fold

higher IAA synthesis under nitrogen starved condition. Therefore, diazotrophic bacteria

boost plant growth by stimulating the production of phytohormone production and

supplying nitrogen. It is further supported that both wild type and mutant increased the

sugarcane biomass when nitrogen was not limiting (Sevilla et al., 2001).

Bacterial endophytes present in root zone have capability to increase the

availability of phosphorus to plant. Growth facilitation in plants is also enhanced by

phosphorus solubilizing endophytic bacteria that solubilize the inorganic phosphorus.

Bacteria release organic acids to solubilize phosphate complexes and trnsform them into

orthophosphate that is taken up and utilized by plant (Oteino et al., 2015). These organic

acids with their carboxyl and hydroxyl groups chelate cation bounded phosphate

(Kpomblekou and Tabatabai, 1994). Recently, it has been demonstrated that bacterial

18

Page 39: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

endophytes produce gluconic acid, possess phosphate solublizing capabilities

approximately 400-1300 mg L-1 accompanied with beneficial effects on Pea sativum plant

growth under limited soluble phosphate conditions (Oteino et al., 2015). Andrade et al.

(2014) found that out of 40 bacterial endophytes isolated from banana trees, 37.5%

isolates have potential to solubilize inorganic phosphate and highest solubilization

capacity was observed in isolate EB-47 and EB-64.

Moreover, siderophore production by bacteria provides competitive advantage to

colonize the tissues of host plant and omit other microbes from ecological niche (Loaces

et al., 2011). However, role of endophytically produced siderophore in plant colonization

is unknown but plays vital role in induction of ISR (Hardoim et al., 2015). Production of

siderophores can inhibit the growth of plant pathogen, thereby improve plant growth

(Sharma and Johri, 2003). Some bacteria produce catecholate-types while other bacteria

produce hydroxamate-type siderophores (Neilands and Nakamura, 1991). In this process,

produce small molecular compounds which have high affinity for iron and cell captures

the iron charged siderophores. This property has been found in many endophytes isolated

from different plants and acts as antagonist of pathogen (Cho et al., 2007; Li et al., 2009).

It has also been studied that most of hetrotrophic endophytic bacteria produce

siderophores in mature plants; however, less than 10% of bacterial endophytic produce

siderophores in leaves and roots of younger plants (Loaces et al., 2011).

2.9.2. Endohytic bacteria as phytohormone producer

Similar to PGPR, bacterial endophytes stimulate plant growth through generation

of phytohormones (Umamaheswari et al., 2013). The synthesis of phytohormone and their

possible role in plant growth promotion by endophytic bacteria have been stated by

copious researchers (Govindarajan et al., 2008; Long et al., 2008; Malfanova et al., 2011).

Bacterial endophytes produce auxins that stimulates cell division, elongation and

differentiation, thus facilitate the plant growth (Shokri and Emtiazi, 2010). Indole acetic

acid has been reported as physiological active hormone in plants, acts as signaling

molecule; regulates plant development processes including tropic responses, organogensis

and cellular responses including cell division, expansion, differentiation and regulation of

genes (Ryu and Patten, 2008). Beneficial effects of endophytic bacteria in rice plants are

associated with their ability to produce IAA that increases the growth of rice seedling and

development under control environment (Etesami et al., 2014). Capability of endophytic

bacteria isolated from the cashew to produce IAA was also checked by Lins et al. (2014)

19

Page 40: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

and found that out of 31 isolates, 17 isolates had the ability to produce IAA in

concentration from 11.79 to 145.85 µg/mL. Indole acetic acid stimulates plant growth by

increasing root length and root growth that is associated with elongation and proliferation

of root hair (Weyens et al., 2009). One of the possibilities is that IAA producing bacteria

colonize the root better compared to other isolates (Kuklinsky-Sobral et al., 2004; Mendes

et al., 2007). Bacteria colonize the plant root by forming attachment to root epidermal cell

and basal zone of emerging root hair where root hair is major zone for primary

colonization. Endophytic bacteria produce many other phytohormones such as abscisic

acid (ABA) (Cohen et al., 2008), gibberellins (GB) (Lucangeli and Bottini, 1997;

Malfanova et al., 2011) and cytokinins (Sgroy et al., 2009). Increased level of GA3 in

maize roots was observed with Azospirillum spp., GB-producing endophytic bacteria that

ultimately enhanced plant growth (Lucangeli and Bottini, 1997). Inoculation of plant

Dendrobium officinale with Sphingomonas paucimobilis ZJSH1 enhanced plant growth

such as shoot fresh weight that might be attributed to production of phytohormones

because higher amount of salicyclic acid, IAA, zeatin c-ZR and GA were recorded in the

inoculated plant acting as both plant growth regulator and driver of systemic acquired

resistance (Yang et al., 2014). Zeatin is member of phytohormone known as cytokinins

that promote the cell division and facilitate the growth of lateral bud (Patel et al., 2012).

Production of gibberellins and auxins are typical characteristics of root associated

endophytes (Shi et al., 2010; Khan et al., 2012; Merzaeva and Shirokikh, 2010) . Higher

level of hormones was observed when sugarcane plant roots and shoot were inoculated

with endophytic bacterial isolates compared to uninoculated control plants. These

bacterial isolates produced high level of GA, IAA, ABA and ZR in L-TRP amended

growth medium and improved plant growth under both axenic and glasshouse conditions

in sugar beet (Shi et al., 2010). Endophytes associated with plant having ability to

produce phytohormones can be important biological tool with widespread agriculture

potentials (Ali and Vora, 2014).

2.9.3. Endophytes as modulator of plant ethylene level

Plant also produces phytohormone ethylene on exposure to various constrainsts

which is promising modulator of normal growth and development in plant (Abeles et al.,

1992). To ameliorate the stress caused by high ethylene level, plants select ACC-

deaminase containing bacteria as endophytic bacteria and minimize deleterious effects

caused by ethylene such as reduced root growth (Hardoim et al., 2008). The bacteria have

20

Page 41: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

ability to lower the endogenously produced ethylene level by producing the ACC

deaminase that play essential role in optimal functioning of bacteria which not only

facilitate the plant growth but also secure the plant against drought stress (Glick, 2013).

Many endophytic bacteria have been reported which contain ACC-deaminase activity

taking part in lowering the ethylene (Long et al., 2008). The endophytic bacteria

containing high ACC deaminase activity proved to be efficient growth stimulator as they

efficiently block the ethylene production and ameliorate plant stress (Cheng et al., 2007).

Bacteria containing high ACC-deaminase activity take ACC before its oxidation through

ACC oxidase, thus lower ethylene level inside the plant (Glick et al., 1998). This enzyme

cleaves the precursor of plant ethylene ACC into ammonia and α-ketobutyrate (Honma

and Shimomura, 1978).

Different stresses enhance ethylene level known to involve in reduction of root

growth and lateral root emergence (Ivanchenko et al., 2008). Few bacteria use ethylene as

nutrient and lower the ethylene synthesis inside the plant. According to Glick (2005),

bacterially mediated IAA enhance the synthesis of ACC-synthase resulting in enhancing

the regulation of ethylene precursor ACC. Moreover, ACC-deaminase activity was

observed in many plant growth facilitating endophytic Burkholderia (Sun et al., 2009;

Gasser et al., 2011), Herbaspirillum (Rothballer et al., 2008) and Pseudomonas (Long et

al., 2008). Mutational study also confirmed the involvement of ACC-deaminase in plant

growth stimulation where gene coding for ACC-deaminase, acdS gene, was deleted in B.

phytofirmans PsJN and resulted in 32% decrease in root length of canola (Sun et al.,

2009). Another study on cut flower also showed that bacterial endophytes also delayed

the senescence of flowers (Ali et al., 2012). Several other researchers have also

demonstrated the use of bacteria having ACC deaminase activity to protect the biomass of

different plants against drought stress (Arshad et al., 2008; Shakir et al., 2012).

2.9.4. Endophytes as a biocontrol agent of phytopathogen

Pathogenic microbes are serious threat to food productivity and ecosystem

sustainability throughout the world. Several bacteria have been found to form endophytic

association with host plant and prevent disease development. The use of endophytic

bacteria for general and specific biological control purpose is wide spread (Kobayashi and

Palumbo 2000; Sturz et al. 2000) and well established (Berg and Hallmann, 2006;

Scherwinski et al., 2008; Malfanova et al., 2011), however, the exact mechanism for

biocontrol is less elucidated. Biocontrol is based on several mechanisms including

21

Page 42: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

antibiosis, induced systemic resistance (ISR) and fight for niches and nutrients (CNN).

Among these, role of ISR was confirmed in plants (Malfanova et al., 2013). Microscopic

observation showed that endophytic bacteria induced phenotypic changes related with

ISR and reduced the disease symptoms at the site where endophytes were present.

Bacterial endophytes colonize the ecological niche similar to plant pathogen thus make

them useful for biocontrol (Berg et al., 2005). Colonization by endophytic bacteria

induces several modifications in cell wall including accumulation of cellulose, pectin and

phenolic compound that leads to generation of barrier at the place of infection caused by

phytopathogen (Benhamou et al., 1998; 2000). Another response of bacterized plant to

attack of phytopathogen is expression of defense associated proteins such as chitinase,

peroxidase and β-1,3-glucanases (Fishal et al., 2010) resulting in decrease of pathogens in

plant. Reduction in disease was also observed in endophytically colonized wheat with B.

subtilis (Liu et al., 2009) and banana plant inoculated with endophytic Burkholderia and

Pseudomonas (Fishal et al., 2010). Melnick et al. (2008) investigated the capability of

Bacilli to colonize and decrease signs of black pod rot in cacao plants induced by

Phytophthora capsici. However, genomic analysis proposed endophytes competition for

iron and colonization but yet it has not been studied in plant.

It has also been investigated that plant-pathogen interaction is greatly influenced

by lipopolysaccharides (LPS) obtained from external cell wall of gram negative bacteria.

Apart from direct effect on plant pathogen, these bacteria also induced systemic

resistance in plant. Lipopolysaccharides have been suggested to be main component

present in cell wall of gram negative bacteria (Van et al., 1998). These LPS producing

endophytic bacteria like Burkholderia cepacia caused protective effect against Nicotianae

tabacum and Phytophthora nicotianae when plants were treated with zoospores of

pathogen. LPS activity acts as elicitor molecule to plant defense response. Enhanced

defensive ability and expression of pathogenesis related protein occur due to inoculation

with lipopolysaccharide producing Burkholderia cepacia against pathogen in Nicotianae

tabacum. Endophytic bacteria released LPS in plant where they interacted with host plant

cell and acted as elicitor molecule (Conventry and Dubery, 2001).

Endophytic bacteria protect the host plant by synthesizing the large amount of

antimicrobial compounds, siderophores and molecules that elicit induced systemic

resistance (Compant et al., 2010). Molecules that play an influential role in biocontrol are

lipopeptides (LPs) (Perez-Garcia et al., 2011). Compound such as fengycins and

22

Page 43: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

surfactins have recently been found as elicitors of ISR (Jordan et al., 2009). Biocontrol of

bacterial wilt pathogen in tomato was observed by endophytic bacteria where plant

treated with isolate BC4 hastened the disease incidence compared to control plant

(Nawangsih et al., 2011). Furthermore, it has been established that growth can be

enhanced in plants by the control of pathogenic bacteria under natural condition. The

enhancement in growth may occur due to i) lack of parasitism and disease ii) lack of

susceptibility to abiotic stresses such as drought and salinity and iii) resistance to frost

(Becan and Hinton, 2006). Therefore, biocontrol is being considered a supplemental or

alternative way to minimize the use of agrochemicals (Welbaum et al., 2004).

2.9.5. Endophytic bacteria as producer of secondary metabolites

Endophytes produce secondary metabolites involved in competition, signaling and

defense mechanisms as well as in regulation and establishment of symbiosis (Schulz and

Boyle, 2005). Endophytes act as chemical synthesizer inside the plant (Owen and

Hundley, 2004). Actually, these metabolites are biologically important compounds and

act as antioxidant, antibacterial, antifungal, antiviral, immunosuppressive, nematicidal

and insectidal agents (Tan and Zou, 2001; Strobel et al., 2004; Gunatilaka. 2006; Verma

et al., 2009; Brader et al., 2014). Kaaria et al. (2012) found that endophytic bacteria

isolated from Kenyan plants produced secondary metabolites that possessed the

antimicrobial activities against human pathogen specially Bacillus substilus. Besides

producing the secondary metabolites, these bacterial endophytes also affect the plant

metabolism. Bacterial endophytes may also modulate the synthesis of plant metabolites

(Brader et al., 2014).

Metabolic signature (alteration in metabolic profiling of plant inoculated with

same strain in repeated trials) showed that modulation of metabolites production which

were vital for colonization and activation of pathways associated with plant defense. A

metabolic signature of grapewine plant inoculated with Enterobacter ludwigii EnVs6

showed the decrease in concentration of esculin, catechin, astringin, pallidol, arbutin,

ampelopsin, D- isohopeaphenol and quadrangularin and increase in vanillic acid (Lopez-

Fernandez, 2015). Inoculation of strawberry plant with the Methylobacterium strain

influenced the synthesis of flavor compounds i.e. furanones in host plant

(Koutsompogeras et al., 2007). Endophytic bacteria with methanol dehydrogenase

transcripts were found in vascular tissue of receptacles and in achenes cell of strawberry

where gene of furanone biosynthesis was expressed (Nasopoulou et al., 2014). Bacterial

23

Page 44: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

endophytes, Candida- tus Burkholderia kirkii protected the host plant against various

pathogen/harbivores by producing metabolites. It has also been observed that genome of

C. Burkholderia kirkii contain gene involved in several metabolites production especially

analogues of C7N aminocyclitols family (Brader et al., 2014). Many members have also

been demonstrated to possess insecticidal and antifungal activity in this family.

2.10. Role of endophytic bacteria in growth stimulation under drought stress

Bacterial endophytes secure the plant growth from the devastating effect of

drought. It has been observed that drought tolerant endophytic bacteria, actinobacteria,

Streptomyces olivaceus DE10, Streptomyces geysiriensis DE27 and Streptomyces

coelicolor DE07 tolerate the drought stress from 0.05 to -0.73 MPa and enhance plant

growth in wheat under stressed environment due to production of phytohormones

(Yandigeri et al., 2012). Bacterial endophytes also minimized the adverse effect of

drought by affecting physiological responses and growth in maize including increase root

and shoot biomass, chlorophyll content, leaf area, photochemical efficiency of PSII and

photosynthesis. Endophytic bacteria Enterobacter sp. FD17 and Burkholderia

phytofirmans strain PsJN also reduced the damage caused by H2O2 compared to control

plants due to production of defense related enzyme superoxide dismutase, peroxidase,

catalase or phenolic compounds that mitigate the oxidative stress caused by drought

(Naveed et al., 2014a). Another study also revealed that bacterial endophytes B.

phytofirmans strain PsJN increased the chlorophyll content, CO2 assimilation rate and

water use efficiency in wheat compared to non-inoculated control plant under drought

stress (Naveed et al., 2014a). Endophytic bacteria B. phytofirmans PsJN remained active

in potato plant and induce variety of genes and pathways showing high metabolic activity

in the plant. However, activity of selected strain is greatly affected under drought stress.

Bacterial strains also sense the stress signals and adjusts its gene pattern accordingly to

cope with drought stress (Sheibani-Tezerji et al., 2015). Inoculation of arabidopsis plant

with Pirifomospora indica enhanced drought tolerance by handling the stress related

genes in the leaves. Drought stress related genes including early response to dehydration1

(ERD1), response to dehydration 29A (RD29A), dehydration-response element binding

protein2A (DREB2A), ANAC072, calcineurin b-like proteiN (CBL)1, phospholipase Dδ,

(PLD), salt-,drought-induced ring finger1 (SDIR1), histone acetyltransferase (HAT) and

cbl-interacting protein kinase3 (CIPK3) were upregulated in Arabidopsis thaliana leaves

colonized with P. indica compared to uninoculated seedling (Sherameti et al., 2008).

24

Page 45: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Overexpression of DREB2A (Sakuma et al., 2006) and ANAC072 (Tran et al., 2004)

improved drought tolerance in Arabidopsis. Gibberellin and absicic acid produced by

endophytic bacteria Azospirillum lipoferum alleviated the drought stress symptoms in

maize.

2.11. Bacterial produced carbonic anhydrase and its role in plant photosynthesis

Endophytic bacteria may possess an enzyme carbonic anhydrase (CA). Bacterial

produced CA enzymes belong to α, β, and δ classes (Capasso and supuran, 2014).

Carbonic anhydrase involves in several physiological processes of life including

respiration, photosynthesis as well as transport of CO2 (Supuran, 2008; 2011). This is key

enzyme in photosynthetic carbon assimilation and any change in its activity directly

influences the photosynthetic CO2 fixation under CO2 limited conditions. It catalyzes the

hydration of CO2 into HCO3 that is physiologically important reaction in all forms of life.

CO2+H2O HCO3- + H+ (Supuran, 2008; 2011)

The relation between plant photosynthesis and CA is widely understood. It plays

important role because uncatalyzed reaction is 104 times slower as compared to CO2 flux

in the photosynthesis (Badger and Price, 1994). Carbonic anhydrase generally converts

CO2 to HCO3- for phosphoenolpyruvate carboxylase (PEPC) in C4 plant during

photosynthesis and converts HCO3- to CO2 and provides CO2 for ribulose-1,5-

bisphosphate carboxylase (Rubsico) reaction during photosynthesis in C3 plants (Wei-

Hong et al., 2014). In C4 plants, CA first converts the CO2 to HCO3 that is fixed by PEPC

to produce the C4 acid. Latterly, these C4 acids are diffused and decarboxylated to

supply CO2 for Rubsico in the bundle sheath cell, consequently Rubisco works close to its

Vmax and represses the photorespiration (Hatch, 1987), indicating the requirement of CA

for C4 plants (Badger & Price, 1994). A study reported that CA activity inhibited in

ethoxyzolamide treated C3 plant caused 80-90% reduction in photosynthesis at low CO2

concentration showing the key role of CA in photosynthesis (Badger and Pfanz, 1995).

The CA also plays important role in stressed conditions (Wei-Hong et al., 2014).

Overexpression of OsCA1 in Arabidopsis enhances the salt tolerance compared to wild

type at seedling stage (Yu et al., 2007). The CA activity increases in the flag leaves

during first period of water stress in drought resistance cultivars but decreases during last

stage of vegetation (Guliyev et al., 2008). Transformants of maize having l0% CA

activity than the wild type showed less CO2 assimilation at ambient pressure of CO2

25

Page 46: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

(Caemmerer et al., 2004). Perez-Martin et al. (2014) studied the role of CA enzyme in the

stomatal and mesophyll conductance and found that CA enzyme had little but vital role in

stomatal conductance in 5 year old olive plant under water stress. They also observed that

CA activity was down regulated under water stress and after recovery, it was again

upregulated. Under drought stress, lower CA activity caused reduction in net

photosynthetic rate (PN) in plant. This happens to an extent that it becomes near limiting

for plant photosynthesis. Studer et al. (2014) also suggested that CA appeared to limit the

photosynthesis in C4 plants. Therefore, it would be an interesting subject to increase the

CO2 assimilation in both C3 and C4 plants by regulating the expression of CA activity.

After a complete discussion of endophytic bacteria, their capacity to tolerate the

drought stress and facilitate plant growth under non-stressed as well as stressed

conditions, it can be speculated that endophytic bacteria can be beneficial for growth

enhancement under drought stress. Carbonic anhydrase can improve the photosynthetic

rate and crop productivity. For the reason, present studies were designated to investigate

the efficient drought tolerant CA containing endophytic bacteria for enhancing

photosynthesis and growth of wheat and maize under drought conditions. Moreover,

influence of endophytic bacteria on Arabidopsis thaliana, a model plant, was also studied

under drought conditions in search of finding the possible mechanism of action.

26

Page 47: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Chapter III

MATERIALS AND METHODS

A number of bacterial endophytes were isolated from different plant parts of

wheat and maize crops and tested for their ability to tolerate PEG-6000 induced water

deficit stress. The endophytic bacteria exhibiting high drought tolerance ability were

analyzed for CA activity. These drought tolerant CA containing endophytic bacteria were

screened for plant growth promotion and colonization efficiency in both wheat and maize

under normal and stressed environment in axenic conditions. Endophytic bacterial

isolates having good capability to survive and improve growth of wheat and maize

seedlings under water deficit conditions were further tested in pot and field conditions.

Furthermore, influence of CA containing bacterial endophytes on gene expression of

Arabidopsis thaliana was also studied. Materials and methods used during the

experiments are detailed as under:

3.1. Collection of plant material

Healthy and disease free wheat and maize plants used in this study were randomly

collected during their growing season from five different locations at University of

Agriculture, Faisalabad. The plants of each crop were uprooted, placed in polyethylene

(poly) bags, kept in ice and immediately transferred to laboratory for further processing.

3.2. Isolation and preservation of endophytic bacteria

For the isolation of putative endophytic bacteria, plants from both crops were

washed thoroughly with running tap water to clear away microbes and adhering soil

particles and separated into different parts including root, shoot and leaves. These plant

parts were further cut into 2-3 cm long pieces with the sterilized blade and transferred

aseptically in Petri plates. Grains of two crops were also collected. Plant tissues were

surface-sterilized by dipping them in 70% ethanol for 30 seconds, 3% sodium

hypochlorite (NaClO) for 3 min and rinsed again four to five times with sterilized

distilled water to eliminate the hypochlorite. To confirm the surface sterility, aliquot of

sterile water used in last wash were spread on Petri plates containing Luria Bertani (LB)

and Tryptic Soy Agar (TSA) media, placed in incubator at 28ºC and observed for 3-7

days. The root, shoot, leaf and grain tissues were macerated using sterile pestle and

mortar, serially diluted in 0.85% NaCl solution to improve the disruption of cell wall.

27

Page 48: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Then, 100 µL of tissue extract was poured on two different media (LB, TSA) in triplicate

and placed in incubator at 28º C for 3-7 days to recover bacterial endophytes. After

incubation, individual bacterial colonies were picked on the basis of appearance and

streaked on media and incubated for three days. Eventually, pure cultures of bacterial

endophytes were attained by repeatedly streaking on respective media and then preserved

in glycerol stock at -80 ºC. A total of one hundred and fifty fast growing isolates from

different tissues of both crop plants were selected.

3.3. Drought tolerance ability of endophytic bacteria

One hundred and fifty bacterial isolates from each crop were tested for their

ability to tolerate water deficit stress by using different levels of PEG-6000 in LB media

(Busse and Bottomley, 1989). For this pupose, inoculum of all the isolates either obtained

from wheat or maize was prepared in 100 mL conical flask containing 50 mL sterile LB

broth and left for 3 days in shaking incubator at 28 ºC and 100 rpm. Bacterial cells from

each isolate were harvested by centrifugating the culture at 4000 × g for 15 min and

uniform cell density (0.5 OD i.e. 107- 108 cells mL-1) was maintained in LB media at 600

nm by spectrophotometer. Freshly prepared bacterial cultures (0.5 mL) were inoculated

into test tubes containing 7 mL LB broth media with different osmotic potentials and left

for 3 days at shaking incubator. All the isolates including uninoculated control were

maintained in triplicate at different osmotic potentials. Osmotic potential of -0.31, -0.61, -

1.09, -1.91 and -3.20 MPa was developed by adding different levels of polyethylene

glycol (0, 10, 20, 30 and 40%) in LB broth media measured by Cryoscopic Osmometer

(OSMOMAT-030-D, Gonotec, Germany). Furthermore, osmotic potential was measured

before and after autoclaving the LB broth media. Drought tolerance ability was measured

at 600 nm by spectrophotometer after 3 days and corresponding population was

calculated by dilution plate technique (data not shown; Wollum, 1982). Isolates

exhibiting more absorbance and higher OD were considered more drought tolerant

compared to other isolates.

3.4. Carbonic anhydrase activity of endophytic bacterial isolates

Isolates having better drought tolerance ability from both crops were further tested

for carbonic anhydrase activity by method as described by Achal and Pan (2011) and

Zhang et al. (2011) with some modifications. Bacterial isolates were cultured in 250 mL

flasks having 100 mL LB media and kept at 28 º C for 3 days. Then, these isolates were

28

Page 49: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

inoculated in 250 mL flask containing 100 mL CA producing medium and incubated in

mechanical shaker at 150 rpm and 32º C. Cell cultures of bacterial isolates were

centrifuged at 8000 × g for 10 minutes and cell pallets were suspended in 1 mL of Tris-

EDTA (pH 8.0) buffer containing 0.01 mg of RNaseI/mL, and kept at 37 ºC for 1 h.

These bacterial lysates were centrifuged and supernatant was used as CA enzyme

solution.

Carbonic anhydrase activity was measured by colorimetric method. The assay

mixture consisted of 0.8 mL tris buffer solution (pH 7.5, 50 mM), 0.1 mL enzyme

solution and 1 mL substrate solution (p-NPA dissolved in acetonitrile). The released p-

nitrophenol was determined at 400 nm by spectrophotometer using p -nitrophenol.

Distilled water was also used as a blank instead of enzyme solution. One unit enzyme

activity represented the amount of enzyme to produce 1 μmol p-nitrophenol per min.

3.5. Screening of endophytic bacteria for growth promotion of plant and drought

tolerance enhancement in growth pouch assay under controlled conditions

Among the isolates, 10 isolates with good drought tolerance and CA activity from

each crop were tested for plant growth promotion and improvement in drought tolerance

capacity in both wheat and maize, respectively under axenic conditions. Grains of two

wheat varieties viz drought tolerant (Fsd-2008, Faisalabad 2008) and drought sensitive

(Uqab-2000, Uqab 2000) were surface-sterilized with 70 % ethanol and 3.5% NaClO for

5 min followed by four to five washings with sterile distilled water. Four pre-germinated

surface-sterilized seeds were dipped in inoculum for 10 minutes and placed in sterile

(autoclaved) growth pouches already containing 10 mL autoclaved nutrient solution at

120º C for 20 minutes. Similarly, surface-sterilized seeds (as described above) of two

maize hybrids tolerant (H1, Monsanto 919) and sensitive (H2, Monsanto 6525) were

dipped in suspensions but in contrast to wheat, three pre-germinated seed of maize were

placed in each growth pouch. Selection of drought tolerant wheat cultivars and maize

hybrids was done on the basis of literature; cultivar or hybrid which showed better grain

yield under drought stress were considered tolerant and other that showed relatively less

yield was selected as sensitive. Inoculum of each isolate was prepared as described in

previous section. Sterilized broth was used for control and 3 repeats were used for each

treatment. Water deficit stress was induced by dissolving various amount of polyethylene

glycol (PEG-6000) into half strength Hoagland solution (-0.04, -1.09 and -1.23 MPa). For

ensuring sterility, nutrient solution containing PEG-6000 was autoclaved at 120º C for 20

29

Page 50: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

min. Approximately, 10 mL of nutrient solution containing different concentrations of

PEG-6000 was added into the growth pouches after 5 days of germination to maintain

water deficit stress. Suitable temperature 25±1 ºC was maintained and light and dark

period was adjusted at 10 and 14 h, respectively for wheat. On the other hand, maize

seedlings were placed at 25 ± 2 ºC and light and dark period was adjusted at a 16 h light

and 8 h dark period, repectively. Plants were collected after 21 days in wheat and 24 days

in maize and data related to root length, shoot length, fresh and dry biomass of root, fresh

and dry bimass of shoot, relative water content, relative membrane permeability,

physiological parameters were recorded. Carbonic anhydrase activity was also measured

in plants. Isolates having better drought tolerance and plant growth promotion were

recognized as efficient isolates in wheat and maize.

Three bacterial isolates from wheat (WR2, WS11, and WL19) and maize (MR17,

MS1, and MG9) were further assessed in both pot as well as in field conditions based on

drought tolerance ability and CA activity as well as growth facilitating activity for wheat

and maize sapling sunder limited water conditions.

3.6. GUS labeling of selected growth promoting bacterial isolates

Based on growth facilitation under screening trial, three potential endophytes from

each crop (wheat and maize) were selected and tagged with glucoronidase A (gusA)

marker gene that form stable insertion in variety of bacteria according to protocol as

defined by Wilson et al. (1995). Construct pCAM110 was used as delivery plasmid DNA

in which gusA gene is under the control of promoter (ptac promoter). For this purpose,

wild type isolates (wheat and maize) and E. coli (Delivery plasmid pCAM110) were

grown at 28 ± 1°C in LB medium until the OD reached to 0.6 at λ 600 nm. Endophytic

bacterial cells of each isolate were then pelleted by centrifugated at 3000 × g (10 min),

washed three times with ice cold deionized water and finally resuspended in 100 μL of

0.85% NaCl (saline buffer). Each cell suspension of 100 μL was mixed well and spread

on the respective plates and placed at 28 ± 1°C for overnight. Bacterial colonies that

exhibited the gusA marker gene were cultured on M9 medium [KH2PO4, 3g; NH4Cl, 1g;

NaCl, 0.5g; Na2HPO4.12H2O, 11g; Fe-EDTA solution, 1 mL; MgSO4, 0.24g; trace

elements solution 1 mL (Alef, 1994) containing SAC (succinate, acetate and citrate) each

at 2 g concentration being dissolved in one litre], rectified with 100 μg mL -1 of XGlcA (5-

bromo-4-chloro-3-indolyl-β-D-glucuronide), 100 μg mL-1 of IPTG (isopropyl-β-D-

galactopyranoside and 100 μg mL-1 of spectinomycin (Sigma, St. Louis, Mo.). Then, the

30

Page 51: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

bacterial isolates were inspected by using an optical microscope.

3.8. Evaluation of selected GUS labeled endophytic bacterial isolates in pot trial

Pot experiments were excuted in the glass house of Institute of Soil &

Environmental Sciences at University of Agriculture, Faisalabad to study the efficacy of

three bacterial isolates (each for wheat and maize) for growth enhancement and yield

stimulation in wheat and maize under different field capacity levels. Selection of drought

tolerant wheat cultivars and maize hybrids was done as described in section 3.5. Fresh

inoculum of each gus labeled isolates (Wheat: WR2, WS11, and WL19 and Maize:

MR17, MS1, and MG9) was prepared in 250 mL LB broth possessing 100 µg mL -1

spectinomycin and placed at 28 ± 1˚C in mechanical shaker at 100 rpm for 48 hours. Cells

were collected by centrifugated at 4 ˚C and 4000 × g for 15 min and OD of the culture was

measured by using the OD meter and adjusted to 106-8 CFU mL-1 to obtain uniform

population for inoculation by adding the harvested cell in sterilized LB medium. Seeds of

both wheat cultivars (Fsd, Faisalabad-2008; Uqab, Uqaab-2000) and maize hybrids (H1,

Monsanto 919; H2, Monsanto 6525) were soaked in 10% sterilized sugar solution and

inoculated with the mixture of inoculum, peat and clay in ratio of 1:6:2. Control (without

bacterial inoculation) treatment was also maintained by inoculating seeds of both crops

with mixture of sterilized broth culure, clay, peat and sugar solution. After inoculation,

seeds were dried in shade for 6-8 hour and were sown at 5 seeds per pot. Seventy two

pots were aligned in accordance to completely randomized design under factorial setting

filled with 10 kg of air- dried, well ground and sieved soil that was collected from farm of

Institute of Soil & Environmental Sciences and tested for physico-chemical attributes.

Plants were thinned to three plants for wheat and one plant for maize after two weeks of

germination and allowed to establish before the start of water deficit stress such as well

watered (100% field capcity: FC), moderately stressed (70% FC) and severely stressed

(40% FC) plants. In both crops, plants were grown at different water holding capacities

by weight. Plants were irrigated with tap water as per moisture requirement. Two wheat

cultivars were applied with recommended dose of NPK as 120, 90 and 60 kg ha -1, and

maize hybrids were applied with 160-100-60 kg ha-1 using urea, diammonium phosphate

(DAP) and murate of potash (MOP) fertilizers, respectively. Full doses of K and P were

applied as basal but N was applied in 3 equal splits (before sowing, vegetated and

reproductive stage of the crop). All the treatments were replicated with 3 repeates. After

inducing stress, photosynthesis system was measured using CIRAS. Colonization by

31

Page 52: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

endophytic bacteria was also checked in different plant parts by dilution plate technique

in both crops. Green leaves were collected from all the treatments to measure carbonic

anhydrase activity, antioxidants, relative water contents (RWC) and electrolyte leakge

(EL). The plants were allowed to grow till maturity, however, growth and yield aatributes

were noted at harvesting.

3.8. Evaluation of selected endophytic bacteria under skipped irrigation system

Potential bacterial isolates were also tested in the field trials at the experimental

site of the Institute of Soil & Environmental Sciences, University of Agriculture,

Faisalabad. Soil samples were analyzed for physico-chemical characteristics (Table 3.1)

before sowing. Wheat (Uqab-2000) and maize (Monsanto 6525) seeds were treated in the

similar pattern as mentioned in pot study. Recommended dose of NPK (120-90-60 kg ha -

1) for wheat and (160-100-60 kg ha-1) maize were applied, respectively, using fertilizers

urea, DAP and MOP, respectively. Nitrogen was supplied in 3 equal splits (1/3 as basal,

1/3 at vegetative and 1/3 at reproductive stage of crop) whereas P and K were applied as

basal dose. Trials were laid down following the randomized complete block design

(RCBD) under factorial arrangements with three replicates. In wheat, irrigaion was done

with water passing through canal and skipped at tillering, flowering and grain filling

stages of crop as per treatment plan whereas control was sustained with recommended

(four) irrigations. In case of maize, drought stress was imposed at vegetative and

reproductive stage whereas a treatment receiving recommended irrigations was also

maintained. Green leaves were collected from all the treatments to measure the carbonic

anhydrase and antioxidants. After inducing stress, photosynthesis system was measured

using CIRAS. The plants were allowed to grow till maturity, however, growth and yield

attributes were noted at harvesting. Morevover, grains of both crops were analyzed for

nutrient contents (NPK).

3.9. Physico-chemical properties of soil

Soil used in pot and field experiment of both crops was analyzed according to

protocols given below.

3.9.1. Textural analysis

Soil texture was exmined using method defined by Moodie et al. (1959). For this

purpose, 40 g soil was taken into plastic bottles (600 mL) and 40 mL of dispersing agent

(2% sodium hexametaphosphate) was incorporated and left for overnight. After that, soil

32

Page 53: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

suspension was mixed with mechanical stirrer and gentally transferred into 1 L

cylinder. Then, brought to volume using deionized water and stirred the suspension with

plunger. The readings were recorded with Bouyoucos hydrometer. Textural class was

assigned using the USDA textural class calculator (USDA, 2011).

3.9.2. Saturation percentage (SP)

For the determination of soil saturated paste, air-dried soil (200 g) was slowly

saturated using distilled water and weighed in a tared china dish. After that, paste was

dried in an oven at 105 ˚C and re-weighed. Saturation percentage (SP) was obtained by

putting values in the following formula (Method 27a, U.S. Salinity Lab. Staff, 1954).

SP(%)=Weight of saturated soil−Weight of oven dried soilWeight of ovendried soil

×100

3.9.3. pH of the saturated soil paste (pHs)

pH of soil saturated paste was examined after preparing paste using pH meter

(Kent Eil 7015, England) according to Method 21a, U.S. Salinity Lab. Staff (1954).

3.9.4. Electrical conductivity of soil extract (ECe)

Water from saturated paste was obtained using vacuum pump through ceramic

filter. Electrical conductivity (EC) of the saturated paste extract was measured by

conductivity meter (Jenway Conductivity Meter Model 4070) following Method 3a and

4b, U.S. Salinity Lab. Staff, 1954.

3.9.5. Organic matter content

Soil organic matter was quantified as per method given by Moodie et al. (1959).

One gram (1g) soil was well blended with 10 mL of IN potassium dichromate (K2Cr2O7)

solution and then 20 mL of concentrated sulphuric acid (H2SO4) was added in 500 mL

flask, agitated and left for 30 min. After that, about 150 mL of distilled water and 25 mL

of 0.5 N FeSO4 (ferrous sulphate) were added. Then, titrated against 0.1 N solution of

KMnO4 till end point. Blank (without soil) was also prepared using same procedure.

3.9.6. Total nitrogen

Soil total nitrogen was measured following the Gunning and Hibbard’s method

using conc. H2SO4 where ammonia was collected into 4% boric acid during distillation

with Kjeldhal’s apparatus. Then, receiver flask was titrated with 0.01 N solution of H2SO4

(Jackson, 1962).

33

Page 54: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 3.1. Phyico-chemical characteristics of the soils used for wheat and maize trials

Characteristics Unit Value

Pots Field

(Wheat)

Field

(Maize)

Textural Class Sandy clay loam Sandy clay loam Sandy clay loam

Saturation percentage % 35 38 37

pHs -- 7.2 7.9 7.4

ECe dS m-1 2.15 2.20 2.19

Organic matter % 0.87 0.77 0.74

Total nitrogen % 0.03 0.05 0.05

Available phosphorus (P) mg kg-1 6.8 7.9 7.4

Extractable potassium mg kg-1 120 115 113

34

Page 55: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

3.9.7. Available phosphorus

Extract of five gram (5 g) air-dried soil was taken using 100 mL 0.5 M sodium

bicarbonate solution (pH 8.5) and filtered with Whatman No. 42 filter paper. Afterwards,

filtrate was taken in 250 mL conical flask. Five milliliter (5 mL) of filtrate was mixed

with 5 mL of color developing reagent (ascorbic acid) and volume (100 mL) was brought

with distilled water. Absorbance was measured at 880 nm wavelength using

spectrophotometer (ANA-720W, Tokyo Photo-electric Company Limited, Japan) while P

concentration was calculated from caliberation curve (Watanabe and Olsen, 1965).

3.9.8. Extractable potassium

Soil extraction was done with 1 N ammonium acetate (pH 7.0) and K was

determined with Jenway PFP-7 flame photometer (England). However, K concentration

was calculated by using caliberation curve (Method 1la, Salinity Lab. staff, 1954).

3.10. Plant analysis

3.10.1. Chemical analysis

Grain samples were ground and wet digested to measure nutrient (N, K and P)

contents as follow.

3.10.2. Digestion

Dried and ground sample (0.1g) was transferred quantitavely in 25 mL conical

flask. Two millimeter of concentrated H2SO4 was added and allowed to stand at room

temperature for overnight. Then, 1 mL of 35% hydrogen peroxide (H2O2) was poured in

flask and kept at 350 ˚C for 20 min on hot plate. Afterwards, flasks were removed, 1 mL

H2O2 was slowly poured and were kept on hot plate for 20 min. This procedure was

carried out until the digestate became colourless. Then, filtered this colourless material

and diluted with distilled water in volumetric flask (50 mL), brought to volume and

preserved for macronutrients estimation (Wolf, 1982).

3.10.3. Nitrogen determination

Nitrogen estimation was done by placing 5 mL of digested solution in digestion

tube on Kjeldhal distillation apparatus. Briefly, 10 mL of NaOH (40%) poured in tube

and started the distillation. After that, distillate was collected in 100 mL receiver flask

containing H3BO3 and stopped the distillation when volume was reached to 50 mL. Then,

35

Page 56: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

removed the receiver flask from apparatus and allowed to cool for few min. At the end,

distillate was treated against 0.01 N standardized H2SO4 till pink color end point.

3.10.4. Phosphorus determination

Five milliliter of sample aliquot was added in Barton’s reagent (10 mL) and

volume was brought to 50 mL using distilled water. These samples were allowed to stand

for 30 min and measured the absorbance on ANA-720W spectrophotometer (Tokyo

Photo-electric Company Limited, Japan) at 410 nm wavelength. Actual P concentration

was calculated by plotting standard curve.

a). Barton reagent

The Barton reagent was processed by mixing the Reagent A and B following the

Ashraf et al. (1992).

Solution A

This solution was made by mixing 25 g ammonium molybdate in distill water

(400 mL).

Solution B

For this, 1.25 g of ammonium metavenadate was added in 300 mL of boiling

water and about 250 mL concentrated HNO3 was added after cooling. Both solutions (A

and B) were well mixed in volumetric flask and brought to 1L with distilled water.

3.10.5. Potassium determination

For K content, fed the sample aliquote to Jenway PFP-7 flame photometer

(England) and its content was measured using a standard curve developed by different

known cocenctation of potash.

3.10.6. Leaf relative water contents (RWC)

The relative leaf water content (RWC) was calculated with the following formula

suggested by Teulat et al., (2003).

RWC= (Fresh weight−Dry weight )(Fully turgid weight−Dry weight)

Three leaf samples were collected early in the morning (8:00 to 10:00 a.m) from

each treatment to determine the relative water content. Fully turgid weight was attained

by placing them at 4 ºC for 24 h and dry weight was noted after oven dried.

36

Page 57: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

3.10.7. Electrolyte leakage

Freshly cut leaf discs were rinsed with deionized water and transferred into test

tube having 5 mL deionized water. Then, tubes were placed on shaker for 4 h at room

temperature. The electrolyte leakage, electrical conductivity was measured with

conductivity meter (Jenway Conductivity Meter Model 4070, England). Then, tubes were

placed in autoclave at 121 ºC and 15 psi for 20 min. After cooling these tubes, electrical

conductivity was recorded and leakage of ions from leaves was calculated following the

formula (Jambunathan, 2010).

%EL= EC before autoclavingEC after autoclaving

× 100

3.10.8. Chlorophyll content

Chlorophyll content in leaves was recorded with the help of SPAD-502 meter

(Konica-Minolta, Japan). Measurements were taken at three points of each leaf. Three

readings were averaged to provide single reading. Readings were taken from all the

repeats of each treatment.

3.10.9. Gaseous exchange parameters

Gases exchange parameters of flag leaves were recorded using portable

photosynthesis systems CIRAS-3, during 9:00 to 12:00 a.m in both pot and field

experiments of wheat and maize at photosynthetic photon flux density of 1200-1400 μmol

m-2 s-1. Fully expanded flag leaf was used to measure CO2 assimilation rate (A), stomatal

conductance (gs), substomatal CO2 conductance (Ci), transpiration rate (E), and water use

efficiency (WUE). Readings were noted from all the repeatsof each treatment.

3.10.10. Carbonic anhydrase activity

The CA activity was measured according to method defined by Dwivedi and

Randhawa (1974). Briefly, leaf samples obtained from wheat and maize were chpped into

small fragments and 200 mg samples were dipped in cystein hydrochloride solution and

incubated for 20 min at 4˚C. After that, leaf samples were blotted and transferred to test

tube containing phosphate buffer (pH 6.8) followed by alkaline bicarbonate and

bromothymol indicator. These test tubes were incubated at 5˚C for 20 min and titrated

against HCl using methyl red as indicator. Results are expressed as mol (CO2) kg−1 (F.M.)

s−1.

37

Page 58: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

3.10.11. Proline content

For the quantification of proline contents, 1 g of leaf tissues was ground with

sulphosalycylic acid (3%) and clarified with filter paper (Whatman No.2). Filtrate was

blended with glacial acetic acid and acid ninhydrin and heated for 1 h in water bath at 100

ºC. After 1 h of heating, reaction was allowed to stopp on an ice bath. Then, extract was

taken with toluene and its absorbance was recorded on spectrophotometer at about 520

nm wavelength. Afterwards, proline content (µg g-1) was calculated using caliberation

curve (Bates et al., 1973).

3.10.12. Total protein content

Protein content in leaves of both crops was measured as method defined by

Bardford (1976). For total protein contents, 200 µL extract of fresh leaves was transferred

into test tube and mixed with 1800 µL deionized water. Then, added 2 mL Bardford

reagent and placed for 10-20 min at room temperature. After that, absorbance was

computed by ANA-720W spectrophotometer, made by Tokyo Photo-electric Company

Limited, Japan, at 595 nm wavelength. Protein content (µg g-1 fresh weight) in leaves was

determined from caliberation curve derived form BSA (bovine serum albumin).

3.10.13. Malondialdehyde content

The lipid peroxidation/ malondialdehyde (MDA) was checked by calculating the

quantity of MDA produced through the reaction of thiobarbituric acid (TBA) as

mentioned by Jambunathan (2010). The reaction mixture (2.5 mL), containing 0.5 mL

leaf extract, 0.5% thiobarbituric acid (TBA) and 20% trichloroacetic acid (TCA), was

heated in fume hood for 30 min at 95 ºC and quickly allowed to cool on ice bath. After

that, absorbance was recorded on 532 and 600 nm. Then, MDA contents (µg g-1 fresh

weight) were figured out by measuring difference in absorbances (A532-A600) following

equation as described by Beer and Lambert’s.

