Subhan Danish Soil Sci 2020 Bzu

EFFECTS OF ACC-DEAMINASE CONTAINING
RHIZOBACTERIA AND BIOCHAR ON WHEAT AND

MAIZE PRODUCTIVITY UNDER DROUGHT STRESS
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENT FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN AGRICULTURE

(SOIL SCIENCE)
By
SUBHAN DANISH
M.Sc. (Hons.) Agriculture – Soil Science
DEPARTMENT OF SOIL SCIENCE
FACULTY OF AGRICULTURAL SCIENCES & TECHNOLOGY
BAHAUDDIN ZAKARIYA UNIVERSITY MULTAN
2020
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“IN THE NAME OF ALLAH
THE MOST BENEFICENT

And
THE MOST MERCIFUL”
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DECLARATION
I hereby declare that the contents of the thesis “EFFECT OF ACC-DEAMINASE
CONTAINING RHIZOBACTERIA AND BIOCHAR ON WHEAT AND
MAIZE PRODUCTIVITY UNDER DROUGHT STRESS” are product of my own
research and no part has been copied from any published source (except references,
standard mathematical or / equations / formulate / protocols, etc.). I further declare that
this work has not been submitted for award of any other degree. The university may
take action if the information/data provided are found inaccurate at any stage. I am
aware that in case of any problem, it will be proceeded as per HEC plagiarism policy.
SUBHAN DANISH
2008-ag-62
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Acknowledgement
All Prays for Allah (SWT) only Who is Merciful and Most Beneficent. Darood-o-Salaam
on last Prophet Muhammad (SAWW) and His Family (Panjtan Pak) they are all true inspirations
for me on every step of my life and hereafter. I read in Quran and believe “There are Signs in
that for people who believe” (Surat al-An’am, 99) so I want to thanks to Allah SWT for electing
me among those who tried to find out (research) in Nature.
I have a lot of people to acknowledge for this Ph.D. research work I did carry out. First
of all, I would like to express gratitude to my supervisor Assoc. Prof. Dr. Muhammad Zafar-ul-
Hye for his support and leadership throughout my work. I want to express my special thanks to my
all respectful teacher Dr. Muhammad Abid, Dr. Abdur Rahim, Dr. Muhammad Arif Ali, Dr.
Farooq Qayyum, Dr. Shahid and Dr. Niaz Ahmed who taught me and make me able to reach
my destination. Financial support for the Ph.D. provided by Higher Education Commission of
Pakistan through INDIGENOUS 5000 PHD FELLOWSHIP PROGRAM, is gratefully
acknowledged. I also thank anonymous referees who gave valuable suggestions to improve the
quality of the dissertation.
I would like to acknowledge to my mother (Shabana Hameed) and especially my Father

Abdul Hameed (Late) who have been so caring and supportive all the time both financially and
emotionally. Without their support, all the way through my life it was not possible for me to reach
this stage.
Subhan Danish
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DEDICATING
THIS THESIS TO
MY LOVING PARENTS
THEY GIVE ME THE DIRECTION TOWARDS SUCCESS AND PRAYS FOR ME IN

EVERY STEP OF LIFE
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LIST OF PUBLICATIONS FROM THE THESIS RESEARCH
1. Danish, S., M., Zafar-ul-Hye, M., Hussain, M., Shaaban, A., Núñez-Delgado, S., Hussain and
M.F., Qayyum, 2019. Rhizobacteria with ACC-deaminase activity improve nutrient uptake,
chlorophyll contents and early seedling growth of wheat under PEG-induced osmotic stress.
International Journal of Agriculture and Biology, 21: 1212‒ 1220.
https://doi.org/10.17957/IJAB/15.1013.
2. Danish, S., and M., Zafar-ul-Hye. 2019. Co-application of ACC-deaminase producing PGPR
and timber-waste biochar improves pigments formation, growth and yield of wheat under
drought stress. Scientific Report, 9: 5999. https://doi.org/10.1038/s41598-019-42374-9.
3. Zafar-ul-Hye, M., S., Danish, M., Abbas, M., Ahmad and T.M., Munir. 2019. ACC Deaminase
Producing PGPR Bacillus amyloliquefaciens and Agrobacterium fabrum along with Biochar
Improve Wheat Productivity under Drought Stress. Agronomy, 9: 343.
https://10.3390/agronomy9070343.
4. Danish, S., M. Zafar-ul-Hye. 2020. ACC deaminase producing bacteria and biochar: Key
mitigators of drought stress in plants. Phyton. 89(2): 217-227.
https://doi.org/10.32604/phyton.2020.08523.
5. Danish, S., M. Zafar-ul-Hye, S. Hussain, M. Riaz and M.F. Qayyum. 2020. Mitigation of
drought stress in maize through inoculation with drought tolerant ACC deaminase containing
PGPR under axenic conditions. Pakistan Journal of Botany, 52(1): 49-60.
http://dx.doi.org/10.30848/PJB2020-1(7).
6. Danish, S., M. Zafar-ul-Hye, F. Mohsin and M. Hussain. 2020. ACC-deaminase producing
plant growth promoting rhizobacteria and biochar mitigate adverse effects of drought stress on
maize growth. PLoS One, 15(4): e0230615. http://dx.doi.org/10.1371/journal.pone.0230615.
7. Danish, S., M. Zafar-ul-Hye, S. Fahad, S. Saud, M. Brtnicky, T. Hammerschmiedt and R.
Datta. 2020. Drought Stress Alleviation by ACC Deaminase Producing Achromobacter
xylosoxidans and Enterobacter cloacae with and without Timber Waste Biochar in Maize.
Sustainability, 12(15): 6286. https://doi.org/10.3390/su12156286.
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TABLE OF CONTENTS
TABLE OF CONTENTS .......................................................................................................... viii
LIST OF TABLES ..................................................................................................................... xvi
LIST OF FIGURES ................................................................................................................... xix
LIST OF ABBREVIATIONS ................................................................................................... xxi
ABSTRACT .................................................................................................................................... 1
CHAPTER 1 .................................................................................................................................. 2
INTRODUCTION .......................................................................................................................... 2
CHAPTER 2 .................................................................................................................................. 6
REVIEW OF LITERATURE ......................................................................................................... 6
2.1. Climatic changes and food demand .................................................................................... 6
2.2. Drought ............................................................................................................................... 6
2.4. Plant growth promoting rhizobacteria ................................................................................ 8
2.5. Biochar ................................................................................................................................ 9
CHAPTER 3 ................................................................................................................................ 12
MATERIALS AND METHODS ............................................................................................... 12
3.1.1. Collection of Rhizosphere Soils ........................................................................................ 12
3.1.2. Isolation, Purification and Selection of Drought Tolerant Isolates ................................. 12
3.1.3. Experimental Details ........................................................................................................ 12
3.1.4. Growth and Chemical Analysis of Plants ......................................................................... 13
3.1.5. Chlorophyll Content.......................................................................................................... 14
3.1.6. Biochemical Characterization of Effective PGPR ............................................................ 14
3.1.7. Identification of PGPR ...................................................................................................... 14
3.1.8. ACC deaminase PGPR ..................................................................................................... 15
3.1.9. Biochar production ........................................................................................................... 15
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3.1.10. Biochar characterization .................................................................................................. 15
3.1.11. Soil characterization ......................................................................................................... 16
3.1.12. Electrolyte Leakage .......................................................................................................... 16
3.1.13. Determination of proline................................................................................................... 16
3.1.14. Gas exchange attributes .................................................................................................... 17
3.1.15. Statistical Analysis ............................................................................................................ 17
CHAPTER 4 ................................................................................................................................ 18
Rhizobacteria with ACC-deaminase Activity Improves Nutrient Uptake, Chlorophyll Contents and
Early Seedling Growth of Wheat under PEG-induced Osmotic Stress ........................................ 18
Abstract .............................................................................................................................................