3.10.14. Catalase in leaves

Catalase activity was performed by measuring the disintegration of H2O2 at 240

nm. The reaction mixture consisted of 2 mL extract which was diluted 200 times using 50

mM solution of potassium phosphate buffer having pH 7.0 and 1 mL 10 mM H2O2.

Decline in absorbance was observed due to H2O2 extinction at 240 nm. The activity was

described as mM H2O2 min-1 mg-1 protein (Cakmak and Marschner, 1992) at 25 ± 2 °C.

38

Page 59: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

3.10.15. Glutathione reductase in leaves

Leaves of both plants grown under normal as well as drought condtions were

homogenized in 50 mM potassium phosphate buffer having pH 7.8 along with 2 mM

EDTA. Then, homogenate was centrifuged and supernatant was used for assay. Three

millimeter of reaction mixture contained 30 µL of sample extract, 0.75 mM 5, 5´-dithiobis

2-nitrobenzoic acid (DTNB), 1 mM oxidized glutathione (GSSG) and 0.1 mM

Nicotinamide adenine dinucleotide phosphate NADPH. Glutathione reductase was

determined by measuring rise in absorbance due to reduction of DTNB to TNB. The

activity of GR was described in µM TNB min-1 mg-1 protein (Smith et al., 1988) at 25 ± 2

°C.

3.10.16. Ascorbate peroxidase in leaves

Ascorbate peroxidase (APX) activity was figured out by subjecting the samples to

spectrophotometer at 290 nm wavelength (Nakano and Asada, 1981). The reaction

mixture consisted of 20 µL plant extract, 660 µL H2O2, 660 µL 50 mM potassium

phosphate buffer having pH 7.0, and 0.5 mM 660 µL ascorbic acid solution. However,

H2O2 was poured at the end to initate the reaction because the reaction wasstarted after the

addition of H2O2. Decline in absorbance was measured for three min. The enzyme activity

APX (mM Ascorbate min-1 mg-1 protein) was described at 25 ± 2 °C.

3.10.17. Total phenolics in leaves

Total phenolics were estimated calorimetrically using the method defined by

Singleton et al. (1999). The reaction mixture (2 mL) was prepared by transferring 20 µL

sample extract, 1580 µL distilled water, 300 µL 1 N Na2CO3 and 100 µL 0.25 N Folin

ciocalteu’s reagent and left in dark for h at room temperature. In Folin-Ciocalteau’s

reagent, phenol reacts with phosphomolybdic acid and formed a blue coloured

complex.Then; absorbance of above stated reaction mixture was measured on

spectrophotometer at 760 nm Total phenolics were expressed in µg g-1.

3.10.18. Total soluble sugars in leaves

To measure the total soluble sugars, anthrone colorimetric method was used as

described by Sadasivam and Manickam, (1992). For this, 200 µL of sample was obtained

from leaf extract and mixed with 1800 µL deionized water followed by 8 mL anthrone

reagent. This solution was heated in hot water for 8 min and afterwards cooled on ice

bath. The absorbance was recorded at 630 nm on spectrophotometer. Amount of soluble

39

Page 60: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

sugars was determined from caliberation curve developed by glucose solutions (Hedge

and Hofreiter, 1962) and expressed in terms of µg g-1.

3.10.19. Colonization of plant tissues

Colonization by endophytic bacteria in root, shoot and leaves were studied by

using method described by Naveed et al. (2014a). For isolation of endophytic bacteria, 3

g of root, 5 g of shoot and leaves from each treatment were surface-sterilized and

macerated in 15 mL saline buffer (0.85% NaCl) solution with autoclaved mortar and

pestle. Suspension was mixed well on rotary shaker for 1 min and then allowed to settle

down at room temperature. Afterwards, serial dilutions were made upto 10 -4 in case of

root and 10-3 in case of shoot and leaves and spread on LB media plates having 100 g mL-

1 spectinomycin, 100 g mL-1 XGlcA, and 100 g mL-1 IPTG as mentioned by Afzal et al.

(2012). Plates were placed at 28 ± 1˚C in an incubator for 48 h and transferred to 4˚C for

three days and measured the blue colonies with colony counter.

3.11. Characterization and identification of selected endophytic bacteria

Selected drought tolerant and growth promoting carbonic anhydrase containing

endophytic bacterial isolates were studied for particular plant growth facilitating

charcteristics. Their PGP activities were studied using the standard protocol as discussed

below:

3.11.1. Indole 3-acetic acid production under normal and stressed environment

To determine the quantity of IAA (indole 3-acetic acid, an auxin) produced by

endophytic bacterial isolates with L-TRP and without L-TRP under non-stressed and

water-deficit stressed conditions, a spectrophotometric technique was followed using

procedure demonstrated by Sarwar et al. (1992). For this, fresh cultures of selected

isolates were prepared in conical flasks having LB media and placed at 28 ± 1 °C in

mechanical shaker (100 rpm) for 72 h. After incubation, broth culture of isolate was

centrifuged at 4 °C for 15 min and 4000 × g, and uniform OD 0.5 was developed in

sterilized distilled water. Then, 20 mL sterilized LB broth was shifted in 100 mL conical

flasks followed by 5 mL of 5% filter-sterilized (0.2 µm membrane filter) L-TRP solution

to achieve the 1 g L-1 solution concentration. For drought stress, 15% PEG-6000 was

added into LB medium coupled with and without substate (L-TRP). Flasks containing LB

broth media were inoculated with 1 mL of inoculum in the presence and absence of

substrate (L-TRP) under normal (non- stressed) and stressed conditions and placed at 28

40

Page 61: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

± 1 ºC in mechanical shaker for 2 days at 100 rpm. Uninoculated control was also

maintained for comparison. Then, culture was filtered using filter paper. Afterwards, 2

mL of freshly prepared Salkowski reagent as colour developing reagent (CDR) was

mixed with 3 mL of culture filterate. After adding CDR, tubes were allowed to stand for

30 min at room temperature. Standard IAA solutions were prepared and colour was

induced using salkowski reagent. The reading was recorded on spectrophotometer using

absorbance of 535 nm and IAA concentration was attained from caliberation curve.

3.11.2 Phosphate solubilization

3.11.2.1 Phosphate solubilization (plate assay)

Bacterial strains isolated from both wheat and maize were studied for their

phosphate (inorganic phosphate) solubilization capacity by growing them on NBRI-PBB

(National Botanical Research Institute Phosphate Bromophenol Blue) medium (Mehta

and Nautiyal, 2001). Loopful quantity of each actively growing bacterial strain cultures

(0.5 OD) were poured at four different sites of Petri plates. All the selected endophytic

bacteria were inoculated on three Petri plates and placed in an incubator for 7 days at 28 ±

1 ºC. After 7 day, isolates showing halo zone around their colonies were able to solubilize

the inorganic phosphate and proved to be efficient P solubilizers.

3.11.2.2. Phosphate solubilization under normal and stressed condition

Endophytic bacteria were also tested for quantification of P solubilization ability

under normal (non-stressed) as well as PEG-6000 induced stressed conditions. For this

purpose, fresh cultures of selected isolates were prepared in flasks having LB media and

placed at 28 ± 1°C in orbital shaker (100 rpm) for 72 h. After incubation, broth culture

was centrifuged using centrifuge machine for 15 min at 4 °C and 4000 × g and uniform

OD 0.5 was maintained in sterilized distilled water. After that, loopful of actively

growing isolates was inoculated in 100 mL conical flasks carrying 50 mL of NBRI-PBB

media both with without 15% PEG-6000 and placed at 30 ± 2 °C on mechanical shaker

for 7 days at 150 rpm. After 7 days incubation, cell culture was centrifuged using

centrifuged machine at 5000 rpm for 10 min to get cell free supernatant of bacterial

isolates. Then, 1 mL of cell free supernatant was blended with 4 mL Reagent B and 20

mL of water and allowed for color development. After 20 min, OD was measured on

spectrophotometer at 882 nm. Blank samples were also run. Soluble P was measured by

following the standard curve of KH2PO4.

41

Page 62: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Picture 1: Phosphorus solubilization by endophytic bacterial isolates

42

Page 63: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

3.11.3. Siderophore production

Bacterial culture being assayed for production of siderophore was spotted on Petri

plates having sterilized CAS (chrom azurol S) agar media. Ten microliters of each

inoculum was spot inoculated on plates with 3 repeats and placed at t 28 ± 1 ºC in an

incubator a for 48-72 h. Similarly, control plates were also processed by spot inoculation

of sterilized broth. Positive results for siderophore were observed by formation of orange

yellow zone along the growth (Schwyn and Neilands, 1997).

3.11.4. Exopolysaccharide (EPS) production

Bacterial inoculants were prepared in 10 mL of customized LB medium

inoculated with 100 µL of culture and placed at shaking incubator. After incubation at 28

± 1 ºC and 100 rpm for 72h, they were adjusted to OD 0.5, loopful of grown cultures was

plated at different locations on Petri plates carrying RCV-glucose media proposed by

Ashraf et al. 2004 and incubated for 48 h at 28 ± 2oC. At the end of incubation, colonies

which had mycoid growth were examined by visual appearance. Mucoidy growth showed

that isolates were considered positive for exopolysaccharides (EPS) production.

3.11.5. Chitinase activity

The potential of these bacteria for chitolytic activity was assayed by growing them

on colloidal chitin LB agar medium (Chernin et al., 1998). For this, loopful of culture of

each isolate, adjusted to 0.5 OD, was poured on four sites of chitin agar Petri plates and

kept for 96 h in an incubator at 28 ± 1 ºC . Plates were observed for zone of clearance

around the inoculated area. Colonies showing the large halo zone around them were

considered as potent chitinase producer.

3.11.6. Catalase activity

Catalase activity in selected bacterial isolates was determined using method

demonstrated by MacFaddin (1980). For this, loopful quantity of bacterial culture to be

tested was poured on slide (microscopic glass slide), afterthat, a drop of H2O2 (35%) was

placed on the bacterial culture with the help of dropper. Bubbles production, on reaction

of H2O2 (35%) addition, was a signal of existence of catalase activity in selected

endophytic bacteria (Diane Hartman, Baylor University, Waco, TX).

3.11.7. Oxidase activity

Isolates to be tested for oxidase activity were grown in LB agar plates at 28 ± 1 ºC

43

Page 64: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Picture 2: Exopolysaachride production by endophytic bacterial isolates

44

Page 65: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

for 24 h. After incubation, a portion of overnight grown colony with inoculation loop was

picked and mashed on filter papers treated with Kovacs reagent (soaked in 1% reagent

and dried). Filter paper was observed for colour change to purple. Change in colour, in a

period of 90 s, from blue to purple was considered positive for oxidase (Steel, 1961).

3.11.8. Organic acid production

Bacterial isolates were tested for the production of oraganic acid as described by

Vincent (1970). For this, freshly prepared bacterial culture was streaked on LB agar

plates possessing blue dye (0.025%) and incubated at 28 ± 1 ºC for 72. At the end of

incubation, media color change to yellow was considered positive for organic acid.

3.11.9. Microbial aggregation ability

The ability of bacteria to aggregate was assessed according to method

demonstrated by Madi and Henis (1989) with certain modifications. The selected isolates

were cultured separately in LB medium at 28ºC for 24 h. The freshly grown cultures were

poured into test tubes and left at room temperature for 20 min. Aggregates were settled

down at the bottom of test tubes and free bacterial cells were present in suspension.

Afterthat, turbidity (cloudiness) of aliquot was recorded (OD1) at 540 nm by

spectrophotometer. Then, vortexed the cell suspensions for 1 min and measured the

turbidity (OD2). The percent aggregate of isolates was expressed as

% aggregation=OD 2−OD 1OD 2

× 100

3.11.10. Survival under starved condition

Ability of isolates to survive under starved conditions was assayed following Tal

and Okon (1985) with few modifications. Briefly, after three days incubation at 100 g and

28 ± 1 ºC, selected bacterial cells were collected by centrifugating at 4000 × g using

centrifuge machine and then suspended in LB broth medium without carbon source

(Trypton). Inoculated culture was again incubated for 13 days at 100 × g and 28 ± 1 ºC

in orbital shaking incubator. Bacterial population (CFU mL-1) was counted with the help

of colony counter using the serial dilution method.

3.11.11. Survival of bacterial inocula in soil

Survival of inoculants was studied using protocol of Fallik and okon (1996) with

some modifications. Ten gram of previously sterilized soil was inoculated with isolates in

45

Page 66: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Picture 3: Catalase production by endophytic bacteria

Picture 4: Oxidase production by endophytic bacteria

46

Page 67: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

test tubes and placed at 28 ± 1 ºC for 30 days. After that, (0.9%) saline water was added

in soil and shaken well for 2 h. Viability of endophytic bacteria was determined through

serial dilution method and given as CFU g-1 of soil.

3.11.12. Cellulase activity

Cellulase activity of endophytic bacterial isolates was also determined by

growing the isolates on plates containing 1% carboxymethyl cellulose. After 2 days

incubation, plates were filled with Gram's iodine staining solution. Plates were washed

with NaCl (1 M) to confirm the cellulose degrading ability and to clarify the halo zone

around bacterial colonies (modified from Yin et al., 2010).

3.11.13 Xylanase activity

Ability to isolate to degrade xylan was determined as described by Roy and Habib

(2009). Isolates were spread on plates containing 0.5% xylan and incubated for 2 days.

After incubation, bacterial colonies producing halo zone were considered positive for

xylanase activity.

3.11.14. Protease activity

For confirmation of protease activity, bacterial isolates were streaked on 10%

gelatin containing agar plates. Plates were incubated for 24 h. Halo zone formed around

bacterial colonies was used as indicator of protease activity (Josephine et al., 2012).

3.11.15. Identification of selected isolates

Sequencing of bacterial isolates (WS7, WS11, WL19, MR17, MS1, MG9, AR4

and AR14) was performed by Macrogen Inc. (Korea). These isolates were identified by

16S r RNA gene sequencing. For phylogenetic tree, the partial sequences of nucleotides

of these isolates were analyzed for sequence smililarity using Basic Local Alignment

Search Tool (BLAST) program on National Center for Biotechnology Information

(NCBI) site and identified on the basis of closest homology. Then, aligned the sequences

using ClustalW alignment and subjected to phylogeny analysis using MEGA 5 software.

3.12. Influence of endophytic bacteria on gene expression in Arabidopsis thaliana

under drought stress

3.12.1. Sample collection and isolation of endophytic bacteria from arabidopsis

Healthy plant samples were collected from already cultivated Arabidopsis plants

47

Page 68: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

in three different soils in the controlled environment of growth chamber of Plant Microbe

Interaction Laboratory, The University of Queensland, Australia. Plant samples were

obtained and cleaned with running tap water. Roots and leaves were separated, surface

sterilized with ethanol and 1 % NaClO for 3 min and afterwards, washed four to five

times with sterile distill water. To check the sterilization efficacy, aliquots from last

washing was poured on LB medium containing Petri plates. These samples were

pulverized in pestle and mortar. Different dilutions were made and spread on plates and

then preserved as described in earlier section (3.2)

3.12.2. Screening of endophytic bacteria for stress tolerance and carbonic anhydrase

activity

All the isolates were screened for the sensitivity of isolates against polyethylene

glycol (PEG-6000). Media (LB broth) with different concentration of PEG were prepared

and inoculated with overnight grown culture. The cultures were placed for 24 h at shaking

incubator at 180 rpm and 28 ˚C and growth was calculated by reading absorbance at 600

nm. All the drought tolerant endophytic bacteria were also screened for carbonic

anhydrase activity as described in earlier section (3.4.).

3.12.3. Screening of selected bacterial isolates for plant growth promotion under

PEG induced water deficit stress

Ten drought tolerant endophytic isolates having higher carbonic anhydrase were

further screened under gnotobiotic conditions to improve Arabidopsis thaliana biomass

under normal and PEG indued water deficit stress conditions in pouch trial. Seed of

arabidopsis ecotype Columbia were sterilized by dipping in ethanol (70%) for 5 min and

50% bleach for 2 min followed by 5 times rinsing with sterile distilled water. Afterthat

seeds were spread on Petri plates having half strength Murashige and Skoog (MS) media

and then placed for 2 days at 4˚C for scarification. After scarification, those Petri plates

were transferred to growth room and kept at 22°C for 10 h of light and 14 h dark for 2

weeks. Inoculum of each isolate was prepared as explained in previous section (3.3).

Sterilized broth was used for control and 3 repeats were used for each treatment. Root of

arabidposis plants were dipped in respective inocula and placed in growth pouch already

containing sterile half strength Hoagland solution. After two weeks, plants were exposed

to water deficit stress induced by various amount of PEG-6000 (polyethylene glycol) i.e,

0, 3 and 5% into half strength Hoagland solution. Then, 10 mL of PEG-6000 solution was

48

Page 69: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Picture 5: Screening of drought tolerant CA containing endophytic bacterial isolates for plant growth promotion in Arabidopsis thaliana under axenic conditions

49

AR14Control

Page 70: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

added into the growth pouch. Suitable temperature (22º C) was maintained and light and

dark period was adjusted at 10 and 14 h, respectively, in growth chamber. Plants were

harvested after one week exposure to drought stress and data related to root and shoot

length, root fresh and dry biomass, shoot fresh and dry weight were recorded

3.12.4. Effect of selected isolates on gene expression in Arabidopsis thaliana under

PEG induced water deficit stress

Arabidopsis seeds (ecotype Columbia) were collected from Plant Microbe

Interaction Laboratory, The University of Queensland, Australia. Seeds were surface-

sterilized, placed on MS media plates as described in section 3.12.3 and scarified for 2

days at 4˚C. Then, plates were placed vertically for 2 weeks in growth room at 10 h light

and 14 h dark. Two isolates (AR1 and AR14) were used. Inoculum of selected isolate was

prepared as explained in earlier section (3.3). Sterilized broth was used for control and 3

repeats were used for each treatment with two technical repeats. After 2 weeks, roots of

arabidposis plants were dipped in respective inocula and spread in Petri plates containing

half strength MS medium without sucrose with 0 and 3 % PEG-6000 for 10 days. Plant

samples were harvested and number of lateral roots, root length, and root and plant fresh

biomass were measured.

3.12.5 RNA extraction

For RNA extraction, arabidopsis leaves were cut and immediately placed in liquid

nitrogen. Leaf samples from both PEG induced water defcit stressed and non-stressed

plants were mashed in liquid nitrogen. However, total RNA from leaf tissues was

obtained using SV Total RNA Isolation System (Promega, Madison, USA) following the

instruction given by manufacturer. The integrity and quality of RNA as well as lack of

genomic DNA was confirmed with agarose gel electrophoresis and using NanoDrop

2000D (NanoDrop Technologies,Wilmington, DE, USA).

3.12.6. Preparation of cDNA and primers sequence

First strand of complementary DNA was synthesized from all the treatments using

kit namely Invitrogen, USA, SuperScript III Reverse Transcriptase kit according to the

instruction described by manufacturer and stored at -20˚C. Most of primer sequences used

in this study was synthesized using Primer express 3 software whereas some primers were

collected from Plant Microbe Interaction Laboratory, The University of Queensland. The

sequences of primers which were used for RT-PCR (real time polymerase chain reaction)

50

Page 71: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

are given in the table (3.2.).

3.12.7. Expression profiling through Real Time PCR

Transcripts levels of arabidopsis gene were determined with RT PCR on sequence

detection system, ABI Prism 7900 (Applied Biosystems), with SYBR Green PCR Master

Mix following the instructions defined by manufacturer i.e. 1µL primer, 5 µL SYBR

Green PCR and 4 µL cDNA. Two biological repeats for all the treatments were prepared.

Actin, internal reference gene, was used for measureing the relative transcript level in

analysis. The level of these internal reference genes did not change during the PEG

induced water deficit stress. Specificity of RT PCR was assured by the presence of single

peak of amplified products in melting curve during analysis of RT PCR and single band

on agarose gel. PCR efficiency of arabidopsis primer pairs was investigated using

programme LinReg as described by Ramakers et al. (2003). Moreover, relative

quantification of each treatment was determined following the method described by Pfaffl

(2001). Relative mRNA value in the given sample was determined using the mean of two

values against reference gene. For gene expression; ratio is expressed in relative

expression level compared with actin internal standard.

3.13. Statistical analysis

Various statistical tools were followed to interpret the data (Steel et al., 1997) and

Duncan’s Multiple Range Tests was used for comparing means (Duncan, 1955).

51

Page 72: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

52

Table 3.2. Primer sequences for arabidopsis used in RT-PCR

Gene Category Primer Name 5’-3’ Designed by

Actin rt_ActinUni_Frt_ Actin 2_Rrt_ Actin 7_Rrt_ Actin 8_R

AGTGGTCGTACAACCGGTATTGTGATGGCATGGAGGAAGAGAGAAACGAGGAAGAGCATTCCCCTCGTAGAGGATAGCATGTGGAACTGAGAA

Bob

AtERF7(AP2/EREBP-type Transporter related to ABA)

At-ERF7_FAt-ERF7_R

CTTCTCCATGAGGAAAGGGAGAGTCTATACCTCGGCTCCTTCACAG

This work

Late embryogenesis abundant protein (LEA) At-LEA-FAt-LEA-R

GCACCGTTGGAGAAACACATCTTGCTCCAGTACCAGCAGAGAC

This work

Putative DRE- binding protein (DREB2A) At_DERB2A_FAt_DERB2A_R

GGAGTGGAGCCGATGTATTGTCCTCGCTCAGCCAATGCTTATC

This work

At_ERF13_FAt_ERF13_R

CAGTTAACGTCGGAGCAGAAGAGGATCCACCGTGAAATCCAACTC

This work

WRKY At-WRKY8_FAt-WRKY8_R

GAAGTTGTCGGTGATGGTTGTGGATGATGATCGGCCTCACTTG

This work

At-WRKY57_FAt-WRKY57_R

CCCTCAGCTACCTCAAGTTCAAGGAGCCTTCTTCTTCTCCTTCACTG

This work

RAB 18 ABA responsive gene At-RAB18-FAt-RAB18_R

AGCTCTAGCTCGGAGGATGATGCATGATGACCTGGCAACTTCTC

This work

At_LTI78_FAt_LTI78_R

GAATCGCCACATTCTGTTGAAGACAGTGGAGCCAAGTGATTGTG

This work

PR1 At_PR-1_FAt_PR-1_R

GTGGCGTGACTCGGTTCGTGTAATTCCCCGGAGGAT

This work

Gene Category Primer Name 5’-3’ Designed by

MYB 15 (MYB domain protein 15) DNA binding and transcription factor

MYB_FMYB_R

CCTGATTGTGTTTCCAAGAAGATTGCCAGCCACTTCTAGGTCATTCG

Nasser

RD22 (Responsive to dehydration) RD22_FRD22_F

ATTGTGCGACGTCTTTGGAGTTGCGTTCTTCTTAGCCACCTC

Lilia

799 Putative cold acclimation protein (dehydrin 10)

ERD10799 and 799 Homolog

AGCTCTTCTTCCTCTTCGAGTGATGCCACTGTTTTCACATGATCTCCTTC

Peer

Cor 47Similar to cold-regulated protein cor47

rt_COR47_Brt_COR47_A

CCACTAGTCCTTTCTTATCTTCCTCTCCCTTCTTCCTCTTCGAGCGATGA

Peer

RD29b(Rreposnive to dessication)

RD29B_FRD29B_R

ATGGAGTCACAGTTGACACGTCCTCTTCTGGGTCTTGCTCGTCATACT

This work

Zat-10 ZAT10_FZAT10_R

GTGTCCAACTCCGAAGGTGCCCATCGAGAATTCAGGGATCG

This work

Page 73: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

53

Page 74: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Chapter IV

RESULTS

Increasing water deficit stress and global concerns for production of copious food

to support expanding human population have gained attention among the scientific

community. They are striving hard to explore innovative approaches for meeting the

challenge. In the present study, several culturable endophytic bacteria were obtained from

wheat and maize plants and tested for their ability to tolerate PEG-induced water deficit

stress. Drought tolerant isolates were further characterized for carbonic anhydrase (CA)

activity. Selected endophytic bacterial isolates with higher CA activity were screened for

growth promotion of wheat (C3 plant) and maize (C4 plant) at different water deficit

stress levels under axenic conditions. Efficient isolates (from wheat and maize) were

further evaluated for improving photosynthetic rate and plant biomass of both crops in pot

and field under non-stressed as well as stressed conditions. Moreover, potential of

drought tolerant CA containing endophytic bacteria was also tested for understanding the

gene expression in Arabidopsis thaliana, a model plant, under water deficit stress. Results

are demonstrated in details as under:

4.1. Drought tolerance ability of endophytic bacteria

Endophytic bacteria isolated from different tissues of wheat and maize plants were

analyzed for their drought tolerance ability at -0.31, -0.61, -1.09, -1.91 and -3.20 MPa

with PEG-6000 induced water deficit stress. Results showed that optical density of

endophytic bacterial isolates decreased with increasing the PEG-6000 induced water

deficit stress. However, isolates differed for their gowth and survial ability at different

water deficit levels. The results of wheat and maize isolates are presented separately.

4.1.1. Drought tolerance ability of endophytic bacterial isolates from wheat

Results regarding drought tolerance ability of 150 endophytic bacterial isolates

exposed to PEG induced water deficit stress (-0.31, -0.61, -1.09, -1.91 and -3.20 MPa)

showed that isolates differed for their cability to tolerate PEG-mediated water deficit

stress. Principal component analysis of endophytic bacteria based on drought tolerance

ability is shown in figure 4.1. First two principal factors explained maximum variation

(F1=78.99% and F2=10.27%). Plot of scores on first and second factor coordinate

showed that fifty endophytic bacterial isolates which scored high values ranging from

54

Page 75: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7-4

-3

-2

-1

0

1

2

3

4

5

6

7(-0.31MPa)

(-0.61MPa)

(-1.09MPa)

(-1.91MPa)

(-3.20MPa)

WR1 WR4WR7WR12

WR15

WR18WR26 WR29

WR32WR35

WR43

WS4 WS5WS9WS16WS18WS20WS24

WS27WS30

WS40WL2

WL7

WL11WL23

WL27

WL28

WL32WL36 WG1

WG3

WG8WG13

WG16WG22WR10

WR17

WR19

WR21WR23

WR33WR44

WS1WS8

WS12

WS14WS19WS21

WS35WS38

WL4

WL12

WL22

WL24

WL29

WL31WL34

WL39

WG6WG9

WG15

WG17

WG23WG25 WR5WR9

WR14

WR22WR27WR31

WR36

WR38

WR41

WR45

WS6

WS10WS13WS25

WS26WS29

WS32

WS33WS36

WS37WL3WL5

WL8 WL14

WL15WL17

WL18WL21

WL25

WL37

WG4

WG7WG11

WG19WL38

WR2

WR3

WR6

WR8WR11

WR13

WR16 WR20

WR24WR25 WR28

WR30 WR34

WR37WR39WR40

WR42WS2 WS3WG10

WS7WS11

WS15WS17

WS22 WS23 WS28WS31

WS34WS39

WL1WL6

WL9 WL10WL13WL16 WL19WL20

WL26WL30

WL33WL35WL40

WG2

WG5WG12

WG14

WG18

WG20WG21 WG24

Biplot (axes F1 and F2: 89.26 %)

F1 (78.99 %)

F2 (1

0.27

%)

Fig. 4.1. Principal component analysis of optical density of endophytic bacteria isolates from wheat at different PEG-6000 induced water deficit stress levels

55

Page 76: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.1 Selected drought tolerant endophytic bacterial isolates from wheat

Sr.

No.

Isolates Sr.

No.

Isolates Sr.

No

Isolates Sr.

No

Isolates Sr.

No.

Isolates

1 WR2 11 WR28 21 WS11 31 WL9 41 WL40

2 WR3 12 WR30 22 WS17 32 WL10 42 WG2

3 WR6 13 WR34 23 WS22 33 WL13 43 WG5

4 WR8 14 WR37 24 WS23 34 WL16 44 WG10

5 WR11 15 WR39 25 WS28 35 WL19 45 WG12

6 WR13 16 WR40 26 WS31 36 WL20 46 WG14

7 WR16 17 WR42 27 WS34 37 WL26 47 WG18

8 WR20 18 WS2 28 WS39 38 WL30 48 WG20

9 WR24 19 WS3 29 WL1 39 WL33 49 WG21

10 WR25 20 WS7 30 WL6 40 WL35 50 WG24

56

Page 77: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

0.668 to 4.918 in first factor coordinate were selected as drought tolerant isolates (Table

4.1). However, second factor coordinate did not produce results that were as clear as the

first factor coordinate as it explained less variation. These fifty isolates were used for

further study.

4.1.2. Drought tolerance enhancing ability of endophytic bacterial isolates of maize

Principle component analysis of optical density of 150 endophytic bacterial

isolates from maize at five PEG-induced water deficit stress levels (-0.31, -0.61, -1.09, -

1.91 and -3.20 MPa) is presented in figure 4.2. Results revealed that ability of isolates to

tolerate PEG-induced water deficit stress varied with different stress levels. First principal

factor (F1) explained 79.18% of variation while 9.26% variation was explained by second

principal factor (F2). Maximum score (4.791) was observed with isolate ML30 while

minimum (-2.748) was observed with isolate MS33 in F1 coordinate. Fifty isolates with

maximum score ranging from 0.537 to 4.791 in F1 (shown in Table 4.2) were selected as

drought tolerant isolates.

4.2. Carbonic anhydrase activity of drought tolerant isolates

4.2.1. Carbonic anhydrase activity of drought tolerant wheat isolates

From the endophytic bacterial isolates of wheat, 50 drought tolerant isolates were

further tested for CA activity. All the isolates varied in CA activity. Ten drought tolerant

isolates (WR2, WS7, WS11, WS22, WS23, WL9, WL13, WL16, WL19 and WL20) with

higher CA activity ranging from 13.03 to 20.23 are shown in figure 4.2. Out of these ten,

isolate WL19 showed significantly higher CA activity followed by WS11 and WS23.

Moreover, isolate WR2 also showed better CA activity which was statistically similar

with isolate WS11, WS23 and WL9. Isolates WS22, WL16 and WL20 possessed

statistically similar CA activity. However, isolate WS7 showed lowest CA activity among

the drought tolerant isolates.

4.2.2. Carbonic anhydrase activity of drought tolerant maize isolates

Similar to endophytic bacteria isolated from wheat, 50 drought tolerant isolates

from maize were analyzed for CA activity. All the isolates varied in CA activity. Ten

drought isolates (MR1, MR3, MR17, MS1, MS7, ML5, ML8, ML15, MG2 and MG9)

with higher CA activity ranging from 13.08 to 21.63% are shown in figure 4.3. Among

these 10 isolates, isolate MG9 showed significantly higher compared to all other isolates.

57

Page 78: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7-3

-2

-1

0

1

2

3

4

5

6

7 (-0.31 MPa)

(-0.61MPa)

(-1.09MPa)(-1.91MPa)

(-3.20MPa)

MR7

MR10

MR16MR23

MR26MR36

MR30MR32

MR35MR38MR41

MS2MS6

MS8MS10MS12

MS21

MS22MS23

MS27MS28

MS30

MS33

MS35

MS38

MS39ML2

ML6

ML9

ML12

ML14ML16ML24

ML27

ML29ML31ML36

ML39MG8MG10MG16

MG17MG20

MR2MR4

MR5MR9

MR12MR13MR14MR18

MR39

MS4MS14MS18MS26

MS31 MS32

MS34

ML4 ML10ML19

ML22 ML33ML34

ML40

MG1

MG7MG11

MG12

MG14MG23

MR11

MR15MR19MR21MR24MR27

MR28MR31

MR44

MR45MS3

MS5MS15MS16

MS19MS25

ML1

ML7

ML13 ML17ML18ML23

ML25

MG5

MG6MG18

MG22 MR1

MR3

MR6MR8MR17MR20

MR22MR25MR29

MR33MR34MR37

MR40MR42

MR43MS1

MS7MS9

MS11

MS13MS17

MS20MS24

MS29

MS36

MS37MS40

ML3ML5ML8

ML11

ML15

ML20

ML21ML26ML28

ML30ML32ML35

ML37

ML38

MG2

MG3

MG4

MG9

MG13MG15

MG19

MG21MG24

MG25

Biplot (axes F1 and F2: 88.44 %)

F1 (79.18 %)

F2 (9

.26

%)

Fig. 4.2. Principal component analysis of optical density of endophytic bacteria isolates from maize at different PEG-6000 induced water deficit stress levels

58

Page 79: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.2. Selected drought tolerant endophytic bacteria isolates from maize

Sr.

No.

Isolates Sr.

No.

Isolates Sr.

No

Isolates Sr.

No

Isolates Sr.

No.

Isolates

1 MR1 11 MR34 21 MS17 31 ML11 41 ML38

2 MR3 12 MR37 22 MS20 32 ML15 42 MG2

3 MR6 13 MR40 23 MS24 33 ML20 43 MG3

4 MR8 14 MR42 24 MS29 34 ML21 44 MG4

5 MR17 15 MR43 25 MS36 35 ML26 45 MG9

6 MR20 16 MS1 26 MS37 36 ML28 46 MG13

7 MR22 17 MS7 27 MS40 37 ML30 47 MG19

8 MR25 18 MS9 28 ML3 38 ML32 48 MG21

9 MR29 19 MS11 29 ML5 39 ML35 49 MG22

10 MR33 20 MS13 30 ML8 40 ML37 50 MG25

59

Page 80: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

WR2 WS7 WS11 WS22 WS23 WL9 WL13 WL16 WL19 WL200

5

10

15

20

25

bc

f

ab

d

ab

cd

efd

a

de

Endophytic bacterial isolates

CA

activ

ity (µ

mol

/mL)

Fig. 4.3. Drought tolerant endophytic bacterial isolates from wheat with high carbonic anhydrase activity

MR1 MR3 MR17 MS1 MS7 ML5 ML8 ML15 MG2 MG90

5

10

15

20

25

de db bc

g

ef

c

f f

a

Endophytic bacterial isolates

CA

act

ivity

(µm

ol/m

L)

Fig. 4.4. Drought tolerant endophytic bacterial isolates from maize with high carbonic anhydrase activity

60

Page 81: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

However, lowest CA activity was recorded with isolate MS7 among the 10 drought

tolerant isolates.

4.3. Screening of selected drought tolerant CA containing endophytic bacteria for

plant growth promotion under axenic conditions

The drought tolerant CA containing endophytic bacteria were further evauated for

plant growth promotion in wheat and maize under gnotobiotic conditions. Two different

studies were conducted on wheat and maize with their respective isolates. The outcomes

of these gnotobiotic studies are given below.

4.3.1 Screening of wheat isolates for growth promotion

4.3.1.1 Root length

Drought stress considerably reduced the root length of non-inoculated plants of

both cultivars (Fsd-2008 and Uqab-2000). Inoculation with drought tolerant CA

containing endophytic bacteria not only alleviated the deleterious effect of water deficit

stress on plant but also significantly improved root length under PEG-induced water

deficit conditions (-1.09 and -1.23 MPa), however, effect was more obvious in Uqab-

2000 (Table 4.3). Under normal conditions (-0.04 MPa), root length was increased up to

43.7, 37.1 and 45.5 % in Fsd-2008 and 48.0, 49.1, 47.4% in Uqab-2000 with isolates

WR2, WS11 and WL19 respectively, compared to their respective uninoculated control

plants. However, minimum increase up to 7.7 and 17.5% was observed in Fsd-2008 and

Uqab-2000 respectively, compared to uninoculated control under normal conditions. At

PEG-imposed water deficit stress (-1.09 MPa), isolates WS11, WR2 and WL19 showed

52.1, 48.5 and 47.8% increase in Fsd-2008 and 57.3, 53.1 and 57.3 % in Uqab-2000

compared to control (un-inoculated plant). Similarly, isolate WL19 showed substantial

increase by 56.7% in Fsd-2008 and 65.0% in Uqab-2000 followed by WR2, WS11 and

WS23 compared to uninoculated control plants in exposure to PEG-induced water deficit

stress (-1.23 MPa). Moreover, minimum increase was observed by 16.9 and 23.5% in

Fsd-2008 and Uqab-2000 respectively, with bacterial inoculation comparison to control at

-1.23 MPa of water deficit stress.

4.3.1.2. Shoot length

Shoot length of wheat significantly improved by the inoculation of drought

tolerant CA containing endophytic bacteria under non-stressed and stressed environments

61

Page 82: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Picture 6: Effect of drought tolerant CA containing endophytic bacteria on root length under normal conditions

Picture 7: Effect of drought tolerant CA containing endophytic bacteria on root length under PEG-induced water deficit conditions

62

Control WL19

Control WR2

Page 83: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Picture 8: Effect of drought tolerant CA containing endophytic bacteria on shoot length under normal conditions

Picture 9: Effect of drought tolerant CA containing endophytic bacteria on shoot length under PEG-induced water deficit conditions

63

Control WL19WR2WS11

Control WL19 WR2WS11

Page 84: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.3. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot length of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

-0.04 MPa -1.09 MPa -1.23 MPaIsolates Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000Root length (cm)Control 16.7±0.18 17.7±0.34 14.2±0.08 14.3±0.34 11.8±0.29 12.3±0.35WR2 24.0±0.16 26.2±0.29 21.1±0.25 21.9±0.22 18.4±0.35 19.5±0.04WS7 20.4±0.40 22.4±0.30 17.3±0.58 18.1±0.24 14.7±0.39 15.2±0.27WS11 22.9±0.36 26.4±0.29 21.6±0.34 22.5±0.26 17.8±0.34 18.6±0.26WS22 18.0±0.35 20.8±0.02 17.4±0.19 18.4±0.29 15.3±0.24 15.6±0.22WS23 22.1±0.24 24.6±0.34 20.7±0.21 20.4±0.32 18.2±0.33 18.7±0.15WL9 20.5±0.43 24.3±0.24 18.7±0.18 19.8±0.39 15.3±0.26 16.9±0.07WL13 19.6±0.67 22.3±0.15 17.0±0.32 18.1±0.25 13.8±0.71 15.4±0.32WL16 22.6±0.57 21.8±0.69 19.3±0.59 19.9±0.24 17.0±0.19 18.3±0.33WL19 24.3±0.29 26.1±0.34 21.0±0.63 22.5±0.33 18.5±0.32 20.3±0.11WL20 20.1±0.21 22.4±0.27 18.2±0.24 18.9±0.22 14.6±0.21 15.8±0.17Shoot length (cm)Control 23.7±0.47 24.8±0.08 19.9±0.64 20.2±0.54 14.3±0.46 15.5±0.63WR2 30.9±1.16 32.7±0.19 26.3±0.48 26.6±0.64 19.3±0.16 22.6±0.66WS7 27.6±0.66 31.9±0.87 25.0±0.55 24.9±0.54 18.5±0.17 20.7±0.27WS11 29.5±1.06 32.9±0.95 26.5±0.20 27.5±0.80 19.8±0.38 21.8±0.90WS22 25.8±0.31 26.1±0.56 22.6±1.04 23.3±0.51 17.7±0.44 19.3±0.33WS23 28.9±0.34 28.5±0.83 24.4±1.28 26.8±0.09 17.4±0.15 20.1±0.59WL9 26.4±0.98 32.3±0.34 24.7±0.76 26.5±0.35 16.3±0.31 19.8±0.38WL13 28.0±0.52 29.4±0.85 23.4±1.37 25.7±0.60 18.2±0.52 18.5±0.52WL16 27.5±0.82 29.1±0.31 25.5±0.89 24.5±0.70 19.5±0.71 20.2±1.10WL19 30.2±1.08 31.8±0.81 26.0±0.86 26.8±0.22 18.8±0.74 22.5±0.29WL20 25.9±0.95 27.7±0.58 23.5±1.95 24.9±0.52 19.1±0.12 19.4±0.75

Note: Least significant difference (LSD): Root length, 0.98; Shoot length, 1.56Mean are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

64

Page 85: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

uninoculated control plants (Table 4.3.). However, increase was more pronounced in

Uqab-2000 than Fsd-2008. Inoculation with isolate WR2 caused significant improvement

of 30.3, 32.1 and 34.9% in Fsd-2008 while 31.8, 31.6 and 45.8% in Uqab-2000 compared

to uninoculated control under normal (-0.04 MPa) and PEG-induced water deficit stress

of -1.09 and -1.23 MPa, respectively. Isolate WS11 improved the shoot length by 24.4,

33.1 and 38.4% in Fsd-2008 and 32.6, 36.1 and 40.6% in Uqab-2000 compared to control

plants under non-stressed as well as stressed conditions, respectively. However, isolate

WS22 showed minimum increase of 13.6 % in Fsd-2008 and 15.3% in Uqab-2000 at -

1.09 MPa of water deficit stress compared to uninoculated control plants. Isolate WL19

also produced significant enhancement in shoot length up to 27.4, 30.6 and 31.4% in Fsd-

2008 while 28.2, 32.6 and 45.1% in Uqab-2000 compared to their respective non-

inoculated control plants at different stress levels.

4.3.1.3. Root fresh weight

The results showed that root fresh weight was substantially decreased in non-

inoculated plants of both cultivars under PEG-induced water deficit conditions (Table

4.4). However, inoculation with drought tolerant CA containing endophytic bacteria

significantly improved root fresh weight under normal (-0.04 MPa) as well as PEG

induced water deficit conditions (-1.23 MPa), particularly in Uqab-2000. Root fresh

weight was improved up to 40.2% in Fsd-2008 and 41.5% in Uqab-2000 by the

inoculation of endophytic bacteria under non-stressed conditions (-0.04 MPa). However,

minimum increase 13.6 and 16.9% was observed in Fsd-2008 and Uqab-2000

respectively, by the inoculation of WS22 compared to uninoculated control plants under

normal conditions. Isolate WL19 and WR2 showed maximum increase by 49.3 and 43.7

in Fsd-2008 and 55.0 and 51.0% in Uqab-2000 compared to uninoculated control plants

under PEG-induced water deficit stress (-1.09 MPa). Similarly, at -1.23 MPa, PEG

induced water deficit stress, isolate WL19 and WR2 caused considerable enhancement in

root fresh biomass with 69.2 and 61.7% increase in Fsd-2008 and 74.2 and 67.3% in

Uqab-2000 followed by WL16 and WS11 compared to uninoculated control plants.

However, isolate WL13 gave minimum increase of 17.9% in Fsd-2008 while isolate

WS22 gave 33.4% increase in Uqab-2000.

4.3.1.4. Shoot fresh weight

Under non-stressed conditions (-0.04 MPa), symbolic increase of 39.8% in shoot

65

Page 86: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.4. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot fresh weight of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

-0.04 MPa -1.09 MPa -1.23 MPaIsolates Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000Root fresh weight (mg)Control 114.22±2.23 134.67±2.65 98.66±1.71 103.63±2.31 47.67±1.54 54.33±2.92WR2 154.56±1.42 190.67±1.93 141.78±1.11 156.56±2.74 77.11±2.31 90.94±2.82WS7 144.00±1.75 161.78±1.93 127.28±2.39 135.11±2.94 69.11±1.83 81.22±2.59WS11 150.55±1.17 187.67±2.03 134.00±1.93 151.78±1.11 73.06±1.84 84.22±0.22WS22 129.78±2.09 157.56±1.94 116.33±2.17 131.78±2.94 60.72±2.22 72.52±2.43WS23 137.11±1.73 158.33±2.70 125.67±1.67 135.11±1.11 64.44±1.06 79.11±3.89WL9 144.83±1.80 171.78±2.94 128.55±1.06 134.33±2.08 66.11±1.64 78.00±2.89WL13 154.00±2.72 186.22±2.22 119.78±2.89 135.31±2.36 56.22±0.78 73.17±2.32WL16 145.94±1.86 177.33±2.55 134.28±1.69 137.33±1.93 74.89±2.70 87.33±1.92WL19 160.22±1.90 183.89±2.22 147.33±2.21 160.67±2.03 80.67±1.93 94.67±2.34WL20 143.39±1.60 165.66±2.55 129.56±2.22 140.44±2.13 60.89±4.24 71.44±2.22Shoot fresh weight (mg)Control 181.91±3.18 190.44±2.42 109.33±2.89 111.00±3.85 53.44±2.45 58.00±3.33WR2 243.89±2.95 267.22±4.01 155.92±2.14 160.56±4.85 81.67±3.34 90.00±3.66WS7 237.39±4.53 231.66±2.89 140.00±3.34 146.11±2.94 73.78±3.81 78.56±2.28WS11 241.94±2.28 260.56±1.47 147.50±3.82 155.00±3.85 83.33±4.41 88.60±2.75WS22 194.17±3.61 206.11±4.01 125.00±2.41 131.11±3.89 60.44±1.50 72.11±3.88WS23 226.11±2.01 245.00±3.85 148.61±1.65 152.78±4.01 76.11±4.45 84.44±4.45WL9 238.33±4.41 237.00±2.78 137.72±2.51 142.50±2.68 67.89±3.28 74.17±3.01WL13 205.56±2.51 231.67±3.34 128.00±4.34 127.22±1.87 62.78±2.94 69.63±1.73WL16 233.44±3.86 237.22±2.95 151.67±3.01 147.22±2.01 78.11±4.07 86.63±3.32WL19 254.44±4.28 272.78±4.45 160.33±3.67 166.11±4.02 83.33±3.34 92.11±2.95WL20 210.00±2.89 221.67±3.64 135.56±2.94 136.66±2.55 69.44±2.22 78.89±3.10

Note: Least significant difference (LSD): Root fresh weight, 6.34; Shoot fresh weight, 9.26Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

66

Page 87: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

fresh weight of Fsd-2008 and 43.2% of Uqab-2000 was recorded with isolate WL19

followed by WR2 and WS11 compared to (uninoculated) control plants (Table 4.4).

However, isolates WL9 and WL16 remained statistically similar for improving the shoot

fresh weight in Fsd-2008 and Uqab-2000 under normal conditions (-0.04 MPa). Shoot

fresh weight also improved by 46.6% in Fsd-2008 and 49.6% in Uqab-2000 with

inoculation of isolate WL19 under PEG-imposed water deficit stress (-1.09 MPa)

compared to uninoculated control plants. In the same way, inoculation significantly

enhanced shoot fresh weight at -1.23 MPa of water deficit stress especially in Uqab-2000.

Shoot fresh weight was also improved by 55.9% in Fsd-2008 plants and 58.8% in Uqab-

2000 plants with isolate WL19 compared to uninoculated control plants under severe

water stress.

4.3.1.5. Root dry weight

Root dry weight of two wheat cultivars (Fsd-2008 and Uqab-2000) significantly

decreased in response to PEG-induced water deficit stress. Inoculation with drought

tolerant CA containing endophytic bacteria significantly enhanced the root dry weight,

especially in Uqab-2000 (Table 4.5). Significant increase of 40.8% in Fsd-2000 and

45.5% in Uqab-2000 with was observed with isolate WL19 under normal conditions (-

0.04 MPa) when compared to their respective uninoculated control plants. However,

isolate WR2, WS11 and WL9 showed statistically similar response in increasing root dry

weight in both genotypes. At PEG-imposed water deficit stress (-1.09 MPa), significant

enhancement of 45.9% in root dry weight was observed in Fsd-2008 and 48.3% in Uqab-

2000 with isolate WL19 followed by WR2, WS23, WS11 compared to control plants.

Inoculation with WL19, WR2 and WS23 showed significant increase up to 49.1, 43.8 and

37.0% in Fsd-2008 while 53.3, 44.9 and 46.6% in Uqab-2000 compared to uninoculated

control plants at PEG-induced water deficit stress (-1.23 MPa). However, minimum

increase of 11.2% was observed with isolate WS22 in both cultivars compared to

uninoculated control at -1.23 MPa of water deficit stress.

4.3.1.6. Shoot dry weight

A momentous enhancement in shoot dry weight was recorded with drought

tolerant CA containing endophytic bacterial inoculation compared to uninoculated control

in both cultivars, especially in Uqab-2000 (Table 4.5). Isolate WL19 showed significantly

higher shoot dry weight which was 32.7 and 38.0% in Fsd-2008 and Uqab-2000, 32.4%

67

Page 88: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.5. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot dry weight of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

Isolates-0.04 MPa -1.09 MPa -1.23 MPa

Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000Root dry weight (mg)Control 16.33±0.34 17.73±0.18 11.90±0.23 13.37±0.38 8.85±0.15 9.50±0.21WR2 20.70±0.67 24.13±0.47 16.07±0.58 18.96±0.54 12.73±0.29 13.77±0.38WS7 19.53±0.42 20.90±0.52 14.55±0.39 16.73±0.72 11.80±0.61 13.27±0.03WS11 21.00±0.50 23.10±0.46 15.82±0.61 18.20±0.42 10.98±0.54 11.67±0.32WS22 18.86±0.57 19.50±0.40 15.10±0.25 19.57±0.33 9.84±0.37 10.57±0.27WS23 20.73±0.44 23.40±0.20 16.01±0.57 17.03±0.38 12.13±0.32 13.93±0.34WL9 19.45±0.43 22.47±0.37 14.80±0.64 16.50±0.12 11.23±0.03 12.73±0.27WL13 18.38±0.46 19.90±0.47 13.60±0.25 15.27±0.77 10.23±0.23 11.50±0.50WL16 21.14±0.50 21.77±0.23 16.06±0.61 17.27±0.43 12.27±0.09 12.57±0.23WL19 23.00±0.58 25.80±0.47 17.37±0.57 19.83±0.62 13.20±0.40 14.57±0.52WL20 20.23±0.39 23.13±0.58 15.67±0.44 18.43±0.29 11.70±0.51 13.10±0.61Shoot dry weight (mg)Control 23.60±0.46 25.74±0.48 17.37±0.45 16.83±0.46 12.67±0.33 12.90±0.26WR2 30.00±0.58 34.13±0.30 23.00±0.29 24.00±0.58 17.50±0.12 18.45±0.51WS7 26.50±0.29 30.00±0.24 21.20±0.47 22.03±0.55 14.53±0.23 15.00±0.46WS11 28.87±0.57 32.90±0.08 23.67±0.20 23.63±0.37 17.89±0.34 18.17±0.33WS22 25.27±0.32 28.37±0.17 19.80±0.36 20.97±0.55 14.77±0.62 16.66±0.12WS23 27.80±0.42 30.80±0.34 22.37±0.32 20.83±0.64 13.41±0.45 15.81±0.40WL9 28.63±0.20 31.50±0.41 21.77±0.49 22.00±0.29 16.33±0.38 17.97±0.58WL13 26.57±0.59 30.83±0.36 20.69±0.16 19.40±0.46 13.33±0.33 13.69±0.16WL16 28.63±0.41 32.43±0.35 23.12±0.65 23.23±0.39 17.67±0.67 18.40±0.31WL19 31.33±0.33 35.53±0.38 24.17±0.44 24.67±0.44 18.40±0.31 19.17±0.33WL20 25.80±0.42 29.33±0.54 20.00±0.58 20.27±0.37 14.37±0.18 16.07±0.43

Note: Least significant difference (LSD): Root dry weight, 1.23; Shoot dry weight, 1.82 Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

68

Page 89: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

in Fsd-2008 and 46.5, 40.4 and 42.6% in Uqab-2000 inoculated with isolate WL19,

WS11 and WR2 compared to uninoculated control plants at -1.09 MPa of water deficit

stress. Inoculation with isolate WL19 significantly enhance shoot dry weight by 45.2% in

Fsd-2008 and 48.6% in Uqab-2000 compared to uninoculated control plants followed by

WR2, WS11 and WL16 at -1.23 MPa.

4.3.1.7. Chlorophyll contents

The results ravealed that PEG-induced water deficit conditions caused

considerable reduction in chlorophyll contents in uninoculated plants, especially in Uqab-

2000 (Table 4.6). Inoculation with drought tolerant CA containing endophytic bacterial

isolates significantly improved the chlorophyll contents compared to uninoculated control

plants, under normal (-0.04 MPa) as well as PEG-induced water deficit stress (- 1.09, -

1.23 MPa) in both cultivars, however, effect of endophytic bacterial inoculation was

much prominent in Uqab-2000. Chlorophyll contents were enhanced by 32.0 and 39.1%

in Fsd-2008 and Uqab-2000, respectively, with isolate WL19 followed by WS23, WR2

and WS7 compared to uninoculated control plants under normal conditions (-0.04 MPa).

At -1.09 MPa, isolate WS22 showed minimum increase in both cultivars. However,

isolate WL19 significantly improved chlorophyll contents in Uqab-2000 by 48.5 % while

43.0% in Fsd-2008 compared to their respective control plants. At -1.23 MPa, inoculation

with isolate WS11, WL19 and WR2 caused significant enhancement of 54.9, 52.6 and

52.1% in Fsd-2008 and 54.0, 55.6 and 52.9% in Uqab-2000 when compared with their

respective control. Moreover, isolate WL9 showed 57.2% increase in Uqab-2000 and

45.9% in Fsd-2008 compared to control plants under stressed conditions (-1.23 MPa).

4.3.1.8 Carbonic anhydrase activity in leaves

Bacterial endophytes and PEG-induced water deficit stress had strong effect on

CA activity. Carbonic anhydrase activity was significantly decreased in non-inoculated

plants under PEG-mediated water deficit conditions, particularly in Uqab-2000 (Table

4.6). Inoculation with drought tolerant CA containing endophytic bacterial isolates

(WL19, WR2 and WS11) significantly increased CA activity under non stressed

conditions (-0.04) in both cultivars compared to uninoculated control. Moreover, under

PEG-induced water deficit stress (-1.09 MPa), inoculation with isolate WL19 followed by

WS23, WR2, WS11 significantly enhanced CA activity, especially in Uqab-2000.

However, less increase in CA activity was detected with isolate WS22 in both

69

Page 90: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.6. Effect of drought tolerant CA containing endophytic bacterial isolates on chlorophyll content and CA activity in leaves of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

-0.04 MPa -1.09 MPa -1.23 MPaIsolates Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000Chlorophyll content (SPAD Value)Control 35.6±0.33 31.9±0.47 27.2±0.61 24.9±0.58 21.1±0.38 18.5±0.49WR2 45.6±0.15 43.2±0.53 38.2±0.15 36.4±0.64 32.1±0.09 28.3±0.76WS7 42.1±0.28 39.3±0.26 36.0±0.09 33.7±0.15 29.9±0.55 26.8±0.72WS11 46.2±0.14 42.7±0.24 38.0±0.58 34.5±0.83 32.7±0.09 28.5±0.72WS22 42.5±0.17 38.7±0.12 35.3±0.29 32.3±0.38 28.3±0.63 23.9±0.55WS23 46.4±0.07 43.6±0.15 37.8±0.52 33.2±0.55 31.4±0.06 27.4±0.61WL9 42.0±0.15 37.7±0.15 35.5±0.03 32.1±0.37 30.8±0.64 29.1±0.85WL13 39.8±0.07 38.8±0.03 36.2±0.18 32.9±1.03 28.8±0.61 25.5±0.67WL16 43.7±0.27 40.3±0.19 37.3±0.26 34.0±0.69 30.4±0.23 27.7±0.35WL19 47.0±0.07 44.4±0.33 38.9±0.09 37.0±0.50 32.2±0.53 28.8±0.83WL20 43.4±0.45 40.8±0.37 35.5±0.61 34.2±0.25 29.7±0.55 27.4±0.29CA activity in leaves (mol CO2 Kg-1 leaf FM s-1)Control 1.259±0.05 1.027±0.04 0.810±0.04 0.760±0.04 0.574±0.04 0.501±0.03WR2 1.762±0.04 1.496±0.03 1.177±0.06 1.127±0.06 0.839±0.03 0.799±0.07WS7 1.448±0.02 1.149±0.04 1.114±0.05 1.012±0.05 0.791±0.02 0.608±0.05WS11 1.694±0.07 1.461±0.04 1.198±0.03 1.148±0.04 0.808±0.06 0.825±0.05WS22 1.268±0.05 1.139±0.07 0.902±0.05 0.859±0.04 0.616±0.03 0.566±0.04WS23 1.450±0.06 1.251±0.06 1.221±0.02 1.171±0.04 0.765±0.04 0.761±0.04WL9 1.505±0.05 1.273±0.04 0.976±0.06 0.956±0.06 0.687±0.03 0.637±0.06WL13 1.449±0.03 1.183±0.03 1.086±0.04 0.903±0.05 0.713±0.04 0.680±0.03WL16 1.310±0.04 1.247±0.03 1.118±0.05 1.048±0.06 0.815±0.07 0.581±0.06WL19 1.865±0.03 1.549±0.06 1.254±0.03 1.204±0.04 0.890±0.06 0.843±0.08WL20 1.308±0.06 1.152±0.02 0.982±0.01 0.872±0.05 0.756±0.04 0.754±0.03

Note: Least significant difference (LSD): Chlorophyll content, 1.29; CA activity, 0.133Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

70

Page 91: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

when compared to their respective uninoculated control. Similarly, isolate WL19 and

WR2 showed significant increase in CA activity up to 68.2 and 59.5% in Uqab-2000 and

55.0 and 46.1% in Fsd-2008 compared to non-inoculated control plants in exposure to

water deficit stress (-1.23 MPa). However, isolate WS23 and WL20 remained statistically

similar in improving the CA activity in both cultivars.

4.3.1.9. Photosynthetic rate

Water deficit stress caused significant reduction in net CO2 assimilation rate of

non-inoculated plants of both wheat cultivars. Inoculation with drought tolerant CA

containing endophytic bacteria enhanced the photosynthetic rate in the leaves of non-

stressed and stressed plants, however, increase was more pronounced in Uqab-2000

(Table 4.7). Isolate WL19 improved photosynthetic rate in Uqab-2000 leaves by 39.4%

and 33.6 % in Fsd-2008 when compared to their respective (non-inoculated) control

under non-stressed conditions (-0.04 MPa). In the same way, isolate WL19 caused 40.0

and 44.2% increase in photosynthetic rate of Fsd-2008 and Uqab-2000 compared to their

uninoculated control plants at -1.09 MPa of water deficit stress. However, isolates WL13

and WL16 showed similar response for improving the photosynthetic rate in both

cultivars compared to control plants. Moreover, significant increase of 43.7% in Fsd-2008

and 47.2% in Uqab-2000 in photosynthetic rate was observed when exposed to water

stress (-1.23 MPa) compared to their respective non-inoculated plants, however, less

increase was observed with isolate WS22 followed by WL13 and WL20 in both cultivars.

4.3.1.10. Transpiration rate

Inoculation of drought tolerant CA containing endophytic bacterial isolates WL19,

WS11 and WR2 increased the transpiration rate in both cultivars, especially in Uqab-

2000 under normal conditions (Table 4.7). Isolate WL19 showed significantly higher

transpiration rate of 23.3% in Fsd-2008 and 28.1% in Uqab-2000 compared to

uninoculated control plants under normal conditions (-0.04 MPa). Transpiration rate was

also increased by 28.3, 30.9 and 31.9% in Fsd-2008 with inoculation of isolates WL19,

WR2 and WS11 and by 37.7, 37.7, 37.1% in Uqab-2000 compared to uninoculated

control plants at -1.09 MPa. Similarly, inoculation with isolate WL19 significantly

improved transpiration rate under PEG induced severe water deficit stress (-1.23 MPa) by

38.3% in Fsd-2008 and 44.5% in Uqab-2000 compared to inoculated control plants

followed by WR2, WS11 and WS23. However, effect of isolate WS22 remained

71

Page 92: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.7. Effect of drought tolerant CA containing endophytic bacterial isolates on photosynthetic and transpiration rate of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

-0.04 MPa -1.09 MPa -1.23 MPaIsolates Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000Photosynthetic rate (μmol CO2 m-2 s-1)Control 9.5±0.10 7.6±0.18 7.0±0.18 5.2±0.15 4.8±0.15 3.6±0.15WR2 12.0±0.17 10.3±0.12 9.0±0.09 7.1±0.06 6.5±0.15 5.0±0.06WS7 10.9±0.12 9.0±0.06 8.5±0.15 6.1±0.13 5.4±0.09 3.7±0.18WS11 12.3±0.10 10.0±0.12 9.4±0.09 6.9±0.09 6.4±0.13 4.8±0.03WS22 11.7±0.09 8.8±0.06 8.9±0.03 6.2±0.17 5.0±0.10 3.6±0.17WS23 11.9±0.07 10.0±0.09 9.1±0.17 7.1±0.15 6.3±0.15 4.9±0.09WL9 10.7±0.17 9.5±0.15 8.1±0.06 6.9±0.06 5.8±0.17 4.3±0.07WL13 11.4±0.19 9.2±0.17 8.6±0.07 6.4±0.13 5.5±0.07 4.1±0.18WL16 11.6±0.15 9.5±0.07 8.8±0.10 6.5±0.07 5.8±0.15 4.1±0.12WL19 12.7±0.09 10.6±0.10 9.8±0.12 7.5±0.12 6.9±0.06 5.3±0.15WL20 11.0±0.18 8.8±0.15 8.1±0.19 6.0±0.15 5.3±0.17 4.0±0.09Transpiration rate (mmol H2O m

-2 s-1)Control 2.18±0.04 2.13±0.02 1.94±0.03 1.83±0.03 1.46±0.02 1.48±0.03WR2 2.60±0.03 2.72±0.01 2.54±0.02 2.52±0.02 2.02±0.02 2.08±0.01WS7 2.49±0.01 2.49±0.02 2.46±0.03 2.45±0.03 1.92±0.01 1.95±0.02WS11 2.67±0.02 2.70±0.02 2.56±0.05 2.51±0.03 2.00±0.03 2.03±0.02WS22 2.28±0.03 2.26±0.01 2.05 ±0.03 1.96±0.04 1.56±0.02 1.57±0.02WS23 2.60±0.01 2.66±0.03 2.50±0.06 2.44±0.03 1.95±0.03 2.10±0.03WL9 2.43±0.05 2.45±0.01 2.31±0.03 2.36±0.02 1.93±0.01 2.04±0.01WL13 2.54±0.02 2.67±0.04 2.47±0.02 2.33±0.01 1.92±0.02 1.90±0.02WL16 2.63±0.03 2.72±0.02 2.35±0.01 2.42±0.03 1.87±0.02 1.96±0.03WL19 2.69±0.01 2.73±0.01 2.49±0.05 2.52±0.02 2.02±0.01 2.14±0.04WL20 2.47±0.02 2.52±0.02 2.38±0.04 2.30±0.04 1.97±0.02 1.95±0.02

Note: Least significant difference (LSD): Photosynthetic rate, 0.37; Transpiration rate, 0.07Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤0.0.05

72

Page 93: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

statistically similar in both cultivars under severe water deficit stress (-1.23 MPa).

4.3.1.11. Stomatal conductance

Results regarding stomatal conductance of two wheat cultivars showed that

stomatal conductance significantly decreased under PEG-induced water deficit stress.

Inoculation with drought tolerant CA containing endophytic bacteria significantly

enhanced the stomatal conductance, especially in Uqab-2000 (Table 4.8). Stomatal

conductance was enhanced by 33.3% in Fsd-2008 and Uqab-2000 with the inoculation of

isolate WL19 under normal conditions (-0.04 MPa) when compared to their respective

uninoculated control plants. However, isolate WL13 followed by WL20 and WS22

showed minimum improvement in stomatal conductance of both cultivars. At PEG-

imposed water stress (-1.09 MPa), significant improvement upto 37.5% in Fsd-2008 and

40.0% in Uqab-2000 in stomatal conductance was detected with inoculation of bacterial

isolates compared to control plants. However, isolate WL19 remained statistically similar

with WR2 in both cultivars at -1.09 MPa of water deficit stress imposed by PEG.

Seedling inoculated with WL19, WR2 and WS23 showed significant increase up to

40.0% in Fsd-2008 and 66.6% in Uqab-2000 compared to uninoculated control plants

under severe water deficit stress (-1.23 MPa). However, minimum increase was observed

with isolate WS22 in both cultivars compared to uninoculated control at -1.23 MPa of

water stress.

4.3.1.12. Substomatal conductance

Contrary to stomatal conductance, substomatal conductace raised with growing

level of PEG-6000 induced water deficit stress. Inoculation of isolates (WL19, WS11 and

WR2) significantly decreased substomatal conductance under normal and stressed

conditions, especially in in Uqab-2000 (Table 4.8). Maximum reduction of 25.4% in Fsd-

2008 and 29.7% in Uqab-2000 was observed by the inoculation of isolate WL19

compared to control plants under normal conditions (-0.04 MPa). However, isolates

WS11 and WR2 remained statistically similar for decreasing the substomatal conductance

in both cultivars. At PEG-induced water stress (-1.09 MPa), isolates WL19 and WR2

showed significant increase of 38.4 and 41.5% in Uqab-2000 followed by 29.6 and 32.8%

in Fsd-2008 compared to their respective controls. Similarly, substomatal conductance

was enhanced by 41.7 and 49.0% in Fsd-2008 and Uqab-2000 respectively, compared to

uninoculated control plants on exposure to PEG-induced water deficit stress (-1.23 MPa).

73

Page 94: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.8. Effect of drought tolerant CA containing endophytic bacterial isolates on stomatal and substomatal conductance of drought tolerant (Fsd-2008) and sensitive (Uqab-2000) wheat cultivars under normal and PEG-induced water deficit conditions

-0.04 MPa -1.09 MPa -1.23 MPaIsolates Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000 Fsd-2008 Uqab-2000Stomatal Conductance (mol H2O m

-2 s-1)Control 0.12±0.002 0.09±0.001 0.08±0.002 0.05±0.002 0.05±0.002 0.03±0.002WR2 0.14±0.003 0.12±0.002 0.11±0.004 0.07±0.003 0.07±0.001 0.05±0.001WS7 0.13±0.001 0.09±0.002 0.09±0.001 0.06±0.001 0.05±0.003 0.03±0.001WS11 0.14±0.002 0.11±0.004 0.09±0.003 0.07±0.001 0.07±0.002 0.05±0.001WS22 0.13±0.001 0.10±0.002 0.09±0.001 0.06±0.004 0.06±0.001 0.04±0.002WS23 0.14±0.001 0.11±0.003 0.10±0.002 0.07±0.000 0.07±0.002 0.05±0.002WL9 0.13±0.002 0.10±0.003 0.09±0.002 0.07±0.001 0.06±0.001 0.04±0.001WL13 0.13±0.003 0.09±0.004 0.09±0.001 0.06±0.004 0.05±0.001 0.04±0.002WL16 0.14±0.002 0.10±0.001 0.09±0.002 0.07±0.003 0.06±0.003 0.05±0.003WL19 0.16±0.002 0.12±0.004 0.11±0.004 0.07±0.002 0.07±0.002 0.05±0.003WL20 0.13±0.001 0.09±0.002 0.09±0.003 0.06±0.001 0.06±0.003 0.04±0.002Substomatal conductance (µmol mol-1)Control 240±3.97 235±4.09 280±5.04 276±4.49 335±2.89 328±4.34WR2 183±2.65 174±5.26 197±4.41 183±2.67 199±4.59 189±2.65WS7 202±4.41 180±2.83 205±3.18 204±2.41 238±4.06 214±2.91WS11 181±4.06 175±2.13 198±5.57 179±4.36 206±4.10 186±4.59WS22 210±5.49 192±3.05 225±2.89 205±3.93 237±3.61 214±2.97WS23 206±3.85 179±2.18 211±2.85 184±4.34 240±3.93 198±3.34WL9 215±4.73 199±4.49 228±3.48 220±5.24 245±6.07 242±2.03WL13 197±3.93 190±4.96 224±4.81 191±2.67 246±5.57 236±5.18WL16 207±1.79 181±6.44 207±5.70 188±2.91 241±3.48 207±3.85WL19 179±2.03 165±3.81 188±4.41 169±5.21 192±5.24 172±2.00WL20 198±2.34 182±1.52 203±2.41 190±3.06 223±4.17 200±4.34

Note: Least significant difference (LSD): Stomatal conductance, 0.006; Substomatal conductance, 11.13Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

74

Page 95: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.3.1.13. Relationship between photosynthetic rate and CA activity exhibited by

drought tolerant endophytic bacterial isolates

Relationship between photosynthetic rate and CA activity in bacteria under

normal as well as PEG-induced water deficit conditions (-1.09 and -1.23MPa) is shown in

figure 4.5 and 4.6 Inoculation of isolates (WL19, WR2, WS11 and WS23) exhibiting

highest CA activity gave considerable increase in photosynthetic rate in both cultivars,

especially in Uqab-2000 on exposure to water stress imposed by PEG. Photosynthetic rate

improved substantially with increasing CA activity and relationship was more

pronounced under water deficit stress Any change in CA activity exhibited by bacteria

significantly affected the photosynthetic rate in both cultivars. Moreover, correlation

analysis showed that photosynthetic rate and CA activity were positively correlated under

normal and stressed condition. However, correlation was more positive under PEG-

induced water deficit conditions. In case of cultivars, correlation was more significant in

drought sensitive cultivars (Uqab-2000) compared to tolerant cultivar (Fsd-2008) at

probability of p ≤ 0.05.

4.3.2. Screening of maize isolates for growth promotion

4.3.2.1. Root length

In the presence of PEG-induced water deficit stress, root length reduced

significantly in the seedling of maize susceptible hybrid (H2) in comparison with tolerant

(H1) hybrid (Table 4.9). However, inoculation of drought tolerant CA containing

endophytic bacterial isolates caused significant increase in root length under non-stresses

(-0.04) and PEG-induced water deficit stress (-1.09 and -1.23 MPa) inboth hybrids. Root

length for H2 increased by 41.3% and for H1 by 37.1% with the inoculation of isolate

MG9 compared to non-inoculated control plants under non-stressed conditions.

Comparable enhancement in root length of both hybrids was also observed with isolate

MG9 followed by MR17 and MS1 at PEG-induced water deficit stress (-1.09 MPa),

however, effect was slightly greater (50.3%) for H2 than for H1 (46.1%). Root length also

increased by 70.0 and 84.5% for H1and H2 hybrids respectively, with the inoculation of

isolate MG9 compared to uninoculated control plants under PEG-induced water deficit

stress (-1.23 MPa).

4.3.2.2. Shoot length

The results revealed that inoculation of drought tolerant CA containing endophytic

75

Page 96: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

10 12 14 16 18 20 220

2

4

6

8

10

12

14

R² = 0.737843012109168

R² = 0.707025417529565

R² = 0.680798158984398

D0=-0.04 MPa Linear (D0=-0.04 MPa) D1=-1.09 MPaLinear (D1=-1.09 MPa) Linear (D1=-1.09 MPa) D2=-1.23 MPa

Carbonic anhydrase activity (µmol/mL)

Phot

osyn

theti

c rat

e (μ

mol

CO2

m-2

s-1)

Fig. 4.5. Relationship between photosynthetic rate and carbonic anhydrase activity exhibited by drought tolerant endophytic bacterial isolates in wheat cv.

Fsd-2008

10 12 14 16 18 20 220

2

4

6

8

10

12

R² = 0.797094952015257

R² = 0.743704869825054

R² = 0.738762049340802

D0=-0.04 MPa Linear (D0=-0.04 MPa) D1=-1.09 MPaLinear (D1=-1.09 MPa) Linear (D1=-1.09 MPa) D2=-1.23 MPa

Carbonic anhydrase activity (µmol/mL)

Phot

osyn

theti

c rat

e (μ

mol

CO2

m-2

s-1)

`

Fig. 4.6. Relationship between photosynthetic rate and carbonic anhydrase activity exhibited by drought tolerant endophytic bacterial isolates in wheat cv.

Uqab-2000

76

Page 97: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Picture 10: Effect of drought tolerant CA containing endophytic bacteria on root length under normal conditions

Picture 11: Effect of drought tolerant CA containing endophytic bacteria on root length under water deficit conditions

77

MG9Control

Control MG9

Page 98: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Picture 12: Effect of drought tolerant CA containing endophytic bacteria on shoot length under normal conditions

Picture 13: Effect of drought tolerant CA containing endophytic bacteria on shoot length under PEG-induced water deficit conditions

78

Control

Control

MG9

MG9

Page 99: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.9. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot length of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG-induced water deficit conditions

-0.04 MPa -1.09 MPa -1.23 MPaIsolates H1 H2 H1 H2 H1 H2Root length (cm)Control 22.6±0.43 20.3±0.44 19.3±0.44 16.3±0.67 13.7±0.38 11.0±0.53MR1 25.9±0.52 23.4±0.46 23.2±0.33 19.9±0.49 18.0±0.58 16.0±0.23MR3 27.7±0.39 25.3±0.52 25.3±0.52 21.0±0.58 18.9±0.72 15.2±0.62MR17 30.3±0.49 29.7±0.49 27.8±0.37 24.7±0.77 23.9±0.57 19.9±0.31MS1 29.7±0.58 28.7±0.33 27.2±0.44 24.0±0.58 23.3±0.52 19.3±0.44MS7 26.3±0.54 24.2±0.62 19.9±0.35 17.8±0.60 14.0±0.56 11.7±0.52ML5 29.7±0.27 27.7±0.17 25.8±0.57 21.0±0.58 19.7±0.41 15.6±0.30ML8 29.0±0.47 25.0±0.51 25.6±0.37 23.3±0.52 21.7±0.33 17.7±0.33ML15 28.3±0.54 26.0±0.74 23.8±0.44 20.3±0.60 16.7±0.44 13.3±0.67MG2 24.2±0.36 23.7±0.44 24.2±0.33 21.7±0.17 18.0±0.17 13.0±0.49MG9 31.0±0.33 28.7±0.62 28.2±0.67 24.5±0.50 23.3±0.67 20.3±0.44Shoot length(cm)Control 34.1±0.73 29.1±0.61 27.4±0.76 23.6±0.70 21.2±0.45 16.5±0.47MR1 39.6±1.32 33.3±1.07 33.1±0.94 30.3±0.46 28.7±0.96 24.0±0.82MR3 42.9±0.97 34.4±1.19 36.3±1.50 27.9±1.21 26.7±0.40 21.8±0.38MR17 44.0±0.27 40.7±0.43 38.8±0.50 34.0±0.65 31.1±0.89 25.3±0.59MS1 42.7±0.86 39.4±0.54 37.5±0.92 32.5±0.74 30.0±1.35 24.5±0.28MS7 38.6±0.69 33.5±0.36 28.7±0.31 26.5±0.58 22.3±0.56 18.2±0.58ML5 40.7±1.49 36.3±0.57 33.4±1.57 26.8±0.39 28.9±0.74 22.9±0.64ML8 44.0±0.93 38.6±0.62 37.7±0.72 31.5±0.98 30.9±0.44 24.3±0.77ML15 40.9±0.69 35.4±0.48 35.8±0.61 28.5±1.19 26.8±0.33 21.1±1.18MG2 37.7±0.73 32.6±0.97 29.2±0.32 29.2±0.46 22.4±0.27 19.8±0.33MG9 44.6±0.78 41.5±1.35 38.9±0.43 34.8±1.06 31.9±1.29 25.8±0.97

Note: Least significant difference (LSD): Root length, 1.43; Shoot length, 2.29Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

79

Page 100: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

normal (non-stressed) and PEG-mediated water deficit environment (Table 4.9). Growth

enhancement in shoot length of H2 was greater compared to H1 hybrid by the inoculation

of endophytic bacterial isolates. Inoculation of isolate MG9 significantly enhanced the

shoot length varying about 42.6% in H2 and 30.7% in H1 compared to their uninoculated

control plants under normal conditions (-0.04 MPa). Inoculation of isolate MS7 also

increased the shoot length under normal conditions; however, increase over control plants

was smaller compared to other isolates. At PEG-induced water deficit stress (-1.09 MPa),

H2 hybrid treated with isolates MG9 and MR17 showed 47.4 and 44.0% increase while

H1 showed 41.9 and 41.6% compared with uninoculated control plants. In addition, shoot

length increased by 50.4 and 56.3% with the inoculation of isolate MG9 followed by

MR17, MS1 and ML8 in both hybrid compared to their respective control at -1.23 MPa.

4.3.2.3. Root fresh weight

Root fresh weight of maize decreased substantially with rising PEG-6000 induced

water deficit stress, however, inoculation of drought tolerant CA containing endophytic

bacterial isolate MG9, MR17, MS1 and ML8 enhanced the root fresh weight under non-

stresses as well as stressed conditions, especially in drought sensitive hybrid H2 (Table

4.10). Maximum root fresh weight (48.4%) was detected with isolate MG9 in H2 while

43.9 % in H1 compared to uninoculated control plants in the absence of PEG (-0.04

MPa). However, minimum increase was detected with isolate ML15 in both hybrids

under normal conditions (-0.04 MPa). Isolate MG9 followed by MR17, MS1 also showed

significant increase in both hybrids in response to water deficit conditions (-1.09 MPa).

Comparable increase (84.0%) in root fresh weight was recorded with isolate MG9 in H2

and 71.9% in H1 hybrid after PEG-treatment (-1.23 MPa). However, isolate MS7 showed

minimum increase in both H2 and H1 hybrid compared to respective uninoculated control

plant at -1.23 MPa mediated with PEG.

4.3.2.4. Shoot fresh weight

Momentous enhancement in shoot fresh biomasst was found in inoculated plants

of maize hybrids (H1 and H2), indicating the beneficial effect of drought tolerant CA

containing endophytic bacteria on shoot fresh weight in contrast to non-inoculated control

plants. However, effect of inoculation on enhancement of shoot fresh weight was more

pronounced in H2 hybrid compared to uninoculated control plants under normal as well

as stressed conditions (Table 4.10). Isolate MS7 showed minimum increase over the

80

Page 101: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

-0.04 MPa -1.09 MPa -1.23 MPaIsolates H1 H2 H1 H2 H1 H2Root fresh weight (g)Control 1.320±0.02 0.923±0.01 0.837±0.03 0.700±0.03 0.570±0.03 0.433±0.01MR1 1.780±0.03 1.267±0.02 1.167±0.02 1.027±0.01 0.867±0.02 0.697±0.01MR3 1.623±0.02 1.173±0.02 1.073±0.02 0.910±0.02 0.773±0.02 0.603±0.02MS17 1.893±0.02 1.367±0.01 1.267±0.01 1.117±0.02 0.967±0.01 0.750±0.03MS1 1.803±0.01 1.320±0.04 1.220±0.02 1.070±0.03 0.920±0.03 0.717±0.02MS7 1.627±0.02 1.193±0.02 1.093±0.02 0.943±0.02 0.673±0.02 0.490±0.01ML5 1.783±0.02 1.150±0.03 1.157±0.03 0.890±0.02 0.857±0.01 0.587±0.02ML8 1.673±0.01 1.257±0.02 1.150±0.02 1.007±0.02 0.750±0.02 0.680±0.02ML15 1.577±0.03 1.097±0.01 0.997±0.01 0.827±0.03 0.697±0.01 0.660±0.03MG2 1.677±0.02 1.074±0.03 1.110±0.01 0.847±0.01 0.674±0.02 0.513±0.01MG9 1.900±0.03 1.370±0.03 1.270±0.03 1.120±0.03 0.980±0.03 0.797±0.02Shoot fresh weight (g)Control 1.870±0.01 1.520±0.02 1.400±0.02 1.063±0.01 0.780±0.02 0.608±0.02MR1 2.210±0.01 1.997±0.02 1.817±0.02 1.470±0.03 1.067±0.01 0.880±0.01MR3 2.113±0.02 1.953±0.03 1.790±0.01 1.407±0.01 1.030±0.02 0.750±0.01MS17 2.370±0.02 2.067±0.02 1.973±0.03 1.627±0.01 1.197±0.03 0.997±0.02MS1 2.340±0.03 2.023±0.01 1.857±0.01 1.540±0.03 1.113±0.02 0.880±0.03MS7 1.953±0.02 1.633±0.02 1.523±0.02 1.250±0.02 0.837±0.03 0.643±0.02ML5 2.207±0.02 1.883±0.02 1.737±0.02 1.440±0.01 0.993±0.01 0.853±0.02ML8 2.307±0.01 2.070±0.01 1.833±0.03 1.380±0.02 1.127±0.02 0.883±0.03ML15 2.237±0.03 1.920±0.02 1.780±0.01 1.440±0.03 0.967±0.02 0.833±0.02MG2 2.117±0.01 1.813±0.03 1.593±0.02 1.267±0.01 0.910±0.03 0.737±0.02MG9 2.487±0.03 2.147±0.02 1.993±0.02 1.673±0.02 1.233±0.01 1.030±0.01

Table 4.10. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot fresh weight of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG-induced water deficit conditions

Note: Least significant difference (LSD): Root length, 0.064; Shoot length, 0.060Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

81

Page 102: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

uninoculated control compared to other isolates in both hybrids. Out of 10 isolates, isolate

MG9 improved the shoot fresh weight by 32.9% in H1 and 41.2% in H2 compared to

uninoculated control under normal conditions (-0.04 MPa). In the same way, isolate MG9

showed 57.4 and 69.4% increase in H2 followed by 42.3 and 58.0% in H1 at -1.09 and -

1.23 MPa of PEG-induced water deficit stress, respectively in comparison with control.

However, isolate MS1 and ML8 remained statistically at par for improving the shoot

fresh weight in both hybrids at PEG-6000 induced water deficit stress (-1.23 MPa).

Moreover, minimum increase over control plants was recorded with isolate MS7 in both

hybrids under severe water deficit stress conditions (-1.23 MPa).

4.3.2.5. Root dry weight

The data explained in table 4.11 demonstrated that inoculation of isolates (MG9,

MR17, MS1 and ML8) significantly enhanced root dry weight of both sensitive and

tolerant hybrids under non-stressed (-0.04 MPa) as well as water deficit conditions (-1.09

and -1.23 MPa). Inoculation of drought tolerant CA containing endophytic bacterial

isolates MG9 and MR17 remained statistically similar for improving the root dry weight

in both hybrids with 39.2% in H1 and 42.3% in H2 compared to respective control plants

at -0.04 MPa. However, isolates MR1 and MS7 showed minimum increase under normal

conditions in H1 and H2 hybrids. Isolate MG9 showed 46.1% increase for H1 and 54.5%

for H2 in the presence of PEG-mediated water deficit stress (-1.09 MPa) compared to

uninoculated control plants. In the same way, at -1.23 MPa of PEG-6000 induced water

deficit stress, inoculation significantly enhanced root dry weight in both hybrids

especially in H2. Root dry weight was improved by 68.9 % in H1 plants and 73.9 % in

H2 plants by the inoculation of isolates MR17 and MG9 compared to uninoculated

control plants. However, smaller increase up to 6.5 and 6.7% was observed in both

hybrids with isolate MS7 compared to respective control plants on exposure to severe

water deficit stress (-1.23 MPa).

4.3.2.6. Shoot dry weight

Water deficit caused noticeable reduction in shoot dry weight of non-inoculated

maize seedlings of both hybrids, especially in H2 hybrid. Contrarily, shoot dry weight

was significantly increased with the inoculation of drought tolerant CA containing

endophytic bacterial isolates under PEG-induced water deficit stress (Table 4.11).

Inoculation of MG9, MR17, MS1 and ML8 enhanced shoot dry weight and improved the

82

Page 103: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.11. Effect of drought tolerant CA containing endophytic bacterial isolates on root and shoot dry weight of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG induced water deficit conditions

Isolates-0.04 MPa -1.09 MPa -1.23 MPa

H1 H2 H1 H2 H1 H2Root dry weight (g)Control 0.158±0.01 0.111±0.00 0.117±0.01 0.077±0.00 0.074±0.00 0.046±0.00MR1 0.185±0.00 0.128±0.00 0.144±0.00 0.098±0.01 0.099±0.00 0.059±0.00MR3 0.202±0.01 0.126±0.01 0.150±0.00 0.091±0.00 0.102±0.01 0.061±0.01MR17 0.219±0.00 0.158±0.00 0.171±0.01 0.115±0.00 0.125±0.00 0.079±0.00MS1 0.214±0.00 0.152±0.00 0.161±0.00 0.110±0.00 0.121±0.00 0.073±0.00MS7 0.179±0.00 0.131±0.00 0.125±0.00 0.084±0.00 0.079±0.00 0.049±0.00ML5 0.203±0.01 0.136±0.00 0.153±0.00 0.096±0.00 0.099±0.01 0.054±0.00ML8 0.211±0.00 0.147±0.01 0.172±0.00 0.117±0.00 0.118±0.00 0.064±0.00ML15 0.188±0.00 0.136±0.00 0.147±0.00 0.083±0.00 0.090±0.00 0.065±0.01MG2 0.194±0.01 0.125±0.00 0.153±0.00 0.096±0.00 0.090±0.00 0.054±0.00MG9 0.220±0.00 0.156±0.00 0.171±0.01 0.119±0.01 0.125±0.01 0.080±0.00Shoot dry weight (g)Control 0.123±0.00 0.095±0.00 0.082±0.00 0.068±0.00 0.057±0.00 0.044±0.01MR1 0.135±0.00 0.107±0.00 0.092±0.00 0.079±0.01 0.069±0.01 0.050±0.00MR3 0.147±0.01 0.114±0.00 0.089±0.00 0.085±0.00 0.074±0.00 0.053±0.00MR17 0.161±0.00 0.128±0.01 0.110±0.01 0.094±0.00 0.081±0.00 0.065±0.00MS1 0.155±0.01 0.125±0.00 0.108±0.00 0.088±0.01 0.078±0.00 0.063±0.01MS7 0.141±0.00 0.105±0.00 0.089±0.00 0.073±0.00 0.060±0.00 0.047±0.00ML5 0.141±0.00 0.114±0.00 0.094±0.00 0.085±0.00 0.078±0.01 0.053±0.00ML8 0.155±0.01 0.125±0.00 0.105±0.01 0.085±0.00 0.080±0.00 0.064±0.01ML15 0.132±0.00 0.116±0.01 0.103±0.00 0.073±0.00 0.066±0.00 0.054±0.00MG2 0.143±0.01 0.103±0.01 0.105±0.00 0.081±0.01 0.067±0.01 0.050±0.00MG9 0.158±0.00 0.130±0.00 0.113±0.00 0.096±0.00 0.082±0.00 0.066±0.01

Note: Least significant difference (LSD): Root dry weight, 0.009; Shoot dry weight, 0.008Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

83

Page 104: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

plant health under non-stressed as well as stressed conditions, although improvement in

shoot dry weight was greater for H2 than H1 hybrid. Significant increase by 36.8, 41.1

and 50.0% was observed in H2 followed by 28.4, 37.8 and 43.8% in H1 hybrid with

isolate MG9 at different PEG-induced water deficit stress levels compared to their

respective non inoculated plants. However, inoculation of isolate MS7 gave significant

increase under normal conditions (-0.04 MPa) but showed non-significant differences

with uninoculated control plants under PEG-mediated water deficit conditions (-1.09 and

-1.23 MPa) in both HI and H2 hybrids of maize.

4.3.2.7. Chlorophyll contents

Data regarding chlorophyll content showed that inoculation with drought tolerant

CA containing endophytic bacterial isolates significantly enhanced the chlorophyll

contents in both hybrids, especially in H2 under normal and stressed conditions (Table

4.12). Inoculation with isolates MG9 followed by MR17 and MS1 significantly increased

chlorophyll contents by 28.8% in H1 and 32.2% in H2 compared to respective control

under non stressed conditions (-0.04). Under PEG-induced water deficit stress (-1.09

MPa), inoculation with isolate MG9 significantly enhanced chlorophyll contents by

39.5% in H2 hybrid. However, minimum increase in chlorophyll content was detected

with isolate MS7 in both hybrids when compared to their (uninoculated) control.

Similarly, isolate MG9 and MR17 showed significant increase in chlorophyll content up

to 47.8 and 44.9% in H2 and that of 40.9 and 39.0 % in H1 compared to non-inoculated

plants at PEG-induced water deficit stress (-1.23 MPa). However, isolate MR3 and ML15

remained statistically similar for improving the chlorophyll contents in both hybrids.