18
4.1. Introduction ....................................................................................................................... 19
4.2. Materials and Methods ...................................................................................................... 21
4.3. Results ............................................................................................................................... 21
4.3.1. Growth attributes .............................................................................................................. 21
4.3.2. Chlorophyll contents ......................................................................................................... 26
4.3.3. Nutrients in shoot .............................................................................................................. 26
4.3.4. PGPR characteristics ........................................................................................................ 31
4.4. Discussion ......................................................................................................................... 32
4.5. Conclusion ........................................................................................................................ 34
CHAPTER 5 ................................................................................................................................ 35
Mitigation of drought stress in maize by inoculation of drought tolerant ACC deaminase containing
PGPR under axenic conditions ..................................................................................................... 35
Abstract ......................................................................................................................................... 35
5.1. Introduction ....................................................................................................................... 35
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5.2.1. Collection of rhizosphere .................................................................................................. 37
5.2.2. Isolation, incubation and purification of isolates ............................................................. 37
5.2.3. Selection of drought-tolerant isolates ............................................................................... 37
5.2.4. Design and site of experiment ........................................................................................... 37
5.2.5. Seeds sterilization and inoculation ................................................................................... 37
5.2.6. Application of Hoagland solution ..................................................................................... 38
5.2.7. Artificial drought stress .................................................................................................... 38
5.2.8. Harvesting and Morphological attributes ........................................................................ 38
5.2.9. Nutrients analysis.............................................................................................................. 38
5.2.11. Molecular identification of effective drought tolerant PGPR........................................... 38
5.2.12. Biochemical characterization of most efficient PGPR ..................................................... 39
5.3. Results ............................................................................................................................... 40
5.3.1. Shoot and root length ........................................................................................................ 40
5.3.2. Shoot fresh and dry weight ............................................................................................... 42
5.3.3. Root fresh and dry weight ................................................................................................. 42
5.3.4. Chlorophyll content .......................................................................................................... 45
5.3.5. N, P and K concentration in shoot .................................................................................... 45
5.4. Discussion ......................................................................................................................... 50
5.5. Conclusion ........................................................................................................................ 51
CHAPTER 6 ................................................................................................................................ 52
Co-application of ACC-deaminase producing PGPR and timber-waste biochar improves pigments

formation, growth and yield of wheat under drought stress ......................................................... 52
Abstract ......................................................................................................................................... 52
6.1. Introduction ....................................................................................................................... 52

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6.2.1. ACC deaminase PGPR ..................................................................................................... 54
6.2.2. Biochar production ........................................................................................................... 54
6.2.3. Biochar characterization .................................................................................................. 54
6.2.4. Pots dimensions and soil characterization ....................................................................... 55
6.2.5. Pots preparation ............................................................................................................... 55
6.2.6. Seeds collection and sterilization...................................................................................... 55
6.2.7. PGPR inoculation ............................................................................................................. 55
6.2.8. Experiment site and treatments ......................................................................................... 55
6.2.9. Seeds sowing and drought ................................................................................................ 55
6.2.10. Reproductive stage harvesting and yield attributes .......................................................... 56
6.2.11. Nutrients analysis.............................................................................................................. 56
6.2.14. Proline............................................................................................................................... 56
6.2.15. Statistical analysis ............................................................................................................ 56
6.3. Results ............................................................................................................................... 56
6.3.1. Shoot length and electrolyte leakage ................................................................................ 56
6.3.2. Yield attributes .................................................................................................................. 58
6.3.3. N, P and K concentration in grains .................................................................................. 60
6.3.4. N, P and K concentration in shoot .................................................................................... 60
6.3.7. Carotenoids and proline ................................................................................................... 65
6.4. Discussion ......................................................................................................................... 69
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6.5. Conclusion ........................................................................................................................ 71
CHAPTER 7 ................................................................................................................................ 72
PGPR capable to produce ACC deaminase and biochar mitigate drought effects in maize......... 72
Abstract ......................................................................................................................................... 72
7.1. Introduction ....................................................................................................................... 72
7.2. Materials and methods ...................................................................................................... 74
7.2.1. Drought-tolerant PGPR .................................................................................................... 74
7.2.2. Characteristics of PGPR................................................................................................... 74
7.2.3. Production of Timber-waste biochar ................................................................................ 75
7.2.4. Characterization of timber-waste biochar ........................................................................ 75
7.2.5. Soil characteristic ............................................................................................................. 75
7.2.6. Polythene bags preparation .............................................................................................. 76
7.2.7. Seed inoculation ................................................................................................................ 76
7.2.8. Seeds Sowing and Drought stress ..................................................................................... 76
7.2.9. Experiment site.................................................................................................................. 76
7.2.10. Application rate of biochar and treatments ...................................................................... 77
7.2.16. Proline and Chlorophyll contents...................................................................................... 77
7.3. Results ............................................................................................................................... 77
7.3.1. Shoot length and shoot dry weight .................................................................................... 77
7.3.2. 100-grains weight and grain yield pot-1............................................................................ 78
7.3.3. N, P and K concentration in grain .................................................................................... 81
7.3.4. Gas Exchange Attributes................................................................................................... 83
7.3.5. Chlorophyll content .......................................................................................................... 84
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7.3.6. Electrolyte leakage............................................................................................................ 88
7.3.7. Carotenoids and Proline ................................................................................................... 88
7.4. Discussion ......................................................................................................................... 90
7.5. Conclusion ........................................................................................................................ 92
CHAPTER 8 ................................................................................................................................ 93
ACC deaminase producing PGPR Bacillus amyloliquefaciens and Agrobacterium fabrum along
with biochar improve wheat productivity under drought stress.................................................... 93
Abstract ......................................................................................................................................... 93
8.3. Results ............................................................................................................................... 98
8.3.1. Plant height, root length and spike length ........................................................................ 98
8.3.2. Grain, straw and biological yield ..................................................................................... 99
8.3.3. Spikelets spike-1, grains spike-1 and 1000 grain weight .................................................... 99
8.3.4. N, P and K concentration in grains ................................................................................ 105
8.3.5. N, P and K concentration in shoot .................................................................................. 105
8.3.6. Gas exchange attributes .................................................................................................. 106
8.3.7. Chlorophyll content ........................................................................................................ 110
8.3.8. Electrolyte leakage.......................................................................................................... 110
8.4. Discussion ....................................................................................................................... 112
8.5. Conclusion ...................................................................................................................... 114
CHAPTER 9 .............................................................................................................................. 115
Sole and combined application A. xylosoxidans and E. cloacae with timber-waste biochar
mitigated the drought induced stress in maize under field condition ......................................... 115
Abstract ....................................................................................................................................... 115
9.1. Introduction ..................................................................................................................... 115
9.2. Materials and methods .................................................................................................... 117
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9.2.1. PGPR strains .................................................................................................................. 117
9.2.2. PGPR characterization ................................................................................................... 117
9.2.3. Biochar production ......................................................................................................... 118
9.2.5. Experimental site and soil characteristic ....................................................................... 118
9.2.6. Seeds collection and inoculation .................................................................................... 118
9.2.7. Field preparation, nutrients and biochar application .................................................... 118
9.2.8. Experimental design and treatment plan ........................................................................ 119
9.2.9. Seeds sowing and Drought stress ................................................................................... 119
9.2.10. Harvesting ....................................................................................................................... 119
9.2.11. Yield attributes ................................................................................................................ 120
9.2.12. Nitrogen, phosphorus and potassium concentration in grain and shoot ........................ 120
9.2.13. Nutrients use efficiency (NUE) ....................................................................................... 120
9.2.14. Gas exchange parameters ............................................................................................... 120
9.2.15. Chlorophyll contents ....................................................................................................... 120
9.2.16. Electrolyte leakage.......................................................................................................... 120
9.2.17. Statistical Analysis .......................................................................................................... 120
9.3. Results ............................................................................................................................. 120
9.3.1. Plant height, cob length and number of grains cob-1...................................................... 120
9.3.2. 1000-grain weight, grain yield and biological yield ...................................................... 121
9.3.3. N, P and K concentration in grain .................................................................................. 121
9.3.4. N, P and K concentration in shoot .................................................................................. 122
9.3.5. Nutrients use efficiency ................................................................................................... 122
9.3.6. Electrolyte leakage and gas exchange attributes............................................................ 125
9.3.7. Chlorophyll content ........................................................................................................ 125
9.4. Discussion ....................................................................................................................... 128
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9.5. Conclusion ...................................................................................................................... 130
SUMMARY ............................................................................................................................. 1351
THESIS CONCLUSION ....................................................................................................... 1354
REFERENCES .......................................................................................................................... 135
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LIST OF TABLES
Table 4.1. Effect of ACC deaminase containing PGPR on shoot length and root length of wheat
seedlings under drought stress .................................................................................... 23
Table 4.2. Effect of ACC deaminase containing PGPR on shoot and root fresh weight of wheat
Table 4.3. Effect of ACC deaminase containing PGPR on shoot and root dry weight of wheat
Table 4.4. Effect of ACC deaminase containing PGPR on chlorophyll a, chlorophyll b and total
chlorophyll in wheat seedlings under drought stress ................................................. 27
Table 4.5. Characterization of most efficient ACC deaminase containing PGPR ....................... 31
Table 5.1. Characterization of ACC deaminase containing PGPR .............................................. 39
Table 5.2. Effect of ACC deaminase containing PGPR on shoot length (cm) and root length (cm)
of maize seedlings under various levels of PEG induced drought.............................. 41
Table 5.3. Effect of ACC deaminase containing PGPR on shoot fresh weight (g) and shoot dry
weight (g) of maize seedlings under various levels of PEG induced drought ............ 43
Table 5.4. Effect of ACC deaminase containing PGPR on root fresh weight (g) and root dry weight
(g) of maize seedlings under various levels of PEG induced drought ........................ 44
Table 5.5. Effect of ACC deaminase containing PGPR on cholorophyll a (mg/g), cholorophyll b
(mg/g) and total cholorophyll (mg/g) synthesis in maize seedlings under various levels
of PEG induced drought.............................................................................................. 46
Table 6.1. Characteristics of soil and timber waste biochar (BC)................................................ 54
Table 6.2. Effect of ACC deaminase containing PGPR in combination with various rates of timber
waste biochar (0BC, 1BC and 2BC) on grains yield pot-1 100-grains weight and straw
yield under various levels of drought (D) ................................................................... 59
waste biochar (0BC, 1BC and 2BC) on grains nitrogen, phosphorus and potassium
concentration under various levels of drought (D) ..................................................... 62
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waste biochar (0BC, 1BC and 2BC) on shoot N, P and K concentration under various
levels of drought (D) ................................................................................................... 63
waste biochar (0BC, 1BC and 2BC) on gas exchange attributes under various levels of
drought (D).................................................................................................................. 66
waste biochar (0BC, 1BC and 2BC) on chlorophyll content under various levels of
drought (D).................................................................................................................. 67
Table 7.1. Pre-experimental characteristics of soil and timber waste biochar (BC) .................... 76
Table 7.2. Effect of ACC deaminase producing PGPR with and without different rates of timber
waste BC biochar (BC0, BC0.75 and BC1.50) on shoot length and shoot dry weight under
various levels of drought ............................................................................................. 79
waste BC (BC0, BC0.75 and BC1.50) on 100-grains weight and grains yield pot-1 under
various levels of drought ............................................................................................. 80
waste BC (BC0, BC0.75 and BC1.50) on grains N, P and K concentration under various
levels of drought ......................................................................................................... 82
waste BC (BC0, BC0.75 and BC1.50) on shoot N, P and K concentration under various
levels of drought ......................................................................................................... 85
waste BC (BC0, BC0.75 and BC1.50) on gas exchange attributes under various levels of
drought ........................................................................................................................ 86
waste BC (BC0, BC0.75 and BC1.50) on chlorophyll content under various levels of
drought ........................................................................................................................ 87
Table 8.1. Characteristics of soil and timber waste biochar (BC)................................................ 96
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Table 8.2. Effect of Agrobacterium fabrum, Bacillus amyloliquefaciens with/without biochar (30
Mg ha-1) on plant height, root length and spike length of wheat cultivated in field
conditions .................................................................................................................. 101
Mg ha-1) on spikelets spike-1, grains spike-1 and 1000 grains weight of wheat cultivated
in field conditions ..................................................................................................... 104
Mg ha-1) on nitrogen, phosphorus and potassium concentration in wheat grains
cultivated in field conditions ..................................................................................... 107
Mg ha-1) on nitrogen, phosphorus and potassium concentration in wheat shoot
cultivated in drought-stressed field conditions ......................................................... 108
Mg ha-1) on gas exchange attributes of wheat cultivated in drought-stressed field
conditions .................................................................................................................. 109
Mg ha-1) on photosynthetic pigments synthesis and electrolyte leakage in wheat leaves
cultivated in drought-stressed field conditions. ........................................................ 111
Table 9.1. Pre-experimental characteristics of soil and timber waste BC.................................. 118
Table 9.2. Effect of sole and combined application of E. cloacae and A. xylosoxidans with biochar
(15 Mg ha-1) on growth and yield of maize cultivated under different level of irrigation
in field conditions ..................................................................................................... 123
(15 Mg ha-1) on grains and shoot nutrients concentration of maize cultivated under
different level of irrigation in field conditions .......................................................... 124
(15 Mg ha-1) on electrolyte leakage, gas exchange attributes and chlorophyll contents
of maize cultivated under different level of irrigation in field conditions ................ 127
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LIST OF FIGURES
Figure 4.1. Phylogenetic tree obtained from 16S rDNA sequence alignment for most effective
drought tolerant ACC deaminase producing isolates collected from wheat rhizosphere
..................................................................................................................................... 28
Figure 4.2. Effect of ACC deaminase containing PGPR on wheat shoot nitrogen concentration
under drought stress. Means sharing the same letter are statistically similar while
different letters are significantly different at p ≤ 0.05. ............................................... 28
Figure 4.3. Effect of ACC deaminase containing PGPR on wheat shoot phosphorus concentration
Figure 4.4. Effect of ACC deaminase containing PGPR on wheat shoot potassium concentration
..................................................................................................................................... 39
Figure 5.2. Effect of ACC deaminase containing PGPR on nitrogen concentration in shoot of
maize seedlings under various levels of PEG induced drought .................................. 47
Figure 5.3. Effect of ACC deaminase containing PGPR on phosphorus concentration in shoot of
Figure 5.4. Effect of ACC deaminase containing PGPR on potassium concentration in shoot of
Figure 6.1. Effect of drought tolerant ACC deaminase containing PGPR and various levels of
timber waste biochar (1BC and 2BC) on shoot length (A) electrolyte leakage (B) in
wheat leaves under various levels of drought (D). Means sharing the same letter are
statistically similar. Error bars represent ± standard deviations. NM = Normal
Moisture; MD = Mild Drought; SD = Severe Drought .............................................. 56
xix
timber waste biochar (1BC and 2BC) on carotenoids (A) proline (B) in wheat leaves
under various levels of drought (D). Means sharing the same letter are statistically
similar. Error bars represent ± standard deviations. NM = Normal Moisture; MD = Mild
Drought; SD = Severe Drought .................................................................................. 67
Figure 7.1. Effect of ACC deaminase producing PGPR with and without different rates of timber
waste BC (BC0, BC0.75 and BC1.50) on electrolyte leakage under various levels of
moisture (Normal Moisture (NM), Mild Drought (MD) and Severe Drought (SD)).
BC0.75 = 0.75% Biochar, BC1.50 = 1.50% Biochar ................................................. 89
waste BC (BC0, BC0.75 and BC1.50) on carotenoids contents under various levels of
moisture (Normal Moisture (NM), Mild Drought (MD) and Severe Drought (SD)).
BC0.75 = 0.75% Biochar, BC1.50 = 1.50% Biochar ................................................. 89
waste BC (BC0, BC0.75 and BC1.50) on proline content under various levels of moisture
(Normal Moisture (NM), Mild Drought (MD) and Severe Drought (SD)). BC0.75 =
0.75% Biochar, BC1.50 = 1.50% Biochar .................................................................. 90
Figure 8.1. Effect of single and combined application of Agrobacterium fabrum, Bacillus
amyloliquefaciens and biochar (30 Mg ha-1) on grains yield (tons acre-1) in wheat grains
cultivated in field conditions. .................................................................................... 102
amyloliquefaciens and biochar (30 Mg ha-1) on straw yield (tons acre-1) in wheat grains
cultivated in field conditions. ................................................................................... 102
amyloliquefaciens and biochar (30 Mg ha-1) on biological yield (tons acre-1) in wheat
grains cultivated in field conditions. ......................................................................... 103
Figure 9.1. Weathering data 2017 .............................................................................................. 119
xx
LIST OF ABBREVIATIONS
ANOVA Analysis of Variance
ACC 1-aminocyclopropane-1-carboxylic acid
BC Biochar
BLAST Basic Local Alignment Search Tool
CH4 Methane
CO2 Carbon dioxide
DF Dworkin and Foster
FC Field Capacity
FW Fresh weight
g Gram
H2 Hydrogen
HgCl2 Mercuric chloride
IAA Indole-3-acetic acid
kg Kilogram
MPa Mega Pascal
MD Mild drought
PLP Pyridoxal 5-phosphate
PEG Polyethylene glycol
PGPR Plant growth promoting rhizobacteria
SD Severe drought
t Ton
TKW Thousand-kernel weight
WHC Water Holding Capacity
WUE Water use efficiency
Yr-1 Per Year
xxi
ABSTRACT
Drought stress is one of the most crucial abiotic stresses which significantly deteriorates crops
productivity. High evaporation and less rainfall especially in arid climate are major causes of
drought. However, to improve crop yield under limited availability of water by environment
friendly and organic amendments has become center of attention for soil scientists. Now a days,
inoculation of plant growth promoting rhizobacteria (PGPR) and use of activated carbon i.e.,
biochar for mitigation of drought stress have become popular due to their potential benefits.
Biochar has potential to improve water holding capacity of soil. Similarly, root elongation by
PGPR inoculation provides the plants with a chance to uptake water and nutrients by increasing
surface area of roots. A lot of work has been documented so far where PGPR and biochar were
used independently as amendments. However, novelty of the current study is the combined use of
ACC deaminase producing PGPR and timber water biochar for alleviation of drought stress in
wheat and maize. For that purpose, most efficient drought tolerant ACC demainase producing
PGPR were isolated, screened out and identified from wheat and maize rhizospheres. From wheat
rhizosphere, the Pseudomonas aeruginosa, Enterobacter cloacae, Achromobacter xylosoxidans
and Leclercia adecarboxylata were included and from maize rhizosphere, Leclercia
adecarboxylata, Agrobacterium fabrum, Bacillus amyloliquefaciens and Pseudomonas
aeruginosa were finalized. The results of pot and field studies showed that the Bacillus
amyloliquefaciens + 30 Mg ha-1 timber waste biochar significantly improved wheat growth,
chlorophyll content, nutrients uptake and yield attributes over control. Similarly, the Enterobacter
cloacae and Achromobacter xylosoxidans with 15 Mg ha-1 timber waste biochar proved
significantly better over control for maize growth, chlorophyll content, nutrients uptake and yield
attributes indices. Conclusively, inoculation of ACC deaminase producing PGPR and timber waste
biochar would be a better approach than their sole applications for alleviation of drought adverse
effects on wheat and maize.
Keywords: Activated carbon, Cereals crops, Growth attributes, Nutrients concentration, Osmotic stress,
PGPR
1
CHAPTER 1
INTRODUCTION
Roots of plants usually show a complex interaction with the microorganisms of the
rhizosphere. The rhizosphere is a site of soil which is under the influence of roots (Sylvia et al.,
1998). However, in the stream of exploration of rhizospheric part of soil and microorganisms in
last century, a group of such bacteria was identified that was capable to promote the growth of
plants via colonization in roots (Kloepper et al., 1980). Such bacteria were referred as plant growth
promoting rhizobacteria (PGPR) (Kloepper, 1980). Later on, it was observed that most of the
rhizobacteria not only promote the productivity of crops but also protect them from abiotic stresses
(Saleem et al., 2007; Saraf et al., 2010; Ngumbi and Kloepper, 2016; Vurukonda et al., 2016).
Certain PGPR have been identified that are capable of producing ACC deaminase (Shah et
al., 1998; Glick, 2004; Shahzad et al., 2013). It has been established that ACC deaminase
containing PGPR are quite effective to mitigate the drought stress by decreasing the ethylene
biosynthesis and accumulation in plants (Mayak et al., 2004; Zahir et al., 2008). The polymeric
ACC deaminase enzyme is dependent on pyridoxal 5-phosphate (PLP) (Honma and Shimomura,
1978) that serve as a sink for ACC (ethylene precursor) (Shah et al., 1998). At the time of
germination, most of seeds or roots release ACC in the rhizosphere. When ACC deaminase
become in contact with ACC, it hydrolyses ACC into α-ketobutyrate and ammonia by binding
itself with the surface of seed (Glick et al., 1997, 2007; Cheng et al., 2007; Naveed et al., 2008a).
The reduction in ACC by its deamination results in less biosynthesis of ethylene that is an
important and beneficial trait of ACC deaminase producing PGPR for plants that are cultivated
under stress environment (Glick, 2004).
Besides inoculation of PGPR, use of activated black carbon named biochar has become
center of attention among scientists of the world as an environment-friendly technique for carbon
(C) sequestration in agricultural lands (Lehmann et al., 2006; Marris, 2006; Lehmann, 2007;
Spokas et al., 2010; Abideen et al., 2020). Studies conducted on Amazonian dark earth called terra
preta showed that biochar is an effective soil amendment to restore soil health (Glaser et al., 2001;
Marris, 2006; Jahan et al., 2020). It has been observed that application of biochar in the soil as an
amendment (Lehmann et al., 2006; Administrator, 2011; Ippolito et al., 2012; Iqbal et al., 2015;
Tian et al., 2016; Jahan et al., 2020) improved soil water holding and cation exchange capacity
(Glaser et al., 2001; Steiner et al., 2008) fertility status, nutrients concentration and organic matter
2
contents (Glaser et al., 2001). Due to effective in C sequestration use of biochar is considered an
environment friendly, innovative and novel approach (Qayyum et al., 2014; Younis et al., 2014a,
2015; Budai et al., 2016; Sattar et al., 2020) for the amelioration of physio-chemical properties of
soil (Glaser et al., 2002).
Biochar (BC) is produced through the process of pyrolysis that is an effective carbon
sequestration technique which can be used for recycling of agricultural and industrial wastes (Chen
et al., 2010). This technique is also very effective for organic production of syngas (CO, H 2 and
CH4) which is considered as a source of energy (Laird et al., 2009). Biochar has a large surface
area, porosity and cation π-bonding mechanisms, on graphene-like structures or either with –C=O
functional groups (Harvey et al., 2011). The pyrolysis temperature and waste feed stocks are
important key factors that decide the pH and surface area of the BC produced (Brown et al., 2006).
Application of BC can also enhance water availability in soil due to its high sorption ability (Yu
et al., 2013). It can significantly increase chlorophyll content, growth and yield attributes of crops
by improving the uptake of water and nutrients under drought stress. In addition, application of
biochar can also decrease electrolyte leakage by alleviation of osmotic stress in plants (Abideen et
al., 2020; Sattar et al., 2020).
Water is an essential part of every organism (Lambers et al., 2008). About 70% of human
water requirement includes irrigation water to grow crops (Wada et al., 2013). This 70% irrigation
water fulfil 40% of the global food demand (Bin Abdullah, 2006). However, climatic models
predicted that the changes in climate were going to increase the frequency and severity of water
deficiency called drought (IPCC, 2007; Farooq et al., 2009). It is predicted that in the near future
(2025’s), 2000 km3 more water will be needed to fulfil the demand for irrigation (Bin Abdullah,
2006). While in the 2050’s, the demand for water to cultivate crops is predicted to rise by 10% as
compared to the current irrigation water availability (Wada et al., 2013).
Among various factors, increasing temperature of the earth due to global warming is
playing an important role in the expansion of drought area over cultivated land (Mir et al., 2012).
An elevated level of atmospheric carbon dioxide (CO2) from 275 in pre-industrial times to 375
ppm today, is the highest increase which has given birth to global warming (IPCC, 2007). This
increase in temperature is continuously decreasing the available resources of water on earth thus
changing the cultivatable land into a barren non-cultivatable area. To meet future need of food and
energy for exponentially increasing population of the world (FAO, 2009; Anjum et al., 2011) under
3
water scarcity and global warming (Kimetu et al., 2009; Kammann et al., 2011), most of the plant
scientists are focusing to introduce solutions for it. The higher rate of evapotranspiration and low
precipitation are also allied indicators that predict the development of drought condition (Mishra
and Cherkauer, 2010). Under drought stress, most of the plants are unable to take up enough
amount of water which is required for optimum growth of plants (Manivannan et al., 2008).