4.3.2.7. Carbonic anhydrase activity

Under PEG-induced water deficit stress, CA activity reduced significantly in the

seedling of maize susceptible (H2) hybrid in comparison with tolerant (H1) hybrid (Table

4.12) However, inoculation of drought tolerant CA containing endophytic bacterial

isolates caused significant enhancement in CA activity of both hybrids under non-stressed

(-0.04) as well as PEG- induced water deficit stress (-1.09 and -1.23 MPa). Under normal

conditions, CA activity for H2 increased by 26.6% and for H1 by 24.0% with the

inoculation of isolate MG9 followed by MR17 and MS1 compared to non-inoculated

plants. Comparable increase in CA activity was also observed with isolate MG9 followed

by MR17 and MS1 for both hybrids, however, effect was slightly greater

84

Page 105: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.12. Effect of drought tolerant CA containing endophytic bacterial isolates on chlorophyll contents and carbonic activity of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG-induced water deficit conditions

-0.04MPa -1.09 MPa -1.23 MPaIsolates H1 H2 H1 H2 H1 H2Chlorophyll content (SPAD Value)Control 40.2±0.31 35.3±0.82 32.6±0.23 29.6±0.55 26.1±0.46 23.8±0.53MR1 48.6±0.54 45.9±0.07 41.4±0.39 39.9±0.08 32.9±0.29 28.3±0.33MR3 47.8±0.74 43.0±0.26 39.6±0.35 39.0±0.03 31.7±0.33 31.5±0.06MR17 49.0±0.87 46.2±0.12 43.9±0.51 41.1±0.15 36.3±0.18 34.5±0.29MS1 50.1±0.06 46.1±0.03 42.1±0.12 40.8±0.17 34.8±0.43 34.7±0.05MS7 44.2±0.32 39.9±0.66 38.2±0.34 35.1±0.53 29.5±0.24 26.0±0.13ML5 49.3±0.23 45.7±0.10 39.6±0.50 39.8±0.26 30.5±0.90 32.7±0.12ML8 49.7±0.31 46.9±0.06 42.2±0.34 40.5±0.06 35.6±0.15 33.5±0.31ML15 47.9±0.14 44.7±0.24 41.7±0.07 38.9±0.08 33.3±0.43 33.2±0.20MG2 47.5±0.43 42.0±0.80 39.6±0.50 35.8±0.15 32.8±0.13 28.0±0.38MG9 51.8±0.30 46.7±0.08 43.9±0.33 41.3±0.12 36.8±0.11 35.2±0.15CA activity (mol CO2 Kg-1 leaf FM s-1)Control 2.309±0.04 2.007±0.05 1.700±0.05 1.630±0.04 1.327±0.05 1.121±0.03MR1 2.442±0.06 2.249±0.02 2.064±0.04 2.060±0.02 1.715±0.04 1.335±0.04MR3 2.370±0.04 2.215±0.05 1.870±0.05 1.851±0.06 1.608±0.05 1.328±0.02MR17 2.762±0.04 2.461±0.03 2.148±0.04 2.171±0.02 1.768±0.04 1.609±0.06MS1 2.694±0.03 2.496±0.03 2.171±0.03 2.154±0.07 1.789±0.04 1.578±0.03MS7 2.375±0.04 2.112±0.06 1.792±0.04 1.842±0.05 1.533±0.02 1.253±0.03ML5 2.505±0.06 2.239±0.02 2.067±0.06 2.058±0.05 1.635±0.06 1.526±0.07ML8 2.649±0.08 2.373±0.06 2.171±0.04 2.161±0.02 1.729±0.03 1.585±0.04ML15 2.543±0.06 2.151±0.03 1.936±0.02 1.916±0.06 1.511±0.03 1.357±0.03MG2 2.450±0.07 2.257±0.05 2.127±0.06 1.889±0.03 1.577±0.01 1.383±0.04MG9 2.86±50.04 2.542±0.07 2.214±0.04 2.217±0.03 1.873±0.04 1.666±0.05

Note: Least significant difference (LSD): Chlorophyll content,1.08; CA activity, 0.13Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

85

Page 106: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

(36.0%) for H2 than for H1 (30.2%) at -1.09 MPa of PEG-induced water deficit stress.

Similarly, in the presence of PEG-mediated water deficit stress of -1.23 MPa, about 41.1

and 48.6% increase was observed in H1 and H2 hybrids respectively, with the inoculation

of isolate MG9 compared to non-inoculated control plants. Moreover, smaller increase of

11.8 and 15.5% was observed with isolate MS7 in H2 and H1 hybrids, respectively, after

PEG-induced stress of -1.23 MPa.

4.3.2.9. Photosynthetic rate

Photosynthetic rate differed significantly between hybrids and reduced more for

sensitive (H2) than tolerant (H1) hybrid under non-stressed and stressed conditions (Table

4.13). However, inoculation with drought tolerant CA containing bacterial isolates

significantly enhanced the photosynthetic rate of both hybrids in the presence as well as

absence of PEG. Increase in photosynthetic rate was greater for H2 than H1 hybrid when

compared to their non-inoculated control plants. Inoculation with isolate MG9 followed

by MR17 and MS1 improved photosynthetic rate by 28.1 and 30. 3% for H1 while 33.6

and 37.8% for H2 compared to their respective control plants under non-stressed (-0.04

MPa) and stressed conditions (-1.09 MPa), respectively. However, inoculation of MR1,

ML5 and ML8 did not give any significant difference for improving the photosynthetic

rate in both hybrids after PEG-imposed water deficit stress of -1.09 MPa. Smaller

increase in photosynthetic rate was observed with isolate MG2 followed by ML5 and

MR1compared to their respective control plants under PEG-induced water deficit stress (-

1.23 MPa). Isolate MS7 did not show any significant effect for improving the

photosynthesis under severe water deficit stress (-1.23 MPa).

4.3.2.10. Transpiration rate

Under normal conditions (-0.04 MPa), significant increase of 26.2% in

transpiration rate for drought tolerant (H1) hybrid and 27.6% for sensitive (H2) hybrid

was recorded with the inoculation of drought tolerant CA containing endophytic bacterial

isolates MR17 followed by MG9 and MS1 compared to their non-inoculated control

plants (Table 4.13). However, inoculation of isolate MS7 did not cause significant

differences for improving transpiration rate of both hybrids under normal conditions (-

0.04 MPa). Under PEG-imposed water deficit stress (-1.09 MPa), inoculation of bacterial

isolates significantly enhanced the transpiration rate by 32.7% in H1 and 33.0 % in H2

compared to uninoculated control plants. Similarly, inoculation significantly enhanced

86

Page 107: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.13. Effect of drought tolerant CA containing endophytic bacterial isolates on photosynthetic and transpiration rate of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG-induced water deficit conditions

-0.04 MPa -1.09 MPa -1.23 MPaIsolates H1 H2 H1 H2 H1 H2Photosynthetic rate (μmol CO2 m

-2 s-1)Control 12.8±0.17 11.0±0.12 9.9±0.18 8.2±0.22 7.2±0.22 5.8±0.15MR1 14.8±0.10 13.9±0.07 11.7±0.07 9.9±0.08 8.5±0.06 6.7±0.12MR3 15.4±0.18 13.3±0.15 10.9±0.18 9.0±0.03 8.6±0.12 6.3±0.05MR17 16.0±0.07 14.2±0.12 12.6±0.21 11.1±0.15 9.0±0.18 7.9±0.10MS1 16.1±0.06 14.1±0.03 12.1±0.12 10.8±0.17 8.9±0.06 7.5±0.11MS7 14.5±0.09 12.9±0.16 10.5±0.13 9.1±0.13 7.7±0.09 6.0±0.13ML5 15.3±0.23 13.7±0.10 10.9±0.18 9.8±0.26 8.9±0.15 6.8±0.03ML8 16.0±0.14 14.9±0.06 12.2±0.34 10.5±0.06 8.6±0.15 7.7±0.05ML15 14.9±0.14 13.7±0.24 11.7±0.07 9.9±0.08 8.5±0.14 6.2±0.20MG2 15.8±0.11 13.7±0.15 11.3±0.18 9.2±0.20 8.0±0.13 6.5±0.06MG9 16.4±0.05 14.7±0.08 12.9±0.33 11.3±0.12 9.6±0.11 8.2±0.15Transpiration rate (mmol H2O m

-2 s-1)Control 3.69±0.07 3.54±0.06 2.75±0.04 2.54±0.07 2.05±0.07 1.85±0.05MR1 3.98±0.06 3.87±0.08 3.08±0.02 2.80±0.06 2.71±0.04 2.47±0.04MR3 3.91±0.04 3.82±0.06 3.04±0.08 2.84±0.07 2.79±0.02 2.55±0.08MR17 4.66±0.08 4.52±0.05 3.46±0.03 3.43±0.29 2.87±0.04 2.46±0.04MS1 4.54±0.03 4.39±0.08 3.48±0.03 3.38±0.09 2.89±0.02 2.66±0.04MS7 3.89±0.02 3.78±0.02 2.98±0.09 3.08±0.05 2.57±0.09 1.98±0.06ML5 4.38±0.07 4.32±0.04 3.38±0.03 2.74±0.03 2.70±0.03 2.62±0.04ML8 4.44±0.06 4.42±0.04 3.44±0.08 3.10±0.01 2.89±0.02 2.58±0.06ML15 3.88±0.07 3.81±0.07 3.08±0.02 2.80±0.06 2.57±0.09 2.12±0.03MG2 3.91±0.03 4.32±0.02 3.02±0.07 2.77±0.02 2.70±0.04 1.95±0.04MG9 4.59±0.05 4.49±0.04 3.65±0.04 3.38±0.05 3.04±0.08 2.76±0.05

Note: Least significant difference (LSD): Photosynthetic rate, 0.07; Transpiration rate, 0.18Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

87

Page 108: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

transpiration rate in both hybrids, especially in H2 under severe water deficit stress (-1.23

MPa). Transpiration rate was improved in H2 plants by 49.1% and that of 48.2% in H1

plants with isolate MG9 compared to uninoculated control plants under PEG-induced

water deficit stress (-1.23 MPa). However, isolates MR1 and MR3 remained statistically

similar for improving the transpiration rate in H1 and H2 hybrids under severe water

deficit conditions.

4.3.2.11. Stomatal conductance

On exposure to PEG-mediated water deficit stress, stomatal conductance of maize

seedlings significantly decreased in non-inoculated plants. However, inoculation of

drought tolerant CA containing endophytic bacterial isolates improved stomatal

conductance under normal (-0.04 MPa) as well as PEG-induced water deficit stress

conditions (-1.09 MPa) in both maize hybrids i.e. drought tolerant (H1) and sensitive (H2)

compared to uninoculated plants (Table 4.14) especially in H2 than H1. Inoculation of

isolate MG9 showed considerable improvement of 33.3% in H1 and 36.3% in H2

compared uninoculated control under normal conditions (-0.04 MPa). Isolate MR17

improved the stomatal conductance in both hybrids compared to uninoculated control

plants under PEG-induced water deficit stress (-1.09 MPa), which was similar to isolates

MG9 and MS1. However, isolate MG2 showed minimum increase of 22.2 % in H1 and

14.2 % in H2 at -1.09 MPa compared to uninoculated control plants. Under severe water

deficit conditions (-1.23 MPa), isolate MG9 gave pronounced enhancement in stomatal

conductance i.e. 57.1% in H1 and 60.0% in H2 compared to control plants.

4.3.2.12. Substomatal conductance

Substomatal conductance showed inverse relationship with the inoculation of

drought tolerant CA containing bacterial isolates for drought tolerant (H1) and sensitive

(H2) hybrids under non-stressed as well as stressed conditions (Table 4.14). Inoculation

with drought tolerant CA containing bacterial isolates significantly reduced the

substomatal conductance in both hybrids. Decrease in substomatal conductance was

greater for H2 than H1 hybrid when compared with their respective uninoculated control

plants under normal conditions (-0.04 MPa) as well as stressed conditions (-1.09 and -

1.23 MPa). Inoculation with isolate MG9, MR17 and MS1 significantly reduced

substomatal conductance under PEG-induced water stress (-1.09 MPa). At greater level of

PEG-mediated water deficit stress (-1.23 MPa), isolate MS7 showed minimum reduction

88

Page 109: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

-0.04 MPa -1.09 MPa -1.23 MPaIsolates H1 H2 H1 H2 H1 H2Stomatal conductance (mol H2O m

-2 s-1)Control 0.15±0.002 0.11±0.005 0.09±0.006 0.07±0.005 0.07±0.004 0.05±0.004MR1 0.16±0.003 0.15±0.003 0.11±0.007 0.09±0.003 0.10±0.004 0.06±0.003MR3 0.16±0.005 0.13±0.003 0.11±0.002 0.08±0.004 0.09±0.002 0.06±0.002MR17 0.18±0.006 0.15±0.002 0.13±0.003 0.10±0.004 0.09±0.004 0.08±0.006MS1 0.18±0.003 0.14±0.003 0.13±0.004 0.10±0.003 0.10±0.004 0.08±0.002MS7 0.16±0.004 0.13±0.005 0.11±0.003 0.09±0.005 0.08±0.003 0.06±0.005ML5 0.17±0.002 0.13±0.003 0.12±0.005 0.09±0.006 0.09±0.003 0.07±0.003ML8 0.17±0.004 0.15±0.004 0.12±0.005 0.08±0.004 0.09±0.006 0.07±0.006ML15 0.16±0.002 0.13±0.004 0.11±0.002 0.09±0.003 0.08±0.005 0.07±0.003MG2 0.16±0.005 0.12±0.002 0.11±0.004 0.08±0.005 0.08±0.003 0.07±0.002MG9 0.20±0.003 0.15±0.002 0.13±0.002 0.10±0.004 0.11±0.003 0.08±0.005Substomatal conductance (µmol mol-1)Control 274±1.23 244±3.04 289±2.04 265±2.10 365±1.78 345±2.89MR1 183±2.21 198±2.13 210±2.15 198±3.20 290±3.89 288±1.32MR3 203±2.10 210±4.05 223±4.01 213±2.13 243±3.20 203±2.45MR17 178±6.03 169±4.67 210±3.06 178±2.07 215±2.94 302±3.20MS1 184±1.78 175±5.32 199±5.29 184±2.89 206±1.78 214±2.02MS7 202±2.40 164±1.80 276±3.05 239±1.07 254±2.89 298±3.04ML5 207±4.08 224±6.03 271±4.44 229±5.02 267±3.45 242±2.45ML8 239±2.87 187±3.20 205±2.33 230±3.33 238±1.23 236±1.32ML15 265±4.32 189±2.90 234±1.59 281±5.23 299±2.89 234±5.23MG2 198±4.32 216±2.89 279±2.07 245±3.68 298±4.13 274±6.32MG9 214±2.21 169±1.78 210±3.30 192±1.50 215±5.09 220±5.00

Table 4.14. Effect of drought tolerant CA containing endophytic bacterial isolates on stomatal substomatal conductance of drought tolerant (H1) and sensitive (H2) maize hybrids under non-stressed and PEG-induced water deficit conditions

Note: Least significant difference (LSD): Stomatal conductance 0.01; Sub stomatal conductance, 8.90Means are given with standard error of three replicates and LSD is statistically significant differences at probability of p ≤ 0.05

89

Page 110: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

90

Page 111: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

in substomatal conductance in both hybrids, especially in H1. However, inoculation of

isolate MG9 significantly lowered the intracellular CO2 concentration in both hybrids,

especially for H2 maize hybrid compared to control plants on exposure to severe water

deficit stress (-1.23 MPa).

4.3.2.13. Relationship between photosynthetic rate and CA activity exhibiting by

drought tolerant endophytic bacteria

Water deficit stress had strong effect on CA activity and photosynthetic rate of

maize seedlings. Relationship between CA activity in bacteria and photosynthetic rate

was calculated for both drought tolerant (H1) and sensitive hybrids (H2) of maize under

normal (-0.04 MPa) as well as PEG induced water deficit conditions (-1.09, -1.23 MPa)

(Fig 4.7. and 4.8.). Photosynthetic rate was significantly influenced with any change in

CA activity in both hybrids, however, relationship was more pronounced on exposure to

water stress developed by PEG. In case of hybrids, H2 (sensitive) showed more positive

relationship than the H1 (tolerant). Significant increase in photosynthetic rate was

observed with the inoculation of isolate MG9, MR17, MS1 and ML8 having high CA

activity in both maize hybrids at PEG-induced water deficit stress (-1.09 and -1.23MPa).

Moreover, correlation analysis also showed that CA activity was positively correlated

with photosynthetic rate in both (H1and H2) hybrids under non-stressed and stressed

condition. However, correlation was stronger under PEG-induced water deficit stress for

drought sensitive hybrid (H2) than for tolerant (H1) hybrid.

4.5. Evaluation of selected Gus labeled endophytic bacterial isolates in pot trial

Three efficient Gus labeled isolates (WL19, WR2 and WS1 from wheat while

MG9, MR17 and MS1 from maize) showed varied response for improving growth in

wheat and maize crops under drought stress in pot trials. Results are described below.

4.5.1. Effect of selected Gus labelled endophytic bacterial isolates on wheat

4.5.1.1. Plant height

Response of wheat cultivars was monitored in the presence as well as absence of

drought tolerant CA producing endophytic bacterial inoculant under different water

deficit environment (Fig. 4.9A). Under non-stressed conditions (100% FC), Uqab-2000

inoculated with all the three isolates i.e. WS11, WL19 and WR2 showed increased plant

height compared to uninoculated control plants. Wheat cultivar Fsd-2008 also showed

91

Page 112: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

10 12 14 16 18 20 22 240

2

4

6

8

10

12

14

16

18

R² = 0.838531764339567

R² = 0.774641803795353

R² = 0.630850239478198

D0=-0.04 MPa Linear (D0=-0.04 MPa) D1=-1.09 MPaLinear (D1=-1.09 MPa) Linear (D1=-1.09 MPa) D2=-1.23 MPa

Carbonic anhydrase actitity (µmol/mL)

Phot

osyn

theti

c rat

e (μ

mol

CO2

m-2

s-1)

Fig. 4.7. Relationship between photosynthetic rate and carbonic anhydrase activity exhibited by drought tolerant endophytic bacterial isolates in maize hybrid

(H1)

10 12 14 16 18 20 22 240

2

4

6

8

10

12

14

16

R² = 0.841650906065255

R² = 0.793622726958695

R² = 0.650181430781647

D0=-0.04 MPa Linear (D0=-0.04 MPa) D1=-1.09 MPaLinear (D1=-1.09 MPa) Linear (D1=-1.09 MPa) D2=-1.23 MPa

Carbonic anhydrase actitity (µmol/mL)

Phot

osyn

theti

c rat

e (μ

mol

CO2

m-2

s-1)

Fig. 4.8. Relationship between photosynthetic rate and carbonic anhydrase activity exhibited by drought tolerant endophytic bacterial isolates in maize hybrid

(H2)

92

Page 113: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

0

20

40

60

80

100

hie-g

k-m jk

p op

c-eb

gh ef

nok-m

de

a

ijc-e

nlm

bca

f-hb-d

mkl

Control WR2 WS11 WL19

Field capacity levels

Pla

nt

hei

gh

t (c

m)

A

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

0

5

10

15

20

fgef

i

gh

lkl

cd a-c

f-h

b-d

ij i

de

a

ij fg

jk ijk

b-dab

ef c-e

ij

h

Control WR2 WS11 WL19

Field capacity levels

Ro

ot

dry

wei

gh

t (

g)

B

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

0

5

10

15

20

25

fgef

i-kg-i

nl-n

c-eab

fgc-e

k-m j-l

b-d a-cd-f ef

mn

h-j

a-ca

efa-c

high

Control WR2 WS11 WL19

Field capacity levels

Sho

ot

dry

wei

gh

t (g

) C

Fig. 4.9. Effect of drought tolerant CA containing endophytic bacteria on plant height (A), root dry weight (B) and shoot dry weight (C) in both wheat cultivars at different field capacity levels

93

Page 114: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

pronounced increase in plant height with endophytic bacterial isolates, though the

increase was slightly smaller than Uqab-2000 at 100% FC. Moreover, plant height was

increased by 18.9 and 24.3% for Fsd-2008 and 24.3 and 27.5% for Uqab-2000 with the

inoculation of isolate WL19 under stressed conditions (70% and 40% FC, respectively)

compared to their respective control plants.

4.5.1.2. Root dry weight

Under water deficit stressed conditions (70 and 40% FC), root dry weight was

significantly decreased in both wheat cultivars (Fsd-2008 and Uqab-2000) (Fig. 4.9B).

However, inoculation of bacterial isolates (WR2, WS11 and WL19) improved the root

dry weight more for Uqab-2000 and Fsd-2008 compared to their respective uninoculated

control plant under non-stressed (100% FC) as well as stressed (70, 40% FC) conditions.

Inoculation of Uqab-2000 with isolate WR2 significantly enhanced the root dry weight by

27.0 and 23.2% for Fsd-2008 under mild stressed conditions (70% FC) compared to

untreated plant. Root dry weight was markedly increased (37.2%) by isolate WL19 for

Uqab-2000 than for Fsd-2008 (31.8%) under severe water deficit conditions (40% FC).

4.5.1.3. Shoot dry weight

Inoculation of wheat cultivars with drought tolerant CA containing endophytic

bacteria helped plant to improve the shoot dry weight under well-waterd condition (100%

FC) and stressed environment in both wheat cultivars (Fsd-2008 and Uqab-2000) (Fig.

4.9C). Improvement in dry weight was observed under non-stressed and mild water

deficit conditions (100%, 70% FC). Inoculation of bacterial isolates WL19 and WR2 also

showed comparable differences for enhancing the shoot dry weight in both Fsd-2008 and

Uqab-2000 under mild water stress condition compared to their respective control.

Moreover, isolate WL19 improved shoot dry weight but did not show significant

difference for Fsd-2008 and Uqab-2000 under severe water stress condition (40% FC).

4.5.1.4. Carbonic anhydrase activity

Carbonic anhydrase activity of plant considerably decreased in both cultivars

under water deficit environements (70 and 40% FC), especially in Uqab-2000 (Fig.

4.10A). Drought tolerant CA containing endophytic bacterial inoculants significantly

enhanced CA activity compared to respective uninoculated control plants, however, effect

of inoculation was more pronounced for Uqab-2000 than Fsd-2008. Under well-watered

condition (100% FC), isolates WL19 showed better CA activity in both wheat cultivars.94

Page 115: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

0

0.5

1

1.5

2

2.5

3

3.5

gh hi

jk

lm

b

ef fgg

k k

cdfg

cd

hij

l

abc

def

hi-l

Control WR2 WS11 WL19

Field capacity levels

Ca

rbo

nic

an

hy

dra

se a

ctiv

ity

(m

ol

CO

2 K

g-1

lea

f F

M s

-1)

A

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

0

5

10

15

20

25

cfg

hij

mn

bc-e ef

gh

jklm

bc-f cd

h

k k

ab

c-e d-f

ikl

Control WR2 WS11 WL19

Field capacity levels

Ph

oto

syn

thet

ic r

ate

mo

l C

O2

m-2

s-1

)

B

Fsd Uqab Fsd Uqab Fsd Uqab70% 40%

0

1

2

3

4

5

6

7

deef

ghi

lmn

a-cc-e

defg

j-l

mn

cd cdde

h

jkkl

a ab

ef ef

ijkl

Control WR2 WS11 WL19

Field capacity levels

Tra

nsp

ira

tio

n r

ate

(mm

ol

H2

O m

-2 s

-1)

C

Fig. 4.10. Effect of drought tolerant CA containing endophytic bacteria on carbonic anhydrase activity (A), photosynthetic rate (B) and transpiration rate (C) in both wheat cultivars at different field capacity levels

95

Page 116: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Moreover, inoculation of isolate WL19 showed 37.7 and 58.3% increase in Fsd-2008 and

53.3 and 66.6% in Uqab-2000 in comparison with control at mild (70% FC) and severe

(40% FC) water deficit stress.

4.5.1.5. Photosynthetic rate

A significant decrease in photosynthetic rate was observed for Uqab-2000 than

Fsd-2008 under limited water supply (Fig. 4.10B). However, inoculation of drought

tolerant CA containing endophytic bacterial isolates (WL19, WR2 and WS11)

significantly increased leaf photosynthetic rate, especially in Uqab-2000 both in the

presence and absence of water deficit stress (100, 70 and 40% FC). Under normal

condition, Uqab-2000 showed significant increase of 21.8% with the inoculation of

isolate WL19 that was 18.7% for Fsd-2008 compared to uninoculated control plants.

Nevertheless, under severe water deficit stress (40% FC), photosynthetic rate was

increased upto 30.1% with isolate WL19 for Fsd-2008 and 35.2% with isolate WS11 for

Uqab-2000 compared to respective control plants.

4.5.1.6. Transpiration rate

Transpiration rate was increased about 22.9% in Uqab-2000 with isolates WL19

followed by 19.7% increase in Fsd-2008 compared to control under normal growth

conditions (100% FC) (Fig. 4.10C). However, isolates showed variable response for

facilitating the transpiration rate under water deficit environement (70% FC) in both

cultivars. Inoculation with drought tolerant bacterial isolate WL19 gave highest increase

of 42.5% for Uqab-2000 and 32.5% for Fsd-2008 compared to respective control plants,

under severe water stress (40% FC). On the other hand, isolate WR2 showed similar

increase for transpiration rate in both cultivars at 40% FC level.

4.5.1.7. Stomatal conductance

Under water stress conditions (40% FC), stomatal conductance was decreased in

both cultivars, however, effect was more pronounced in Uqab-2000 compared to Fsd-

2008 (Fig. 4.11A). Inoculation of isolate WL19 improved the stomatal conductance in

both cultivars and showed 21% increase when compared to their non-inoculated control

plants under normal growth conditions. Similarly, enhancement in stomatal conductance

was observed with the inoculation of drought tolerant endophytic bacterial inoculant

WL19 compared to control (un-inoculated) plants under water stress conditions 70% FC)

96

Page 117: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

cf

h i

mno

b

cd de

gh

klm

b b

fg

kln

ab

cdef

jkl

Control WR2 WS11 WL19

Field capacity levels

Sto

ma

tal

con

du

cta

nce

(mo

l H

2O

m-2

s-1

)

A

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

0

20

40

60

80

100

fhi

jk mn o

abd

gh hi jk kl

cd de efi

m lm

aa

f fgij jk

Control WR2 WS11 WL19

Field capacity levels

Rel

ati

ve

wa

ter

con

ten

t(%

)

B

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

0

10

20

30

40

50

h-j hef

d

ba

kh-j gh

fg

c c

i-k jkgh

e

cb

k jkhi

ef ef e

Control WR2 WS11 WL19

Field capacity levels

Ele

ctro

lyte

lea

ka

ge

(%)

C

Fig. 4.11. Effect of drought tolerant CA containing endophytic bacteria on stomatal conductance (A), relative water content (B) and electrolyte leakage (C) in both wheat cultivars at different field capacity levels

97

Page 118: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Inoculation of endophytic bacterial isolate WL19 gave significant improvement of 39.1%

for Uqab-2000 and 29.7% for Fsd-2008 compared to uninoculated control plants under

severe water deficit conditions (40% FC).

4.5.1.8. Relative water content (RWC)

Relative water content, indicator of plant water status, decreased more in Uqab-

2000 than Fsd-2008 (Fig. 4.11B) under water deficit conditions. However, inoculation of

drought tolerant CA containing endophytic bacterial inoculants (WR2, WS11 and WL19)

significantly improved RWC in both cultivars under normal (100% FC) and stressed

condition (70, 40% FC) but increase was more in Uqab-2000. In normal growth

conditions, relative water content (RWC) was significantly increased by 18.6% in Uqab-

2000 and 14.8% in Fsd-2008 compared to uninoculated control plants. Inoculation with

isolate WL19 facilated the RWC by15.7 and 24.8 % in Fsd-2008 and by 21.1 and 30.6 %

in Uqab-2000 under severe water stress (40% FC) in comparison with control.

4.5.1.9. Electrolyte leakage (EEL)

In contrast to RWC, electrolyte leakage (EEL) was increased under water deficit

stress conditions (70 and 40% FC) (Fig. 4.11C). Decrease for Uqab-2000 was greater for

Fsd-2008. Inoculation with drought tolerant CA containing endophytic bacterial isolates

reduced the electrolyte leakage in both cultivars. Isolate WL19 showed 17% reduction in

electrolyte leakage under normal (100% FC) and mild stressed conditions (70% FC) in

Uqab-2000. Under severe water stress (40% FC), significant reduction of 32. 7% for Fsd-

2008 was observed and 37.3% for Uqab-2000 compared to control plants.

4.5.1.10. Proline content

Concentration of proline in the leaves of wheat genotypes was comparable for

entire treatments; however, it was more for the plants inoculated with isolate WL19 under

water deficit (70% FC) conditions in comparison with control (Fig. 4.12A). Moreover,

greater accumulation was observed for Fsd-2008 compared to Uqab-2000. Under normal

conditions, isolate WR2, WS1 and WL19 showed variable response for proline

accumulation in both the cultivars. A similar response for proline content was observed

under mild water stress (70% FC). However, inoculation with isolate WL19 significantly

enhanced the proline content by 20.1% in Fsd-2008 and 25.2 % in Uqab-2000 compared

to uninoculated control plants under sever water stress (40% FC).

98

Page 119: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

1.0

3.0

5.0

7.0

9.0

11.0

13.0

15.0

mnp

h

k

c

e

lm no

f

j

b

d

lop

g

j

b

d

kno

f

i

a

c

Control WR2 WS11 WL19

Field capacity levels

Pro

lin

e co

nte

nt

(µm

ol

g-1

)

A

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

0

3

6

9

12

15

18

21

24

lm jk

g f

b

a

mn m

h g

d

b

m kli h

cb

nlm

ijgh

ed

Control WR2 WS11 WL19

Field capacity levels

Mel

an

ald

ehy

de

con

ten

t

(μm

ol.

g-1

fw

)

B

Fsd Uqab Fsd Uqab Fsd Uqab100% 70% 40%

0

3

6

9

12

15

18

21

24

fg

ij h-jkl

no

bcde ef

ghjk

mn

de de cd

hikl

n

a

bde

fg

ijlm

Control WR2 WS11 WL19

Field capacity levels

G

rain

yie

ld (

g p

ot-

1)

C

Fig. 4.12. Effect of drought tolerant CA containing endophytic bacteria on proline content (A), melanaldehyde (B) and grain yield (C) in both wheat cultivars at different field capacity levels

99

Page 120: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.5.1.11. Melanaldehyde content

Melanaldehyde (MDA) content was increased as intensity of water deficit stress

increased; however, increase was more in Uqab-2000 compared to Fsd-2008 (Fig. 4.12B).

Inoculation of isolate WS11 showed slight decrease in MDA contents of both cultivars

under normal conditions compared to isolates WL19 and WR2. However, isolates

exhibited different response for lowering the MDA content under mild stress conditions

(70% FC). Under sever water deficit conditions, isolate WL19 gave considerable

reduction of 30.2 and 34.9% in Fsd-2008 and Uqab-2000, respectively, under severe

water stress (40% FC).

4.5.1.12. Grain yield

Inoculation of endophytic bacterial isolates (WR2, WS11 and WL19) gave

significant increase in grain yield, especially in Uqab-2000 (Fig 4.12C). Isolates WR2

and WL19 facilitated the grain yield compared to uninoculated control plants in both

cultivars under non-stressed (100% FC) and under water deficit (70% FC) stress. Under

severe water deficit stress (40% FC), isolate WL19 showed 32.6 and 34.4% increase in

grain yield in both Fsd-2008 and Uqab-2000, respectively, in comparison with control

plants.

4.5.1.13. Colonization of plant tissues

The results regarding colonization of different tissues of both wheat cultivars

(Fsd-2008 and Uqab-2000) with Gus labelled drought tolerant CA containing endophytic

bacterial isolates are shown in figure 4.13. The isolates i.e. WR2, WS11 and WL19

efficiently colonized the root, shoot and leaf tissues of both cultivars however,

colonization was slightly greater for Fsd-2008 than Uqab-2000. Colonization of tissues by

isolates decreased under mild (70% FC) and severe (40% FC) stress compared to normal

growth conditions (100% FC). In case of root, higher CFU of isolate WR2 was observed

for Fsd-2008 than Uqab-2000 followed by WL19 and WS11 under normal (100% FC) as

well as water deficit conditions (70 and 40% FC). Inoculation of isolate WR2 and WL19

efficiently colonized the shoot tissues than WS11, though, their number decreased as

intensity of water deficit stress increased in both the cultivars. On the other hand, less

CFU was recorded in shoot tissues of Fsd-2008 and Uqab-2000 compared to root tissues.

100

Page 121: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

100% 70% 40% 100% 70% 40%Fsd-2008 Uqab-2000

0E+00

1E+05

2E+05

3E+05

4E+05

5E+05

6E+05

7E+05 WR2 WS11 WL19

Root tissues

CF

U/g

fre

sh b

iom

ass

A

100% 70% 40% 100% 70% 40%Fsd-2008 Uqab-2000

0E+00

1E+03

2E+03

3E+03

4E+03

5E+03

6E+03

7E+03

8E+03 WR2 WS11 WG9

Shoot tissues

CF

U/g

fre

sh b

iom

ass

B

100% 70% 40% 100% 70% 40%Fsd-2008 Uqab-2000

0E+00

1E+03

2E+03 WR2 WS11 WL19

Leaf tissues

CF

U/g

fre

sh b

iom

ass

C

Fig. 4.13. Colonization of root (A), shoot (B) and leaf (C) tissues with drought tolerant CA containing endophytic bacteria in both wheat cultivars at different field capacity levels

101

Page 122: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Minimum viable cell number was found with inoculants in leaf tissues of both cultivars

compared to root and shoot tissues. However, among the isolates, isolate WL19

efficiently colonized the leaf tissue of Fsd-2008 than Uqab-2000 under non-stressed

(100% FC) and under water deficit (70 and 40% FC) conditions.

4.5.1.14. Characterization of selected bacterial isolates for IAA production under

normal and stressed conditions

Selected drought tolerant CA containing endophytic bacterial isolates (WR2,

WS11, and WL19) were also characterized for IAA production under normal and PEG-

induced water deficit conditions both in the presence as well as in absence of the L-

tryptophan (L-TRP) with reference to time (1-6 days) (Fig. 4.14). Results showed that

PEG-induced water deficit conditions significantly reduced IAA synthesis, especially in

the absence of substate (L-TRP). Moreover, isolates WR2, WS11and WL19 enhanced the

IAA synthesis under normal conditions, though; increase was higher with L-TRP under

non-stressed and PEG-induced water deficit conditions. However, among the isolates,

highest 1AA production was observed with isolate WL19 in the presence of substrate (L-

TRP) followed by L-TRP+ PEG, without L-TRP and PEG at day 3 and showed slight

decrease in IAA at day 6. Isolate WS11 also showed maximum IAA production at day 3

with L-TRP while IAA substantially decreased at day 5 and 6. However, minimum

accumulation was observed with isolate WR2 and WS11 at day 3 under PEG-induced

water deficit conditions.

4.5.1.15. Characterization of selected bacterial isolates for P solubilization under

normal and stressed conditions

Results showed that P- solubilization was increased with increasing time and

maximum P solubilization was observed at day 7. Selected isolates (WR2, WS11, and

WL19) efficiently enhanced P solubilization under normal (no-stresses) and PEG-6000

induced water deficit stress conditions (Fig. 4.15). Interestingly, greater P-solubilization

was observed under PEG-6000 induced water deficit stress with isolates. Among the

isolates, isolate WR2 gave maximum P solubilization followed by isolate WL19 and

WS11 under PEG-6000 induced water deficit stress than the normal conditions at day 7.

102

Page 123: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

WL19

1 2 3 4 5 60

10

20

30

40

50

60

70

80 Growth in L-TRP Growth without L-TRP PEG+L-TRP

PEG+without L-TRP

Days

IAA

pro

duct

ion

(μg

mL

-1)

WS11

1 2 3 4 5 60

5

10

15

20

25

30

35

40

Growth in L-TRP Growth without L-TRP PEG+L-TRPPEG+without L-TRP

Days

IAA

pro

duct

ion

(μg

mL

-1)

WR2

1 2 3 4 5 60

10

20

30

40

50

60

Growth in L-TRP Growth without L-TRP PEG+L-TRP

PEG+without L-TRP

Days

IAA

pro

duct

ion

(μg

mL

-1)

Fig. 4.14. IAA production of drought tolerant CA containing endophytic bacterial isolates with reference to time

103

Page 124: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

1 2 3 4 5 6 70

20

40

60

80

100

NBRIPNBRIP+PEG

Days

Tri

calc

ium

pho

spha

te so

lubi

lizat

ion

(µg/

ml) WL19

1 2 3 4 5 6 70

20

40

60

80

100

NBRIPNBRIP+PEG

Days

Tri

calc

ium

pho

spha

te so

lubi

lizat

ion

(µg/

ml) WS11

1 2 3 4 5 6 70

20

40

60

80

100

NBRIPNBRIP+PEG

Days

Tri

calc

ium

pho

spha

te so

lubi

lizat

ion

(µg/

ml) WR2

Fig. 4.15. P-solubilization of drought tolerant CA containing endophytic bacterial isolates with reference to time

104

Page 125: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.5.2. Study the selected Gus labeled endophytic bacterial isolates on maize

4.5.2.1. Plant height

In response to water deficit stress (40%), plant height of maize hybrids viz

tolerant (H1) and sensitive (H2) was significantly improved by the inoculation of drought

tolerant CA containing endophytic bacterial isolates compared to uninoculated control

plants (Fig. 4.16A). However, increase was more pronounced in H2 than H1 hybrid.

Inoculation with isolate MG9 significantly improved plant height by 14.8 and 18.5% in

H1 and H2 respectively, compared to respective control under well-watered conditions

(100%FC), though response was statistically similar in both (H1, H2) hybrids. Under

water deficit (70 and 40% FC) conditions, isolate MG9 showed 17.2 and 20.3% increase

for H1 and 20.5 and 25.8 % for H2, respectively, compared to respective control.

4.5.2.2. Root dry weight

A substantial reduction in root dry weight of both non inoculated plants of both

drought tolerant (H1) and sensitive (H2) hybrid of maize was observed under limited

water supply (Fig. 4.16B). Inoculation of drought tolerant CA containing endophytic

bacterial inoculants enhanced root dry weight in both hybrids, however, increase was

more for sensitive than for tolerant hybrid. In H2 hybrid, significant increase of 25.0, 27.3

and 34.4% was observed with isolate MG9 and that of 17.9, 25.8 and 30.7% in H1,

respectively, compared to corresponding control under non-stressed (100% FC) and under

water deficit conditions (70 and 40% FC).

4.5.2.3. Shoot dry weight

Shoot dry biomass of maize hybrids (H1 and H2) was increased both in the

presence as well as absence of drought tolerant CA producing endophytic bacterial

inoculants under different water deficit environment (Fig. 4.16C). Under normal growth

conditions (100% FC), plant of both hybrids inoculated with isolate MG9 showed similar

response for improving shoot dry weight. However, H2 plant also showed momentous

increase in shoot dry biomass in the presence of endophytic bacterial isolates (MR17,

MS1 and MG9) and increase was slightly greater than H1 at 70% FC. Moreover, 18.8

and 30.3% increase in shoot dry weight was observed for H1 and H2, respectively, with

isolate MR17 compared to their control plants under stressed condition (40% FC).

105

Page 126: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

H1 H2 H1 H2 H1 H2100% 70% 40%

40

60

80

100

120

140

160

180

efhi ij

kl

no

bcd de

gh

l m

bcd

e-g fg

k

n

a abcd de

jkkl

Control MR17 MS1 MG9

Field capacity levels

Pla

nt

hei

gh

t (c

m) A

H1 H2 H1 H2 H1 H2100% 70% 40%

0

5

10

15

20

25

30

35

cdgh

ij

o o

b cd-f

hij-l

n

bcd

fg e-g

mn l-n

a bc

de

jk k-m

Control MR17 MS1 MG9

Field capacity levels

Ro

ot

dry

wei

gh

t (

g)

B

H1 H2 H1 H2 H1 H2100% 70% 40%

40

60

80

100

120

effg

ghij

lm

abc cd

ef

jk jk

ab bc

d-f efh

k

a abbc

de

hijk

Control MR17 MS1 MG9

Field capacity levels

Sh

oo

t d

ry w

eig

ht

(g) C

Fig. 4.16. Effect of drought tolerant CA containing endophytic bacteria on plant height (A), root dry weight (B) and shoot dry weight (C) in both maize hybrids at different field capacity levels

106

Page 127: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.5.2.4. Carbonic anhydrase activity

Inoculation with drought tolerant CA containing endophytic bacterial inoculants

stimulated CA activity in the leaf of maize hybrids both in the presence and absence of

stressed conditions (Fig. 4.17A). Significant increase in CA activity was observed with

isolate MG9, however, isolate MR17 and MG9 showed varied response in both hybrids

under normal conditions (100/%FC). Inoculation of isolate MG9 caused 18.4%

improvement in H1 and 24.1% in H2 compared to control plants under mild water deficit

conditions (70% FC) compared to control plants. Similar to this, isolate MG9 also gave

highest CA activity under severe water deficit conditions (40% FC) in both hybrids

compared to control.

4.5.2.5. Photosynthetic rate

Isolates showing higher CA activity in leaves of maize hybrids, also caused

marked increase in photosynthetic rate compared to uninoculated control plants under

normal as well as stressed conditions (Fig 4.17B). Isolate (MR17, MS1, and MG9)

showed variable response for both hybrids compared to control plants under normal

growth conditions (100% FC). However, inoculation of H2 with isolate MG9

significantly increased the photosynthetic rate by 22.9 and 30.2% and by 19.1 and 28.9%

for H1under mild and severe stressed conditions (70 and 40% FC), respectively,

compared to control plant.

4.5.2.6. Transpiration rate

Drought tolerant CA containing endophytic bacterial isolates also facilitated the

transpiration under non-stressed (100%FC) and stressed (70%, 40% FC) conditions in

drought tolerant (H1) and drought sensitive (H2) maize hybrid in a pot conditions. Under

normal plant growth (100% FC) and mild stressed conditions (70% FC), isolates MR17

showed minimum increase whereas isolate MG9 gave maximum in both hybrids (H1 and

H2) compared to respective control plants. However, increase was greater for H2

compared to H1 hybrid (Fig. 4.17C). Under severe water deficit conditions (40% FC),

isolate MG9 increased the transpiration rate by 22.2 % for H2 and 26.4% for H1

compared to control plants.

107

Page 128: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

H1 H2 H1 H2 H1 H2100% 70% 40%

0

1.5

3

4.5

6

gh hi

jkl m

bfg fg g

ijl

cd ef cd

hk k

abc de f

h ij

Control MR17 MS1 MG9

Field capacity levels

Ca

rbo

nic

an

hy

dra

se a

ctiv

ity

(m

ol

CO

2 k

g-1

lea

f f

.w s

-1)

A

H1 H2 H1 H2 H1 H2100% 70% 40%

0

5

10

15

20

25

30

f-hh-j

j-ll-n

pqq

abd-g

g-i g-i

m-oop

b-e a-ce-g

i-k

o no

ab-d c-f e-g

k-mm-o

Control MR17 MS1 MG9

Field capacity levels

Ph

oto

syn

thet

ic r

ate

mo

l C

O2

m-2

s-1

)

B

H1 H2 H1 H2 H1 H2100% 70% 40%

0

2

4

6

8

e fgi

j

mo

bcd

ghi

kl lm

d cdfg h

kn

a abef gh

jk-m

Control MR17 MS1 MG9

Field capacity levels

Tra

nspi

rati

on r

ate

mm

ol H

2O m

-2 s

-1)

C

Fig. 4.17. Effect of drought tolerant CA containing endophytic bacteria on carbonic anhydrase activity (A), photosynthetic rate (B) and transpiration rate (C) in both maize hybrids at different field capacity levels

3.5.2.7. Stomatal conductance108

Page 129: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

The results regarding stomatal conductance showed that inoculation with drought

tolerant CA containing endophytic bacterial inoculants MR17, MS1 and MG9 facilitated

the stomatal conductance under non-stressed and stressed conditions (Fig. 4.18A).

However, increase was more for H2 compared to H1 hybrids. Under normal conditions

(100% FC), stomatal conductance enhanced by 18.5 and 31.2% in H1 and H2,

respectively, in comparison with non-inoculated control plants. At mild water stress (70%

FC), isolates also showed comparable differences but response varied among isolates in

both hybrids. However, under severe water stress conditions (40% FC), inoculation with

isolate MG9 showed 25.7% increase in H2 that was smaller than that in H1 (20.9%) in

comparison with control plants.

3.5.2.8. Relative water content (RWC)

Response of maize hybrids was monitored for RWC in the presence of drought

tolerant CA producing endophytic bacterial inoculant under different moisture regimes

(Fig. 4.18B). Under normal conditions (100% FC), H2 planta inoculated with isolate

MG9 had increased RWC by 28.6% and while 19.9% H1 compared to control

(uninoculated) plants. However, H2 plants also showed momentous increase in RWC in

the presence of endophytic bacterial isolates, though increase was slightly smaller than

that H1 plants in mild water stress (70% FC). Moreover, RWC was increased by 66.6%

for H2 and 38.8% for H1 with the inoculation of isolate MG9 under stressed condition

(40% FC) compared to their respective uninoculated plants.

4.5.2.9. Electrolyte leakage (EEL)

In response to mild and severe water deficit conditions (70 and 40% FC),

electrolyte leakage significantly enhanced in both maize hybrids (Fig. 4.18C). However,

inoculation with bacterial isolates (MR17, MS1 and MG9) reduced the electrolyte

leakage, more for H2 compared to H1 hybrid under non-stressed and stressed conditions.

Moreover, isolate MR17 significantly decreased the electrolyte production by 19.6%,

which was greater than H1 under mild stressed conditions (70% FC) compared to

untreated plant. Electrolyte leakage was markedly decreased (30.0%) by isolate MG9 for

H2 hybrid than for H1 hybrid (27.9%) under severe water deficit conditions (40% FC).

109

Page 130: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

H1 H2 H1 H2 H1 H2100% 70% 40%

0.00

0.20

0.40

0.60

cdf f

gjk

l

bc de

fgh

ij

b bc

f

hikl

ab

cde

gh-j

Control MR17 MS1 MG9

Field capacity levels

Sto

ma

tal

con

du

cta

nce

(m

ol

H2

O m

-2 s

-1) A

H1 H2 H1 H2 H1 H2100% 70% 40%

0

20

40

60

80

cd

g g

h-jk

l

bcd de

gh

ij

b

ef efg g

j

a

c cdf

ghi

Control MR17 MS1 MG9

Field capacity levels

Rel

ativ

e w

ater

con

tent

(%)

B

H1 H2 H1 H2 H1 H2100% 70% 40%

0

10

20

30

40

50

60

mnjk

f-h

cdb

a

nolm

i-kg-i

d

b

o

j-l h-jef

cb

om k-m

fg f-hde

Control MR17 MS1 MG9

Field capacity levels

Ele

ctro

lyte

lea

ka

ge

(%

)

C

Fig. 4.18. Effect of drought tolerant CA containing endophytic bacteria on stomatal conductance (A), relative water content (B) and electrolyte leakage (C) in both maize hybrids at different field capacity levels

110

Page 131: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.5.2.10. Proline content

Inoculation of both maize hybrids with drought tolerant CA containing endophytic

bacterial isolates did not reveal any considerable difference in proline content under

normal (100% FC) growth conditions, however, isolates helped plants to improve the

proline content under stressed (70 and 40% FC) conditions (Fig 4.19A). Isolate MG9

enhanced proline content in both hybrids under mild stress (70% FC) compared to other

isolates. Nevertheless, pronounced increase in proline accumulation was observed with

isolates MR17, MS1 and MG9 under severe water deficit conditions (40% FC) especially

in H2 than non-inoculated control plants.

4.5.2.11. Melanaldehyde (MDA) content

Content of MDA, indicator of lipid peroxidation, was increased with increasing

water deficit stress in both hybrids (Fig. 4.19B). Inoculation of isolates reduced MDA

content, however, reduction was marked for H2 compared to H1. Under normal

conditions (100% FC), isolate MS1 showed 15.0 and 22.6% reduction in MDA content of

H1 and H2, respectively, compared to control plants. Comparable differences for MDA

content was observed under mild water stress (70% FC) with bacterial inoculation.

However, inoculation of isolate MG9 significantly reduced MDA content by 19.6% in H1

and 23.9 % in H2 compared to uninoculated control plants under severe water stress (40%

FC).

4.5.2.12. Grain yield

Grain yield was substantially increased in the presence of drought tolerant CA

producing endophytic bacterial inoculant under non-stressed as well as stressed

conditions (Fig. 4.19C). Under normal conditions (100% FC), inoculation showed

different response for improving grain yield in both hybrids. However, H2 showed

significant increase (19.8%) in grain yield in the presence of endophytic bacterial isolates,

though increase was slightly greater than H1 at 70% FC. Grain yield was also increased

by 21.5 % for H2 and 38.0% for H1 compared to their respective non-inoculated plants

under non-stressed (100% FC) as well as stressed (70 and 40% FC) conditions.

Inoculation of maize hybrid H2 with isolate H1 with isolate MG9 under stressed

condition (40% FC) compared to respective control.

111

Page 132: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

H1 H2 H1 H2 H1 H2100% 70% 40%

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

ijk

fh

ce

i

k

f-h gh

b

cd

ijjk

degh

b

de

ik

defg

a

c

Control MR17 MS1 MG9

Field capacity levels

Pro

lin

e co

nte

nt

(µm

ol

g-1

)

A

H1 H2 H1 H2 H1 H2100% 70% 40%

0

20

40

60

80

mnjk

ghe

d

a

nkl

efhi

efd

o no

ijgh

el

b

om lm

jkfg

de

Control MR17 MS1 MG9

Field capacity levels

Mel

an

ald

ehy

de

con

ten

t

mo

l g

-1)

b

B

H1 H2 H1 H2 H1 H2100% 70% 40%

0

20

40

60

80

100

cd

efg

hij

l

bc

e

gh

j

a

d

fg g

k

a

c

ef

ghij

Control MR17 MS1 MG9

Field capacity levels

G

rain

yie

ld (g

pot

-1)

C

Fig. 4.19. Effect of drought tolerant CA containing endophytic bacteria on proline content (A), melanaldehyde content (B) and grain yield (C) in both maize hybrids at different field capacity levels

112

Page 133: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.5.2.13. Colonization of plant tissues

The data present in figure. 4.20 showed that colonization efficiency of Gus

labelled drought tolerant CA containing endophytic bacterial isolates (MR17, MS1, MG9)

decreased from root to leaf tissues in both maize hybrids under non-stressed as well as

stressed conditions, although, colonization was slightly greater for drought tolerant hybrid

(H1) than sensitive (H1). Furthermore, colonization efficiency by isolates decreased with

increasing stress i.e. mild (70% FC) and severe (40% FC) water stress compared to

normal growth condition (100% FC). Maximum CFU was detected in root tissues with

isolate MR17 and MG9 for H1 than H2 under non-stressed as well as stressed conditions.

Isolate MR17 efficiently colonized the shoot tissue followed by MS1 and MG9 in H1

under different water regimes (100, 70 and 40% FC). On the other hand, all the three

isolates showed similar response for colonizing the shoot tissues except for MG9 which

showed highest CFU at 40% FC. Smaller CFU of isolates was found in leaf tissues of

both hybrids. Among the isolates, isolate MG9 efficiently colonized the leaf tissue of H1

than H2 at 100, 70 and 40% FC.

4.5.2.14. Characterization of selected bacterial isolates for IAA production under

normal and stressed conditions

The results regarding IAA production by drought tolerant CA containing

endophytic bacterial isolates (MR17, MS1 and MG9) showed that IAA production

increased with increasing time in the presence of L-TRP under non-stressed as well as

PEG-6000 induced water deficit stress (Fig. 4.21). Maximum IAA synthesis was

observed at day 3. All the isolates (MR17, MS1and MG9) enhanced the IAA synthesis

under normal growth conditions and PEG-6000 induced water deficit stress with L-TRP.

Highest 1AA production was observed with isolate MG9 in the presence of substrate (L-

TRP) followed by L-TRP+ PEG, without PEG and PEG at day 3. Isolate MS1also

showed maximum IAA production at day 3 in the presence of substrate (L-TRP).

4.5.1.15. Characterization of selected bacterial isolates for P solubilization under

normal and stressed conditions

Drought tolerant CA containing endophytic bacterial isolates interestingly

enhanced P-solubilization under PEG-induced water deficit stress (Fig. 4.22). Isolates

MG9 and MR17 showed higher P- solubilization under stressed conditions than non-

stressed conditions at day 6 and 7, indicating the ability of isolates to solubilize P under

113

Page 134: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

100% 70% 40% 100% 70% 40%H1 H2

0E+00

1E+05

2E+05

3E+05

4E+05

5E+05

6E+05

7E+05

8E+05

9E+05 MR17 MS1 MG9

Root tissues

CF

U/g

fre

sh b

iom

ass

A

100% 70% 40% 100% 70% 40%H1 H2

0E+00

1E+03

2E+03

3E+03

4E+03

5E+03

6E+03

7E+03

8E+03 MR17 MS1 MG9

Shoot tissues

CF

U/g

fre

sh b

iom

ass

B

100% 70% 40% 100% 70% 40%H1 H2

0E+00

1E+03

2E+03

MR17 MS1 MG9

Leaf tissues

CF

U/g

fres

h bi

omas

s

C

Fig. 4.20. Colonization of root (A), shoot (B) and leaf (C) tissues with drought tolerant CA containing endophytic bacteria in both maize hybrids at different field capacity levels

114

Page 135: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

MR17

1 2 3 4 5 60

10

20

30

40

50

60

70Growth in L-TRP Growth without L-TRP PEG+L-TRP

PEG+without L-TRP

Days

IAA

pro

du

ctio

n (

μg

mL

-1)

MS1

1 2 3 4 5 60

5

10

15

20

25

30

35Growth in L-TRP Growth without L-TRP PEG+L-TRPPEG+without L-TRP

Days

IAA

pro

duc

tio

n (

μg

mL

-1)

MG9

1 2 3 4 5 60

5

10

15

20

25

30

35

40Growth in L-TRP Growth without L-TRP PEG+L-TRPPEG+without L-TRP

Days

IAA

pro

duc

tion

(μg

mL

-1)

Fig. 4.21. IAA production of drought tolerant CA containing endophytic bacterial isolates with reference to time

115

Page 136: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

1 2 3 4 5 6 70

20

40

60

80

100

NBRIPNBRIP+PEG

Days

Tri

calc

ium

pho

spha

te s

olub

iliz

atio

n (µ

g/m

l)

MR17

1 2 3 4 5 6 70

20

40

60

80

100

NBRIPNBRIP+PEG

Days

Tri

calc

ium

pho

spha

te so

lubi

lizat

ion

(µg/

ml)

MS1

1 2 3 4 5 6 70

20

40

60

80

100

NBRIP

NBRIP+PEG

Days

Tri

calc

ium

pho

spha

te so

lubi

lizat

ion

(µg/

ml)

MG9

Fig. 4.22. P-solubilization of drought tolerant CA containing endophytic bacterial isolates with reference to time

116

Page 137: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.6. Evaluation of selected endophytic bacterial isolates in field trial

Selected drought tolerant CA containing endophytic bacterial isolates (WR2,

WS11 and WL19 for wheat while MR17, MS1 and MG9 for maize) showed different

response for improving wheat (Uqab-2000) and maize (H2) growth under field condition.

Results are described as below.

4.6.1. Evaluation of selected endophytic bacterial isolates for wheat

4.6.1.1. Number of tillers

Inoculation of drought tolerant CA containing endophytic bacterial isolates (WR2,

WS11 and WL19) significantly improved number of tillers compared to uninoculated

control, under normal growth conditions and where water deficit stress was imposed by

holding irrigation at different development stages viz tillering, flowering and grain filling

(Fig. 4.23A). Withdrawal of irrigation at tillering significantly reduced the number of

tillers; however, isolate WL19 showed considerable increase (14.3%) in number of tillers

at tillering. On the other hand, isolate WR2 and WS11 performed better where water

deficit stress was imposed at flowering by holding irrigation. Moreover, isolate WL19

showed 13% increase in comparison with control (un-inoculated) plant under stressed

conditions where irrigation was hold on grain filling.

4.6.1.2. Carbonic anhydrase activity

Carbonic anhydrase activity in the leaves significantly enhanced with isolate

WL19 under non-stressed and stressed conditions where irrigation was hold on either

tillering, flowering or during grain filling phase (Fig. 4.23B). A considerable increase of

26.6 and 32.7% was observed with isolate WL19 where water deficit conditions were

imposed by cut-off irrigations at tillering or grain filling. However, isolate WS11 showed

pronounced enhancement in CA activity compared to uninoculated plants, where water

stress was induced at flowering.

4.6.1.3. Photosynthetic rate

Under field conditions, photosynthetic rate was markedly influenced when cutoff

the irrigation either at tillering or grain filling phase (Fig. 4.23C). Inoculation with

endophytic bacterial isolates caused considerable increase in photosynthetic rate where

wheat plants were normally irrigated or stressed conditions were induced at various

development stages of wheat crop. However, WL19 inoculated plant showed pronounced

117

Page 138: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling200

270

340

410

480

e

j

gf

ab

i

decdc

hi

d d

a

gh

e

bc

Control WR2 WS11 WL19

Nu

mb

er o

f ti

ller

s (m

-2) A

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling0

1

2

3

4

5

6

c

ghi

k

b

ef ef

j

b

fg

cd

ij

a

cd de

hi

Control WR2 WS11 WL19

CA

aci

tiv

ty

(mo

l C

O2

Kg

-1 l

eaf

FM

s-1

)

B

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling0

5

10

15

20

25

c

hg

j

b

gef

i

b

g

cd

i

a

fde

h

Control WR2 WS11 WL19

Pho

tosy

nthe

tic

rate

(μm

ol C

O2

m-2

s-1

)

C

Fig.4.23. Effect of drought tolerant CA containing endophytic bacteria on number of tillers (A), carbonic anhydrase activity (B) and photosynthetic rate (C) of wheat under water deficit stress

118

Page 139: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

increase in photosynthetic rate.

4.6.1.4. Transpiration rate

A considerable decline in transpiration rate was detected when water deficit stress

was applied by holding the irrigation at grain filling phase in comparison with unstressed

and stressed plants (withdrawal of irrigation at tillering or flowering) (Fig 4.24A).

However, inoculation of isolate WL19 improved the transpiration rate by 14.9% when

irrigation was cutoff at flowering and by 17.2% when water deficit stress was induced by

holding the irrigation at grain filling. Inoculation of isolates caused pronounced increase

in transpiration rate when stress was imposed by withdrawing the irrigation at tillering

compared to control plants.

4.6.1.5. Water use efficiency (WUE)

Reduced irrigation either at tillering, flowering or during grain filling phase

lowered the WUE with respect to plants raised under non-stressed growth conditions (Fig.

4.24B). However, minimum value of WUE was observed where irrigation was

withdrawal at tillering or cutoff at grain filling. Inoculation with drought tolerant

endophytic bacterial isolates did not show any significance difference for facilitating the

WUE under non-stressed and stressed conditions.

4.6.1.6. Grain yield

Grain yield declined significantly under stressed conditions when water deficit

stress was simulated at different development phases of wheat by withholding the

irrigation (Fig. 4.24C). However, inoculation with drought tolerant CA containing

endophytic bacterial isolates improved the grain yield under non-stressed as well as

stressed conditions (Skipping irrigation at tillering, flowering or grain filling). Under

normal condition, isolate WL19 showed significant increase in comparison with

uninoculated control plants. Moreover, isolate WL19 and WS11 also caused significant

improvement in grain yield where water deficit conditions were induced by withholding

irrigation at tillering or flowering compared to uninoculated control plants. However,

isolate WL19 showed 30% increase in comparison with control when cutoff irrigation at

grain filling.

119

Page 140: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling3

5

7

9

f

cde

i

e

bc

h

d

b b

g

d

ab

f

Control WR2 WS11 WL19

Tra

nsp

ira

tio

n r

ate

(mm

ol

H2

O m

-2 s

-1)

A

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling1.00

2.00

3.00

4.00

b

g

d

fg

a

e-g

c

ef

b

fg

c

e-g

a

e

cd

e

Control WR2 WS11 WL19

Wat

er u

se e

ffic

ienc

y (A

/E) B

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling0

1

2

3

4

5

c-e

hi f-ii

b

e-hde

e-g

bc

c-ebc

g-i

a

de

b-d

ef

Control WR2 WS11 WL19

Gra

in Y

ield

(M

g h

a-1

) C

Fig. 4.24. Effect of drought tolerant CA containing endophytic bacteria on transpiration rate (A), water use efficiency (B) and grain yield (C) of wheat under water deficit stress

120

Page 141: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.6.1.7. Catalase contents

A significant increase in catalase content was detected when water deficit stress

was induced by holding the irrigation at tillering stage or grain filling compared to

unstressed and stressed plants (irrigation skipped at flowering) (Fig 4.25A). However,

inoculation of isolate WL19 decreased the catalase when irrigation skipped at tillering

and when stress was induced by holding the irrigation during grain filling. Moreover,

inoculation of isolate WL19 also gave significant decrease in catalase content when

irrigation was stopped at flowering compared to uninoculated control plants.

4.6.1.8. Ascorbate peroxidase (APX) contents

Reduced irrigation either at tillering, flowering or during grain filling phases

increased the amount of ascorbate peroxidase with respect to plants raised under non-

stressed conditions (Fig 4.25B). Inoculation with endophytic bacterial isolates

significantly reduced ascorbate peroxidase compared to uninoculated control when plant

grown under normal growth conditions as well as stressed conditions. All the three

isolates (WR2, WS11 and WL19) reduced the ascorbate peroxidase content where

irrigation skipped at development phases (tillering or grain filling). However, significant

decrease in ascorbate peroxidase content was observed with isolate WL19 by reduced

irrigation at flowering stage compered to uninoculated control plants.

4.6.1.9. Glutathione reductase (GR) contents

Glutathione reductase contents were significantly increased under stressed

environment when water deficit stress was inuced at various phases viz tillering,

flowering or grain filling by withholding the irrigation (Fig. 4.25C). However, inoculation

with drought tolerant CA containing endophytic bacterial isolates reduced the glutathione

reductase content under stressed conditions (skipping irrigation either at tillering,

flowering or during grain filling with respect to uninoculated controls). Under normal

condition, inoculation did not give any significant decrease compared to uninoculated

control plants. However, inoculation of isolates WL19 and WS11 caused significant

reduction in glutathione reductase where water deficit conditions were induced by

withholding irrigation at tillering and flowering stage compared to uninoculated control

plants. However, isolate WS11showed significant decrease in comparison with control

when reduced the irrigation at grain filling.

121

Page 142: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling20

40

60

80

100

120

140

160

f

d

gh

a

fg

e

hi

bc

j

e

ij

ab

h

e

j

cd

Control WR2 WS11 WL19C

ata

lase

(µM

H2

O2

min

-1 m

g-1

pro

tein

)

A

Norm

al ...

Skip

ped

...

Skip

ped

...

Skip

ped

a...100

200

300

400

500

600

f

bc

a

g

c

d

b

h

cd

b

gh

c

e

b

Control WR2 WS11 WL19

Asc

orb

ate

Per

ox

ida

se(µ

M a

sco

rba

te m

in-1

mg

-1 p

rote

in) B

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling20

40

60

80

100

h

a

g

bc

hi

b

i

cd

h

f

h

f

hi

de

j

ef

Control WR2 WS11 WL19

Glu

thio

ne r

educ

tase

(µM

TN

B m

in-1

mg

-1 p

rote

in) C

Fig. 4.25. Effect of drought tolerant CA containing endophytic bacteria on catalase (A), ascorbate peroxidase (B) and glutathione reductase (C) of wheat under water deficit stress

122

Page 143: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.6.1.10. Total protein contents

Inoculation of drought tolerant CA containing endophytic bacterial isolates (WR2,

WS11 and WL19) significantly improved total protein contents compared to uninoculated

control, where water deficit stress was imposed by holding the irrigation at various

development stages (Fig. 4.26A). Withdrawal of irrigation at tillering or grain filling,

significantly reduced the total protein, however, inoculation of isolates (WR2, WS11 and

WL19) showed marked increase in total protein contents. On the other hand, isolate WR2

and WS11 performed better where water deficit stress was imposed at flowering by

skipping irrigation. Isolates depicted the pronounced increase incomparison to

uninoculated control plant under normal conditions

4.6.1.11. Total soluble sugars

Drought tolerant CA containing endophytic bacterial isolates improved total

soluble sugars in contrast to uninoculated control, where water deficit stress was imposed

by holding the irrigation at various development phases of wheat (Fig. 4.26B).

Withdrawal of irrigation at tillering or grain filling significantly reduced the total soluble

sugars, however, inoculation of isolates showed significant increase in total soluble

sugars. Under normal conditions and water deficit stress condition during flowering,

isolates also showed considerable increase compared to uninoculated control plant.

4.6.1.12. Total phenolic contents

Data regarding total phenolics showed that phenolic contents increased under

stressed conditions (holding the irrigation at tillering or grain filling) but inoculation of

drought tolerant CA containing endophytic bacterial isolates (WR2, WS11 and WL19)

significantly reduced total phenolics compared to uninoculated control, where water

deficit stress was imposed by holding the irrigation at tillering, flowering and grain filling

(Fig. 4.26C). Minimum phenolic contents were observed where plants were grown under

normal growth conditions and stress was imposed at flowering. Inoculation of isolate

WL19 caused marked decrease in phenolic contents where holding the irrigation during

tillering, flowering or grain filling.

123

Page 144: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling20

40

60

80

100

d

i

e

h

c

gh

b

f

a

f

b

g

b

h

d

f

Control WR2 WS11 WL19

To

tal

pro

tein

(u

g g

-1)

A

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling0

1

2

3

4

5

b

h

e

i

ab

ee

h

a

f

c

g

a

e

d

g

Control WR2 WS11 WL19

To

tal

solu

ble

su

ga

rs

(u

g g

-1)

B

Normal irrigation Skipped at tillering Skipped at flowering Skipped at grain filling100

150

200

250

300

350

400

g

c

a

d

hi

e

c

g

ij

ef

b

fg

j

g

d

h

Control WR2 WS11 WL19

To

tal

ph

en

oli

cs

(ug

g-1

)

C

Fig. 4.26. Effect of drought tolerant CA containing endophytic bacteria on total protein (A), total soluble sugars (B) and total phenolic content (C) of wheat under water deficit stress

124

Page 145: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.6.1.13. Grain nitrogen (%)

A significant reduction in nitrogen content was recorded when water deficit stress

was applied by holding the irrigation at tillering or grain filling in comparison with

unstressed and stressed plants (irrigation skipped at flowering) (Fig 4.27A). However,

inoculation of isolates WR2 and WL19 improved the grain nitrogen when cutoff the

irrigation during tillering or grain filling. Inoculation of isolate WL19 gave significant

increase in grain nitrogen where watering was stopped at grain filling compared to

uninoculated control plants.

4.6.1.14. Grain phosphorus (%)

Reduced irrigation during various developmental stages lowered grain phosphorus

with respect to plants raised under non-stressed growth conditions (Fig. 4.27B). However,

inoculation with drought tolerant CA containing endophytic bacterial isolates improved

grain phosphorus compared to control plants under normal as well as reduced irrigation at

different developmental phases. Moreover, inoculation of isolates performed statistically

similar for increasing the grain phosphorus when holding the irrigation during grain

filling.

4.6.1.15. Grain potassium (%)

Grain potassium decreased significantly when water deficit stress was mediated

during tillering, flowering or grain filling phases of wheat by withholding the irrigation

(Fig. 4.27C). However, inoculation with drought tolerant CA containing endophytic

bacterial isolates improved the grain potassium content under stressed conditions

(skipping irrigation at different developmental stages). Under normal condition,

inoculation of isolates WS11 and WL19 showed marked increase in grain potassium in

comparion to respective uninoculated control plants. In the same way, isolate WR2,

WS11 and WL19 also caused significant increase in grain potassium where water deficit

conditions were induced by holding the irrigation during tillering or grain filling in

comparison with uninoculated control plants. Isolate WL19 also showed significant

increase in grain potassium content in contrast to respective non-inoculated control plant

where cutoff the irrigation at flowering.

125

Page 146: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Nor

mal

i...

Skip

ped at..

.

Skip

ped a...

Skip

ped at .

..0.5

1

1.5

2

2.5

c

hi

fg

i

b

g

cd

gh

b

e de

fg

a

ef

bc

fg

Control WR2 WS11 WL19

Gra

in n

itro

gen

(%

)

A

Nor

mal

i...

Skip

ped

a...

Skip

ped

a...

Skip

ped

at ...

0.1

0.2

0.3

0.4

0.5

0.6

0.7

d

hi i

k

b

efg

j

c

fg fg

j

a

f f

j

Control WR2 WS11 WL19

Gra

in P

ho

sph

oru

s (%

)

B

Norm

al...

Skip

ped...

Skip

ped.

..

Skip

ped ...0.1

0.15

0.2

0.25

0.3

0.35

b-d

hi

fg

i

b

gh

c-e

gh

a

gh

d-f

hi

a

e-g

bc

g

Control WR2 WS11 WL19

Gra

in p

ota

ssiu

m (

%)

C

Fig. 4.27. Effect of drought tolerant CA containing endophytic bacteria on grain nitrogen (A), phosphorus (B) and potassium (C) of wheat under water deficit stress

126

Page 147: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.6.2. Evaluation of selected endophytic bacterial isolates for maize

4.6.2.1. Number of grains per cob

A marked enhancement in the number (No.) of grains was observed by the

inoculation of drought tolerant CA containing endophytic bacterial isolates (MR17, MS1

and MG9) compared to uninoculated control plants in the presence of water deficit stress

condition which was imposed by holding irrigation at vegetative stage (Fig. 4.28A).

Inoculation with isolate MG9 improved the grain per cob by 27.2% when cutoff the

irrigation at vegetative stage and by 26.8% when stressed conditions were induced by

holding the irrigation at reproductive stage compared to respective uninoculated control

plants. Inoculation of isolate MG9 gave marked increase in grains also where plants were

normally irrigated compared to non-inoculated control plants.

4.6.2.2. Carbonic anhydrase activity

Inoculation improved the CA activity under non-stressed and stressed conditions

(skipped irrigation at vegetative stage or reproductive stage) in comaprison with

respective controls (Fig. 4.28B). However, overall marked reduction in CA activity was

observed when irrigation skipped at reproductive stage. Under normal condition, all the

endophytic bacterial isolates showed marked increase in comparison with respective

uninoculated control plants. In the same way, all the isolates also showed marked increase

in CA activity under stressed conditions. Isolate MG9 caused significant improvement

(25.1%) in CA activity where water deficit conditions were induced by withholding

irrigation at vegetative and 34.2% at reproductive stage compared to non-inoculated

control plants.

4.6.2.3. Photosynthetic rate

Water deficit conditions had strong effect on CA activity and photosynthesis.

Photosynthetic rate decreased under water deficit conditions (where holding the irrigation

at vegetative stage or reproductive stage). Nevertheless, inoculation with isolate MG9

significantly enhanced photosynthetic rate under non-stressed as well as stressed

conditions where irrigation was skipped at vegetative or reproductive stage (Fig. 4.28C).

A considerable increase of 23.7% was observed with isolate MG9 where water deficit

conditions were imposed by holding irrigation at reproductive stage. On the other hand,

all the isolates showed significant increase in

127

Page 148: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Normal irrigation Skipped at vegetative stage

Skipped at reproductive stage

100

200

300

400

500

c-ef-h

j

c cd

ef

c

c-eij

a

b

c-e

Control MR17 MS1 MG9

( N

um

ber o

f g

ra

ins

per c

ob

)

A

Normal irrigation Skipped at vegetative stage

Skipped at reproductive stage

0

2

4

6

8

ce

h

b

d

ef

b

d

g

a

c

f

Control MR17 MS1 MG9

Ca

rb

on

ic a

nh

yd

ra

se a

cit

ivty

(mo

l C

O2

kg

-1 l

ea

f fw

s-1

)

B

Normal irrigation Skipped at vegetative stage Skipped at reproductive stage

0

5

10

15

20

25

30

35

40

c-e

gh

k

b

ef

j

bcc-e

j

a

d-e

g-i

Control MR17 MS1 MG9

Ph

oto

syn

thet

ic r

ate

(μm

ol

CO

2 m

-2 s

-1)

C

Fig. 4.28. Effect of drought tolerant CA containing endophytic bacteria on no. of grains per cob (A), carbonic anhydrase activity (B) and photosynthetic rate (C) of maize under water deficit stress

128

Page 149: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

photosynthesis in comparison with respective uninoculated control plants, where

withdrawaing the irrigation at vegetative stage. Under normal condition, isolates MG9

and MR17 showed significant increase in comparison with respective uninoculated

control plants.

4.6.2.4. Transpiration rate

Inoculation with drought tolerant CA containing endophytic bacterial isolates

significantly improved transpiration rate compared to uninoculated control, where water

deficit stress was imposed by holding the irrigation during vegetative or reproductive

stage (Fig. 4.29A). Irrigation withdrawal at reproductive stage significantly reduced the

transpiration rate; however, isolate MS1 and MG9 showed pronounced increase in

transpiration rate. Under normal conditions and water deficit stress condition during

vegetative stage, all the endophytic bacterial isolates showed considerable enhancement

in transpiration rate compared to control (uninoculated) plant.

4.6.2.5. Water use efficiency (WUE)

Water stress at vegetative or reproductive stage significantly reduced the water

used efficiency as shown in figure 4.29B. Inoculation of drought tolerant CA containing

endophytic bacterial isolates did not show any statistically significant difference for

facilitating the WUE under non-stressed as well as stressed conditions where water deficit

stress was imposed by cutoff the irrigation during vegetative or reproductive stage.

However, inoculation of isolate MG9 showed maximum increase in WUE under normal

condition with respect to non-inoculated control plant.

4.6.2.6. Grain yield

Grain yield was considerably decreased under skipped irrigation at vegetative and

reproductive stage (Fig. 4.29C). However, inoculation with drought tolerant CA

containing endophytic bacterial isolates (MR17, MS1and MG9) improved the grain yield

under non-stressed and stressed conditions (vegetative and reproductive stage). Under

normal condition, isolate MG9 showed significant increase in comparison with respective

uninoculated control plants. Similarly, isolate MG9 caused significant enhancement

(32.6%) in grain yield where water deficit conditions were induced by holding irrigation

at reproductive stage compared to uninoculated control plants. All the isolates did not

show any significant differences where irrigation was withhold at vegetative stage

although they did increase the grain yield with respect to their non-inoculated control.

129

Page 150: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Normal irrigation Skipped at vegetative stage Skipped at reproductive stage

0

2

4

6

8

10

12

c-fgh

l

ab

ce

k

abc

j

a

c-f

g-i

Control MR17 MS1 MG9

Tra

nsp

ira

tio

n r

ate

(mm

ol

H2

O m

-2 s

-1)

A

Normal irrigation Skipped at vegetative stage

Skipped at reproductive stage

2

3

4

5

bc-e

ef

abb-d

de

b bc

g

abc

e-g

Control MR17 MS1 MG9

Wa

ter

use

eff

icie

ncy

(A

/E)

B

Normal irrigation Skipped at vegetative stage Skipped at reproductive stage0

3

6

9

13

c

e

g

ab

c

e

bc

f

a

c

e

Control MR17 MS1 MG9

( Gra

in y

ield

( to

ns h

a-1)

C

Fig. 4.29. Effect of drought tolerant CA containing endophytic bacteria on transpiration rate (A), water use efficiency (B) and grain yield (C) of maize under water deficit stress

130

Page 151: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.6.2.7. Catalase contents

A marked decrease in catalase was observed by the inoculation of drought tolerant

CA containing endophytic bacterial isolates (MR17, MS1and MG9) in the presence of

water deficit stress conditions which were imposed by holding the irrigation at both

developmental stages (vegetative or reproductive) compared to unstressed (Fig. 4.30A).

Inoculation with isolate MG9 resulted in decrease in amount of catalase when holding the

irrigation at vegetative stage and when water stressed conditions were induced by cutoff

the irrigation at reproductive stage in comparison with respective uninoculated control

plants. Inoculation with isolates did not give significant decrease where plants were

normally irrigated compared to uninoculated control plants.

4.6.2.8. Ascorbate peroxidase (APX) contents

Change in ascorbate peroxidase content was detected under water deficit

conditions by skipping irrigation at vegetative stage and reproductive stage (Fig. 4.30B).

However, marked increase in ascorbate peroxidase was observed when irrigation skipped

at reproductive stage. Inoculation with drought tolerant CA containing endophytic

bacterial isolates reduced the ascorbate peroxidase under non-stressed and stressed

conditions (skipping irrigation at vegetative and reproductive stage). Under normal

condition, isolate MS1 did not show significant decrease compared to respective

uninoculated control plants. However, isolate MS1 and MG9 caused significant reduction

in ascorbate peroxidase content where water deficit conditions were induced by

withholding irrigation at vegetative and at reproductive stage compared to uninoculated

control plants.

4.6.2.9. Glutathione reductase (GR) contents

Water deficit conditions had strong effect on glutathione reductase. Glutathione

reductase increased under water deficit conditions where irrigation was withhold at

vegetative or reproductive stage. However, glutathione reductase was significantly

reduced with isolate MG9 under non-stressed and stressed conditions where irrigation

was skipped at vegetative or reproductive stage (Fig. 4.30C). A considerable decrease in

GR content was recorded with isolate MR17 and MG9 where water deficit conditions

were imposed by holding irrigation at reproductive stage.

131

Page 152: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Normal irrigation Skipped at vegetative stage

Skipped at reproductive stage

50

100

150

de

c

a

de

d

c

de

d

b

e

d

c

Control MR17 MS1 MG9

Ca

tala

se

M H

2O

2 m

in-1

mg

--1

pro

tein

)

A

Normal irrigation Skipped at vegetative stage

Skipped at reproductive stage

20

40

60

80

100

120

e

bc

a

f

d

b

ef

d

c

f

d

c

Control MR17 MS1 MG9

Asc

orb

ate

pero

xid

ase

M a

sco

rb

ate

min

-1 m

g-1

pro

tein

)

B

Normal irrigation Skipped at vegetative stage

Skipped at reproductive stage

30.00

60.00

90.00

120.00

e

c

a

ef

dc

ef

d

b

f

d

c

Control MR17 MS1 MG9

Glu

tath

ion

e r

ed

ucta

se

M T

NB

min

-1 m

g-1

pro

tein

)

C

Fig. 4.30. Effect of drought tolerant CA containing endophytic bacteria on catalase (A), ascorbate peroxidase (B) and glutathione reductase (C) content of maize under water deficit stress

132

Page 153: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.6.2.10. Total protein contents

Inoculation with drought tolerant CA containing endophytic bacterial isolates

significantly improved total protein contents compared to uninoculated control under

normal as well as water deficit stress generated by withholding irrigation at vegetative or

reproductive stage (Fig. 4.31A). Irrigation withdrawal at reproductive stage, significantly

reduced the total protein, however, isolate MG9 showed significant increase in total

protein. Under non-stressed and water deficit stress conditions during vegetative stage

isolate MG9 showed considerable increase in comparison with uninoculated control plant.

Moreover, isolates MR17 and MS1 also performed better where plants were normally

irrigated and water deficit stress conditions imposed by holding irrigation at vegetative

stage in comparison with respective control plants.

4.6.2.11. Total soluble sugars

Inoculation caused significant difference in total soluble sugars under water deficit

conditions where water deficit stress was induced by withholding irrigation at

developmental stages (vegetative, reproductive) (Fig. 4.31B). Water deficit conditions at

vegetative and reproductive phases enhanced the total soluble sugars with respect to

plants raised under non-stressed conditions. However, inoculation with isolates also

enhanced total soluble sugars under stressed conditions by withholding irrigation at

vegetative as well as reproductive stage.

4.6.2.12 Total phenolic contents

Total phenolics were considerably increased under skipped irrigation at

developmental stages (vegetative and reproductive) (Fig. 4.31C). However, inoculation

with drought tolerant CA containing endophytic bacterial isolates (MR17, MS1or MG9)

decreased the total phenolics under non-stressed as well as stressed conditions (vegetative

or reproductive phases).Under normal condition, isolates did not show significant

decrease compared to respective control (uninoculated) plants. Contrarily, isolate MG9

caused significant reduction in total phenolics where water deficit conditions were

induced by withholding irrigation at reproductive stage compared to uninoculated control

plants. Moreover, all the isolates performed statistically similar where irrigation was

withhold at vegetative stage although isolate did decrease the total phenolics compared to

uninoculated control.

133

Page 154: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Normal irrigation Skipped at vegetative stage Skipped at reproductive stage

0

20

40

60

80

d

g

j

c

f

i

b

e

i

a

e

h

Control MR17 MS1 MG9

To

tal

Pro

tein

(u

g g

-1) A

Normal irrigation Skipped at vegetative stage

Skipped at reproductive stage

0.00

0.50

1.00

1.50

2.00

2.50

h

fg

d

gh

ef

b

g

dec

g

d

a

Control MR17 MS1 MG9

To

tal

So

lub

le s

ug

ars

(u

g g

-1)

B

Normal irrigation Skipped at vegetative stage Skipped at reproductive stage

30

50

70

90

110

130

150

e

bc

a

e

d

b

e

d

b

e

d d

Control MR17 MS1 MG9

To

tal

ph

eno

lics

(u

g g

-1)

C

Fig. 4.31. Effect of drought tolerant CA containing endophytic bacteria total protein contents (A), total soluble sugars (B) and total phenolic contents (C) of maize under water deficit stress

134

Page 155: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.6.2.13. Grain nitrogen (%)

Statstically significant increase in grain nitrogen was recorded by the inoculation

of drought tolerant CA containing endophytic bacterial isolates (MR17, MS1 and MG9)

in the presence of water deficit stress conditions which was imposed by holding the

irrigation during vegetative or reproductive stage compared to unstressed conditions (Fig

4.32A). Inoculation of isolate MS1 improved the grain nitrogen by 19.3% when holding

the irrigation at vegetative stage and by 15.4% when stressed conditions were induced by

reducing irrigation at reproductive stage compared to respective uninoculated control

plants. Inoculation of isolates also gave marked increase in grain nitrogen where plants

were normally irrigated compared to uninoculated control plants.

4.6.2.14. Grain phosphorus (%)

Change in phosphorus content was observed under water deficit conditions by

skipping irrigation at vegetative stage and reproductive stage (Fig. 4.32B). However,

marked reduction in phosphorus content was observed when irrigation cutoff at

reproductive stage. Inoculation of drought tolerant CA containing endophytic bacterial

isolates improved the phosphorus content under non-stressed as well as stressed

conditions (skipping irrigation during vegetative or reproductive phases). Under normal

condition, isolates MG9 and MS1 showed significant increase compared to their

respective control (uninoculated) plants. Similarly, isolate MG9 also caused significant

improvement (12.2%) in grain phosphorus where water deficit conditions were induced

by withholding irrigation at vegetative and 16.5% at reproductive stage incomparison

with uninoculated control plants.

4.6.2.15. Grain potassium (%)

Water deficit conditions caused considerable reduction in grain potassium

concentration. Grain potassium content decreased with water deficit conditions. However,

inoculation with drought tolerant CA producing bacterial isolates improved its value but

remained statistically at par with non-inoculated control plants in improving the grain

potassium content under normal as well as stressed conditions where holding irrigation at

developmental stages (vegetative or reproductive) (Fig.4.32C). A considerable increase

was observed with isolate MG9 where water deficit conditions were imposed by holding

irrigation at reproductive stage.

135

Page 156: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Norm

al ir

riga

tion

Skip

ped

at v

eget

ativ

e stag

e

Skip

ped

at re

prod

uctive

sta

ge

1

1.5

2

2.5

3

3.5

d

h hi

c

g cd

b

ef-g

a

eff-g

Control MR17 MS1 MG9

Gra

in n

itro

gen

(%

)

A

Norm

al ir

rigat

ion

Skip

ped

at v

eget

ativ

e stag

e

Skip

ped

at re

prod

uctiv

e stag

e

0

0.2

0.4

0.6

0.8

cf

i

bde

g

b

e

h

a

cd

g

Control MR17 MS1 MG9

Gra

in p

ho

sph

oru

s(%

)

B

Normal irrigation Skipped at vegetative stage

Skipped at reproductive stage

0.5

1

1.5

2

bc

ef

h

a

de

g

ab

e

g

a

cd

fg

Control MR17 MS1 MG9

Gra

in p

ota

ssiu

m(%

)

C

Fig. 4.32. Effect of drought tolerant CA containing endophytic bacteria on grain nitrogen (A), phosphorus (B) and potassium (C) of maize under water deficit stress

136

Page 157: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.7. Evaluation of potential endophytic bacterial isolates for gene expression in

Arabidopsis thaliana under PEG-induced water deficit conditions

4.7.1. Screening of endophytic bacterial isolates based on drought tolerance ability,

CA activity and plant growth promotion

One hundred and fifty morphotypes of endophytic bacteria were isolated from

Arabidopsis thaliana and screened for PEG-induced water deficit stress as well as CA

activity at Plant Microbe Interaction Laboratory, The University of Queensland,

Australia. Ten drought tolerant and CA containing endophytic bacterial isolates were

screened for plant growth promotion (data are not shown here). Two drought tolerant CA

producing plant growth facilitating isolates were selected for plant growth promotion and

gene expression study.

4.7.2. Effect of endophytic bacterial isolates on plant growth of Arabidopsis thaliana

4.7.2.1. Root length

Under PEG-induced water deficit conditions, root growth of Arabidopsis thaliana

was decreased significantly compared to uninoculated control plants (Fig. 4.33A).

However, inoculation with drought tolerant CA containing endophytic bacterial isolates

(AR4 and AR14) improved the root length under normal (0% PEG) as well as stressed

conditions (3% PEG). Significant increase of 30.7% was observed with isolate AR4 under

PEG-mediated stress conditions compared to uninoculated control plants.

4.7.2.2. Number of lateral roots

Number of lateral roots was significantly higher in plant inoculated with AR4 and

AR14 than the non-inoculated control plants under normal (0%) as well as PEG induced

water deficit conditions (3%) (Fig. 4.33B). A positive effect on no. of laterals roots (30.6

and 58.2%) were observed in plants inoculated with isolate AR14 compared to non-

inoculated plants under normal as well as stressed conditions.

4.7.2.3. Root fresh weight

Inoculation with drought tolerant CA containing endophytic bacterial isolates

improved the root fresh weight under normal (0% PEG) as well as stressed conditions

(3% PEG) (Fig. 4.33C). Inoculated plants showed 24.9 and 36.9% increase over control

plants under normal conditions. Under PEG-induced water deficit conditions, root fresh

weight was considerably increased in inoculated plants compared to uninoculated control

137

Page 158: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Picture 14: Effect of drought tolerant CA containing endophytic bacteria on Arabidopsis thaliana growth under normal conditions

Picture 15: Effect of drought tolerant CA containing endophytic bacteria on Arabidopsis thaliana growth under PEG-induced water deficit conditions

138

Control AR14

Control AR4

Page 159: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Control AR4 AR140

1

2

3

4

5

6

7

8

90% PEG 3% PEG

Endophytic bacteria

Root

length

(cm)

A

Control AR4 AR140

1

2

3

4

5

6

7

8

9

100% PEG 3% PEG

Endophytic bacteria

Root f

resh w

eight (

mg)

C

Control AR4 AR140

5

10

15

20

250% PEG 3% PEG

Endophytic bacteria

Numb

er of l

ateral

roots

B

Control AR4 AR140

5

10

15

20

25

30

0% PEG 3% PEG

Endophytic bacteria

Shoo

t fresh

weigh

t (mg)

D

Fig. 4.33. Effect of drought tolerant CA containing endophytic bacterial isolates (AR4, AR14) on root length (A) number of lateral roots (B), root fresh weight (C) and shoot fresh weight (D) in Arabidopsis thaliana under normal (0%) as well as PEG-mediated water deficit conditions (3%)

139

Page 160: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

plants. Root fresh weight was increased by 75.7% in arabidopsis plants treated with AR14

on exposure to PEG-6000 induced water deficit stress.

4.7.2.4. Shoot fresh weight

Shoot fresh weight of Arbaidopsis thaliana decreased significantly under stressed

environment (Fig. 4.33D). However, inoculation of drought tolerant CA containing

endophytic bacterial isolates (AR4, AR14) facilitated the shoot fresh weight compared to

non-inoculated control plants under non-stressed as well as stressed conditions. Plants

inoculated with isolate AR14 showed significant increase in shoot fresh weight under

PEG-induced water deficit stress compared to non-inoculated plants.

4.7.3. Effect of selected isolates on gene expression and transcriptional response of

Arabidopsis thaliana

4.7.3.1. Expression pattern of dehydration responsive protein (RD22)

To explore whether difference in drought tolerance could be due to RD 22,

expression of gene encoding RD22 was followed. Results showed that inoculation of

isolates (AR4 and AR14) modified the expression of RD22 under PEG-mediated water

deficit stress (Fig. 4.34 A). The expression level of RD22 was downregulated in plant

inoculated with isolates AR4 and AR14 under water stress (3% PEG) in comparison with

control.

4.7.3.2. Expression pattern of dehydration responsive element (RD29B)

The result regarding drought tolerance induced by RD29B showed that expression

of RD29B was enhanced by plant inoculated with isolates AR4 and AR14 compared to

control (uninoculated) plants under non-stressed (0%) as well as PEG-mediated water

stress (3%) (Fig.4. 34B). Overexpression of RD29B was detected with the inoculation of

AR4 compared to non-inoculated control after PEG treatment.

4.7.3.3. Expression pattern of late embryogenesis (LEA)

Overexpression of LEA protein was observed in both inoculated and non-

inoculated plant under PEG-imposed water deficit stress (Fig. 4.34C). The results of RT-

PCR analysis clearly showed increased expression of LEA in plant treated with AR4 and

AR14 compared to non-inoculated plants.