When plants are cultivated in a limited supply of water, they produce a higher level of
ethylene called stress ethylene (Mayak et al., 2004; Zahir et al., 2008). Severe drought stimulates
the 1-aminocyclopropane-1-carboxylic acid (ACC) which is an ethylene precursor in higher plants
through the methionine pathway and increases ethylene accumulation (Wang et al., 2003a). Due
to the accumulation of ethylene stem becomes thick along with a significant reduction in
elongation (Knight and Crocker, 1913). Less supply of energy and limited water availability at
imbibition phase significantly decrease the germination of seeds as well as higher ethylene
accumulation under stress environment (Taiz and Zeiger, 2010; Aroca, 2012).
Stomatal closure, high transpiration rate, less biological nitrogen fixation, inhibition of
abscisic acid activity and evoking of physiological responses are some other major drawbacks of
higher ethylene accumulation in the plants (Tamimi and Timko, 2003; Wang et al., 2003a; Tanaka
et al., 2005). The changes in the physiological and biochemical processes due to drought stress
can decrease the productivity of crops up to 50% (Hoekstra et al., 2001; Anjum et al., 2011).
Drought stress also decreases the duration of the growing cycle in crops by disturbing their
phenology (Desclaux and Roumet, 1996). Less uptake of water results in the loss of turgor
decreases in leaf water potential, enzymes impairment and cell division (Kiani et al., 2007; Farooq
et al., 2009; Hussain et al., 2009; Taiz and Zeiger, 2010). Reduction in the uptake of nitrogen (N),
phosphorus (P), potassium (K) and other nutrients in shoot and root is generally observed under
drought stress that plays a vital role in the reduction of crops productivity (Subramanian et al.,
2006). The plants which are grown in the drought stress usually show a low leaf area which
decreases the intake of CO2. This reduction in CO2, impairs the ATP and carboxylation enzymes
destroying the photosynthesis mechanism (Yamane et al., 2003).
Among different crops, wheat is widely cultivated due to their nutritional importance.
Wheat is rich in protein (8-12%) and carbohydrates (55%) content that fulfil 20% of the daily
4
human diet requirement (Bos et al., 2005). Worldwide trade of wheat has made it an economic
crop (FAO, 2003). Cultivation of wheat under osmotic stress significantly decreases its growth
and yield (Singh and Chaudhary, 2006), while the demand of wheat is enhancing at the rate of
1.6% annum-1 (Ortíz-Castro et al., 2008). Similarly, maize (Zea mays L.) is 3rd important cereal
crop which is also cultivated worldwide. The share of maize in cereals production is 62% (Farhad
et al., 2011). As a nutritional diet, grains of maize are rich in starch (78%), protein (10%), fibre
(8.5%), oil (4.8%) and sugar (3.1%) which also help to decrease the cholesterol level in humans
blood (Chaudhry, 1983; Chaudhary et al., 2014). However, cultivation of maize under drought
stress can decrease up to 17% yield (loss of 24 million tons yr-1) as compared to well-watered
production (Edmeades et al., 1993).
Sole applications of biochar and PGPR have been already investigated (Zahir et al., 2011;
Stearns et al., 2012; Naz et al., 2013). The present study was conducted to elucidate whether or
not the combined application of ACC-deaminase producing PGPR and timber waste BC enhances
wheat and maize growth and productivity under drought stress. It is hypothesized that the use of
ACC deaminase containing PGPR and timber waste BC may be an effective and environmental
friendly approach to mitigate drought stress in wheat and maize.
The objectives of the study were as follow;
1. Isolation and screening of drought-tolerant ACC deaminase containing PGPR from maize and
wheat rhizosphere.
2. Molecular identification of most effective drought tolerant ACC deaminase containing PGPR.
3. Production and characterization of timber waste BC.
4. Comparative analysis of wheat and maize growth and productivity with special reference to
the combined role of ACC-deaminase producing PGPR and timber waste BC under drought
stress both in pot culture and field conditions.
5
CHAPTER 2
REVIEW OF LITERATURE
2.1. Climatic changes and food demand

Continuous elevation in carbon dioxide level in the atmosphere due to continuously
changing climatic conditions has exacerbated the situation for the production of crops in the
agriculture sector (Peters et al., 2012). It is predicted that at the end of 21st century, the temperature
of our earth will be increased by 2oC as compared to the era of 1850-1900 (Zandalinas et al., 2018).
Increasing temperature of the earth due to the elevation in carbon dioxide level is playing an
important role in the expansion of the drought area (Mir et al., 2012). However, an increase in
yield of crops despite drought conditions is imperative to fulfil the increasing demand for food for
the rapidly increasing world population (Hunter, 2016).
2.2. Drought
Most of the abiotic stresses produced due to change in climate impose severe consequences
on the yield of crops (Amonette and Joseph, 2009). Among all abiotic stresses (heat, cold, drought,
salinity, oxidative stress and nutrient deficiency) (Awasthi et al., 2014), drought is considered most
crucial one (Arshad et al., 2008). Drought stress is very common in worldwide arid and semi-arid
areas. The susceptibility of drought covers more than 1/2 of the earth with each passing year
(Hewitt, 1997; Kogan, 1997; Wilhite, 2000). Moreover, climate change is going to create the worst
situation in this regard (Anjum et al., 2011b; Griffin et al., 2013; Mehran et al., 2017; Zhang et al.,
2017; Saikia et al., 2018). It is predicted that the demand for irrigation water is expected to increase
to 10% by 2050 (Wada et al., 2013).
2.3. Drought effects on plants
Despite other negative effects (stomatal closure, high transpiration rate, less biological
nitrogen fixation, inhibition of abscisic acid activity and evoking of physiological responses)
(Tanaka et al., 2005), a higher level of endogenous ethylene is a negative effect too produced due
to drought (Ali et al., 2012). The ethylene act as a signalling molecule in response to any biotic
and abiotic stress (Iqbal et al., 2011).
On the other hand, for optimum growth of any plant, photosynthesis has basic role (Anjum
et al., 2011a). When plants are cultivated in drought stress, the reduction in the rate of
6
photosynthesis severely affect the yield of crops (Siddique et al., 1999). To cope with drought
stress, plants usually close their stomata to conserve the water of aerial parts (Deng et al., 2005).
According to Flexas et al. (2004), the reduction in intracellular CO2 flux in leaves provides the
early indication of stress in plants due to which conductance of stomata is decreased.
A field experiment on chickpea was conducted by Ouji et al. (2016) to explore the possible
damages that can be caused by drought stress. They cultivated chickpea genotypes (Béja 1,
Bouchra, Neyer and Kasseb) and provided artificial drought stress by skipping the irrigation at
flowering and pod formation stage. They found that stress generated by drought significantly
decreased the biological yield, 100-seed weight, seed number/plant, grain yield/plant and grain
yield per m2. They stated that the varieties that had potential to tolerate drought stress might be
effective to minimize the losses of yield.
In one of the experiments, Kilic and Tacettin (2010) examined the influence of drought on
yield and other growth attributes of wheat. They noted that drought stress significantly induced
negative effects on the flowering stage, chlorophyll synthesis, filling of grains, spikelets and tillers
formation. They also observed a negative correlation of days with maturity by increasing drought
stress. Similar results were noted by Giunta et al. (1993). They observed a decrease in N harvesting
index in drought condition.
An experiment on bread wheat in Karaj, Iran was accrued out by Paknejad et al. (2007)
under drought stress to examine possible negative effects on drought on wheat chlorophyll content,
chlorophyll fluorescence and grain yield. They observed that osmotic stress significantly reduced
the chlorophyll fluorescence. The change in chlorophyll fluorescence significantly affected the
grain yield of wheat under osmotic stress. On the basis of results, they concluded that relative
water contents directly affected the chlorophyll fluorescence in wheat.
In another experiment, Kimurto et al. (2003) noted that cultivation of wheat under osmotic
stress showed a significant reduction in the yield. They observed that drought stress decreased
16.9% ear length, 14.3% spikelets head-1 and 22.4% 1000-kernel weight in wheat plants. They
suggested the cultivation of drought tolerant wheat varieties under stress conditions. An
experiment was conducted by Plaut et al. (2004) on drought tolerant (Suneca) and drought
susceptible (Batavia) varieties of wheat to examine the most susceptible stages of wheat against
drought stress. According to Plaut et al. (2004), the deficiency of water to the wheat plants did not
7
disturb the number of kernels but significantly damaged dry weight production on each kernel.
Leaf dry matter and the relative contribution of the stem were less affected by drought in drought
tolerant wheat variety as compared to drought susceptible one. They noted that thousand-kernel
weight and weight of kernels are more susceptible among all the attributes of wheat in drought
induced stress.
2.4. Plant growth promoting rhizobacteria

Application of PGPR is also getting importance in overcoming drought stress (Saikia et
al., 2018). The plant root system is a habitat for millions of PGPR that form a complex ecological
community and affects the growth and productivity of crops (Berg, 2009; Schmidt et al., 2014).
These PGPR can secrete multiple metabolites and enzymes which are helpful in mitigating the
effects of biotic and abiotic stresses (Ngumbi and Kloepper, 2016). As water uptake ability of
plants is dependent too on their roots elongation, drought resistant PGPR can improve 40% root
system of plants (Marasco et al., 2013). In addition, PGPR group containing ACC-deaminase is
the most remarkable in enhancing the resistance against drought and promoting the health of plants
(Shakir et al., 2012; Cherif et al., 2015).
In a study on velvet bean under drought stress, Saleem et al. (2018) examined the
effectiveness of ACC deaminase containing PGPR (Enterobacter spp. and Bacillus sp.). They
observed that the plants which were inoculated with PGPR produced a relatively small amount of
ethylene as compared to those which were not inoculated. According to Saleem et al. (2018), the
significant improvement in shoot and root dry mass, might be because of decrease in the production
of ethylene in the plants under drought stress.
In another experiment conducted by Zafar-ul-Hye et al. (2014), a significant decrease in
the productivity of maize under drought and salinity stress condition was observed. They
inoculated ACC-deaminase containing rhizobacteria (Pseudomonas syringae and Pseudomonas
fluorescens) as an amendment and observed a significant improvement in cobs plant-1, number of
grains cob-1, number of grain rows cob-1, number of grains cob-1, cob length, 1000-grain weight,
plant height and grain yield of maize plants. According to them the inoculation of ACC deaminase
containing PGPR could be an effective tool to mitigate the salinity and drought stress by increasing
the uptake of NPK when used in combination with inorganic fertilizers.
8
Inoculation of ACC deaminase-containing rhizobacteria, Bacillus subtilis (LDR2),
Rhizophagus irregularis (Ri), and Ensifer meliloti (Em) under three levels of irrigation water on
Trigonella foenum-graecum was studied by Barnawal et al. (2013). They observed that under
drought stress, the synthesis of chlorophyll was decreased, while, proline concentration was
significantly enhanced in the plants. According to Barnawal et al. (2013), the reduction in
biosynthesis and accumulation of ethylene played an imperative role in decreasing the stress,
generated through drought in those plants which were inoculated with PGPR. However, the better
uptake of nutrients and enhancement in the nodulation were also allied factors that contributed
besides ethylene reduction for the improvement in yield of the crop under drought stress.