140

Page 161: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Control AR4 AR140

2

4

60% PEG 3% PEG

Endophytic bacteria

Relat

ive fol

d chan

geRD22 A

Control AR4 AR140

400

800

1200

1600

20000% PEG 3% PEG

Endophytic bacteria

Relativ

e fold c

hange

LEA C

Control AR4 AR140

2

4

6

8 0% PEG 3% PEG

Endophytic bacteria

Relativ

e fold c

hange

RD29B B

Control AR4 AR140

20

40

60 0% PEG 3% PEG

Endophytic bacteria

Relativ

e fold c

hange

RAB18 D

Fig. 4.34. Effect of drought tolerant CA containing endophytic bacterial isolates (AR4, AR14) on stress responsive genes RD22 (A), RD29B (B), LEA (C) and RAB18 (D) in Arabidopsis thaliana under normal (0%) as well as PEG-mediated water deficit conditions (3%)

141

Page 162: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.7.3.4. Expression pattern of dehydrin (RAB18)

Expression level of representative gene RAB18 was the highest in inoculated plan

compared to uninoculated plants under PEG-mediated water deficit stress (Fig. 4.34D).

Dehydrin RAB18 also differently expressed in inoculated and non-inoculated plants

under normal condition.

4.7.3.5. Expression of dehydration-response element binding protein 2A (DREB2A)

In exposure to PEG induced water deficit conditions, transcripts level of DREB2A

enhanced in both inoculated as well as uninoculated control plants (Fig. 4.35A).

Inoculation with drought tolerant CA containing endophytic bacterial isolates modified

the expression of DREB2A. Increased transcript level was observed in Arabidopsis

thaliana plant inoculated with AR4 and AR14 compared to non-inoculated under PEG-

mediated water deficit condition.

4.7.3.6. Expression of defense related gene (PR1.2.)

Results regarding defense related gene showed that PR1.2. gene differentially

expressed when inoculated with bacterial isolates (AR4 and AR14) under non-stressed

growth conditions (Fig. 4.35B). Under PEG-mediated water deficit stress (3%),

expression level of PR1.2. was also upregulated in plant treated with isolates (AR4 and

AR14), however, effect was smaller under normal conditions.

4.7.3.7. Expression of WRKY57 transcription factors

The analysis of RT-PCR showed that WRKY57 gene was not differentially

expressed in control as well as in plant treated with drought tolerant CA containing

endophytic bacterial isolates (AR4 and AR14) under normal growth conditions as shown

in figure 4.35C. However, expression of drought responsive gene WRKY57 was down

regulated in plant treated with AR4 and AR14 in comparison with uninoculated control

plants under PEG- imposed water stressed conditions.

4.7.3.8. Expression of WRKY8 transcription factors

Under normal growth conditions (0% PEG), constitutive expression of WRKY8

transcription factors were upregulated by the inoculation of drought tolerant CA

producing endophytic bacterial isolates (AR4 and AR14) compared to control

(uninoculated) plants (Fig. 4.35D). In the same way, in exposure to water stressed

142

Page 163: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Control AR4 AR140

50

100

1500% PEG 3% PEG

Endophytic bacteria

Relat

ive fo

ld cha

nge

DREB2A A

Control AR4 AR140

20

40

60

800% PEG 3% PEG

Endophytic bacteria

Relativ

e fold c

hange

WRKY 57 C

Control AR4 AR140

2

4

6

8

10 0% PEG 3% PEG

Endophytic bacteria

Relativ

e fold c

hange

PR1.2 B

Control AR4 AR140

5

10

15

20 0% PEG 3% PEG

Endophytic bacteria

Relativ

e fold c

hange

WRKY 8 D

Fig. 4.35. Effect of drought tolerant CA containing endophytic bacterial isolates (AR4, AR14) on DREB2A (A) PR1.2 (B) WRKY57 (C) and WRKY 8 (D) in Arabidopsis thaliana under normal (0%) as well as PEG-mediated water deficit conditions (3%)

143

Page 164: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

conditions, inoculation of isolates (AR4 and AR14) showed overexpression of WRKY8

in comparison with untreated plants.

4.7.3.9. Expression pattern of C2H2–Zinc finger protein (Zat 10)

Transcripts of Zat 10 were significantly deceased in plant assisted with AR14

compared to uninoculated control plants under normal growth conditions (0% PEG) (Fig.

4.36A). However, Zat 10 transcripts were elevated in leaves of non-inoculated control

plants on exposure to PEG-induced water deficit stress. Moreover, expression of Zat 10

was downregulated in plant inoculated with isolates (AR4 and AR14) and this effect was

more pronounced in plant treated with isolate AR4 under PEG-induced water deficit

stress (3%).

4.7.3.10. Expression pattern of dehydrins (COR47)

The result regarding drought tolerance induced by COR 47 showed that

expression level of COR47 dehydrins was enhanced in plant inoculated with AR4 and

AR14 compared to uninoculated control plants under non-stressed (0%) as well as PEG-

mediated water stress (3%) (Fig. 4.36B.). Overexpression of COR 47 was detected with

the inoculation of AR4 and AR14 compared to uninoculated control under water deficit

stress (3% PEG).

4.7.3.11. Expression of ethylene responsive transcription factor 7(AtERF 7)

The AtERF7 transcripts were accumulated in Arabidopsis thaliana leaves under

PEG- mediated water deficit stress (Fig. 4.36C). The AtERF 7 transcripts were induced

through dehydration in non-inoculated plants under PEG-6000 induced water deficit

stress (3%). Significant reduction in AtERF 7 transcripts was recorded in plant inoculated

with AR4 and AR14 compared to uninoculated control plants under PEG-induced water

deficit stress (3%). Moreover, inoculation with drought tolerant CA containing

endophytic bacterial isolates (AR4 and AR14) also downregulated the expression of

AtERF 7 under normal conditions.

4.7.3.12. Expression pattern of dehydrins (LTI78)

The expression of LT178 exposed to water deficit stress was evaluated by RT-

PCR, in the presence as well as absence of drought tolerant CA producing endophytic

144

Page 165: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

bacterial isolates. AR4 and AR14) (Fig. 4.36D). Under normal growth conditions (0%

PEG), constitutive expression of LTI78 dehydrins transcription factors remained similar

145

Page 166: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Control AR4 AR140

2

4

6

8

10

12

14

16 0% PEG 3% PEG

Endophytic bacteria

Relat

ive fol

d chan

ge Zat 10

A

Control AR4 AR14

-20

-15

-10

-5

0

5

10

15

200% PEG 3% PEG

Endophytic bacteria

Relat

ive fol

d chan

ge

AtERF7C

146

Page 167: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Control AR4 AR140

20

40

60 0% PEG 3% PEG

Endophytic bacteria

Relat

ive fol

d chan

ge

COR 47B

Control AR4 AR140

20

40

60

80

100 0% PEG 3% PEG

Endophytic bacteria

Relat

ive fo

ld cha

nge

LTI78D

Fig. 4.36. Effect of drought tolerant CA containing endophytic bacterial isolates (AR4, AR14) on transcription factors and gene Zat 10 (A), COR47 (B), AtERF7 (C) and LTI78 (D) in Arabidopsis thaliana under normal (0%) as well as PEG- mediated water deficit conditions (3%).

147

Page 168: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

for inoculated as well as uninoculated control plants. However, on exposure to water

stressed conditions imposed by PEG (3%), inoculation of AR14 showed overexpression

of LTI78 in comparison with untreated control plants.

4.7.3.13. Expression pattern of MYB domain protein 15 (MYB 15)

The analysis of RT-PCR showed that expression of MYB15 gene remained

statistically similar in both inoculated and uninoculated plants under normal growth

conditions (Fig. 4.37A). However, inoculation of drought tolerant CA containing

endophytic bacterial isolates differentially expressed the MYB15 under PEG-mediated

water deficit conditions. Plant treated with AR4 and AR14 showed markedly lower

expression of MYB15 compared to uninoculated control plants under PEG-induced water

deficit conditions (3%).

4.7.3.14. Expression pattern of abscisic acid dependent dehydrins (ERD10)

Abscisic acid responsive gene (ERD 10) was upregulated in non-inoculated and

incoulated plants under PEG-induced water deficit stress (3%) (Fig. 4.37B). Expression

of ERD10 in arabidopsis plant inoculated with AR4 and AR14 did change over

uninoculated control plants under stressed conditions.

4.7.3.15. Expression of ethylene responsive factor (ERF 13)

To analyze whether the expression of ERF13 enhances water deficit stress

tolerance, compared the expression of ERF13 in inoculated as well as non-inoculated

plants (Fig. 4.37C). Expression of ERF transcript was more in non-inoculated plants

under non-stressed (0%) as well as PEG-induced water deficit stress (3%). Inoculation

with drought tolerant CA containing endophytic bacterial isolates (AR4 and AR14)

showed varied response in expressing the ERF13 under normal as well as PEG-induced

water deficit stress. However, inoculation of plant with isolate AR14 showed marked

downregulation of ERF13 under non-stressed as well stressed conditions in comparison

with uninoculated plants.

148

Page 169: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Control AR4 AR140

2

4

6

8

10

0% PEG 3% PEG

Endophytic bacteria

Relativ

e fold c

hange

MYB15 A

Control AR4 AR140

2

4

6 0% PEG 3% PEG

Endophytic bacteria

Relat

ive fo

ld ch

ange

ERF 13 C

Control AR4 AR140

10

20

30

40 0% PEG 3% PEG

Endophytic bacteria

Relativ

e fold c

hange

ERD 10 B

Fig. 4.37. Effect of drought tolerant CA containing endophytic bacterial isolates (AR4, AR14) on transcription factors and gene MYB15 (A), ERD10 (B), ERF13 (C) in Arabidopsis thaliana under normal (0%) as well as PEG-mediated water deficit conditions (3%)

149

Page 170: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

4.8. Characterization and identification of endophytic bacterial isolates

Selected drought tolerant CA containing endophytic bacterial isolates (WR2,

WS11, WL19, MR17, MS1, MG9, AR4 and AR14) were tested for various plant growth

promoting characters as shown in table 4.15. Results demonstrated that all the isolates

had ability to solubilize the phosphorus. Isolate WL19, MG9 and AR14 were found

efficient for P solubilization compared to other isolates. Siderophore production was

observed in all the isolates except MS1. Among the isolates, isolate WS11 and MR17

were found more efficient for siderophore production. Exopolysaacrharide production

was also found in all the selected isolates, while isolate WL19, MG9 and AR4 were more

efficient in exopolysacchride production. Selected isolates were also found positive for

catalase and oxidase activity where maximum oxidase activity was observed in isolate

AR14. All the isolates had ability to produce organic acids. Among isolates, WL19, MSI,

MG9 and AR14 were more efficient in organic acid production. Cellulase activity was

also present in all the isolates except MS1. Xylanase activity was absent in all isolates

except MS1. Protease activity also varied among the isolates. Maximum aggregation

ability was observed with isolate WR2 while minimum was observed with isolate MS1.

Selected drought tolerant endophytic bacteria except AR4 and AR14 were tested for their

survival in sterilized soil. Among the isolates, isolate MR17 showed maximum

population 6.7 CFU × 105 mL-1 followed by WL19 6.1 CFU × 105 mL-1.

Selected endophytic bacterial isolates WR2, WS11 and WL19 from wheat were

diagonosed as different strains of Bacillus on the basis of 16S rRNA gene sequence

similarities in Genbank (Fig 4.38). Endophytic bacterial isolates MR17, MS1 and MG9

from maize also showed clostest match to different Bacillus spp. However, isolates AR4

and AR14 from Arabidopsis were identified as Microbacterium and Psychrobacter sp.,

respectively (Fig 4.38).

150

Page 171: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Table 4.15: Characterization of selected drought tolerant endophytic bacterial isolates

(+) represents presence (-) shows absence and ND shows no detection of characteristics All the characters are average of three replicates

151

Characteristics WR2 WS11 WL19 MR17 MS1 MG9 AR4 AR14

P-solubilization + + ++ ++ + ++ + +++

Siderophore production ++ +++ + +++ - + + ++

Exopolysaccharide production + ++ +++ ++ ++ +++ +++ ++

Chitinase ++ ++ +++ ++ ++ +++ - -

Catalase + + + + + + + +++

Oxidase - - + - - + - ++

Organic acid production + + ++ + ++ ++ + ++

Aggregation 6.7 3.7 5.3 4.3 1.4 4.9 2.2 3.6

Starvation Test

(CFU ×104 mL-1)

4.3 1.2 3.9 1.8 4.2 1.6 1.1 2.2

Survival in soil

(CFU × 105 mL-1)

4.3 5.2 6.1 6.7 4.9 5.8 ND ND

Cellulase + + + + - + + +

Xylanase - - - + - - ND ND

Protease + - ++ + - ++ ND ND

Page 172: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Bacillus sp. WL19

Bacillus sp. MG9

Bacillus sp. WR2

Bacillus sp. WS11

Bacillus sp. MR17

Psychrobacter sp. AR14

Microbacterium sp. AR4

Bacillus sp. MS1

100

100

10099

100

0.05

Fig. 4.38. Identification of selected endophytic bacterial isolates on the basis of 16S rRNA gene sequence similarities

152

Page 173: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Chapter V

DISCUSSION

Drought impairs plant growth and crop productivity throughout the world

especially in the arid and semiarid regions. In this study, number of endophytic bacterial

isolates were isolated from two different crops (wheat and maize) and tested for their

capacity to tolerate the drought stress. Selected drought tolerant isolates were also

screened for CA activity. The drought tolerant bacterial endophytes containing high CA

activity were further screened for plant growth promotion under non-stressed as well as

PEG-6000 induced water deficit stress in axenic conditions. Three efficient endophytic

bacterial isolates (WR2, WS11, WL19 from wheat while MR17, MSI and MG9 from

maize) were evaluated in pot and field trials. These isolated were also characterized for

plant growth facilitating activities and identified. Moreover, effect drought tolerant

endophytic bacterial isolates on gene expression was studied to understand the

mechanism underlying the drought tolerance.

5.1. Drought tolerance ability of bacterial endophytes

Potential of endophytic bacteria isolated from wheat and maize plants was

assessed under PEG-induced water deficit stress (-0.36, -0.61, -1.09, -1.91 and -3.20

MPa). Among the 150 isolates from each crop, 50 isolates showed growth under normal

(-0.36 MPa) as well as stressed conditions (-3.20 MPa). Growth of isolates at -3.20 MPa

depicts their capacity to survive in severe water deficit conditions which might be due to

production of exopolysaccharides (EPS), catalases and oxidases. Production of EPS

occurs by bacteria in response to stress conditions (Roberson and Firestone 1992).

Presumably, EPS provide microenvironment which holds more water and desiccated

gently than the enclosing environment, thus protect the bacterial strain from fluctuation in

water potential and drying (Hepper, 1975). Generation of exopolysacchrides has been

reported to protect Azospirillum brasilense Sp245 cell from the desiccation (Konnova et

al., 2001). Furthermore, production of oxidases and catalases is helpful for protecting the

nucleic acid from stress induced disintegration and regulating cellular metabolism in

stressed environment (Boumahdi et al., 1999). Catalases scavenge hydrogen peroxide

(H2O2) and maintain the cellular metabolism, thus reduced the PEG induced cell damage

(Hussain et al., 2015). Rise in supply of soluble sugars, free amino acids, protein, and

EPS in bacteria under water scarce conditions may also be attributed to their survival

153

Page 174: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

ability (Vardharajula et al., 2011). Earlier, it has also been described that drought tolerant

rhizobia accumulate the osmolytes and modulate the cell morphology for its survival

under stressed environment (Busse and Bottomley, 1989). Results of present study are

similar to Yandigeri et al. (2012) and Abolhasani et al. (2010) where they found the

growth of endophytic actinobacterial isolates at -0.73 MPa and Shinorhizobium sp. at -3.5

MPa, respecitively.

5.2. Carbonic anhydrase activity of drought tolerant isolates

Isolates which showed higher survival ability under PEG-induced water deficit

stress were tested for CA activity. Out of 50, 10 isolates (from each crop) showing high

CA activity were further selected. Increase in CA activity at initial growth phase might be

due to phenomenon of sparking effect, where CO2 is needed to fulfill biosynthetic

demand and is vital for the organism to overcome lag phase (Dean and Ward, 1992).

Zhang et al. (2011) also reported that synthesis of CA is closely related to cell growth.

Bacillus mucilaginosus captured the atmospheric CO2 through bacterially produced CA

(Zhang et al., 2010). These results are in accordance with Ramanan et al. (2009) where

they purified and characterized the plant type CA from Bacillus subtilis

5.3. Screening of CA producing drought tolerant endophytic bacteria for growth

promotion in wheat and maize seedlings under PEG-imposed water deficit

stress in axenic conditions

Plants are usually exposed to different abiotic stress environments, among them,

drought is the major problem associated with growth and development of plants, affecting

agricultural demand throughout the world. In the current study, potential of drought

tolerant CA producing endophytic bacterial isolates was checked for growth in wheat and

maize growth. Among the isolates, isolate WL19, WS11 and WR2 showed significant

increase in wheat growth whereas isolate MG9, MR17 and MS1 proved to be efficient

isolates for improving maize growth. However, effect was more obvious in drought

sensitive cultivar and hybrid of both crops (Uqab-2000 and H2) compared to tolerant ones

(Fsd-2008 and H1). Isolates improved the plant growth by stimulating root length and

root growth under normal (-0.04 MPa) as well as PEG-induced water deficit conditions (-

1.23 MPa). Expansion of root growth might be attributed to auxin (IAA) production by

endophytic bacterial isolates. Synthesis of auxin affects the root system by improving size

and number of adventitious roots (Moreno-Gutierrez et al., 2012). Bacterial induced

154

Page 175: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

changes in root may be attributed to increase in the root surface, resulting in improved

water and nutrient uptake that may cause positive influences on growth of plant (Somers

et al., 2004). Moreover, improved root growth has been suggested to enhance phosphorus

uptake (Jones and Darrah, 1994). There are some reports which suggest that inoculation

of seed with Pseudomonas putida GR12-2 increase the seedling root length up to 2-3 fold

compared to non-inoculated control (Glick et al., 1986; Caron et al., 1995).

In the present study, endophytic bacterial isolates (WR2, WS11, WL19 for wheat

and MR17, MS1 and MG9 for maize) significantly increased the shoot fresh biomass

under non-stressed as well as stressed conditions, especially in Uqab-2000 and H2 hybrid.

Significant enhancement in shoot fresh weight by the endophytic bacterial isolates might

be attributed to CA activity that provides higher photoassimilates to plants through

increasing CO2 assimilation rate in both wheat and maize plants. Moreover, under

drought stress, endogenous level of plant hormone ethylene increases and results in

decreases root and shoots growth. Bacterial may also rescue the plant from stress and

regulate the normal growth by producing ACC-deaminase that degrade ACC, ethylene

precursor (Mayak et al., 2004).

Moreover, inoculation of drought tolerant CA containing endophytic bacteria

isolated from wheat and maize improved chlorophyll content, CA activity, CO2

assimilation rate (A), transpiration rate (E), stomatal conductance (gs) and substomatal

CO2 conductance (Ci) in both crops, however, increase was greater in sensitive cultivars

Uqab-2000 and H2 hybrid of wheat and maize, respectively, compared to non-inoculated

control plants. Increase in photosynthetic rate might be due to increase in CA activity by

the inoculation of drought tolerant CA producing endophytic bacteria. Association

between photosynthetic rate and CA activity was confirmed by Khan (1994) and XinBin

et al. (2001) in higher plants. Afroz et al. (2005) found improvement in net CO2

assimilation rate due to stimulatory effect of CA activity by the application of gibberellin

IIIGA3 in mustard. Inhibition in CA activity with the application of ethoxyzolamide

caused 80-90% decrease in photosynthesis at lower CO2 concentration, indicating the role

of C3 in photosynthesis (Badger and Pfanz, 1995). However, bacterially synthesized CA

and its role in photosynthesis rate is still need to be explored. Furthermore, CA activity

has been reported for regulating the stomatal conductance and protecting the plant from

adverse environmental conditions (Wei-Hong et al., 2014). Naveed et al. (2014b) also

found similar results where inoculation with endophytic bacterial strains PsJN improved

155

Page 176: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

the chlorophyll content, photosynthesis and efficacy of PSII of Mazruka cultivar in maize

compared to other control treatments.

Based on above observation, it may be concluded that endophytic bacterial

isolates WR2, WS11 and WL19 proved to be efficient for improving growth of wheat and

MR17, MS1 and MG9 for maize, respectively under non-stressed as well as PEG-induced

water deficit stress. In case of cultivars and hybrid, effect of endophytic bacterial

inoculation was more prominent in Uqab-2000 and H2 than Fsd-2008 and H1.

5.4. Evaluation of selected endophytic bacterial isolates for wheat and maize in pot

trials

Drought tolerant CA containing plant growth promoting isolates (WR2, WS11

and WL19 from wheat and MR17, MS1 and MG9 from maize) were selected from in

vitro screening and tagged with Gus marker to study their response for plant growth

promotion and colonization efficiency under non-stressed as well as water deficit stress

conditions (100, 70 and 40% FC). Isolates showed varied response for improving

photosynthesis and plant biomass of wheat and maize under normal (100% FC) as well as

water deficit (70 and 40% FC) conditions. Severe water deficit stress (40% FC) markedly

reduced all the growth parameters. However, inoculation of drought tolerant CA

containing bacterial endophytes improved plant growth under non-stressed and stressed

conditions, especially in sensitive cultivars (Uqab-2000 and H2 hybrid) by modulating

their root and shoot growth with different growth promoting activities including

production of phytohormones, P-solubilization, synthesis of phytohormones, ACC-

deaminase activity and production of siderophore. The root and shoot growth was

increased in wheat and maize plants exposed to water deficit stress (40% FC) which is

attributed to impact of CA activity on photosynthesis that not only enhances the

photosynthesis but also improves plant growth. It is assumed that plant excretes more

photoassimilates due to higher photosynthetic rate and attracts more microbial community

that performs beneficial function and improves plant growth. Moreover, increase in root

growth might be due to IAA production. Indole acetic acid enhances root formation by

stimulating cell division, enhancing cell enlargement and increasing surface area of root

(Dey et al., 2004; Gray and Smith, 2005). Production of auxin has been noticed as major

tool for facilitating the early growth in wheat plant (Khalid et al., 2004) in conjunction

with phosphrous solubilization (Rajput et al., 2013). In our experiments, isolates WL19

and MG9 exhibiting more indole acetic acid production under normal as well as stressed

156

Page 177: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

condition showed more root growth and shoot biomass compared to other isolates.

Moreover, these isolates showed higher P solubilization under stressed condition than

non-stressed condition indicating that P-solubilization enhances in response to stress.

However, increase was more in sensitive cultivars than tolerant. These observations are in

line with Forchetti et al. (2010) where drought sensitive genotype B59 showed dramatic

increase in root biomass in the presence of endophytic bacterial strain under water stress.

CA activity was decreased in both crops at 70% and 40% FC especially in drought

sensitive cultivar and hybrid which could be attributed to inhibition of stress-activated

enzymes or other metabolic dysfunctions (Hopkins, 1995). These results confirm the

findings of Guliyev et al. (2008) where CA activity was decreased in Garagilchig and

Giymatli under limited water supply but contradicts the findings of those who reported

that CA activity decreased in wheat in response to mild water deficit stress and increased

in response to severe water deficit conditions mediated by PEG. Findings of present study

are also in line with the research findings described by Talaat and Shawky (2014) where

they found that CA activity also decreased in Giza 168 and Sids 1 under salinity stress

(4.7 and 9.4 dS m-1). However, inoculation with drought tolerant CA producing

endophytic bacteria enhanced the CA activity. Colonization of wheat plant with

mycorhizae inoculation enhanced the CA activity under salinity stress (Talaat and

Shawky, 2014).

Photosynthesis is the major physiological response of plants under water scarce

conditions due to stomatal closure and inhibition of enzyme activity involved in

photosynthesis (Tataeizadeh, 1998). Plant inoculated with drought tolerant CA containing

endophytic bacteria improved the chlorophyll contents, photosynthetic rate and stomatal

and substomatal conductance. Better performance of CA activity and higher

photosynthetic rate was observed by Talaat and Shawky (2014) with mycorhizae

inoculation. The positive effect might be accounted for increased accumulation of

carbohydrate in grain, most probably due to enhanced CO2 assimilation under stressed

conditions (Talaat and Shawky, 2014). Many researchers have described improvement in

photosynthetic rate by inoculation of growth facilitating bacteria (Vardharajula et al.,

2011; Yandigeri et al., 2012). Moreover, inoculation of osmotolerant bacteria facilitated

the chlorophyll content of wheat under drought (Chakraborty et al., 2013).

The relative water content (RWC), indicator of water deficit (Fisher, 2000)

decreased in many plants under water scarce conditions (Liu et al., 2002). However,

157

Page 178: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

inoculation with drought tolerant CA containing bacterial endophytes (WL19 and MG9)

enhanced RWC in both crops especially in Uqab-2000 and H2 hybrid. Inoculated plants

developed effective root system and enhanced water uptake (Dodd et al., 2010). On the

other hand, electrolyte leakage enhanced under water deficit stress (40%FC) in non-

inoculated plants, though the inoculation of isolates WL19 and MG9 reduced the damage

caused by drought compared to uninoculated control. A direct correlation has been found

between water stress and membrane damage but bacterial inoculation reduced the adverse

effect of drought on membrane (Vardharajula et al., 2011).

Proline contents were increased in both crops (wheat, maize) under drought stress

(70 and 40% FC). These osmoprotectants or compatible solutes support plants to

protecting their enzyme activity under limited moisture conditions (Saravanakumar et al.,

2011). Proline serves as protective osmolyte and scavenger of hydroxyl radical which

protects macromolecules from denaturation (Kishor et al., 1995). However, inoculation of

bacterial endophytes improved the proline content in wheat and maize. Increased proline

contents (Theocharis et al., 2012) and enhanced accumulation of free aminaocid and

sugar (Vardharajula et al., 2011) was observed with beneficial bacteria. Reactive oxygen

species inducing lipid peroxidation are generated under stress conditions (Sgherriet et al.,

2000). Results revealed that endophytic bacterial inoculation (WL19 and MG9)

minimized the damage caused by malondialdehyde (MDA) by decreasing MDA contents

in both crops under non-stressed (100% FC) and water deficit conditions (40% FC).

On the basis of these studies and their findings, it can be speculated that drought

tolerant CA containing endophytic bacteria not only improve the wheat and maize growth

but also mimic the adverse impacts of water deficit stress on plant growth. These

beneficial bacteria increased the photosynthetic rate and plant biomass in both cultivars of

wheat (Uqab-2000 and Fsd-2008) and maize hybrid (H1 and H2). However, effect of

inoculation was more obvious in Uqab-2000 and H2 hybrid. Moreover, respective isolates

vigorously colonized different tissues (root, shoot and leaves) under well watered and

limited water conditions. Therefore, selected bacterial strains seems to be valuable for

growth promotion under limited water conditions. However, potential of tested isolates

under field needs to be explored.

5.5. Interaction between endophytic bacterial population and plant tissues

Results regarding colonization of different tissues of both wheat cultivars (Fsd-

158

Page 179: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

2008 and Uqab-2000) and maize hybrids (H1 and H2) with Gus labelled drought tolerant

CA containing endophytic bacterial isolates revealed that colonization of wheat and

maize with their respective isolates decreased as the water deficit stress increased, most

likely due to water deficit stress and its effect on bacterial isolates. Colonization of tissues

by isolates decreased under mild (70% FC) and severe (40% FC) stress compared to well

watered (100% FC) conditions. Moreover, reltively less CFU was recorded in leaf tissue

of wheat (Fsd-2008 and Uqab-2000) and maize (H1 and H2) compared to shoot and root

tissues under normal as well as stressed conditions. These findings are in line with

Gusmaini et al. (2013) where they reported that population of endophytic bacterial

consortia was higher in root tissue compared to stem and leaves. Similarly, more diverse

bacterial population was found in root than the stem of rice (Wang et al., 2016). Direct

contact and close vicinity made the greater chance to get access into root of plants.

Microorganisms can enter from soil to root and their population ranged from 105-107 CFU

g-1 fresh weight (Hallmann, 2001).

However, interaction between endophytic bacterial population and plant cultivar

showed that endophytic population was greatly decreased in drought sensitive cultivars of

both wheat and maize crops (Uqab-2000 and H2) compared to drought tolerant cultivars

(Fsd-2008 and H1). These results are in line with Naveed et al. (2014b) where less

population of endophytic bacteria (Enterobactor sp. FD 17 and Burkholderia

phytofirmans PsJN) was observed in Mazruka than in Kaleo genotypes under well

watered as well as drought stress conditions. Under stressed condition, variety of

physiological alterations result in change in root exudation that influences the

performance of strain (Bais et al., 2006). Similarly, endophytic bacterial strain showed

more persistence in shoot tissue of two maize cultivars (Peso and Morignon) compared to

other cultivars (Naveed et al. 2013).

5.6. Evaluation of selected endophytic bacterial isolates in field trial

Limited supply of water is one of the severe environmental constraints that badly

affect the crop productivity. Therefore, plant adaptability to water deficit stress is required

to stimulate crop production. Wheat and maize are important cereals throughout the

world. Improving productivity of wheat and maize by drought tolerant CA containing

endophytic bacteria is one promising area for ensuring food security. Potential of selected

drought tolerant CA containing endophytic bacterial isolates (WL19, WS11,WR2 from

wheat while MG9, MR17, MS1 from maize) were evaluated in field conditions for

159

Page 180: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

facilitating the growth, CA activity, photosynthesis, yield and antioxidants activity of

drought sensitive cultivar in wheat and drought sensitive hybrid in maize where water

deficit stress was induced by cutoff the irrigation at different growth stages in wheat

( tillering, flowering or grain filling) and maize (vegetative or reproductive). Water

deficit-mediated yield reduction has been suggested by many researchers that depends on

severity and period of drought (Farooq et al., 2009). Limited moisture supply at critical

growth stage, negatively influences crop growth in comparison to non-stressed control

plants. Irrigation skipped at tillering and grain filling phase of wheat and reduced

irrigation at reproductive stage of maize negatively influence the photosynthetic rate and

crop yield. In maize, water deficit stress at vegetative stage caused 25-60% reduction

(Atteya et al., 2003) and 63-87% at reproductive stage (Kamara et al., 2003). Similarly,

plant shows variable response to limited moisture supply at different growth stages

(Gupta et al., 2001) because unfavorable conditions badly effect expansion of

reproductive tissues and yield (Mogensen et al., 1985; Kettlewell et al., 2010).

Inoculation of drought tolerant CA containing endophytic bacteria significantly

improved the growth and yield of both crops at all growth stages, especially at tillering

and grain filling in wheat and reproductive stage in maize. Naveed et al. (2014a) found

that the growth of wheat was increased with endophytic bacterial inoculation especially

where irrigation withdrawal at tillering or flowering phases of wheat. Growth promoting

activities known to be involved in this process are generation of phytohormones, ACC

deaminase activity and siderophore production. Inoculation of drought tolerant

endophytic S. olivaceus DE10 improved the number of tillers, grain yield and biomass of

wheat (Yandigeri et al., 2012).

Our results also revealed that CA activity decreased when wheat and maize plants

were subjected to water deficit conditions. Recent investigations on wheat depicted that

CA activity, at flag leaf stage, rasied to its maximum value and after that lowered to its

minimum value before going to rise at wheat dough stage. Furthermore, CA activity

decreased in awn, glume and grain of Garagilchig and Giymatli cultivars under water

limited environment in comparison with well watered environment at milk and dough

stage (Guliyev et al., 2008). However, CA activity was significantly higher at milk stage

in ear parts compared to leaf tissues (Li et al., 2004).

Significant decrease in chlorophyll content, photosynthetic assimilation rate,

stomatal conductance and rate of transpiration was noticed when drought stress was

160

Page 181: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

imposed at tillering, grain filling and reproductive stage in wheat and maize, respectively;

might be attributed to decrease in CA activity. Khan et al. (2008) found that

photosynthetic rate was decreased with decreasing CA activity under Cd stress in wheat,

indicating the importance of CA in maintaining the photosynthesis at different levels of

Cd. However, inoculation with drought tolerant CA containing endophytic bacteria

enhanced the chlorophyll contents, photosynthetic rate, stomatal conductance and

substomatal conductance. Earlier reports have suggested that increased photosynthetic

rate was due to increased CA activity in Chickpea (Hayat et al., 2012) which regulated

the constant supply of CO2 for Rubisco (Okabe et al., 1980). The increased

photosynthesis has been attributed to increase plant growth that ultimately resulted in

better production of carbohydrates (photoassimilates) and increased dry matter (Hayat et

al., 2012). Stomatal conductance was also affected by endophytic bacteria might be due to

their presence in stomatal cell of plant (Compant et al., 2005) and associated with

different compounds synthesized by bacteria such as coronatine and jasmonate (Brader et

al. 2014). In the present study under field conditions, water use efficiency significantly

decreased under stressed condition compared to normal conditions but inoculation of

drought tolerant CA containing bacteria did not show significant difference with respect

to un-inoculated control in both wheat and maize crops. The findings are in accordance to

outcomes of Naveed et al. (2014a).

Skipped irrigation at any growth stage also affected the protein contents and total

soluble sugars. However, inoculation of drought tolerant CA containing endophytic

bacteria improved the protein contents and soluble sugars compared to uninoculated

plants. Greater accumulation of sugar in stressed plants might be as a result of hydrolysis

of starch to sugars. The outcomes of present study are in line with Naveed et al. (2014a)

where they found that protein contents were increased by endophytic bacteria.

Antioxidants activity (glutathione reductase, GR; ascorbate peroxidase, APX; and

catalase) also increased in both crops under water deficit stress. Antioxidant activity

usually increases in plants under drought and acts as adaptational reaction (Reddy et al.,

2004). In the current experiment, antioxidant content in leaves of plants treated with

endophytic bacteria was significantly reduced compared to uninoculated control plants

under skipped irrigation. These outcomes are aligned with Upadhyay et al. (2012) where

they found that CAT, GR and APX activity decrease in plants treated with bacterial

strains compared to control plants under salinity stress.

161

Page 182: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

. Inoculation with drought tolerant CA containing endophytic bacterial isolates

(WL19, WR2, WS11 from wheat and MG9, MR17 and MS1 from maize) improved their

mineral nutrition under non-stressed as well as stressed conditions. The increase might be

attributed to IAA production that enhanced mineral uptake by stimulating the root growth

(Gray and Smith, 2005). Bacterial inoculation also enhances the P solubilization that

ultimately improves the P availability through organic acid production and phosphatase

activity (Rodriguez et al., 2006). Significant enhancement in nutrient concentration has

been suggested by bacterial inoculation (Nadeem et al., 2006). Inoculation of maize plant

with Pseudomonas tolaasii IEXb enhanced P content by 56% in maize than control plants

(Viruel et al., 2014). Increased phosphorus has been reported by Psuedomonas sp. in

many crop species (Gusain et al., 2015, Fankem et al., 2015, Babana and Antoun, 2006).

It may be concluded from the above discussion that drought tolerant CA

containing endophytic bacteria not only improve plant growth but also mitigate the

adverse effect of drought in both crops. These bacterial isolates alleviate the deleterious

impacts of drought by improving CA activity, photosynthetic rate, inducing protein

accumulation, decreasing antioxidants contents as well as enhancing nutrient acquisition.

In fact, present study provides novel information about plant stimulation under water

deficit condition.

5.7. Influence of drought tolerant CA containing endophytic bacteria on plant

growth promotion and gene expression

Water deficit stress is a major limitation that affects food production around the globe.

Improvement of water deficit stress tolerance using drought tolerant endophytic bacteria

is considered as promising strategy to overcome this environmental limitation. However,

biochemical and molecular mechanisms controlling plant/endophyte interaction under

limites supply of water remain unclear. In the current study, two drought tolerant CA

containing endophytic bacterial isolates were tested to insight the mechanisms underlying

the water deficit stress tolerance enhancement in Arabidopsis thaliana plant when treated

with endophytic bacterial isolates. Inoculated plant showed greater root length, lateral

root development, fresh biomass of root and shoot compared to uninoculated plant. Such

diverse impacts have also been stated by Poupin et al. (2013). They showed that plant

growth facilitating bacteria enhanced growth parameters and accelerated the growth rate

in Arabidopsis thaliana. Increase in root growth might be due to IAA production.

Interestingly, IAA acid produced by PGPB plays a vital role in growth stimulating traits

162

Page 183: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

of plant and is required for proficient rhizosphere colonization in Arabidopsis (Zuniga et

al., 2013). Auxin are involved in lateral root development and elongation of hypocotyls

(Overvoorde et al., 2010). In this study, results showed that endophytic bacterial isolates

enhanced drought tolerance which might be related to acceleration of transcriptional

response also with long term regulation of several genes. Expression pattern of

dehydration responsive protein and element RD22 and RD29B was greatly modified by

the inoculation of endophytic bacterial isolates. This can be attributed to faster sensing of

PEG-induced water deficit stress through dehydration signaling. Transcriptional response

of RD22 was also upregulated in root of arabidopsis plant inoculated with Burkholderia

phytofirmans PsJN (Pinedo et al., 2015)

Results of present study showed that expression of P.R.1.2. was enhanced by the

inoculation of endophytic bacteria. Our outcomes are aligned with Ryu et al. (2007)

where inoculation of Psuedomonas chlororaphis O6 induced resistance against pathogen.

Several WRKY transcription factors (WRKY57, WRKY8) were also differentially

expressed in plants inoculated with AR4 and AR14. However, WRKY transcription

factor are involved in pathogen induced stress and drought stress (Seki et al., 2002: Chen

et al., 2012). It is interesting that expression of ethylene responsive factor, At ERF7, ERF

13 and Zat 10 was repressed in endophytes treated plants compared with non-inoculated

plants during PEG treatment. These research findings are similar to Seki et al. (2002).

Based on all research experiments and their outcomes, it can be perposed that

drought tolerant CA containing endophytic bacteria are beneficial for wheat and maize

not only under well watered but also under water deficit conditions. Improvement in

photosynthesis and plant biomass of wheat and maize especially in sensitive cultivars,

most likely because of drought tolerant CA containing endophytic bacterial isolates

Bacillus sp. (WR2, WS11 and WL19 from wheat while MR17, MS1 and MG9 from

maize). Moreover, these bacterial isolates posses P-solublization and IAA producing traits

which play dundamental role in growth promotion. Thus, endophytic bacterial isolates

proved to be suitable for enhancing photosynthesis and growth of wheat and maize under

water deficit stress conditions. Endophytic bacterial strains Psychrobacter sp. (AR14) and

Microbacterium sp. (AR4) influenced the expression of different genes and transcription

factors under stressed conditions in Arabidopsis. However, different field experiments are

suggested to check their performance and extensive evaluation in field. Molecular studies

are required to confirm role of bacterially synthesized CA in photosynthesis.

163

Page 184: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Chapter V

SUMMARY

Water deficit stress is a major hazard to the production of food crop, affecting

agricultural demand throughout the world. Enhancement in plant growth is required to

sustain global food production under water limited condition. Drought tolerant

endophytic bacteria have recently been reported for facilitating the plant growth and crop

productivity under water deficit conditions. Being, relatively simple and cost effective,

usage of endophytic bacteria has emerged as promising tool for improving the plant

growth. Ability to colonize the interior of plant as well as production of growth

promoting compounds (indole acetic acid, polysaccharides and stress hormone) in plants

has made these bacteria highly valuable for agricultural research. Moreover, CA

producing ability of endophytic bacteria seems to be beneficial for improving

photosynthetic rate and biomass under stressed conditions. Thus, a number of

experiments were carried out to screen the efficient drought tolerant CA containing plant

growth promoting endophytic bacteria for wheat and maize under water stress. The

selected drought tolerant CA containing endophytic bacterial isolates (WR2, WS11,

WL19 from wheat and MR17, MS1, MG9 from maize) were tested in pot and field for

wheat and maize. Moreover, potential of endophytic bacterial isolates was also evaluated

for gene expression in Arabidopsis thaliana a model plant, to understand the mechanism

of drought tolerance by bacterial endophytes.

Results of these studies are summarized as below:

1. One hundred and fifty isolates were isolated from different tissues of wheat and

maize crop and assayed for drought tolerance. Fifty drought tolerant isolates from

each crop were analyzed for CA activity. Ten drought tolerant isolates with higher

CA activity from each crop were selected for growth promotion in wheat (C3) and

maize (C4).

2. All the 10 drought tolerant CA containing endophytic bacterial isolates were

screened for plant growth promotion in drought tolerant and drought sensitive

cultivars of wheat (Fsd-2008 and Uqab-2000) and hybrids of maize (H1 and H2).

Inoculation significantly enhanced the root length, shoot length, root and shoot

dry weight, chlorophyll content, CA activity, photosynthetic rate transpiration

rate, stomatal and substomatal conductance under normal (-0.04 MPa) as well as

164

Page 185: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

PEG induced water deficit conditions (-1.09, -1.23 MPa) in both crops. In wheat,

isolates WR2, WS11and WL19 significantly enhanced the growth, CA activity,

chlorophyll content and photosynthetic rate in both cultivars, however, increase

was more for Uqab-2000 than Fsd-2008. However, minimum increase CA activity

and photosynthetic rate was observed with isolate WS22 in both wheat cultivars.

In maize, isolates MR17, MS1 and MG9 showed significant increase in growth,

CA activity, chlorophyll content, photosynthetic rate, stomatal and substomatal

conductance in both hybrids, though, increase was more pronounced for H2 than

H1. However, isolate MS7 and MG2 showed smaller increase for above

parameters in both hybrids.

3. Selected drought tolerant CA containing endophytic bacterial isolates (WR2,

WS11, and WL19 and MR17, MS1 and MG9) were labeled with Gus marker and

evaluated in pot for improving photosynthesis and plant growth of wheat and

maize. The endophytic bacterial inoculants improved photosynthesis and plant

yield under normal (100% FC) as well stressed conditions (70 and 40% FC) in

both wheat cultivars (Fsd-2008 and Uqab-2000) and maize hybrids (H1 and H2).

However, increase was more in drought sensitive cultivar in wheat (Uqab-2000)

and hybrid in maize (H2). These isolates efficiently colonized the different tissues

of wheat and maize, however, their CFU decreased with increasing distance from

root to leaves as well as with increasing water stress (70 and 40% FC). Isolates

WL19 and MG9 proved to be efficient for IAA production and P-solubilization

under normal and PEG- mediated water deficit conditions compared to other

isolates.

4. Selected bacterial isolates (WR2, WS11, and WL19 from wheat and MR17, MS1

and MG9 from maize) were also evaluated in field conditions for improving

physiology, yield and mineral contents of wheat and maize. The endophytic

bacterial isolates improved photosynthetic rate, yield and mineral contents in

irrigated plants as well as in plants where irrigation was skipped at tillering,

flowering, or grain filling stage of wheat as well as vegetative or reproductive

stage of maize. Moreover, decrease in antioxidants contents was also observed by

the inoculation of isolates under irrigated and stressed conditions.

5. Drought tolerant CA containing endophytic bacteria were isolated and screened

for plant growth promotion at Plant Microbe interaction Laboratory, The

University of Queensland. Two drought tolerant CA containing plant growth

165

Page 186: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

promoting endophytic bacteria were selected for gene expression study. Drought

stress modified the expression of several stress related genes in Arabidopsis

thaliana. However, inoculation with drought tolerant CA containing endophytic

bacteria AR4 and AR14 not only enhanced the growth of Arabidopsis thaliana but

also stimulated the expression of stress related gene and transcription factors

under water deficit stress. Expression of RD29B, LEA, and DREB2A was

upregulated whereas as expression of AtERF7, ERF13 and Zat 10 was

downregulated by the inoculation of isolates compared to uninoculated control

plants.

6. From the= research studies, it can be proposed that inoculation with WR2, WS11,

and WL19 could be beneficial for improving photosynthesis and productivity of

wheat (C3) under water deficit conditions while MR17, MS1 and MG9 proved to

be efficient for maize (C4) under waterlimited conditions. Moreover, inoculation

of isolates (AR4 and AR14) could mitigate the drought stress by influencing the

gene expression of Arabidopsis thaliana under water deficit conditions.

Inoculation with these isolates can increase crop yield and enhance net benefits by

efficient utilization of water resources which ultimately may help to alleviate

poverty through sustainable crop production and seems achieving the target of

food security.

166

Page 187: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Future directions

Carbonic anhydrase encoding gene in endophytic bacteria may be identified and

need to generation of CA negative mutant may be helpful to confirm the role of

bacterially produced CA in photosynthesis

Gene expression studies should may be conducted to find out the exact

mechanism of drought tolerance by endophytic bacteria in both wheat and maize

Ways to improve colonization efficiency of endophytic bacteria in reproductive

tissues of plants need to be explored

Potential of endophytic bacterial isolates containing CA activity should be studied

for other corps under different abiotic stresses viz drought or salinity

Multistrain inoculation of drought tolerant CA containing endophytic bacteria

may be studied to get maximum benefits.

REFERENCES

167

Page 188: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Abeles, F.B., P.W. Morgan and Jr. M.E. Saltveit. 1992. Ethylene in plant biology (2nd

Ed). San Diego, Academic Press.

Abolhasani, M., A. Lakzian, A. Tajabadipour and G. Haghnia. 2010. The study salt and

drought tolerance of Sinorhizobium bacteria to the adaptation to alkaline

condition. Aust. J. Basic Appl. Sci. 4: 882-886.

Achal, V. and X. Pan. 2011. Characterization of urease and carbonic anhydrase producing

bacteria and their role in calcite precipitation. Curr. Microbiol. 62: 894-902.

Adams, P. and J. Kloepper. 1996. Seed borne bacterial endophytes in different cotton

cultivars. Phytopathology 86: 97

Afroz, S., F. Mohammad, S. Hayat and M.H. Siddiqui. 2005. Exogeneous application of

gibberellic acid counteracts the ill effects of sodium chloride in mustard. Turk. J.

Biol. 29: 233-236.

Afzal, M., S. Yousaf, T.G. Reichenauer and A. Sessitsch. 2012. The inoculation method

affects colonization and performance of bacterial inoculant strains in the phy-

toremediation of soil contaminated with diesel oil. Int. J. Phytorem. 14: 35-47.

Agarwal, V.K. and J.B. Sinclair. 1996. Principles of seed pathology. Lewis Pub., Boca

Raton, Florida.

Alef, K. 1994. Biologische Bodensanierung-Methodenbuch. Wiley-VCH, Weinheim,

Germany.

Ali S, T.C. Charles and B.R. Glick. 2012. Delay of flower senescence by bacterial

endophytes expressing ACC deaminase. J. Appl. Microbiol. 5: 1139-1144.

Ali, M.M. and D. Vora. 2014. Bacillus Thuringiensis as endophyte of medicinal plants:

Auxin producing biopesticide. Int. Res. J. Environ. Sci. 3: 27-31.

Allahverdiyev, T. 2015. Effect of drought stress on some physiological traits of durum

(Triticum durum Desf.) and bread (Triticum aestivum L.) wheat genotypes. J.

Stress Physiol. Biochem. 11: 29-38.

Almeselmani, M., F. Abdullah, F. Hareri, M. Naaesan, M.A. Ammar, O.Z. Kanbar and A.

Saud. 2011. Effect of drought on different physiological characters and yield

component in different Syrian durum wheat varieties. J. Agri. Sci. 3: 127-133.

Amaresan, A., V. Jayakumar, K. Kumar and N. Thajuddin. 2012. Isolation and

characterization of plant growth promoting endophytic bacteria and their effect on

tomato (Lycopersicon esculentum) and chilli (Capsicum annuum) seedling

growth. Ann. Microbiol. 62: 805-810.

168

Page 189: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Andrade, L.F., G.L.O.D.D. Souza, S.  Nietsche,  A.A. Xavier, M.R. Costa, A.M.S.

Cardoso, M.C.T. Pereira and D.F.G.S. Pereira. 2014. Analysis of the abilities of

endophytic bacteria associated with banana tree roots to promote plant

growth. Microb. Genet. Genom. Mol. Biol. J. Microbiol. 52: 27-34.

Andrade, L.F., G.L.D. Souza, S. Nietsche, A.A. Xavier, M.R. Costa, A.M. Cardoso, M.C.

Pereira and D.F.  Pereira. 2014. Analysis of the abilities of endophytic bacteria

associated with banana tree roots to promote plant growth. J. Microbiol. 52: 27-

34.

Anjum, S., X.Y. Xie, L.C. Wang, M.F. Saleem, C. Man and L. Wang. 2011.

Morphological, physiological and biochemical responses of plants to drought

stress. Afr. J. Agri. Res. 6: 2026-2032.

Anjum, S.A., L.C. Wang, J. Salhab, I. Khan and M. Saleem. 2010. An assessment of

drought extent and impacts in agriculture sector in Pakistan. J. Food Agri.

Environ. 8: 1359-1363.

Anjum, S.A., M.F. Saleem, M.A. Cheema, M.F. Bilal and T. Khaliq. 2012. An

assessment to vulnerability, extent, characteristics and severity of drought hazard

in Pakistan. Pak. J. Sci. 64: 138-145.

Apel, K. and H. Hirt. 2004. Reactive oxygen species: metabolism, oxidative stress, and

signal transduction. Annu. Rev. Plant Biol. 55: 373-399.

Arshad, M., B. Shaharoona and T. Mahmood. 2008. Inoculation with Pseudomonas spp.

containing ACC deaminase partially eliminates the effects of drought stress on

growth, yield, and ripening of pea (Pisum sativum L.). Pedosphere 18: 611-620.

Ashraf, M. and N.A. Akram. 2009. Improving salinity tolerance of plants through

conventional breeding and genetic engineering: an analytical comparison.

Biotechnol. Adv. 27: 744-52.

Ashraf, M., S.H. Berge and O.T. Mahmood. 2004. Inoculating wheat seedling with

exopolysaccharides producing bacteria restricts sodium uptake and stimulates

plant growth under salt stress. Biol. Fertil. Soils 40: 57-162.

Ashraf, M.Y., A.H. Khan and A.R. Azmi. 1992. Cell membrane stability and its relation

with some physiological process in wheat. Acta Agron. Hung. 41: 182-191.

Atteya, A.M. 2003. Alteration of water relations and yield of corn genotypes in response

to drought stress. Bulg. J. Plant Physiol. 29: 63-76.

169

Page 190: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Babana A. and H. Antoun. 2006. Effect of tilemsi phosphate rock-solubilizing

microorganisms on phosphorus uptake and yield of field-grown wheat (Triticum

aestivum L.) in Mali. Plant Soil 287:51-58.

Bacon, C.W. and D.M. Hinton. 2006. Bacterial endophytes: the endophytic niche, its

occupants, and its utility. p. 155-194. In: S.S. Gnanamanickam (ed.) Plant-

associated bacteria. Springer, Netherlands.

Badger, M.R. and G.D. Price. 1994. The role of carbonic anhydrase in photosynthesis.

Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 369-392.

Badger, M.R. and H. Pfanz. 1995. Effect of carbonic anhydrase inhibition on

photosynthesis by leaf pieces of C3 and C4 plants. Aust. J. Plant Physiol. 22: 45-

49.

Bais, H.P., T.L. Weir, L.G. Perry. S. Gilroy and J.M. Vivanco. 2006. The role of root

exudates in rhizosphere interactions with plants and other organisms. Annu. Rev.

Plant Biol. 57: 233-266. 

Balsanelli, E., R.V. Serrato, V.D. Baura, G. Sassaki, M.G. Yates, L.U. Rigo, F.O.

Pedrosa, E.M. de Souza and R.A. Monteiro. 2010. Herbaspirillum seropedicae

rfbB and rfbC genes are required for maize colonization. Environ. Microbiol. 12:

2233-2244.

Barraquio, W.L., L. Revilla and J.K. Ladha. 1997. Isolation of endophytic diazotrophic

bacteria from wetland rice. Plant Soil 194: 15-24.

Bartels, D. and R. Sunkar. 2005. Drought and salt tolerance in plants. Crit. Rev. Plant

Sci. 24: 23-58.

Bashan, Y. and L.E. de-Bashan. 2005. Fresh-weight measurements of roots provide

inaccurate estimates of the effects of plant growth-promoting bacteria on root

growth: a critical examination. Soil Biol. Biochem. 37: 1795-1804.

Bates, L.S., R.P. Waldern and I.D. Teare. 1973. Rapid determination of free proline for

water status studies. Plant Soil 39: 205-207.

Beattie, G.A. and S.E. Lindow. 1995. The secret life of foliar bacterial pathogens on

leaves. Annu. Rev. Phytopathol. 33: 145-172.

Benhamou, N., J.W. Kloepper and S. Tuzun. 1998. Induction of resistance against

Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial

strain: ultrastructure and cytochemistry of the host response. Planta 204: 153-168.

Benhamou, N., S. Gagne, D.L. Quere and L. Dehbi. 2000. Bacterial-mediated induced

resistance in cucumber: beneficial effect of the endophytic bacterium Serratia

170

Page 191: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

plymuthica on the protection against infection by Pythium ultimum. Biochem. Cell

Biol. 90: 45-56.

Bent, E., and C. Chanway. 1998. The growth-promoting effects of a bacterial endophyte

on lodgepole pine are partially inhibited by the presence of other rhizobacteria.

Can. J. Microbiol. 44: 980-988.

Berg, G. and J. Hallmann. 2006. Control of plant pathogenic fungi with bacterial

endophytes. In: B. Schulz, C. Boyle and T. Sieber (eds.) Microbial Root

Endophytes. Springer‐Verlag. Berlin. p. 53-69.

Berg, G., L. Eberl and A. Hartmann. 2005. The rhizosphere as a reservoir for

opportunistic human pathogenic bacteria. Environ. Microbiol. 7: 1673-1685.

Bochner, B. 1989. Breathprints at the microbial level. ASM News. 55: 536-539.

Bohm, M., T. Hurek and B. Reinhold-Hurek. 2007. Twitching motility is essential for

endophytic rice colonization by the N2-fixing endophyte Azoarcus sp. strain

BH72. Mol. Plant Microb. Interact. 20: 526-533.

Boumahdi, M., P. Mary and J.P. Hornez. 1999. Influence of growth phases and

desiccation on the degrees of unsaturation of fatty acids and the survival rates of

rhizobia. J. Appl. Microbiol. 87: 611-619.

Brader, G., S. Compant, B. Mitter, F. Trognitz and A. Sessitsch. 2014. Metabolic potential

of endophytic bacteria. Curr. Opin. Bitotech. 30-37.

Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Ann. Biochem.

72: 248-254.

Brandi, A., P. Pietroni, C.O. Gualerzi and C.L. Pon. 1996. Post-transcritional regulation

of CspA expression in Escherichia Coli. Mol. Microbiol. 19: 231-240.

Busse, M.D. and P.J. Bottomley. 1989. Growth and nodulation responses of Rhizobium

meliloti to water stress induced by permeating and non-permeating solutes. Appl.

Environ. Microbiol. 10: 2431-2436.

Caemmerer, S.V., T. Lawson, K. Oxborough, N.R. Baker, T.J. Andrews and C.A. Raines.

2004. Stomatal conductance does not correlate with photosynthetic capacity in

transgenic tobacco with reduced amounts of Rubisco. J. Exp. Bot. 55: 1157-1166.

Cakmak, I. and H. Marschner. 1992. Magnesium deficiency and high light intensity

enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione

reductase in bean leaves. Plant Physiol. 98: 1222-1227.

171

Page 192: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Capasso, C. and C.T. Supuran. 2015. An overview of the alpha-, beta-and gamma-

carbonic anhydrases from Bacteria: can bacterial carbonic anhydrases shed new

light on evolution of bacteria? J. Enzyme Inhibit. Med. Chem. 30: 325-332.

Caron, M., C.L. Patten and S. Ghosh. 1995. Effects of plant growth promoting

rhizobacteria Pseudomonas putida GR-122 on the physiology of canola roots.

Proc. Plant Growth Regul. Soc. America. 7: 18-20.

Castro-Sowinski, S., Y. Herschkovitz, Y. Okon and E. Jurkevitch. 2007. Effects of

inoculation with plant growth-promoting rhizobacteria on resident rhizosphere

microorganisms. FEMS Microbiol. Lett. 276: 1-11.

Chakraborty, U., B.N. Chakraborty, A.P. Chakraborty and P.L. Dey. 2013. Water stress

amelioration and plant growth promotion in wheat plants by osmotic stress

tolerant bacteria. World J. Microbiol. Biotechnol. 29: 789-803.

Chang, W.S., M.V.D. Mortel, L. Nielsen, G.N.D. Guzman, X. Li and H.J. Halverson.

2007. Alginate production by Pseudomonas putida creates a hydrated

microenvironment and contributes to biofilm architecture and stress tolerance

under water limiting conditions. J. Bacteriol. 189: 8290-8299.

Chanway, C.P., M. Shishido, J. Nairn, S. Jungwirth, J. Markham, G. Xiao and F.B. Holl.

2000. Endophytic colonization and field responses of hybrid spruce seedlings after

inoculation with plant growth promoting rhizobacteria. For. Ecol. Manag.133: 81-

88.

Chaves, M.M., J. Flexas and C. Pinheiro. 2009. Photosynthesis under drought and salt

stress: regulation mechanisms from whole plant to cell. Ann. Bot. 103: 551-560.

Chaves, M.M., J.P. Maroco and J.S. Pereira. 2003. Understanding plant response to

drought-from genes to the whole plant. Funct. Plant Biol. 30: 239-64.

Chen, L.G., Y. Song, S.J. Li, L.P. Zhang, C.S. Zou, D.Q. Yu. 2012. The role of WRKY

transcription factors in plant abiotic stresses. Biochem. Biophys. 1819: 120-128.

Chernin, L.S., M.K. Winson, J.M. Thompson, S. Haran, B.W. Bycroft, I. Chet, P.

Williams and G.S.A.B. Stewart. 1998. Chitinolytic activity in Chromobacterium

violaceum: substrate analysis and regulation by Quorum sensing. J. Bacteriol. 180:

4435-4441.

Chi, F., S.H. Shen, H.P. Cheng, Y.X. Jing, Y.G. Yanni and F.B. Dazzo. 2005. Ascending

migration of endophytic Rhizobia, from roots to leaves, inside rice plants and

assessment of benefits to rice growth physiology. Appl. Environ. Microb. 71:

7271-7278.

172

Page 193: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Chiarini. L., A. Bevivino, C. Dalmastri, S. Tabacchioni and P. Visca. 2006. Burkholderia

cepacia complex species: health hazards and biotechnological potential. Trends

Microbiol. 14: 277-286.

Chinnusamy, V., J. Zhu and J.K. Zhu. 2007. Cold stress regulation of gene expression in

plants. Trends Plant Sci. 12: 444-451.

Cho, K.M., S.Y. Hong, S.M. Lee, YH. Kim, G.G. Kahng, Y.P. Lim, H. Kim and H.D.

Yun. 2007. Endophytic bacterial communities in ginseng and their antifungal

activity against pathogens. Microb. Ecol. 54: 341-351.

Ciais, P., M. Reichstein, N. Viovy, A. Granier, J. Ogee, V. Allard, M. Aubinet, N.

Buchmann, C. Bernhofer, A. Carrara, F. Chevallier, N.D. Noblet,  A.D. Friend, P.

Friedlingstein, T. Grunwald, B. Heinesch, P. Keronen, A. Knohl, G. Krinner, D.

Loustau, G. Manca, G. Matteucci, F. Miglietta,  J.M. Ourcival, D. Papale, K.

Pilegaard, S. Rambal, G. Seufert, J.F. Soussana,  M.J. Sanz, E.D. Schulze, T.

Vesala and R. Valentini. 2003. An unprecedented reduction in the primary

productivity of Europe during 2003 caused by heat and drought. Nature 437: 529-

33.

Cocking, E. 2003. Endophytic colonization of plant roots by nitrogen-fixing bacteria.

Plant Soil 252: 169-175.

Cohen, A.C., R. Bottini and P. Piccoli. 2008. Azospirillum brasilense Sp. 245 produces

ABA in chemically-defined culture medium and increases ABA content in

arabidopsis plants. Plant Growth Regul. 54: 97-103.

Cominelli, E., L. Conti, C. Tonelli and M. Galbiati. 2013. Challenges and perspectives to

improve crop drought and salinity tolerance. New Biotechnol. 30: 355-361.

Compant, S., B. Mitter, J.G. Colli-Mull, H. Gangl and A. Sessitsch. 2011. Endophytes of

grapevine flowers, berries, and seeds: identification of cultivable bacteria,

comparison with other plant parts, and visualization of niches of colonization.

Microb. Ecol. 62: 188-197.

Compant, S., B. Reiter, A. Sessitsch, J. Nowak, C. Clement and E.A. Barka. 2005.

Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium

Burkholderia sp. Strain PsJN. Appl. Environ. Microbiol. 71: 1685-1693.

Compant, S., C. Clement and A. Sessitsch. 2010. Plant growth-promoting bacteria in the

rhizo- and endosphere of plants: their role, colonization, mechanisms involved and

prospects for utilization. Soil Biol. Biochem. 42: 669-78.

173

Page 194: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Coombs, J.T. and C.M.M. Franco. 2003. Visualization of an endophytic streptomyces

species in wheat seed. Appl. Environ. Microbiol. 69: 4260-4262.

Cornic, G. 2000. Drought stress inhibits photosynthesis by decreasing stomatal aperture:

Not by affecting ATP synthesis. Trend Plant Sci. 5: 187-188.

Cornic, G., J. Ghasghaie, B. Genty and J.M. Briantais. 1992. Leaf photosynthesis is

resistant to a mild drought stress. Photosynthetica 27: 295-309.

Costa, J.M. and J.E. Loper. 1994. Characterization of siderophore production by the

biological control agent Enterobacter cloacae. Mol. Plant Microb. Interact. 7:

440-448.

Coventry, H.S. and I.A. Dubery. 2001. Lipopolysaccharides from Burkholderia

cepacia contribute to an enhanced defensive capacity and the induction of

pathogenesis-related proteins in Nicotiana tabacum. Phys. Mol. Plant

Pathol. 58:149-158.

Das, A. and A. Varma. 2009. Symbiosis: the art of living. p. 1-28. In A. Verma and A.C.

Kharkwal (eds.) Symbiotic fungi principles and practice. Springer, Berlin.

Dean, C.R. and O.P. Ward. 1992. The use of EDTA or polymixin with lysozyme for the

recovery of intracellular products from Eschericia coli. Biotechnol. Tech. 6: 133-

138.

de-Bashan L.E., J.P. Hernandez and Y. Bashan. 2012. The potential contribution of plant

growth promoting bacteria to reduce environmental degradation-A comprehensive

evaluation. Appl. Soil Ecol. 61: 171-189.

Deng, X., L. Shan, S. Inanaga and M. Inoue. 2004. Water saving approaches for

improving wheat production. J. Sci. Food Agri. 85: 1379-1388.

Dey, R., K.K. Pal, D.M. Bhatt and S.M. Chauhan. 2004. Growth promotion and yield

enhancement of peanut (Arachis hypogaea L.) by the application of plant growth

promoting rhizobacteria. Microbiol. Res. 159: 371-394.

Di Fiore, S., and M. Del Gallo. 1995. Endophytic bacteria: their possible role in the host

plant. p. 3-14. In: I. Fendrik I, M.D. Gallo, J. Vanderleyden and M. de Zamaroczy

(eds.) Azospirillum VI and related microorganisms, NATO ASI Series G:

ecological sciences. Springer-Verlag, Berlin, Heidelberg.

Dodd, I.C., N.Y. Zinovkina, V.I. Safronova and A.A. Belimov. 2010. Rhizobacterial

mediation of plant hormone status. Ann. Appl. Biol. 157: 361-379.

174

Page 195: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Dong, Z., M.J. Canny, M.E. McCully, M.R. Roboredo, C.F. Cabadilla, E. Ortega and R.

Rodes. 1994. A nitrogen-fixing endophyte of sugarcane stems. A new role for the

apoplast. Plant Physiol. 105: 1139-1147.

Dorr, J., T. Hurek and B. Reinhold‐Hurek. 1998. Type IV pili are involved in plant-

microbe and fungus-microbe interactions. Mol. Microbiol. 30: 7-17.

Doty, S.L., B. Oakley, G. Xin, J.W. Kang, G. Singleton, Z. Khan, A. Vajzovic and T.S.

James. 2009. Diazotrophic endophytes of native black cottonwood and willow.

Symbiosis 47: 23-33.

Dreesen, P.E., H.J.D. Boeck, I.A. Janssens and I. Nijs. 2012. Summer heat and drought

extremes trigger unexpected changes in productivity of a temperate

annual/biannual plant community. Environ. Exp. Bot. 79: 21-30.

Dudeja, S.S., R. Giri, R. Saini, P. Suneja-Madan and E. Kothe. 2012. Interaction of

endophytic microbes with legumes. J. Basic Microbiol. 52: 248-60.

Dulai, S., I. Molnar, J. Pronay, A. Csernak, R. Tarnai and M. Molnarlang. 2006. Effects

of drought on photosynthetic parameters and heat stability of PSII in wheat and in

Aegilops species originating from dry habitats. Acta Biol. Szeged. 50: 11-17.

Duncan, D.B. 1955. Multiple range and multiple F-test. Biometrics.11: 1- 42.

Dwivedi, R.S. and N.S. Randhawa. 1974. Evolution of rapid test for the hidden hunger of

zinc in plants. Plant Soil 40: 445-451.

Edwards, D.C. and T.B. Mckee. 1997. Characteristics of 20th century drought in the

United States at multiple time scales. Climatol. rep. 97-2, Colorado State Univ.,

Fort Collins, Colorado.

Egener, T., T. Hurek and B.R. Hurek. 1999. Endophytic expression of nif genes of

Azoarcus sp. strain BH 72 in Oryza sativa roots. Mol. Plant Microb. Interact. 12:

813-819.

Elbeltagy, A., H. Suzuki and K. Minamisawa. 2001. Endophytic colonization and in

planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species.

Appl. Environ. Microbiol. 67: 5285-5293.

Elbeltagy, A., K. Nishioka, H. Suzuki, T. Sato, Y. Sato and H. Morisaki, H. Mitsui and K.

Minamisawa. 2000. Isolation and characterization of endophytic bacteria from

wild and traditionally cultivated rice varieties. Soil Sci. Plant Nutr. 46: 617-629.

Etesami, H., H.M. Hosseini and H.A. Alikhani. 2014. Bacterial biosynthesis of 1-

aminocyclopropane-1-caboxylate (ACC) deaminase, a useful trait to elongation

175

Page 196: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

and endophytic colonization of the roots of rice under constant flooded conditions.

Physiol. Mol. Biol. Plant. 20: 425-434.

Fallik, E. and Y. Okon. 1996. Inoculants of Azospirillum brasilense: biomass production,

survival and growth promotion of Setaria italic and Zea mays. Soil Biol.

Biochem. 28: 123-126.

Fankem H., G.V.T. Chakounte, L. Ngonkot, H.L. Mafokoua, D.T. Dondjou, C. Simo, D.

Nwaga and F.X. Etoa. 2015. Common bean (Phaseolus vulgaris L.) and soya bean

(Glycine max) growth and nodulation as influenced by rock phosphate solubilizing

bacteria under pot grown conditions. Int. J. Agric. Policy Res. 5: 242-250.

Farooq, M., A. Wahid, N. Ahmad and S.A. Asad. 2010. Comparative efficacy of surface

drying and re-drying seed priming in rice: changes in emergence, seedling growth

and associated metabolic events. Paddy Water Environ. 8: 15-22.

Farooq, M., A. Wahid, N. Kobayashi, D. Fujita and S.M.A. Basra. 2009. Plant drought

stress: effects, mechanisms and management. Agron. Sust. Develop. 29: 185-212.

Fishal, E.M., S. Meon and W.M. Yun. 2010. Induction of tolerance to fusarium wilt and

defense-related mechanisms in the plantlets of susceptible Berangan Banana pre-

inoculated with Pseudomonas sp. (UPMP3) and Burkholderia sp. (UPMB3). Agri.

Sci. China 9: 1140-1149.

Fisher, D.B. 2000. Long distance transport. p. 730-784. In: B.B. Buchanan, W. Gruissem

and R. Jones (ed.) Biochemistry and molecular biology of plants. Am. Soci. Plant

Biol. Rockville.

Fleury D., J.S. Stephen, H. Kuchel and P. Langridge. 2010. Genetic and genomic tools to

improve drought tolerance in wheat. J. Exp. Bot. 61: 3199-3210.

Flexas, J. and H. Medrano. 2002. Drought-inhibition of photosynthesis in C-3 plants:

Stomatal and nonstomatal limitation revisited. Ann. Bot. 89: 183-1890.

Flexas, J., J. Bota, F. Loreto, G. Cornic and T.D. Sharkey. 2004. Diffusive and metabolic

limitations to photosynthesis under drought and salinity in C3 plants. Plant Biol.

6: 1-11.

Forchetti, G, O. Masciarelli, S. Alemano, D. Alvarez and G. Abdala. 2007. Endophytic

bacteria in sunflower (Helianthus annuus L.): isolation, characterization, and

production of jasmonates and abscisic acid in culture medium. Appl. Microbiol.

Biotechnol. 76: 1145-1152.

Forchetti, G., O. Masciarelli, M.J. Izaguirre, S. Alemano, D. Alvarez and G. Abdala.

2010. Endophytic bacteria improve seedling growth of sunflower under water

176

Page 197: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

stress, produce salicylic acid, and inhibit growth of pathogenic fungi. Curr.

Microbiol. 61: 485-93.

Furnkranz, M., B. Lukesch, H. Muller, H. Huss, M. Grube and G. Berg. 2011. Microbial

diversity inside pumpkins: microhabitat-specific communities display a high

antagonistic potential against phytopathogens. Microb. Ecol. doi:10.1007/s00248-

011-9942-4.

Gagne-Bourque, F., B.F. Mayer, J.B. Charron, H. Vali, A. Bertrand and S. Jabaji. 2015.

Accelerated growth rate and increased drought stress resilience of the model

grass Brachypodium distachyon colonized by Bacillus subtilis B26. PLoS ONE

10(6): e0130456. doi:10.1371/journal. pone.0130456

Gasser, I., M. Cardinale, H. Muller, S. Heller, L. Eberl, N. Lindenkamp, C. Kaddor, A.

Steinbuchel and G. Berg. 2011. Analysis of the endophytic lifestyle and plant

growth promotion of Burkholderia terricola ZR2-12. Plant Soil 347: 125-136.

Glick, B.R. 2005. Modulation of plant ethylene levels by the bacterial enzyme ACC

deaminase. FEMS Microbiol. Lett. 251: 1-7.

Glick, B.R. 2013. Bacteria with ACC deaminase can promote plant growth and help to

feed the world. Microbiol. Res. 169: 30-39.

Glick, B.R., D.M. Penrose and J.P. Li. 1998. A model for the lowering of plant ethylene

concentrations by plant growth-promoting bacteria. J. Theoretical. Biol. 190: 63-

68.

Glick, B.R., H.E. Brooks and J.J Pasternak. 1986. Physiological effects of plasmid DNA

transformation of Azotobacter vinelendi. Can. J. Microbiol. 32: 145-148.

Gomez-Lama Cabanas, C., E. Schiliro, A. Valverde-Corredor and J. Mercado-Blanco.

2014. The biocontrol endophytic bacterium Pseudomonas fluorescens PICF7

induces systemic defense responses in aerial tissues upon colonization of olive

roots. Front. Microbiol. 5:427.10.3389/fmicb.2014.00427

Gosal, S.S., S.H. Wani and K.S. Manjit. 2009. Biotechnology and drought tolerance. J.

Crop Imp. 23: 19-54.

Govindarajan, M., J. Balandreau, S.W. Kwon, H.Y. Weon and C. Lakshminarasimhan.

2008. Effects of the inoculation of Burkholderia vietnamensis and related

endophytic diazotrophic bacteria on grain yield of rice. Microb. Ecol. 55: 21-37.

Gray, E.J. and D.L. Smith. 2005. Intracellular and extracellular PGRP: commonalities and

distinctions in the plant-bacterium signaling processes. Soil Biol. Biochem. 37:

395-412. 

177

Page 198: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Guliyev, N., S. Bayramov and H. Babayev. 2008. Effect of water deficit on Rubisco and

carbonic anhydrase activities in different wheat genotypes. p. 1465-1468. In: J.F.

Allen, E. Gantt, J.H. Golbeck, and B. Osmond (ed.) Photosynthesis. Energy from

the Sun: 14th Int. Cong. Photosyn. Springer.

Gunatilaka, A.A.L. 2006. Natural products from plant-associated microorganisms:

distribution, structural diversity, bioactivity, and implications of their

occurrence. J. Nat. Prod. 69:509-526.

Gupta, G., J. Panwar, M. Akhtar and P. Jha. 2012. Endophytic nitrogen-fixing bacteria as

biofertilizer. p. 183-221. In: E. Lichtfouse (ed.) Sustainable agriculture reviews.

Springer, Netherlands.

Gupta, N.K., S. Gupta and A. Kumar. 2001. Effect of water stress on physiological

attributes and their relationship with growth and yield of wheat cultivars at

different stages. J. Agron. Crop Sci. 186: 55-62.

Gusain, Y.S., R. Kamal, C.M. Mehta, U.S. Singh and A.K. Sharma. 2015. Phosphate

solubilizing and indole-3-acetic acid producing bacteria from the soil of Garhwal

Himalaya aimed to improve the growth of rice. J. Environ. Biol. 36: 301-307.

Gusmaini, S.A. Aziz, A. Munif, D. Sopandie and N. Bermawie. 2013. Isolation and

selection of endophytic bacteria consortia from medicinal plant (Andrographis

Paniculata) as plant growth promoting agents. J. Agron. 12:113-121.

Hallmann, J., A. Quadt-Hallmann, W. Mahaffee, J. Kloepper. 1997. Bacterial endophytes

in agricultural crops. Can. J. Microbiol. 43: 895-914.

Hallmann, J. 2001. Plant interaction with endophytic bacteria. p.87-119. In: M.J. Jeger

and N.J Spence (eds). Biotic interaction in plant pathogen association. CAB

International, USA.

Harb, A., A. Krishnan, M.M.R. Ambavaram and A. Pereira. 2010. Molecular and

physiological analysis of drought stress in arabidopsis reveals early responses

leading to acclimation in plant growth. Plant Physiol. 154: 1254-1271.

Hardoim, P., L.V. Overbeek and J.V. Elsas. 2008. Properties of bacterial endophytes and

their proposed role in plant growth. Trends Microbiol. 16: 463-471.

Hardoim, P.R., A. Campisano, M. Doring and A. Sessitsch. 2015. The hidden world

within plants: ecological and evolutionary considerations for defining functioning

of microbial endophytes. Microbiol. Mol. Biol. Rev. 79: 293-320.

Harris, D., R.S. Tripathi and A. Joshi. 2002. On farm seed priming to improve crop

establishment and yield in dry direct seeded rice. p. 231-240. In: S. Pandey, M.

178

Page 199: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Mortimer, L. Wade, T.P. Tuoung, K. Lopes and B. Hardy (ed.) Direst seedlings:

Research strategies and opportunities. Int. Res. Inst. Manila, Philippines.

Hatch, M.D. 1987. C4 photosynthesis: a unique blend of modified biochemistry, anatomy

and ultrastructure. Biochim. Biophys. Acta. 895: 81-106.

Haupt-Herting, S. and H.P. Fock. 2000. Exchange of oxygen and its role in energy

dissipation during drought stress in tomato plants. Physiol. Plant 110: 489-495.

Havaux, M. O. Canaani and S. Malkin. 1986. Photosynthetic response of leaves to water

stress, expressed by photoacoustics and related methods I. Probing the

photoacoustic method as a indicatoor for water stress in vivo II. The effect of

rapid drought on electron transport and the relative activities of the two

photosystems. Plant physiol. 82: 827-839.

Hayat, Q., S. Hayat, M.N. Alyemeni and A. Ahmad. 2012. Salicylic acid mediated

changes in growth, photosynthesis, nitrogen metabolism and antioxidant defense

system in Cicer arietinum L. Plant Soil Environ. 58: 417-423.

Hayat, R., S. Ali, U. Amara, R. Khalid and I. Ahmed. 2010. Soil beneficial bacteria and

their role in plant growth promotion: a review. Ann Microbiol. 60: 579-598.

Hedge, J.E. and B.T. Hofreiter. 1962. Methods in Carbohydrate Chemistry (17th Ed.).

Academic Press, New York.

Hepper, C.M. 1975. Extracellular polysaccharides of soil bacteria. In: N. Walker (ed) Soil

microbiology, a critical review. Wiley, New York, p. 93-111.

Hisar, O., P. Beydemir, I. Gulçin, P.A. Hisar, T. Yanik and O.I. Kufrevioglu. 2005. The

effects of melatonin hormone on carbonic anhydrase enzyme activity in rainbow

trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo. Turk. J. Vet. Anim.

Sci. 29: 841-845.

Holliday, P. 1989. A dictionary of plant pathology. Cambridge Univ. Press, Cambridge.

Honma, M. and T. Shimomura. 1978. Metabolism of 1-aminocyclopropane-1-carboxylic

acid. Agric. Biol. Chem. 43: 1825-1831.

Hopkins, W.G. 1995. Introduction to plant physiology, John Wiley and Sons, New York.

Huang, J. 1986. Ultrastructure of bacterial penetration in plants. Annu. Rev. Phytopathol.

24: 141-157.

Hussain, M., M.A. Malik, M. Farooq, M.Y. Ashraf and M.A. Cheema. 2008. Improving

drought tolerance by exogenous application of glycine betaine and salicyclic acid

in sunflower. J. Agron. Crop Sci. 194: 193-199.

179

Page 200: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Hussain, M.B., Z.A. Zahir, H.N. Asghar and M. Asghar. 2015. Can catalase and

exopolysaccharides producing rhizobia ameliorate drought stress in wheat? Int. J.

Agric. Biol. 16: 3-13.

Iniguez, A.L., Y. Dong and E.W. Triplett. 2004. Nitrogen fixation in wheat provided by

Klebsiella pneumoniae 342. Mol. Plant Microbiol. Interact. 17: 1078-1085.

Iniguez, A.L., Y. Dong, H.D. Carter, B.M.M. Ahmer, J.M. Stone and E.W. Triplett. 2005.

Regulation of enteric endophytic bacterial colonization by plant defenses. Mol.

Plant Microb. Interact. 18: 169-178.

Ivanchenko, M.G., G.K. Muday and J.G. Dubrovsky. 2008. Ethylene-auxin interactions

regulate lateral root initiation and emergence in Arabidopsis thaliana. Plant J. 55:

335-347.

Jackson, M.L. 1962. Soil chemical analysis. Prentice Hall, Inc. Englewood Cliffs, New

York.

Jajarmi, V. 2009. Effects of water stress on germination indices in seven wheat cultivars.

World Academy Sci. Eng. Technol. 37: 105-106.

Jambunathan, N. 2010. Determination and detection of reactive oxygen species (ROS),

lipid peroxidation, and electrolyte leakage in plants. p. 291-297. In: R. Sunkar

(ed.) Plant Stress tolerance, methods in molecular biology Humana press,

Springer, New York Dordrecht, Heidelberg, London.

James, E.K., E.L. Olivares, A.L.M.D. Oliveira, F.B.J.D. Reis, L.G.D. Silva and V.M.

Reis. 2001. Further observations on the interaction between sugar cane and

Gluconobacter diazotrophicus under laboratory and greenhouse conditions. J.

Exp. Bot. 52: 747-760.

Jasim, H.S., M. Idris, A. Abdullah and A.A.H. Kadhum. 2014. Effects of

physicochemical soil properties on the heavy metal concentrations of Diplazium

esculentum (medicinal plant) from the UKM and Tasik Chini, Malaysia. Int. J.

Chem. Tech. Res. 6: 5519-5527.

Jha, P.N. and A. Kumar. 2007. Endophytic colonization of Typha australis by a plant

growth-promoting bacterium Klebsiella oxytoca strain GR-3 J. Appl. Microbiol.

103: 1311-1320.

Jones D.L. and P.R. Darrah. 1994. Role of root derived organic acids in the

mobilization of nutrients from the rhizosphere. Plant and Soil 166: 247-257.

Jordan, A., S. Haidacher, G. Hanel, E. Hartungen, L. Mark, H. Seehauser, R.

Schottkowsky, P. Sulzer and T.D. Mark. 2009. A high resolution and high

180

Page 201: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

sensitivity proton-transfer-reaction time-offlight mass spectrometer (PTR-TOF-

MS). Int. J. Mass Spectrom. 286: 122-128.

Josephine, F.S., V.S. Ramya, N. Devi, S.B. Ganapa, K.G. Siddalingeshwara, N.

Venugopal and T. Vishwanatha. 2012. Isolation, production and characterization

of protease from Bacillus Sp. isolated from soil sample. J. Microbiol. Biotech.

Res. 2: 163-168.

Kaaria, P., V. Matiru and M. Ndungu. 2012. Antimicrobial activities of secondary

metabolites produced by endophytic bacteria from selected indigenous Kenyan

plants. Afric. J. Microbiol. Res. 6: 7253-7258.

Kamara A.Y., A. Menkir, B. Badu-Apraku and O. Ibikunle. 2003. The influence of

drought stress on growth, yield and yield components of selected maize

genotypes. J. Agr. Sci. 141: 43-50.

Kaya, M.D., G. Okcu, M. Atak, Y. Cikili and O. Kolsarici. 2006. Seed treatments to

overcome salt and drought stress during germination in sunflower (Helianthus

annuus L.). J. Agron. 24: 291-295.

Kettlewell, P.S., W.L. Heath and I.M. Haigh. 2010. Yield enhancement of droughted

wheat by film antitranspirant application: Rationale and evidence. Agric. Sci. 1:

143-147.

Khalid, A., M. Arshad and Z.A. Zahir. 2004. Screening plant growth promoting

rhizobacteria for improving growth and yield of wheat. J. Appl. Microbiol. 46:

473-480.

Khan, A.L., M. Hamayun, S.M. Kang, Y.H. Kim, H.Y. Jung, J.H. Lee and I. Lee. 2012.

Endophytic fungal association via gibberellins and indole acetic acid can improve

plant growth under abiotic stress: an example of  Paecilomyces formosus LHL10

C. Microbiol. 12: 3. doi: 10.1186/1471-2180-12-3.

Khan, N.A. 1994. Variation in carbonic anhydrase activity and its relationship with

photosynthesis and dry mass of mustard. Photosynthetica 30: 317-320.

Khan, N.A., S. Singh, N.A. Anjum and R. Nazar. 2008. Cadmium effects on carbonic

anhydrase, photosynthesis, dry mass and antioxidative enzymes in wheat

(Triticum aestivum) under low and sufficient zinc. J. Plant Interact. 3: 31-37.

Kim, Y.C., B.R. Glick, Y. Bashan and C.M. Ryu. 2012. Enhancement of plant drought

tolerance by microbes. p. 383-413. In: R. Aroca (ed.) Plant responses to drought

stress: from morphological to molecular features. Springer-Verlag, Berlin,

Heidelberg.

181

Page 202: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Kishor, P.B.K., Z. Hong, G.H. Miao, C.A.A. Hu and D.P.S. Verma.1995. Overexpression

of Δ1-pyrroline-5-carboxylate synthetase increases proline production and confers

osmotolerance in transgenic plants. Plant Physiol. 108: 1387-1394.

Kloepper, J.W. and M.N. Schroth. 1978. Plant growth promoting rhizobacteria on

radishes. p. 879-882. In: Proc. 4th Int. Conf. Plant Path. Bact. Angers, France.

Kobayashi, D.Y. and J.D. Palumbo. 2000. Bacterial endophytes and their effects on plants

and uses in agriculture. In: C.W. Bacon and J.F. Jr. White (eds.) Microbial

endophytes. Dekker, New York, p. 199-233.

Konnova, S.A., O.S. Brykova, O.A. Sachkova, I.V. Egorenkova and V.V. Ignatov. 2001.

Protective role of the polysaccharide containing capsular components of

Azospirillum brasilense. Microbiol. 70: 436-440.

Koutsompogeras, P., A. Kyriacou and I. Zabetakis. 2007. The formation of 2,5-dimethyl-

4-hydroxy-2H-furan-3-one by cell-free extracts of Methylobacterium

extorquens and strawberry (Fragaria x ananassa cv. Elsanta). Food Chem. 104:

1654-1661.

Kpomblekou, K. and M.A. Tabatabai. 1994. Effect of organic acids on release of

phosphorus from phosphate rocks. Soil Sci. 158: 442-453.

Kuklinsky-Sobral, J., W. Araujo, R. Mendes, I. Geraldi, A. Pizzirani-Kleiner and J.

Azevedo. 2004. Isolation and characterization of soybean-associated bacteria and

their potential for plant growth promotion. Environ. Microbiol. 6: 1244-1251.

Labuschagne, N., T. Pretorius and A.H. Idris. 2010. Plant growth promoting rhizobacteria

as biocontrol agents against soil-borne plant diseases. p. 211-230. In: D.K.

Maheshwari (ed.) Plant growth and health promoting bacteria, microbiology

monographs. Springer, Verlag, Berlin, Heidelberg.

Ladha, J.K. and P.M. Reddy. 1995. Extension of nitrogen fixation to rice: Necessity and

possibilities. Geo J. 35: 363-372.

Lamb, T.G., D.W. Tonkyn and D.A. Kluepfel. 1996. Movement of Pseudomonas

aureofaciens from the rhizosphere to aerial plant tissue. Can. J. Microbiol. 42:

1112-1120.

Lee, S., M. Flores-Encarnacion, M. Contreras-Zentella, L.Garcia- Flores, J.E. Escamilla

and C. Kennedy. 2004. Indole-3-acetic acid biosynthesis is deficient in

Gluconacetobacter diazotrophicus strains with mutations in cytochrome C

biogenesis genes. J. Bacteriol. 186: 5384-539.

182

Page 203: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Lepetz, V., M. Massot, D.S. Schmeller and J. Clobert. 2009. Biodiversity monitoring:

some proposals to adequately study species’ responses to climate change.

Biodivers. Conserv. 18: 3185-3203.

Li, X., J. Hou, K. Bai, X. Yang, J. Lin, Z. Li and T. Kuang. 2004. Activity and

distribution of carbonic anhydrase in leaf and ear parts of wheat (Triticum

aestivum L.). Plant Sci.166: 627-632s.

Li, Z. J., Q.H. Luo. W. M. Wu, and L. Han. 2009. The effects of drought stress on

photosynthetic and chlorophyll fluorescence characteristics of Populus euphratica

and P. pruinosa. Arid Zone Res. 24: 5-9.

Lins, M.R.C.R., J.M. Fontes, N.M. Vasconcelos, D.M.S. Santos, O.E. Ferriera, J.L.

Azevedo, J.M. Araujo and G.M.S. Lima. 2014. Plant growth promoting potential

of endophytic bacteria isolated from cashew leaves. Afr. J. Biotech. 13: 3360-

3365.

Lisar, S.Y.S., R. Motafakkerazad, M.M. Hossain and I.M.M. Rahman. 2012. Water

Stress in Plants: Causes, effects and responses. In: I.M.M. Rahman (ed.) Water

Stress, In Tech, New York, USA, doi: 10.5772/39363.

Liu, B., H. Qiao, L. Huang, H. Buchenauer, Q. Han, Z. Kang and Y. Gong. 2009.

Biological control of take-all in wheat by endophytic Bacillus subtilis E1R-j and

potential mode of action. Biol. Cont. 49: 277-285.

Liu, Y., G. Fiskum and D. Schubert. 2002. Generation of reactive oxygen species by

mitochondrial electron transport chain. J. Neurochem. 80: 780-787.

Loaces, I., L. Ferrando and A.F. Scavino. 2011. Dynamics, diversity and function of

endophytic siderophore-producing bacteria in rice. Microb. Ecol. 61: 606-618.

Long, H.H., D.D. Schmidt and I.T. Baldwin. 2008. Native bacterial endophytes promote

host growth in a species-specific manner; phytohormone manipulations do not

result in common growth responses. PLoS ONE 3:2702.

doi:10.1371/journal.pone.0002702

Lopez-Fernnndez, S., P. Sonego, M. Moretto, M. Pancher, K. Engelen, I. Pertot and A.

Campisano. 2015. Whole-genome comparative analysis of virulence genes unveils

similarities and differences between endophytes and other symbiotic bacteria.

Front. Microbiol. 6:419. doi: 10.3389/fmicb.2015.00419.

Lucangeli, C. and R. Bottini. 1997. Effects of Azospirillum spp. on endogenous

gibberellin content and growth of maize (Zea mays L.) treated with uniconazole.

Symbiosis 23: 63-72.

183

Page 204: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Luquet, D., A. Vidal, M. Smith and J. Dauzat. 2005 ‘More crop per drop’: how to make it

acceptable for farmers? Agric. Water Manag. 76:108-119.

MacFaddin, J.F. 1980. Biochemical tests for identification of medical bacteria. Williams

and Wilkins, Baltimore, USA.

Madhaiyan, M., S. Poonguzhali, J. Ryu and T. Sa. 2006. Regulation of ethylene levels in

canola (Brassica campestris) by 1-aminocyclopropane- 1-carboxylate deaminase-

containing Methylobacterium fujisawaense. Planta 224: 268-278.