Same kind of results in wheat were also noted by Shakir et al. (2012), when they used ACC
deaminase containing PGPR under drought stress. They stated that ACC deaminase containing
PGPR enhanced the length of root and shoot, biomass of root and shoot and lateral number of
roots. According to them, the better elongation of roots provided the basis for the better uptake of
nutrients and water that played an imperative role in the improvement of yield. The activity of
ACC deaminase decreased the synthesis of ethylene in wheat plants grown under drought stress
that was a key factor for the improvement in roots elongation.
In another study, Arshad et al. (2008) observed the positive results of ACC deaminase
containing PGPR in pea (Pisum sativum L.) regarding the mitigation of drought-induced stress.
According to them, the yield of pea was significantly decreased due to drought stress without
PGPR. However, the PGPR inoculated plants gave more yield at flowering and pod stages
respectively. Arshad et al. (2008) stated that PGPR inoculation decreases ethylene by confirming
it with the classical triple response assay. According to them, the decrease in ethylene might be
one of the reason for an increase in the yield of pea plants. Similar result regarding mitigation of
drought stress was also observed by Zahir et al. (2008) when they applied Pseudomonas
fluorescens biotype G (ACC-5) to pea plants. They observed that bacterial isolates significantly
enhanced the fresh and dry weight, root and shoot length, number of leaves plant-1 and water use
efficiency of pea plants.
2.5. Biochar
Biochar is a black carbon compound which is a good source of nutrients. It is produced
through pyrolysis at high temperature under low or no supply of oxygen (Lehmann, 2007; Singh
et al., 2010; Qayyum et al., 2014). The physio-chemical properties of BC depend on the nature of
9
waste material used and temperature of the pyrolysis (Glaser et al., 2002; Navia and Crowley,
2010). High surface area and pore spaces of BC structure improve soil water and nutrients holding
capacity. (Gundale and DeLuca, 2006; Hartmann et al., 2006; Amonette and Joseph, 2009;
Warnock, 2009).
Regarding biochar, Kammann et al. (2011) carried out an experiment with BC as an
amendment against the drought to cultivate pseudo-cereal Chenopodium quinoa. They stated that
the rate of biochar 200 Mg ha−1 enhanced the growth. Biochar application decreased the drought
stress and enhanced the growth of the crop by making water bioavailable. In another study, Yu et
al. (2013) used biochar as water holding agent in loamy soil and noted that the biochar significantly
enhanced the water holding capacity (WHC). The increase in the WHC was one of the major
reasons for enhancing the productivity of crops in loamy soils.
In another study, Shafie et al. (2012) explored the effect of pyrolysis temperature on
sorption of water and nutrients, they observed that BC produced at high temperature of 400 oC
gave better results regarding water retention and nutrients sorption as compared to the biochar
produced at 300 and 350 oC. Keshavarz et al. (2016) also produced maple hardwood biochar at
350 oC and used it against 3 levels of drought. They found that the application of biochar at 1%
rate enhanced WUE while 2% application rate of biochar did not produce any significant increase
as compared to 1% rate.
Exploring water holding capacicty of soil, Keshavarz et al. (2016) conducted an
experiment and applied biochar as an amendment in soil. They cultivated Silybum marianum L.
applying three different levels of BC (0, 1 and 2%) and observed that the addition of BC at higher
rates enhanced the WHC of soil but this water holding was not sufficient for better growth of
Silybum marianum L. under drought stress.
In an experiment, Kammann et al. (2011) used biochar at the rate of 0, 100 and 200 Mg ha-
1
in sandy soil to examine the best application rate of biochar for mitigation of drought stress in
Chenopodium quinoa wild type. For the introduction of drought stress, they maintained the WHC
at the rate of 60% (control) and 20% (drought stress). They observed that a significant
improvement in the growth of plant where 100 Mg ha-1 biochar was used as an amendment.
However, no marginal difference between 100 and 200 Mg ha-1 biochar was observed. On the
basis of the results, they stated that higher application rate of biochar is less economical. That is
10
why, 100 Mg ha-1 is sufficient for the cultivation of Chenopodium quinoa Wild in sandy soils
under drought stress.
According to Mulcahy et al. (2013) application of biochar at the rate of 30% (v/v) in sandy
soil improved the growth of Lycopersicon esculentum seedlings significantly. On the basis of
results, Mulcahy et al. (2013) suggested the utilization of biochar as an amendment against low
water availability for better growth of crops. They also concluded with facts that application of 15-
30% biochar is sufficient to grant resistance to Lycopersicon esculentum seedlings against wilting.
The findings of Blackwell et al. (2010) also supported the use of biochar as an amendment
to minimize the negative effects of drought in plants. They made woody biochar of low nutrients
status and applied it in soils at the rate of 1 Mg ha-1 in dry wheat land of Western Australia and
South Australia. Blackwell et al. (2010) observed that the application of BC significantly enhanced
the bioavailability of nutrients and water that played an imperative role in the improvement of
crops yield. On the basis of their results, they stated that the application of biochar not only had
the potential to enhance the uptake of water but it decreased the application rate of inorganic
fertilizer without disturbing the yield of crops.
In another experiment Akhtar et al. (2014) examined the quality, physiology and yield of
tomato plants by the application of biochar under different water regimes. They observed that the
application of biochar (5%) in soils as an amendment significantly enhance the water contents in
drought condition. Akhtar et al. (2014) argued that the improvement in the water content of soils
plays an important role in the modification of quality and physiology of tomato plants. Biochar
addition in the soil also enhanced the yield of tomato by increasing the soil water holding capacity.
They concluded that the mixing of biochar in soil is an effective approach to mitigate the water
stress.
11
CHAPTER 3
MATERIAL AND METHODOLOGY
3.1. Materials and Methods
3.1.1. Collection of Rhizosphere Soils
Soils of wheat rhizosphere were collected from two different sites viz., Old Shujabad Road
(30.11°N and 71.43°E) and Akram Abad (30.16°N and 71.29°E) in Multan, Pakistan and brought
into the laboratory. A spatula was used to remove the adhering rhizospheric soil from wheat roots
which was homogenized manually for isolation of PGPR.
3.1.2. Isolation, Purification and Selection of Drought Tolerant Isolates
Serial dilutions with distilled water (10-1 to 10-7) were made by taking 1.0 g homogenized
rhizospheric soils. For the isolation of PGPR containing ACC deaminase, Dworkin and Foster
(DF) minimal salt media was prepared having ACC as the main source of nitrogen (Dworkin and
Foster, 1958). For the growth of PGPR, incubation in an automated chamber (HP400S, Ruihua
Co., Ltd., Wuhan, China) was done at 25°C for 48 h. There were 45 bacterial colonies which were
initially isolated as described by Jalili et al. (2009). For the purpose of purification, all the isolates
were grown multiple times on DF media. The purified strains were grown on DF media in which
20% polyethylene glycol (PEG, Biotechnology grade purchased from ShangHai Biochem. Co.,
Ltd., Shanghai, China) was added to create artificial osmotic stress (analogue of drought) (Huang
et al., 2018; Paul et al., 2018). The isolates which were able to grow in the presence of 20% PEG
in DF media were selected as drought tolerant PGPR. Out of forty-five, 23 wheat rhizosphere
isolated strains were selected as drought-tolerant PGPR which were able to grow on 20%
polyethylene glycol (PEG) containing DF media.
3.1.3. Experimental Details
A hydroponic glass jar (3-inch diameter, 6-inch length) experiment was conducted on wheat under
axenic conditions in the laboratory of Soil Microbiology and Biochemistry, Department of Soil
Science, Bahauddin Zakariya University (BZU), Multan, Pakistan. Seeds of local wheat variety
Galaxy-2013 (obtained from BZU research farm, Multan, Pakistan) were selected and subjected
to surface sterilization by dipping in HgCl2 (0.1%) for 5 min. After that, seeds were washed three
times with autoclaved water (Sadiq and Ali, 2013). On each sterilized filter papers (Whatman’s
No. 40), three healthy seeds were placed on which respective PGPR inocula were poured while
un-inoculated seeds were taken as control. Inoculated and un-inoculated seeds were subjected to
12
three levels of PEG (0, 10 and 20%), mixed in water to induce artificial osmotic (drought) stress
of -0.05, -0.63 and -0.87 MPa, respectively (Piwowarczyk et al., 2014; Paul et al., 2018). The
experiment was laid out following completely randomized design with the factorial arrangement
and replicated three times. Finally, the seeds were rolled in 2 filter papers and placed in a sterilized
glass jar in such a way that seeds were not under submerged conditions. Twenty mL water level
was maintained in each jar throughout the experiment. All the macro and micronutrients were
applied four times; at every 5th day in the form of 5 mL Hoagland solution (Hoagland and Arnon,
1950) starting from sowing. The composition of Hoagland solution was
Components Stock Solution g L-1
Macronutrients
2M KNO3 202
1M Ca(NO3).4H2O 472
Iron Chelate 15
2M MgSO4.7H2O 493
1M NH4NO3 80
1M KH2PO4 (pH to 6.0) 136
Micronutrients
H3BO3 2.86
MnCl2.4H2O 1.81
ZnSO4.7H2O 0.22
CuSO4.5H2O 0.051
H3MoO4.2H2O 0.09
Na2MoO4.2H2O 0.12
3.1.4. Growth and Chemical Analysis of Plants

Wheat seedlings were grown for 21 days in jars containing solutions of PEG. Root and shoot
lengths of all three plants in each jar were measured and averaged. Likewise, root and shoot dry
weight and seedling dry weight was recorded following standard procedures. For determination of
dry weight, shoot and root samples were oven dried at 70°C for 48h. For nitrogen determination
in the wheat shoot, H2SO4 digestion was done by using digestion mixture (Jones et al., 1991).
After that, distillation of samples was performed on Kjeldahl’s distillation apparatus (Van
13
Schouwenberg and Walinge, 1973). For determining phosphorus and potassium concentration in
the wheat shoot, digestion of samples was done with HNO3-HClO4 diacid mixture (Chapman and
Pratt, 1961). Phosphorus was analyzed in digested samples by a yellow colour method on a
spectrophotometer at 420 nm wavelength (Jones et al., 1991). Potassium was determined on flame
photometer by following method described by Nadeem et al. (2013).
3.1.5. Chlorophyll Content
Chlorophyll contents in fresh leaves were extracted by using acetone (80%) as described by Arnon
(1949). Absorption of each sample was taken on a spectrophotometer (HITACHI U-2000, Beijing,
China) at 663 and 645 nm wavelength. The chlorophyll a, chlorophyll b and total chlorophyll were
finally calculated by using the equations as follows;
Cholorophyll a (mg g-1 f.wt) = 12.7 (OD 663) – 2.69 (OD 645) V (1)
1000 (W)
Cholorophyll b (mg g-1 f.wt) = 22.9 (OD 645) – 4.68 (OD 663) V (2)
1000 (W)
Total Cholorophyll (mg g-1 f.wt) = Chlorophyll a + Chlorophyll b (3)
Where,
V = final volume made
W = gram of fresh leaf sample.
3.1.6. Biochemical Characterization of Effective PGPR
Among twenty-three, the most effective four strains were selected for further studies. The ACC-
deaminase activity of four selected PGPR strains was examined following the procedure stated by
Honma and Shimomura (1978) and El-Tarabily (2008). The production of indole acetic acid (IAA)
by drought tolerant PGPR was examined with and without L-tryptophan (L-TRP; Sigma, Shanghai,
China) as described by Glickmann and Dessaux (1995). Isolates were grown on Pikovskaya’s
medium (Pikovskaya, 1948) to evaluate the ability to solubilize the phosphorus. The potassium
solubilizing activity of isolates was examined following the method described by Setiawati and
Mutmainnah (2016). The characteristics of selected drought tolerant ACC deaminase containing
PGPR is provided in Table 3.5.
3.1.7. Identification of PGPR
After confirmation of ACC deaminase production and other plant growth promoting traits of
PGPR, molecular identification of most efficient drought tolerant ACC deaminase producing
14
PGPR was carried out through 16S rRNA gene sequencing by using PCR primers 1492R 5' (TAC
GGY TAC CTT GTT ACG ACT T) 3' and 27F 5' (AGA GTT TGA TCM TGG CTC AG) 3'. The
gene sequencing primers were 907R 5' (CCG TCA ATT CMT TTR AGT TT) 3' and 785F 5' (GGA
TTA GAT ACC CTG GTA) 3'. By using BLAST analysis, 16S rRNA gene sequences were aligned
and relationships were deduced (Siddikee et al., 2010). The most effective drought tolerant ACC
deaminase containing rhizobacterial strains were identified as Leclercia adecarboxylata,
Agrobacterium fabrum, Bacillus amyloliquefaciens and Pseudomonas aeruginosa (Figure 4.1).
For confirmation of AcdS gene responsible for the production of ACC deaminase, NCBI gene
bank was consulted that confirmed that B. amyloliquefaciens (NCBIa), A. fabrum (NCBIb) and P.
aeruginosa (NCBIc) have AcdS gene while work is yet continued on L. adecarboxylata. However,
biochemical analysis confirmed that L. adecarboxylata has the ability to produce ACC deaminase
(Table 3.5).
3.1.8. ACC deaminase PGPR
Out of 23, four most efficient drought-tolerant ACC-deaminase producing PGPR identified as
Leclercia adecarboxylata, Agrobacterium fabrum, Bacillus amyloliquefaciens and Pseudomonas
aeruginosa were screened out after a laboratory trial in the Department of Soil Science, Bahauddin
Zakariya University Multan, Pakistan. These PGPR strains were able to grow at the osmotic
potential -0.78MPa generated through 20% polyethylene Glycol 6000 (PEG). The DF minimal salt
medium was used to grow the strains (Dworkin and Foster, 1958).
3.1.9. Biochar production
For the production of biochar, timber waste was collected from local timber market. The timber
waste was initially sun-dried and then pyrolyzed at 389 oC for 80 min in an especially designed
pyrolyzer as described by Qayyum et al. (2014). All the pyrolyzed material (biochar) was then
crushed in a grinder and passed through 2mm sieve. Finally, the fine powder of timber waste
biochar (BC) was stored in airtight plastic jars (Qayyum et al., 2014).
3.1.10. Biochar characterization
The pH and ECe of BC were determined by mixing the BC and water with the ration 1:20 (w/v)
as described by Qayyum et al. (2014). Di-acid (HNO3: HClO4) digestion (Chapman and Pratt,
1961) of biochar was done for the analysis of total phosphorus following yellow color method on
spectrophotometer (Jones et al., 1991), and those of potassium and sodium on flame photometer
(Nadeem et al., 2013). For the determination of nitrogen, H2SO4 digestion (Jones et al., 1991) was
15
done followed by distillation on Kjeldahl’s distillation apparatus (Van Schouwenberg and
Walinge, 1973). The volatile matter and ash content of biochar were analyzed according to
Qayyum et al. (2012) by heating the biochar in muffle furnace at 450 °C and 550 °C respectively.
The fixed carbon in biochar was assessed using the equation given by (Noor et al., 2012):
Fixed Carbon (%) = 100 - (% Volatile Matter + % Ash Content) (1)
3.1.11. Soil characterization
The soil texture was determined by hydrometer method (Gee and Bauder, 1986) which was sandy
loam (USDA triangle) with mixed hyperthermic Haplocambids. The organic matter in soil was
determined by Walkley (1935). The total nitrogen in soil was determined using the equation given
by Richards (Richards, 1954):
Total N (%) = Soil Organic Matter / 20 (2)
For extractable soil P determination, Olsen and Sommers (Olsen and Sommers, 1982) method was
used. However, the extractable K in soil was determined according to the method described by
Nadeem et al. (2013).
3.1.12. Electrolyte Leakage
The electrolyte leakage (EL) was determined following the procedure given by Lutts et al. (1996).
The leaves were washed with deionized water and then cut using a steel cylinder having a diameter
of 1 cm. One gram of uniform size leaf pieces were immersed in a test tube containing deionized
water (20ml) and incubated at 25 oC for 24h. The EC (EC1) was determined using pre-calibrated
EC meter. The second EC (EC2) was noted heating the test tubes in a water bath at 120 oC for 20
min. The final value of EL was calculated using the equation as follows:
Electrolyte Leakage (EL) = EC1 / EC2 × 100 (3)
3.1.13. Determination of proline
For proline assessment in wheat leaves, methodology stated by Bates et al. (1973) was followed.
The proline was extracted from fresh (0.1g) leaves in 2ml of 40% methanol. After extraction, the
1ml mixture of glacial acetic acid and 6M orthophosphoric acid (3:2 v/v) was mixed in 1 ml extract
along with 25 mg ninhydrin. Then the solution was incubated at 100 oC for 60 min. After cooling
down, 5ml Toluene was added. For the estimation of proline contents, absorbance was noted on
spectrophotometer at 520 nm wavelength.
16
3.1.14. Gas exchange attributes
Leaf gas exchange parameters (net photosynthetic rate, net transpiration rate and stomatal
conductance) were determined with the help of Infra-Red Gas Analyzer (CI-340 Photosynthesis
system, CID, Inc. USA) by joining 4 leaves of wheat. On a sunny day, the readings were taken
between 10:30 and 11:30 AM at saturating intensity of light (Nazar et al., 2014).
3.1.15. Statistical Analysis
Statistical analysis of wheat morphological traits and biochemical attributes was done using
standard statistical procedures (Steel et al., 1997) on SPSS 18.0 software. All the treatments were
compared using two-way ANOVA followed by LSD test at p ≤ 0.05.
17
CHAPTER 4
Rhizobacteria with ACC-deaminase Activity Improves Nutrient Uptake,
Chlorophyll Contents and Early Seedling Growth of Wheat under PEG-
induced Osmotic Stress
Abstract
Drought stress is the leading constraint impairing the wheat growth across the globe. The 1-
aminocyclopropane-1-carboxylate (ACC) deaminase producing plant growth promoting
rhizobacteria (PGPR) has the potential to mitigate the negative effects of drought stress on
crops. This study was carried out to investigate the role of newly evolved ACC-deaminase
containing PGPR in improving the early wheat growth under polyethylene glycol (PEG)
induced osmotic (drought) stress. Out of 45 strains isolated from wheat rhizosphere, 23
strains were found as drought-tolerant PGPR, which were able to grow on 20% PEG
containing Dworkin and Foster (DF) media. Among these 23, the 4 most effective ACC-
deaminase producing PGPR were selected, identified and characterized. Among these 4
strains, Leclercia adecarboxylata and Agrobacterium fabrum were newly reported and
Bacillus amyloliquefaciens and Pseudomonas aeruginosa were earlier reported drought
tolerant ACC deaminase PGPR. Wheat seeds inoculated with above mentioned strains of
PGPR and un-inoculated dry seeds were grown in hydroponic culture under three levels of
PEG-induced drought stress i.e., 0 (-0.05 MPa), 10 (-0.63 MPa) and 20% (-1.87MPa). PEG-
induced osmotic stress, 20% PEG level was more severe, which decreased root and shoot
lengths and dry weights, and seedling dry weight due to decreased chlorophyll contents and
low uptake of nitrogen, phosphorus and potassium. However, seeds inoculation with PGPR,
especially newly reported strains L. adecarboxylata and A. fabrum, substantially improved
seedling growth under osmotic stress due to elevated nutrients uptake and high chlorophyll
contents. More wheat growth, under optimal and less than optimal conditions, subjected to
L. adecarboxylata and A. fabrum application was linked with their higher ACC-deaminase
and IAA production potential. In conclusion, newly reported drought tolerant ACC
deaminase containing PGPR L. adecarboxylata and A. fabrum have the potential to improve
early wheat growth under osmotic stress due to higher ACC-deaminase and IAA production
potential.
18
Keywords: ACC-deaminase; Growth hormones; Molecular identification; PGPR; Polyethylene
glycol 6000
4.1. Introduction
In recent times scarcity of water due to change in the climatic conditions is a serious threat to
sustainable crop production (Sivakumar, 2011; Hussain et al., 2018). Out of 75% available water
for humans, 10–30% is consumed by plants as transpiration in both irrigated and rainfed
agriculture (Wallace, 2000). The demand for irrigation water is expected to increase ~10% in year
2050s and ~14% in 2080s (Wada et al., 2013), which is already 70% of the global water
consumption (Abdullah, 2006). Drought stress is the leading abiotic factor that disturbs the
biochemical and physiological processes in crops leading to a considerable decrease in crop yield
(Farooq et al., 2014; Hussain et al., 2018).
Wheat (Triticum aestivum L.) is a widely cultivated cereal crop which fulfils up to 20% food
requirements of the daily human diet (Bos et al., 2005). It is also a staple diet for Pakistani
residents. According to the FAO report, 1/5th part of the worldwide wheat production is traded
which makes it an important economic crop (Kao et al., 2015). It is expected that up to 2020s,
wheat demand will be enhanced at the rate of 1.6%/annum (Ortiz et al., 2008). Among several
other factors, drought stress is the leading constraint limiting wheat productivity around the globe
(Farooq et al., 2014, 2015); as both vegetative and reproductive stages of wheat are equally
sensitive to drought stress (Farooq et al., 2014; Tack et al., 2014; Hussain et al., 2016). It also
results in reduction of chlorophyll contents (Nikolaeva et al., 2010) due to poor uptake of nitrogen
in wheat (Shabbir et al., 2015). Most of the plants demonstrate molecular and cellular level
responses towards drought stress (Kaur and Asthir, 2017). The roots of plants usually show first
response against drought by sending signals through abscisic acid for reduction in stomatal
conductance (Nezhadahmadi et al., 2013). In this way, drought stress results in decreased CO2
diffusion inside leaves due to poor conductance of mesophyll which results in photosynthesis
impairment (Flexas et al., 2008).