Madi, L. and Y. Henis. 1989. Aggregation in Azospirillum brasilense Cd: conditions and

factors involved in cell-to-cell adhesion. Plant Soil 115: 89-98.

Mahaffee, M.F. and J.W. Klopper. 1997. Department of plant pathology, Alabama

Agriculture experiment station, Biological control institute, Auburn University,

AL. 26849, U.S.A

Mahaffee, W.F., E.M. Bauske, J.W.L.V. Vuurde, M.V.D. Wolf, M.V.D. Brink and J.W.

Kloepper. 1997. Comparative analysis of antibiotic resistance, immunofluorescent

colony staining, and a transgenic marker (bioluminescence) for monitoring the

environmental fate of a rhizobacterium. Appl. Environ. Microbiol. 63: 1617-1622

Majeed, A., M.K. Abbasi, S. Hameed, A. Imran and N. Rahim. 2015. Isolation and

characterization of plant growth-promoting rhizobacteria from wheat rhizosphere

and their effect on plant growth promotion. Front. Microbiol. 6:198. doi:

10.3389/fmicb.2015.00198

Malfanova, N., F. Kamilova, S. Validov, A. Shcherbakov, V. Chebotar, I. Tikhonovich

and B. Lugtenberg. 2011. Characterization of Bacillus subtilis HC8, a novel plant-

beneficial endophytic strain from giant hogweed. Microb. Biotech. 4: 523-532.

Malfanova, N., F. Kamilova, S. Validov, V. Chebotar and B. Lugtenberg. 2013. Is L-

arabinose important for the endophytic lifestyle of Pseudomonas spp.? Arch

Microbiol. 195: 9-17.

Malinowski, D.P., G.A. Alloush and D.P. Belesk. 2000. Leaf endophyte Neotyphodium

coenophialum modifies mineral uptake in tall fescue. Plant Soil Sci. 227: 115-126.

Manivannan, P., C.A. Jaleel, R. Somasundaram and R. Panneerselvam. 2008.

Osmoregulation and antioxidant metabolism in drought-stressed Helianthus

annuus under triadimefon drenching. C.R. Biol. 331: 418-425.

Marasco, R., E. Rolli, B. Ettoumi, G. Vigani, F. Mapelli, S. Borin, A.F. Abou-Hadid,

U.A. El-Behairy, C. Sorlini, A. Cherif, G. Zocchi and D. Daffonchio. 2012. A

184

Page 205: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

drought resistance-promoting microbiome is selected by root system under desert

farming. PLoS ONE 7: e48479 doi: 10.1371/journal.pone.0048479.

Marasco, R., E. Rolli, G. Vigani, S. Borin, C. Sorlini, H. Ouzari, G. Zocchi and

D. Daffonchio. 2013. Are drought-resistance promoting bacteria cross-compatible

with different plant models? Plant Signal. Behav. 8: e26741 doi:

10.4161/psb.26741.

Mary, J.G., J.C. Stark, K.O. Brien and E. Souza, 2001. Relative sensitivity of spring

wheat grain yield and quality parameters to moisture deficit. Crop Sci. 41: 327-

335.

Mayak, S., T. Tirosh and B.R. Glick. 2004. Plant growth-promoting bacteria that confer

resistance to water stress in tomato and pepper. Plant Sci. 166: 525-530.

Mazhar, M., M. Nawaz, A.I. Mirza and K. Khan. 2015. Socio-political impacts of

meteorological droughts and their spatial patterns in Pakistan. South As. Stud. 30:

149-157.

McCully M.E. 2001. Niches for bacterial endophytes in crop plants: a plant biologist’s

view. Aust. J. Plant. Physiol. 28: 983-990

Mehta, S. and C.S. Nautiyal. 2001. An efficient method for qualitative screening of

phosphate solubilizing bacteria. Curr. Microbiol. 43: 57-58.

Melloul, M., D. Iraqi, M. El Alaoui, G. Erba, S. Alaoui, M. Ibriz and E. Elfahime. 2014.

Identification of differentially expressed genes by cDNA-AFLP technique in

response to drought Stress in Triticum durm. Food Technol. Biotechnol. 52: 479-

488.

Melnick, R., N. Zidack, B. Bailey, S. Maximova, M. Guiltinan and P. Backman. 2008.

Bacterial endophytes: Bacillus spp. from annual crops as potential biological

control agents of black pod rot of cacao. Biol. Cont. 46: 46-56.

Mendes, R., A.A. Pizzirani-Kleiner, W.L. Araujo and J.M. Raaijmakerset. 2007.

Diversity of cultivated endophytic bacteria from sugarcane: genetic and

biochemical characterization of Burkholderia cepacia complex isolates. Appl.

Environ. Microbiol. 73: 7259-7267.

Meneses, C.H.S.G., L.F.M. Rouw, J.L. Simoes-Araujo, M.S. Vidal and J.I. Baldani. 2011.

Exopolysaccharide production is required for biofilm formation and plant

colonization by the nitrogen-fixing endophyte Gluconacetobacter diazotrophicus.

Mol. Plant-Microb Interact. 24: 1448-58.

185

Page 206: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Merzaeva, D.V. and I.G. Shirokikh. 2010. The production of auxins by the endophytic

bacteria of winter rye. Appl. Biochem. Microbiol. 46: 44-50.

Milosevic, T., N. Milosevic and I. Glisic. 2012. Effect of tree conduce on the precocity,

yield and fruit quality in apricot on acidic soil. Rev. Cien. Agron. 43: 177-183.

Mishra, V. and K. A. Cherkauer. 2010. Retrospective droughts in the crop growing

season: Implications to corn and soybean yield in the Midwestern United States,

Agri. For. Meteorol. 50: 1030-1045.

Misra, A.K. 2014. Climate change and challenges of water and food security. Int. J. Sust.

Built Environ. 3: 153-165.

Mittler, R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7:

405-410.

Mogensen, V.O., H.E. Jensen and M.A. Rab. 1985. Grain yield, yield components,

drought sensitivity and water use efficiency of spring wheat subjected to water

stress at various growth stages. Irrigation Sci. 6: 131-140.

Moller, I.M., P.E. Jensen and A. Hansson. 2007. Oxidative modifications to cellular

components in plants. Annu. Rev. Plant Physiol. 58: 459-481.

Montanez, A, A.R. Blanco, C. Barlocco and M. Beracochea. 2012. Characterization of

cultivable putative endophytic plant growth promoting bacteria associated with

maize cultivars (Zea mays L.) and their inoculation effects in vitro. Appl. Soil

Ecol. 58: 21-28.

Montesinos, E. 2003. Development, registration and commercialization of microbial

pesticides for plant protection. Int. Microbiol. 6: 245-252.

Moodie, C.D., H.W. Smith and R.A. McCreery. 1959. Laboratory Manual for Soil

Fertility. Department of Agronomy, State College of Washingtion Pullman,

Washington, USA. p. 1-75.

Moreno-Gutierrez, C., G. Battipaglia, P. Cherubini, M. Saurer, E. Nicolas, S. Contreras

and J.I. Querejeta. 2012. Stand structure modulates the long-term vulnerability of

Pinus halepensis to climatic drought in a semiarid Mediterranean ecosystem. Plant

Cell Environ. 35: 1026-1039.

Morrissey, J.P., J.M. Dow, G.L. Mark and F.O. Gara. 2004. Are microbes at the root of a

solution to world food production. EMBO Rep. 5: 922-926.

Mujtaba, S.M. and S.M. Alam. 2002. Drought phenomenon and crop growth.

http://www.Pakistaneconomist. com/issue2002/issue13/i&e4.htm

186

Page 207: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Muthukumarasamy, R., I. Cleenwerck, G. Revathi, M. Vadivelu, D. Janssens, B. Hoste,

K.U. Gum, K. Park, C.Y. Son, T. Sa and J. Caballero-Mellado. 2005. Natural

association of Gluconoacetobacter diazotrophicus and diazotrophic Acetobacter

peroxydans with wetland rice. Syst. Appl. Microbiol. 28: 277-286.

Nadeem, S.M., Z.A. Zahir, M. Naveed, M. Arshad and S.M. Shahzad. 2006. Variation in

growth and ion uptake of maize due to inoculation with plant growth promoting

rhizobacteria under salt stress. Soil Environ. 25: 78-84.

Nagarajkumar, M., R. Bhaskaran and R. Velazhahan. 2004. Involvement of secondary

metabolites and extracellular lytic enzymes produced by Pseudomonas

fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight

pathogen. Microbiol. Res. 159: 73-81.

Nakano, Y. and K. Asada. 1981. Hydrogen peroxide is scavenged by ascorbate-specific

peroxidase in spinach chloroplasts. Plant Cell Physiol. 22: 867-280.

Nasopoulou, C., J. P. Pohjanen, J.J. Koskimaki, I. Zabetakis and A.M. Pirttila. 2014.

Localization of strawberry (Fragaria × ananassa) and Methylobacterium

extorquens genes of strawberry flavour biosynthesis in strawberry tissue by in situ

hybridization. J. Plant Physiol. 171: 1099-1105.

Navari-izzo, F. and N. Rascio. 1999. Plant response to water deficit conditions. Handbook

of plant and crop stress. Marcel Dekker Inc., New York, p 231-270.

Naveed, M., M.B. Hussain, Z. A. Zahir, B. Mitter and A. Sessitsch. 2014a. Drought

stress amelioration in wheat through inoculation with Burkholderia phytofirmans

strain PsJN. Plant Growth Regul. 73:121-131.

Naveed, M., B. Mittera, T.G. Reichenauer, K. Wieczorekc and A. Sessitsch. 2014b.

Increased drought stress resilience of maize through endophytic colonization by

Burkholderia phytofirmans PsJN and Enterobacter sp. FD17. Environ. Exp. Bot.

97: 30-39.

Nawangsih, A.A., I. Damayanti, S. Wiyono and J.G. Kartika. 2011. Selection and

characterization of endophytic bacteria as biological control agents of tomato

bacteria wilt disease. Hayati. 18: 66-70.

Nawaz, A., M. Farooq, S.A. Cheema, A. Yasmeen and A. Wahid. 2013. Stay green

character at grain filling ensures resistance against terminal drought in wheat. Int.

J. Agri. Biol. 15: 1272-1276.

Neilands, J.B. and K. Nakamura. 1991. In: CRC handbook of microbial iron

chelates. Winkelmann G, editor. Florida: CRC Press; 1991. p. 1-14.

187

Page 208: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Nezhadahmadi, A., Z.H. Prodhan and F. Golam. 2013. Drought tolerance in wheat. Sci.

World J. doi:10.1155/2013/610721.

Nogueira, R.J.M.C., J.A.P.M. Moraes, H.A. Burity and E.B. Neto. 2001. Modifications in

vapor diffusion resistance of leaves and water relations in barbados cherry plants

under water stress. R. Bras. Fisiol. Veg. 13: 75-87.

Nogues, S. and N.R. Baker. 2000. Effects of drought on photosynthesis in Mediterranean

plants grown under enhanced UV-B radiation. J. Exp. Bot. 51: 1309-1317.

Nonami, H. 1998. Plant water relations and control of cell elongation at low water

potentials. J. Plant Res. 111: 373-382.

Okabe, K., A. Lindlar, M. Tsuzuki and S. Miyachi. 1980. Effects of carbonic anhydrase

on ribulose 1,5-bisphosphate carboxylase and oxygenase. FEBS Lett. 114: 142-

144.

Okunishi, S., K. Sako, H. Mano, A. Imamura and H. Morisaki. 2005. Bacterial flora of

endophytes in the maturing seed of cultivated rice (Oryza sativa). Microb.

Environ. 20: 168-177.

Oteino, N., R.D. Lally, S. Kiwanuka, A. Lloyd, D. Ryan, K.J. Germaine and D.N.

Dowling. 2015. Plant growth promotion induced by phosphate solubilizing

endophytic Pseudomonas isolates. Front. Microbiol. 6: 1-9.

Overvoorde, P., H. Fukaki, T. Beeckman. 2010. Auxin control of root development. Cold

Spring Harb. Perspect. Biol. 2: 1-16.

Owen, N.L. and N. Hundley. 2004. Endophytes the chemical synthesizers inside plants.

Sci. Prog. 87: 79-99.

Pakistan Economic Survey, 2014-15. Government of Pakistan, Finance Division.

Economic Adviser Wing, Islamabad.

Parry, M.L., O.F. Canziani, J.P. Palutikof, P.J.V.D. Linden and C.E. Hanson. Climate

Change 2007: Impacts, adaptation and vulnerability. Contribution of working

group II to the fourth assessment report of the intergovernmental panel on climate

change. Cambridge Univ. Press, Cambridge, United Kingdom.

Patel, P.P., P.M. Rakhashiya, K.S. Chudasama and V.S. Thaker. 2012. Isolation,

purification and estimation of zeatin from Corynebacterium aurimucosum. Eur. J.

Exp. Biol. 1: 1-8.

Perez-Garcia, A., D. Romero and A.D. Vicente. 2011. Plant protection and growth

stimulation by microorganisms: biotechnological applications of Bacilli in

agriculture. Curr. Opin. Biotechnol. 22:187-193. 

188

Page 209: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Perez-Martin, A., C. Michelazzo, J.M. Torres-Ruiz, J. Flexas, J.E. Fernandez, L.

Sebastiani and A. Diaz-Espejo. 2014. Regulation of photosynthesis and stomatal

and mesophyll conductance under water stress and recovery in olive trees:

correlation with gene expression of carbonic anhydrase and aquaporins. J. Exp.

Bot. 65: 3143-3156.

Pillay, V.K. and J. Nowak. 1997. Inoculum density, temperature, and genotype effects on

in vitro growth promotion and epiphytic and endophytic colonization of tomato

(Lycopersicon esculentum L.) seedlings inoculated with a Pseudomonas

bacterium. Can. J. Microbiol. 43: 354-361.

Pinedo, I., T. Ledger, M. Greve and M.J. Poupin. 2015. Burkholderia phytofirmans PsJN

induces long-term metabolic and transcriptional changes involved in Arabidopsis

thaliana salt tolerance. Front plant Sci. 6: 466.

Poupin, M.J., T. Timmermann, A. Vega, A. Zuniga and B.G. Alez. 2013. Effects of the

plant growth-promoting bacterium Burkholderia phytofirmans PsJN throughout

the Life Cycle of Arabidopsis thaliana. PLoS ONE 8: e69435.

doi:10.1371/journal.pone.0069435

Prasad M.P. and S. Dagar. 2014. Identification and characterization of endophytic

bacteria from fruits like avacado and black grapes. Int. J. Curr. Microbiol. App.

Sci. 3: 937-947.

Price, A.H., J.E. Cairns, P. Horton, H.G. Jones and H. Griffiths. 2002. Linking drought-

resistance mechanisms to drought avoidance in upland rice using a QTL approach:

progress and new opportunities to integrate stomatal and mesophyll responses. J.

Exp. Bot. 53: 989-1004.

Puente, M.E., C.Y. Li and Y. Bashan. 2009a. Rock-degrading endophytic bacteria in

cacti. Environ. Exp. Bot. 66: 389-401.

Puente, M.E., C.Y. Li, and Y. Bashan. 2009b. Endophytic bacteria in cacti seeds can

improve the development of cactus seedlings. Environ. Exp. Bot. 66: 402-408.

Quadt-Hallmann, A., N. Benhamou and J.W. Kloepper. 1997. Bacterial endophytes in

cotton: mechanisms of entering the plant. Can. J. Microbiol. 43: 577-582.

Raaijmakers, J.M., and D.M. Weller. 2001. Exploiting the genetic diversity of

Pseudomonas spp: characterization of superior colonizing P. fluorescens strain

Q8r1-96. Appl. Environ. Microbiol. 67: 2545-2554.

189

Page 210: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Rajput, L., A. Imran, F. Mubeen and F.Y. Hafeez. 2013. Salt-tolerant PGPR

strain Planococcus rifietoensis promotes the growth and yield of wheat (Triticum

aestivum L.) cultivated in saline soil. Pak. J. Bot. 45: 1955-1962.

Ramakers, C, J.M. Ruijter, R.H. Deprez, A.F. Moorman . 2003. Assumption-free analysis

of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett..

339: 62-6.

Ramanan, R., K. Kannan, S.D. Sivanesan and T. Chakrabarti. 2009. Bio-sequestration of

carbon dioxide using carbonic anhydrase enzyme purified from Citrobacter

freundii. W. J. Microbiol. Biotechnol. 25: 981-987.

Ramesh, R., A.A. Joshi and M.P. Ghanekar. 2009. Pseudomonads: major antagonistic

endophytic bacteria to suppress bacterial wilt pathogen, Ralstonia solanacearum

in the eggplant (Solanum melongena L.). World J. Microbiol. Biotechnol. 25: 47

55.

Ramos, H.J.O., M.G. Yates, F.O. Pedrosa and E.M. Souza. 2011. Strategies for bacterial

tagging and gene expression in plant-host colonization studies. Soil Biol.

Biochem.  43: 1626-1638.

Randall, D.A., R.A. Wood, S. Bony, R. Colman, T. Fichefet, J. Fyfe, V. Kattsov, A.

Pitman, J. Shukla, J. Srinivasan, R.J. Stouffer, A. Sumi and K.E. Taylor. 2007.

Climate models and their evaluation. In: S. Solomon, D. Qin, M. Manning, Z.

Chen, M. Marquis, K.B. Averyt, M. Tignor and H.L. Miller (ed.) Climate Change

2007: The Physical Science Basis. Contribution of Working Group I to the Fourth

Assessment Report of the Intergovernmental Panel on Climate Change.

Cambridge Univ. Press, Cambridge, United Kindom, New York, USA.

Raven, J.A. 1995. Photosynthetic and non-photosynthetic roles of carbonic anhydrase in

algae and cyanobacteria. Phycologia 34: 93-101.

Raza, M.A.S., M.F. Saleem, M.Y. Ashraf, A. Ali and H.N Asghar. 2012. Glycine betaine

applied under drought improved the physiological efficiency of wheat (Triticum

aestivum L.) plant. Soil Environ. 31: 67-71.

Reddy, A.R., K.V. Chaitanya and M. Vivekanandan. 2004. Drought induced responses of

photosynthesis and antioxidant metabolism in higher plants. J. Plant Physiol. 161:

1189-1202.

Reguera, M., Z. Peleg and E. Blumwald. 2012. Targeting metabolic pathways for genetic

engineering abiotic stress-tolerance in crops.  Biochim. Biophys. Acta.1819: 186-

194.

190

Page 211: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Reinhold-Hurek, B. T. Hurek T. 1998. Interactions of gramineous plants with Azoarcus

spp. and other diazotrophs: Identification, localization, and perspectives to study

their function. Crit. Rev. Plant Sci. 17: 29-54.

Reinhold-Hurek, B., T. Maes, S. Gemmer, M.Van. Montagu and T. Hurek. 2006. An

endoglucanase is involved in infection of rice roots by the not cellulose

metabolizing endophyte Azoarcus sp. strain BH72. Mol. Plant-Microb. Interact.

19: 181-188.

Roberson, E.B. and M.K. Firestone. 1992. Relationship between desiccation and

exopolysaccharide production in soil Pseudomonas sp. Appl. Environ. Microbiol.

58: 1284-1291.

Rodriguez, H., R. Fraga, T. Gonzalez and Y. Bashan. 2006. Genetics of phosphate

solubilization and its potential applications for improving plant growth-promoting

bacteria. Plant Soil 287: 15-21.

Roncato-Maccari, L.D.B., H.J.O. Ramos, F.O. Pedrosa, Y. Alquini, L.S. Chubatsu, M.G.

Yates, L.U. Rigo, M.B.R. Steffens and E.M. Souza. 2003. Endophytic

Herbaspirillum seropedicae expresses nif genes in gramineous plants. FEMS

Microbiol. Ecol. 45: 39-47.

Rosenblueth, M. and E. Martinez-Romero. 2006. Bacterial endophytes and their

interactions with hosts. Mol. Plant Microb. 19: 827-837.

Rothballer, M., B. Eckert, M. Schmid, A. Fekete, M. Schloter, A. Lehner, S. Pollmann

and A. Hartmann. 2008. Endophytic root colonization of gramineous plants by

Herbaspirillum frisingense. FEMS Microbiol. Ecol. 66: 85-95.

Roy, N. and M.R. Habib. 2009. Isolation and characterization of xylanase producing

strain of Bacillus cereus from soil. Iran. J. Mirobiol. 2: 49-53.

Ryan, R.P., K. Germaine, A. Franks, D.J. Ryan and D.N. Dowling. 2008. Bacterial

endophytes: recent developments and applications. FEMS Microbiol. Lett. 278: 1-

9.

Ryu, C.M., B.R. Kang, S.H. Han, S.M. Cho, J.W. Kloepper, A.J. Anderson and Y.C.

Kim. 2007. Tobacco cultivars vary in induction of systemic resistance

against Cucumber mosaic virus and growth promotion by Pseudomonas

chlororaphis O6 and its gacS mutant. Eur. J. Plant Pathol. 119: 383-390.

Ryu, R.J. and C.L. Patten. 2008. Aromatic amino acid-dependent expression of indole-3-

pyruvate decarboxylase is regulated by TyrR in Enterobacter cloacae UW5. J.

Bacteriol. 190: 7200-7208.

191

Page 212: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Sadasivam, S. and A. Manickam. 1992. Biochemical methods for agricultural sciences.

Wiley Eastern Ltd., New Delhi, India.

Safarnejad, A. 2004. Characterization of somaclones of Medicago sativa L. for drought

tolerance. J. Agric. Sci. Technol. 6: 121-127.

Sakuma, Y., K. Maruyama, Y. Osakabe, Q. Feng, M. Seki, K. Shinozaki and K.

Yamaguchi-Shinozaki. 2006. Functional analysis of an arabidopsis transcription

factor, DREB2A, involved in drought-responsive. Plant Cell 18: 1292-309.

Saravanakumar, D., M. Kavino, T. Raguchander, P. Subbian and R. Samiyappan. 2011.

Plant growth promoting bacteria enhance water stress resistance in green gram

plants. Acta Physiol. Plant 33:203-209.

Sarwar, M., M. Arshad, D.A. Martens and W.T. Jr. Frankenberger. 1992. Tryptophan

dependent biosynthesis of auxins in soil. Plant Soil. 147: 207-215.

Scherwinski, K., R. Grosch and G. Berg. 2008. Effect of bacterial antagonists on lettuce:

active biocontrol of Rhizoctonia solani and negligible, short-term effects on non-

target microorganisms. FEMS Microbiol. Ecol. 64: 106-16.

Schmid, M., J.I. Baldani and A. Hartmann. 2006. The genus Herbaspirillum. The

Prokaryotes 5: 141-50.

Schroeder, J.I., G.J. Allen, V. Hugouvieux, J.M. Kwak and D. Waner. 2001. Guard cell

signal transduction. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 627-658.

Schulz, B. and C. Boyle. 2005. The endophytic continuum. Mycol. Res. 109: 661-686.

Schulz, B. and C. Boyle. 2006. What are endophytes? p. 1-13. In: B. Sculz, C. Boyle and

T.N. Sieber (eds.) Microbial root endophytes. Springer-Verlag, Berlin,

Heidelberg.

Schwyn, B. and J.B. Neilands. 1987. Universal chemical assay for the detection and

determination of siderophores. Anal. Biochem. 160: 47-56.

Seki, M., M. Narusaka, J. Ishida, T. Nanjo, M. Fujita, Y. Oono, A. Kamiya, M.

Nakajima, A. Enju, T. Sakurai, M. Satou, K. Akiyama, T. Taji, K. Yamaguchi-

Shinozaki, P. Carninci, J. Kawai, Y. Hayashizaki and K. Shinozaki. 2002.

Monitoring the expression profile of 7000 arabidopsis genes under drought cold

and high-salinity stresses using a full length cDNA Mircoroarray. Plant J. 279-

292.

Sessitsch, A., B. Reiter, U. Pfeifer and E. Wilhelm. 2002. Cultivation-independent

population analysis of bacterial endophytes in three potato varieties based on

192

Page 213: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

eubacterial and Actinomycetes-specific PCR of 16S rRNA genes FEMS

Microbiol. Ecol. 39: 23-32.

Sevilla, M., R.H. Burris, N. Gunapala and C. Kennedy. 2001. Comparison of benefit to

sugarcane plant growth and 15N incorporation following inoculation of sterile

nitrogen fixation outside and inside plant tissues19 plants with Acetobacter

diazotrophicus wild-type and Nif- mutant strains. Mol. Plant-Microb. Interact. 14:

358-366.

Sgherri, C.L.M., M. Maffei and F. Navari-Izzo. 2000. Antioxidative enzymes in wheat

subjected to increasing water deficit and rewatering. J. Plant Physiol. 157: 273-

279.

Sgroy, V., F. Cassan, O. Masciarelli, M.F. Del Papa, A. Lagares and V. Luna. 2009.

Isolation and characterization of endophytic plant growth-promoting (PGPB) or

stress homeostasis-regulating (PSHB) bacteria associated to the halophyte

Prosopis strombulifera. Appl. Microbiol. Biotech. 85: 371-381.

Shakir, M.A., B. Asghari and M. Arshad. 2012. Rhizosphere bacteria containing ACC

deaminase conferred drought tolerance in wheat grown under semi-arid climate.

Soil Environ. 31: 108-112.

Sharma, A. and B.N. Johri. 2003. Growth promoting influence of siderophore-producing

Pseudomonas strains GRP3A and PRS9 in maize (Zea mays L.) under iron

depriving conditions. Microbiol. Res. 158: 243-248.

Sheibani-Tezerji, R., T. Rattei, A. Sessitsch, F. Trognitz and B. Mitter. 2015.

Transcriptome profiling of the endophyte Burkholderia phytofirmans PsJN

indicates sensing of the plant environment and drought stress. mBio 6:e00621-15.

doi:10.1128/mBio.00621-15.

Sheramati, I, S. Tripathi, A. Varma and R. Oelmuller. 2008. The root-colonizing

endophyte Piriformaspora indica confers drought tolerance in arabidopsis by

stimulating the expression of drought stress-related genes in leaves. Mol. Plant

Microb. Interact. 2: 799-807

Shi, Y., K. Lou, and C. Li. 2010. Growth and photosynthetic efficiency promotion of

sugar beet (Beta vulgaris L.) by endophytic bacteria. Photosyn. Res. 105: 5-13.

Shinozaki, K. and K. Yamaguchi-Shinozaki. 2007. Gene networks involved in drought

stress response and tolerance. J. Exp. Bot. 58: 221-227.

193

Page 214: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Shinozaki, K., K. Yamaguchi-Shinozaki and M. Seki. 2003. Regulatory network of gene

expression in the drought and cold stress responses. Curr. Opin. Plant Biol. 6:

410-417.

Shokri, D. and G. Emtiazi. 2010. Indole-3-acetic acid (IAA) production in symbiotic and

non-symbiotic nitrogen-fixing bacteria and its optimization by Taguchi design.

Curr. Microbiol. 61: 217-225.

Silva, E.C., R.J.M.C. Nogueira, A.D.N. Azevedo and V.F. Saints. 2003. Stomatal

behavior and potential leaf water on three woody species grown under water

stress. Acta. Bot. Bras. 17: 231-246.

Singleton, V.L., R. Orthofer and R.M. Lamuela-Raventos. 1999. Analysis of total phenols

and other oxidation substrates and antioxidants by means of Folin-Ciocalteu

reagent. Methods Enzymol.1299: 152- 178.

Sly, W.S. and P.Y. Hu. 1995. Human carbonic anhydrases and carbonic anhydrase

deficiencies. Annu. Rev. Biochem. 64: 375-401.

Smith, I.K., T.L. Vierheller and C.A. Thorne. 1988. Assay of glutathione reductase in

crude tissue homogenates using 5, 5´-dithiobis (2-nitrobenzoic acid). Anal.

Biochem. 175: 408-413.

Solomon, S.D., D. Qin, M. Manning, Z. Chen, M. Marquis, K.B. Averyt, M. Tignor and

H. L. Miller. 2007. Climate Change 2007: The Physical Scientific Basis:

Contribution of working group I to the fourth assessment report of the

intergovernmental panel on climate change, Cambridge Univ. Press, New York,

996 p.

Somers, E., J. Vanderleijden and M. Srinivasan. 2004. Rhizosphere bacterial signalling: a

love paradebeneath our feet. Crit. Rev. Microbiol. 30: 205-240.

Sorensen, J. and A. Sessitsch. 2006. Plant-associated bacteria-lifestyle and molecular

interactions. p. 211-236. In: J.D. van Elsas, J.K. Jansson, J.K. Trevors (eds.)

Modern soil microbiology. CRC Press.

Steel, K.J. 1961. The oxidase reaction as a toxic tool. J. Gen. Microbiol. 25: 297-306.

Steel, R.G.D., J.H. Torrie and D.A. Dicky. 1997. Principles and Procedures of Statistics-

A Biometrical Approach. (3rd Ed.) McGraw Hill Book International Co.,

Singapore. p. 204-227.

Stikic, R., Z. Jovanovic and L. Prokic. 2014. Mitigation of plant drought stress in a

changing climate. Botanica SERBICA 38: 35-42.

194

Page 215: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Strobel, G., B. Daisy, U. Castillo and J. Harper. 2004. Natural products from endophytic

microorganisms. J. Nat. Prod. 67: 257-268.

Studer, A.J., A. Gandin, A.R. Kolbe, L. Wang, A.B. Cousins and T.P. Brutnell. 2014. A limited role for carbonic anhydrase in C4 photosynthesis as revealed by a ca1ca2 double mutant in maize. Plant. Physiol. 165:608-617.

Sturz, A.V. and J. Nowak. 2000. Endophytic communities of rhizobacteria and the

strategies required to create yield enhancing associations with crops. Appl. Soil

Ecol. 15:183-190.

Sturz, A.V., B.R. Christie and J. Nowak. 2000. Bacterial endophytes: potenti al role in

developing sustainable systems of crop production. Crit. Rev. Plant Sci. 19: 1-30.

Sun, Y., Z. Cheng and B. Glick. 2009. The presence of a 1-aminocyclopropane-1-

carboxylate (ACC) deaminase deletion mutation alters the physiology of the

endophytic plant growth-promoting bacterium Burkholderia phytofirmans PsJN.

FEMS Microbiol. Lett. 296: 131-136.

Supuran, C.T. 2008. Carbonic anhydrases: novel therapeutic applications for inhibitors

and activators. Nat. Rev. Drug Discov. 7: 168-81.

Supuran, C.T. 2011. Carbonic anhydrase inhibitors and activators for novel therapeutic

applications. Future Med. Chem. 3: 1165-80.

Suzuki, T., M. Shimizu, A. Meguro, S. Hasegawa, T. Nishimura and H. Kunoh. 2005.

Visualization of infection of an endophytic Actinomycete Streptomyces galbus in

leaves of tissue-cultured Rhododendron. Actinomycetologica 19: 7-12.

Sziderics, A.H., F. Rasche, F. Trognitz, A. Sessitsch and E. Wilhelm. 2007. Bacterial

endophytes contribute to abiotic stress adaptation in pepper plants (Capsicum

annuum L.) Can. J. Microbiol. 53: 1195-1202.

Taiz, L. and E. Zeiger. 2010. Plant Physiol (5th Ed.). Sinauer Assoc. Inc. Publ.

Massachusetts, USA.

Tal, S. and Y. Okon. 1985. Production of the reserve material poly- beta-hydroxybutyrate

and its function in Azospirillum brasilense Cd. Can. J. Microbiol. 31: 608-613.

Talaat, N.B. and B.T. Shawky. 2014. Protective effects of arbuscular mycorrhizal fungi

on wheat (Triticum aestivum L.) plants exposed to salinity. Environ. Exp. Bot. 98:

20-31.

195

Page 216: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Tan, R.X. and W.X. Zou. 2001. Endophytes: a rich source of functional metabolites. Nat.

Prod. Rep. 18: 448-459.

Tataeizadeh, Z. 1998. Drought-induced responses in plant cells. Int. Rev. Cytol. 182: 193-

247.

Teulat, B., N. Zoumarou-Wallis, B. Rotter, H.B.S. Bahri and D. This. 2003. QLT for

relative water content in field grown barley environment and their stability across

the Mediterranean environments. Theor. Appl. Genet. 108:181-188.

Theocharis, A., S. Bordiec, O. Fernandez, S. Paquis, S. Dhondt-Cordelier, F. Baillieul, C.

Clement and E.A. Barka. 2012. Burkholderia phytofirmans PsJN primes Vitis

vinifera L. and confers a better tolerance to low nonfreezing temperatures. Mol.

Plant Microbe Interact. 25: 241-249.

Thomashow, L.S. and D.M. Weller. 1996. Current concepts in the use of introduced

bacteria for biological disease control: mechanisms and antifungal metabolites. p.

187-235. In: G. Stacey and N. Keen (eds.) Plant-microbe interaction. Champman

and Hall, New York.

Timmusk, S. 2003. Mechanism of action of the plant growth promoting Bacterium

Paenibacillus polymyxa. Comprehansive summaries of uppsala dissertations from

the Faculty of Science and Technology 908. Acta Univ. Upsaliensis Uppsala,

Sweden. p 40.

Timmusk, S. and E. Nevo. 2011. Plant root associated biofilms: perspectives for natural

product mining. p. 285-300. In: D.K. Maheshwari (ed.) Bacteria in agrobiology:

Plant nutrient management. Springer-Verlag, Berlin, Heidelberg.

Timmusk, S. and E.G.H. Wagner. 1999. The plant growth-promoting rhizobacterium

Paenibacillus polymyxa induces changes in Arabidopsis thaliana gene expression:

a possible connection between biotic and abiotic stress responses. Mol. Plant

Microb. Int.12: 951-959.

 Timmusk, S. and L. Behers. 2012. Rhizobacterial application for sustainable water

management on the areas of limited water resources. Irrig. Drainage Syst. Eng. 1:

e111. doi: 10.4172/2168-9768.1000e111 

Timmusk, S., I.A. Abd El-Daim, L. Copolovici, T. Tanilas, A. Kannaste, L. Behers, E.

Nevo, G. Seisenbaeva, E. Stenstrom and U. Niinemets. 2014. Drought-tolerance

of wheat improved by rhizosphere bacteria from harsh environments: enhanced

biomass production and reduced emissions of stress volatiles. PLoS ONE 9:

e96086. doi:10.1371/journal.pone.0096086

196

Page 217: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Timmusk, S., I.A.A. El-Daim, L. Copolovici, T. Tanilas, A. Kannaste, L. Behers, E.

Nevo, G. Seisenbaeva, E. Stenstrom and U. Niinemets. 2011. Drought-tolerance

of wheat improved by rhizosphere bacteria from harsh environments: enhanced

biomass production and reduced emissions of stress volatiles. PLoS ONE 6:

e17968. 10.1371/journal.pone.0017968

Timmusk, S., N. Grantcharova and E.G.H. Wagner. 2005. Paenibacillus polymyxa

invades plant roots and forms biofilms. Appl. Environ. Microbiol. 71: 7292-7300.

Tiwari A, P. Kumar, S. Singh and S.A. Ansari. 2005. Carbonic anhydrase in relation to

higher plants. Photosynthetica 43: 1-11.

Tran, L.S.P., K. Nakashima, Y. Sakuma, S.D. Simpson, Y. Fujita, and K. Maruyama.

2004. Isolation and functional analysis of Arabidopsis stress-inducible NAC

transcription factors that bind to a drought-responsive cis-element in the early

responsive to dehydration stress 1 promoter. Plant Cell 16: 2481-2498.

Tripp, B.C., K. Smith and J.G. Ferry. 2001. Carbonic anhydrase new insights for an

ancient enzyme. J. Biol. Chem. 276: 48615-48618.

U.S. Salinity Lab. Staff. 1954. Diagnosis and improvement of saline and alkali soils.

USDA Hand Book No. 60. Washington. DC, USA. 160p.

UmaMaheswari, T., K. Anbukkarasi, T. Hemalatha and K. Chendrayan. 2013. Studies on

phytohormone producing ability of indigenous endophytic bacteria isolated from

tropical legume crops. Int. J. Curr. Microbiol. App. Sci. 2: 127-136.

United States Department of Agriculture (USDA). 2011. Natural resources conservation

service. http://soils.usda.gov/technical/aids/investigations/texture/ (accessed on

25.09.2011).

Upadhyay, R., K. Ramalakshmi, L.J.M. Rao. 2012. Microwave assisted extraction of

chlorogenic acids from green coffee beans. Food Chem. 130: 184-188.

Van, L.L.C., P.A.H.M. Bakker and C.M.J. Pieterse. 1998. Systemic resistance induced by

rhizophere bacteria. Annu. Rev. Phytopath. 36 453-483.

Vardharajula, S., S.Z. Ali, M. Grover, G. Reddy and V. Bandi. 2011. Drought-tolerant

plant growth promoting Bacillus spp.: effect on growth, osmolytes, and

antioxidant status of maize under drought stress. J. Plant Interact. 6: 1-14.

Verma, V.C., S.K. Gond, A. Mishra, A. Kumar, R.N. Kharwar and A.C. Gange. 2009.

Endophytic actinomycetes from Azadirachta indica A. Juss.: isolation, diversity

and antimicrobial activity. Microb. Ecol. 57: 749-756.

197

Page 218: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Vessey, J.K. 2003. Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 255:

571-586.

Vincent, J.M. 1970. A manual for the practical study of root nodule bacteria (First Ed.)

Oxford Publ. Int. Biolog. Program, p. 164.

Viruel, E., L.E. Erazzu, L.M. Calsina, M.A. Ferrero, M.E. Lucca, F. Sineriz. 2014.

Inoculation of maize with phosphate solubilizing bacteria: effect on plant growth

and yield. J. Soil Sci. Plant Nutr. 14: 819-831.

Wahid, A., S. Gelani, M. Ashraf and M.R. Foolad. 2007. Heat tolerance in plants: an

overview. Environ. Exp. Bot. 61: 199-223.

Walter, J., C. Beierkuhnlein, R. Hein, J. Nagy, U. Rascher, E. Willner and A. Jentsch.

2011. Do plants remember drought? Evidence for drought memory in grasses.

Environ. Exp. Bot. 71: 34-40.

Wang, W., B. Vinocur and A. Altman. 2003. Plant responses to drought, salinity and

extreme temperatures: towards genetic engineering for stress tolerance. Planta

218:1-14.

Wang, W., Y. Zhai, L. Cao, H. Tan and R. Zhang. 2016. Endphytic bacterial and fungal

microbiota in sprouts, roots and stem of rice (Oryza sativa L.). Mirobiol. Res.

188-189:1-8.

Watanabe, F.S. and S.R. Olsen. 1965. Test of an aerobic acid method for determination of

phosphorus in water and NaHCO3 extracts. Soil Sci. Soc. Am. Proc. 29: 677-678.

Wei-Hong, S., W. Yan-You, S. Zhen-Zhen, W. Qiu-Xia and W. Xin-Yu. 2014.

Enzymatic characteristics of higher plant carbonic anhydrase and its role in

photosynthesis. J. Plant Stud. 3: 39-44.

Welbaum, G.E., A.V. Sturz, Z. Dong and J. Nowak. 2004. Managing soil microorganisms

to improve productivity of agro-ecosystems. Crit. Rev. Plant Sci. 23: 75-193.

Weller, D.M. 1988. Biological control of soil borne plant pathogens in the rhizosphere

with bacteria. Annu. Rev. Phytopathol. 26: 379-407.

Weyens, N., D.V. Lelie, S. Taghavi, L. Newman and J. Vangronsveld. 2009. Exploiting

plant-microbe partnerships to improve biomass production and remediation.

Trend. Biotech. 27: 591-598.

Wilson, K.J., A. Sessitsch, J.C. Corbo, K.E. Giller, A.D.L. Akkermans and R.A.

Jefferson. 1995. Glucuronidase (GUS) transposons for ecological and genetic

studies of rhizobia and other gram-negative bacteria. Microbiology 141: 1691-

1705.

198

Page 219: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Wolf, B. 1982. The comprehensive system of leaf analysis and its use for diagnosting

crop nutrient status. Comm. Soil Sci. Plant Anal. 13:1035-1059.

Wollum, A.G. 1982. Cultural methods for soil microorganisms. p. 781-802. In: A.L.

Page, R. H. Miller and D. R. Keeney (ed.) Methods of soil analysis. Part 2.

Chemical and microbiological properties (2nd Ed.). Am. Soc. Agron. Madison,

Wis.

Wood, A.J. 2005. Eco-physiological adaptations to limited water environments. p.1-13.

In: M.A. Jenks and P.M. Hasegawa (ed.) Plant abiotic stress. Blackwell Publish.

Ltd. UK.

XinBin, D., Z. RongXian, L. Wei, XiaMing, C. ShuQing. 2001. Effect of carbonic

anhydrase in wheat leaf on photosynthetic function under low CO2 concentration.

Sci. Agric. Sinica. 34: 97-100.s

Yamaguchi-Shinozaki, K. and K. Shinozaki. 2005. Organization of cis-acting regulatory

elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci. 10:

88-94.

Yandigeri, M.S., K.K. Meena, D. Singh, N. Malviya, D.P. Singh, M.K. Solanki, A.K.

Yadav and D.K. Arora. 2012. Drought-tolerant endophytic actinobacteria promote

growth of wheat (Triticum aestivum) under water stress conditions. Plant Growth

Regul. 68: 411-420.

Yang, J., J.W. Kloepper and C.M. Ryu. 2009. Rhizosphere bacteria help plants tolerate

abiotic stress. Trends Plant Sci. 14: 1-4.

Yang, S., X. Zhang, Z. Cao, K. Zhao, S. Wang, M. Chen and X. Hu. 2014. Growth-

promoting Sphingomonas paucimobilis ZJSH1 associated with Dendrobium

officinale through phytohormone prod of production and nitrogen fixation.

Microb. Biotechnol. 7: 611-20.

Yin, L., H. Lin and Z. Xiao. 2010. Purification and characterization of a cellulase from

Bacillus subtilis YJ1. J. Marine Sci. Technol. 18: 466-471.

You, M., T. Nishiguchi, A. Saito, T. Isawa, H. Mitsui and K. Minamisawa. 2005.

Expression of the nifH gene of a Herbaspirillum endophyte in wild rice species:

daily rhythm during the light-dark cycle. Appl. Environ. Microbiol. 71: 8183-

8190.

Yu, S., X.X. Zhang, Q.J. Guan, T. Takano and S.K. Liu. 2007. Expression of a carbonic

anhydrase gene is induced by environmental stresses in Rice (Oryza sativa L.).

Biotech. Lett. 29: 89-94.

199

Page 220: Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/11611/1/Ana... · Web viewstrain PsJN increased the chlorophyll content, CO 2 assimilation rate and water use efficiency

Zachow, C., J. Fatehi, M. Cardinale, R. Tilcher and G. Berg. 2010. Strain-specific

colonization pattern of Rhizoctonia antagonists in the root system of sugar beet.

FEMS Microbiol. Ecol. 74: 124-35.

Zachow, C., R. Tilcher and G. Berg. 2008. Sugar beet-associated bacterial and fungal

communities show a high indigenous antagonistic potential against plant

pathogens. Microb. Ecol. 55: 119-129.

Zakria, M., J. Njoloma, Y. Saeki and S. Akao. 2007. Colonization and nitrogen-fixing

ability of Herbaspirillum sp. strain B501 gfp1 and assessment of its growth-

promoting ability in cultivated rice. Microb. Environ. 22: 197-206.

Zhang, Z., B. Lian, W. Hou, M. Chen, X. Li and Y. Li. 2011. Bacillus mucilaginosus can

capture atmospheric CO2 by carbonic anhydrase. Afr. J. Microbiol. Res. 5: 106-

112.

Zhang, Z., B. Lian, W. Hou, M. Chen, X. Li, W. Shen and Y. Li. 2010. Optimization of

nutritional constituents for carbonic anhydrase production by Bacillus

mucilaginosus K02. Afr. J. Biotechnol. 10: 8403-8413.

Zlatev, Z. and I. Yordanov. 2004. Effects of soil drought on photosynthesis and

chlorophyll fluorescence in common bean plants. Bulg. J. Plant Physiol. 30: 3-18.

Zuniga, A., M.J. Poupin, R. Donoso, T. Ledger and N. Guiliani, R.A. Gutierrez and B.

Gonzalez. 2013. Quorum Sensing and indole-3-ccetic acid degradation play a role

in colonization and plant growth promotion of Arabidopsis

thaliana by Burkholderia phytofirmans PsJN. Mol. Plant. Microb. Interact. 26:

546-553.

200