Furthermore, drought stress enhances the synthesis of ethylene in plants by stimulating the
ethylene precursor 1-aminocyclopropane-1-carboxylic acid (Dubois et al., 2018). Less elongation
and radial swelling of the stem are primary indications of higher ethylene accumulation (Abeles et
al., 1992). At early stages of crop growth, higher level of ethylene decreases the supply of energy
19
and water at the imbibition phase (Taiz and Zeiger, 2010; Aroca, 2012). Other negative effects of
higher ethylene concentration include stomatal closure, higher transpiration, inhibition of
signalling pathway of abscisic acid and less nitrogen fixation (Tamimi and Timko, 2003; Tanaka
et al., 2005).
Nowadays, scientists are working on various strategies for the protection and improvement of
crops productivity under drought. For the generation of drought stress in hydroponic culture,
change in osmotic potential is considered as an effective tool (Paul et al., 2018). This change in
osmotic potential of nutrients solution can be achieved by the application of high molecular weight
(4000- 8000) polyethylene glycol (PEG) that decrease the availability of water to the plants
without any other negative effect (Huang et al., 2018). Likewise, the application of plant growth
promoting rhizobacteria (PGPR) is also getting importance in overcoming drought stress (Saikia
et al., 2018). The plant root system is a habitat for millions of PGPR that form a complex ecological
community and affects the growth and productivity of crops (Berg, 2009; Schmidt et al., 2014).
These PGPR can secrete multiple metabolites and enzymes to mitigate biotic and abiotic stresses
(Ngumbi and Kloepper, 2016). As water uptake ability of plants is dependent on their roots
elongation, inoculation of drought resistant PGPR can improve 40% root system of plants
(Marasco et al., 2013). In addition, PGPR group containing ACC-deaminase is the most
remarkable in enhancing the resistance against drought and to promote the health of plants (Shakir
et al., 2012; Cherif et al., 2015).
ACC-deaminase is a polymeric enzyme which is dependent on pyridoxal 5-phosphate (PLP)

(Karthikeyan et al., 2004). It is established that the PGPR containing ACC deaminase can reduce
the accumulation of ethylene by breaking cyclo-propanoid amino acid ACC (ethylene precursor),
into intermediate compounds, ammonia and α-ketobutyrate (Glick et al., 1999). The use of ACC-
deaminase containing PGPR in improving drought tolerance of cereal crops including wheat is
well reported (Shakir et al., 2012; Zafar-ul-Hye et al., 2014). However, the isolation, identification
and characterization of new drought tolerant PGPR strains from wheat rhizosphere is a pragmatic
option to search more efficient strains to induce drought tolerance in wheat. Therefore, this
hydroponic study was conducted to evaluate the efficacy of two newly isolated strains i.e., L.
adecarboxylata and A. fabrum with high ACC-deaminase and IAA production potential to improve
20
the nutrient uptake, chlorophyll contents and early seedling growth of wheat under PEG induced
osmotic stress.

See chapter 3 section 3.1 subsection. The identification of PGPR are given in Figure 4.1.
4.3. Results
4.3.1. Growth attributes
Different levels of PEG and inoculation with PGPR had a significant effect on root and shoot
lengths (Table 4.1), dry weights and seedling dry weight of wheat (Table 4.2-4.3); while their
interaction was non-significant in this regard. Under drought stress, 20% PEG level, in particular,
wheat seedlings observed minimum root and shoot lengths, dry weights and seedling dry weight
against the maximum values of these traits recorded under 0% PEG solution. Nonetheless seed
inoculation with different PGPR, L. adecarboxylata and A. fabrum, significantly improved these
traits compared with un-inoculated seeds. Maximum increase of 40.4% in shoot length was noted
as compared to control where L. adecarboxylata was applied as an inoculum. In the case of root
21
length, A. fabrum, B. amyloliquefaciens and P. aeruginosa remained statistically alike to each
other and performed significantly best as compared to control. Maximum increase of 115% in root
length was noted as compared to control where B. amyloliquefaciens was applied as an inoculum.
Likewise, inoculation with L. adecarboxylata and A. fabrum improved root and shoot dry weights
against control. Seed inoculation with L. adecarboxylata observed 36 and 60% more shoot and
root dry weights of wheat. In case of seedling dry weight, L. adecarboxylata and A. fabrum
performed significantly better than control. Maximum increase of 37.5% in seedling dry weight
was noted as compared to control where L. adecarboxylata was applied as an inoculum.
22
Table 4.1. Effect of ACC deaminase containing PGPR on shoot length and root length of
wheat seedlings under drought stress
PEG0 PEG10 PEG20 Mean PEG0 PEG10 PEG20 Mean
PGPR
Shoot Length (cm) Root Length (cm)
BC
Control 18.70 15.47 12.70 15.62 7.47 6.63 4.20 6.10 DE
BbW6 16.37 15.30 14.67 15.44 BC 13.80 11.00 9.70 11.50 A-E
BbW12 17.40 17.57 12.73 15.90 BC 10.60 11.33 10.07 10.67 A-E
AbW4 17.77 13.63 13.07 14.82 BC 12.87 9.37 7.87 10.03 A-E
CbW4 21.20 18.20 7.83 15.74 BC 11.23 10.00 7.70 9.64 A-E
AbW1 23.53 22.57 19.70 21.93 A 11.40 11.27 9.93 10.87 A-E
BbW9 18.97 18.37 16.80 18.04 A-C 13.20 9.70 9.30 10.73 A-E
AbW9 9.87 9.60 6.40 8.62 D 8.78 8.64 5.76 7.73 B-E
AbW8 16.67 15.90 11.40 14.66 BC 12.67 9.54 9.10 10.44 A-E
AB
CbW5 20.07 20.30 17.47 19.28 12.20 11.07 10.13 11.13 A-E
AbW16 18.60 17.13 16.90 17.54 A-C 12.20 10.17 10.87 11.08 A-E
CbW3 16.20 16.13 14.10 15.48 BC 13.65 13.27 12.30 13.07 A
CbW2 21.40 18.17 18.37 19.31 AB 14.80 10.93 9.77 11.83 A-D
BbW14 17.80 16.47 13.00 15.76 BC 14.55 12.33 11.30 12.73 A-C
AbW3 16.57 16.40 14.43 15.80 BC 12.90 7.50 7.27 9.22 A-E
BbW8 16.37 14.40 14.50 15.09 BC 10.10 9.50 9.63 9.74 A-E
AbW20 18.40 17.00 7.50 14.30 BC 11.23 10.40 7.53 9.72 A-E
CbW6 18.30 20.90 13.40 17.53 A-C 11.77 9.47 8.87 10.03 A-E
AbW5 17.50 19.97 17.50 18.32 A-C 14.50 13.50 10.47 12.82 AB
BbW4 19.43 19.80 9.50 16.24 BC 9.40 5.33 3.50 6.08 E
BbW10 14.77 12.67 12.57 13.33 CD 10.00 7.80 4.80 7.53 C-E
AbW11 17.33 16.60 15.77 16.57 BC 10.93 10.00 10.13 10.36 A-E
AbW2 17.60 16.17 14.47 16.08 BC 11.17 10.77 9.40 10.44 A-E
CbW7 15.90 16.70 13.40 15.33 BC 10.00 9.77 6.95 8.91 A-E
Mean 17.78 A 16.89 A 13.67 B 11.73 A 9.97 B 8.61 C
Means sharing the same letter are statistically similar while different letters are significantly
different at p ≤ 0.05.
23
Table 4.2. Effect of ACC deaminase containing PGPR on shoot and root fresh
weight of wheat seedlings under drought stress
Shoot Fresh Weight (g) Root Fresh Weight (g)
PGPR Various levels of PEG
HI
Control 1.07 0.88 0.72 0.89 0.15 0.12 0.11 0.13 HI
BbW6 1.79 1.25 1.03 1.36 A-F 0.25 0.18 0.14 0.19 A-F
BbW12 1.61 1.32 0.97 1.30 B-G 0.23 0.19 0.14 0.18 B-G
AbW4 1.31 0.93 0.82 1.02 F-I 0.19 0.13 0.12 0.14 F-I
CbW4 1.88 1.24 1.03 1.39 A-E 0.27 0.18 0.15 0.20 A-E
AbW1 2.12 1.74 1.13 1.66 A 0.30 0.25 0.16 0.24 A
BbW9 1.90 1.35 0.93 1.39 A-E 0.27 0.19 0.13 0.20 A-E
AbW9 0.83 0.48 0.30 0.54 J 0.12 0.07 0.04 0.08 J
AbW8 1.48 0.92 0.60 1.00 G-I 0.21 0.13 0.08 0.14 G-I
CbW3 1.85 1.51 0.98 1.44 A-D 0.26 0.21 0.14 0.20 A-D
AbW16 1.55 1.24 0.87 1.22 C-H 0.22 0.17 0.12 0.17 B-H
C-H
CbW5 1.59 1.23 0.67 1.16 0.22 0.17 0.10 0.16 C-H
CbW2 2.00 1.54 1.13 1.56 AB 0.28 0.22 0.16 0.22 AB
BbW14 1.49 1.06 0.69 1.08 E-I 0.21 0.15 0.10 0.15 E-I
AbW3 1.34 1.01 0.62 0.99 F-I 0.19 0.14 0.09 0.14 G-I
BbW8 1.70 1.05 0.57 1.11 D-I 0.24 0.15 0.08 0.16 D-I
AbW20 1.65 1.13 0.68 1.16 C-H 0.23 0.16 0.10 0.16 C-H
CbW6 1.72 1.54 1.14 1.46 A-C 0.24 0.22 0.16 0.21 A-C
AbW5 1.64 1.41 1.13 1.40 A-E 0.23 0.20 0.16 0.20 A-E
BbW4 1.84 1.04 0.56 1.15 C-H 0.26 0.15 0.08 0.16 C-H
BbW10 1.22 0.69 0.45 0.79 IJ 0.17 0.10 0.06 0.11 IJ
AbW11 1.96 1.31 0.84 1.37 A-E 0.28 0.18 0.12 0.19 A-F
AbW2 1.39 1.09 0.76 1.08 D-I 0.20 0.15 0.11 0.15 E-I
CbW7 1.50 1.06 0.74 1.10 D-I 0.21 0.15 0.10 0.16 D-I
Mean 1.60 A 1.17 B 0.81 C 0.23 A 0.16 B 0.11 C
Means sharing the same letter are statistically similar while different letters are
significantly different at p ≤ 0.05.
24
Table 4.3. Effect of ACC deaminase containing PGPR on shoot and root dry weight
of wheat seedlings under drought stress
Shoot Dry Weight (g) Root Dry Weight (g)
PGPR Various level of PEG
CD
Control 0.15 0.07 0.04 0.09 0.008 0.011 0.002 0.007 C
BbW6 0.13 0.11 0.09 0.11 B-D 0.015 0.011 0.011 0.012 A-C
BbW12 0.11 0.09 0.07 0.09 CD 0.015 0.012 0.012 0.013 A-C
AbW4 0.18 0.13 0.11 0.14 A-D 0.016 0.008 0.007 0.010 A-C
CbW4 0.20 0.13 0.11 0.15 A-D 0.017 0.011 0.009 0.012 A-C
AbW1 0.22 0.19 0.16 0.19 A 0.020 0.016 0.011 0.016 A
BbW9 0.21 0.16 0.10 0.16 A-C 0.018 0.012 0.008 0.013 A-C
AbW9 0.09 0.13 0.02 0.08 D 0.011 0.011 0.008 0.010 A-C
AbW8 0.16 0.09 0.07 0.11 B-D 0.013 0.008 0.006 0.009 BC
CbW3 0.14 0.13 0.13 0.13 A-D 0.012 0.011 0.011 0.011 A-C
AbW16 0.11 0.12 0.09 0.11 B-D 0.010 0.010 0.008 0.009 A-C
B-D
CbW5 0.13 0.11 0.08 0.11 0.011 0.010 0.007 0.009 A-C
CbW2 0.22 0.16 0.14 0.17 AB 0.019 0.014 0.012 0.015 AB
BbW14 0.13 0.11 0.06 0.10 CD 0.011 0.009 0.005 0.008 BC
AbW3 0.13 0.12 0.08 0.11 B-D 0.011 0.010 0.007 0.009 A-C
BbW8 0.13 0.12 0.06 0.10 CD 0.011 0.010 0.005 0.009 BC
AbW20 0.14 0.13 0.07 0.11 B-D 0.012 0.011 0.006 0.010 A-C
CbW6 0.18 0.16 0.13 0.16 A-C 0.012 0.014 0.011 0.012 A-C
AbW5 0.16 0.13 0.12 0.14 A-D 0.013 0.011 0.010 0.012 A-C
BbW4 0.20 0.13 0.07 0.13 A-D 0.017 0.009 0.005 0.010 A-C
BbW10 0.15 0.14 0.13 0.14 A-D 0.013 0.006 0.004 0.008 C
AbW11 0.15 0.11 0.11 0.12 A-D 0.012 0.009 0.009 0.010 A-C
AbW2 0.17 0.10 0.09 0.12 A-D 0.008 0.009 0.010 0.009 BC
CbW7 0.13 0.10 0.10 0.11 B-D 0.011 0.008 0.008 0.009 A-C
Mean 0.15 A 0.12 B 0.09 C 0.013 A 0.011 B 0.008 C
Means sharing the same letter are statistically similar while different letters are
significantly different at p ≤ 0.05.
25
4.3.2. Chlorophyll contents
Interaction among PGPR and PEG levels had a significant effect on chlorophyll a, b and total
chlorophyll contents of wheat seedlings. Seedlings obtained from seeds inoculated with L.
adecarboxylata and A. fabrum showed higher chlorophyll a, b and total chlorophyll contents at 0%
PEG while un-inoculated seeds and inoculated seeds with B. amyloliquefaciens had the minimum
chlorophyll a, b and total chlorophyll contents at 20% PEG. Nonetheless, wheat seedlings obtained
from seeds inoculated with L. adecarboxylata and A. fabrum produced higher chlorophyll a, b and
total chlorophyll contents under well-watered (0% PEG) and drought conditions (10 and 20%
PEG) compared with un-inoculated control (Table 4.4). Maximum increase of 142, 123 and 205%
in total chlorophyll content was noted where A. fabrum, L. adecarboxylata and A. fabrum were
inoculated at 0, 10 and 20% PEG levels, respectively.
4.3.3. Nutrients in shoot
Interactive effect of PGPR and drought stress (PEG levels) was significant for shoot nitrogen (N),
phosphorus (P) and potassium (K) concentration in wheat. PEG-induced drought stress
substantially reduced NPK uptake while seed inoculation with different PGPR improved NPK
concentration under well-watered and stressed condition compared with control. Seeds inoculation
with A. fabrum observed higher shoot N concentration at 0% PEG while seedlings obtained from
un-inoculated seeds recorded the minimum shoot N concentration under 10 and 20% PEG levels
(Figure 4.2). Maximum increase of 207 and 133% in shoot N concentration compared with control
was noted where inoculated seeds with L. adecarboxylata were sown with 10 and 20% PEG levels.
Likewise, seed inoculation with L. adecarboxylata, A. fabrum and P. aeruginosa resulted in higher
shoot P concentration while un-inoculated seeds grown under 20% PEG had minimum shoot P
concentration in wheat (Figure 4.3). Moreover 122, 77 and 220% more shoot P concentration was
observed in seedlings obtained from inoculated seeds with L. adecarboxylata, B.
amyloliquefaciens and A. fabrum sown at 0, 10 and 20% PEG, respectively against un-inoculated
control. Seeds inoculation with L. adecarboxylata, A. fabrum and B. amyloliquefaciens strains
resulted in maximum shoot K concentration at 0% PEG while un-inoculated seeds remained poor
in this regard at 10 and 20% PEG (Figure 4.4). Maximum increase of 159 and 307% in shoot K
concentration was noted with inoculation of A. fabrum compared with control at 0 and 10% PEG,
respectively.
26
Table 4.4. Effect of ACC deaminase containing PGPR on chlorophyll a, chlorophyll b and total chlorophyll in wheat
seedlings under drought stress
Chlorophyll a (mg g ) -1 Chlorophyll b (mg g-1) Total Chlorophyll (mg g-1)
PGPR Various levels of PEG
PEG0 PEG10 PEG20 Mean PEG0 PEG10 PEG20 Mean PEG0 PEG10 PEG20 Mean
i-w m-z y-A I-K m-w o-y yz HI l-z
Control 0.32 0.29 0.13 0.25 0.21 0.19 0.08 0.16 0.53 0.47 o-C 0.21 B-D 0.41 HI
BbW6 0.33 h-w 0.28 n-z 0.23 p-A 0.28 H-K 0.22 l-v 0.19 o-y 0.16 q-y 0.19 F-I 0.55 k-y 0.47 p-C 0.39 s-C 0.47 G-I
BbW12 0.40 d-r 0.23 q-A 0.23 q-A 0.28 H-K 0.27 f-q 0.15 r-y 0.15 s-y 0.19 E-I 0.66 e-s 0.38 t-C 0.38 u-C 0.47 G-I
AbW4 0.39 e-s 0.33 h-w 0.22 r-A 0.31 G-J 0.26 h-r 0.23 k-u 0.15 r-y 0.21 D-H 0.65 g-u 0.55 j-x 0.38 t-C 0.53 F-H
CbW4 0.44 c-n 0.31 k-x 0.26 o-z 0.34 G-I 0.30 d-n 0.21 m-w 0.18 p-y 0.23 D-F 0.75 d-o 0.52 l-z 0.44 r-C 0.57 FG
AbW1 0.77 a 0.62 a-c 0.23 p-A 0.54 AB 0.49 a 0.43 a-c 0.23 k-u 0.38 A 1.26 a 1.05 a-c 0.46 o-B 0.92 A
b-k k-w n-z GH c-k l-w o-y DE c-k
BbW9 0.48 0.31 0.27 0.35 0.33 0.21 0.19 0.24 0.81 0.52 l-z 0.46 q-C 0.60 FG
AbW9 0.29 m-z 0.19 u-A 0.13 x-A 0.20 K 0.20 n-x 0.13 u-z 0.09 x-z 0.14 I 0.49 n-A 0.32 x-D 0.22 A-D 0.34 I
AbW8 0.57 b-d 0.50 b-i 0.42 d-o 0.50 A-C 0.39 a-d 0.34 b-i 0.29 d-o 0.34 AB 0.96 b-d 0.84 b-i 0.71 d-r 0.84 A-C
CbW3 0.55 b-e 0.34 g-v 0.05 A 0.31 G-J 0.38 b-e 0.23 j-u 0.04 z 0.22 D-G 0.93 b-e 0.57 i-x 0.09 D 0.53 F-H
AbW16 0.54 b-f 0.51 b-h 0.37 f-t 0.47 A-D 0.37 b-g 0.35 b-h 0.25 h-s 0.33 B 0.92 b-g 0.85 b-h 0.62 h-w 0.80 A-C
CbW5 0.55 b-f 0.35 g-u 0.16 v-A 0.35 GH 0.38 b-f 0.24 i-u 0.11 v-z 0.24 D-F 0.93 b-f 0.58 h-x 0.27 y-D 0.59 FG
CbW2 0.78 a 0.51 b-g 0.38 e-t 0.56 A 0.49 a 0.32 d-l 0.26 h-r 0.36 AB 1.28 a 0.83 c-j 0.64 g-u 0.92 AB
BbW14 0.41 d-p 0.39 e-t 0.29 l-z 0.36 F-H 0.28 d-p 0.27 g-q 0.20 n-x 0.25 D 0.69 d-r 0.65 f-t 0.49 n-A 0.61 F
AbW3 0.40 d-q 0.16 w-A 0.12 z-A 0.23 JK 0.28 e-p 0.11 w-z 0.08 yz 0.16 I 0.68 e-r 0.26 z-D 0.20 CD 0.38 I
BbW8 0.49 b-j 0.43 d-o 0.42 d-o 0.45 C-F 0.34 b-j 0.30 d-n 0.29 d-o 0.31 BC 0.84 b-i 0.73 d-q 0.71 d-r 0.76 C-E
AbW20 0.45 c-n 0.27 n-z 0.21 s-A 0.31 G-J 0.31 d-m 0.19 o-y 0.14 s-z 0.21 D-H 0.76 d-n 0.46 q-C 0.35 v-D 0.52 F-H
CbW6 0.47 c-l 0.33 g-w 0.30 k-y 0.37 E-H 0.32 d-l 0.23 k-u 0.21 m-w 0.25 D 0.79 c-l 0.56 j-x 0.51 m-z 0.62 EF
AbW5 0.65 ab 0.43 d-o 0.29 m-z 0.46 B-E 0.45 ab 0.30 d-o 0.20 n-x 0.31 BC 1.10 ab 0.73 d-q 0.48 n-B 0.77 B-D
BbW4 0.33 i-w 0.22 r-A 0.21 t-A 0.25 I-K 0.23 k-u 0.15 r-y 0.14 t-z 0.17 G-I 0.55 j-x 0.38 u-C 0.35 w-D 0.43 HI
BbW10 0.46 c-m 0.37 e-t 0.33 g-w 0.39 D-G 0.31 d-m 0.26 h-r 0.23 k-u 0.27 CD 0.77 d-m 0.63 h-u 0.56 j-x 0.65 D-F
AbW11 0.46 c-m 0.37 e-t 0.32 j-w 0.38 D-G 0.32 d-m 0.26 h-r 0.21 m-w 0.26 CD 0.77 d-m 0.63 h-u 0.53 l-z 0.64 D-F
AbW2 0.45 c-n 0.38 e-t 0.33 h-w 0.39 D-G 0.29 d-o 0.25 h-t 0.21 l-w 0.25 D 0.75 d-p 0.63 h-v 0.54 k-y 0.64 D-F
CbW7 0.42 d-o 0.32 j-w 0.23 q-A 0.32 G-I 0.28 e-p 0.21 l-w 0.15 r-y 0.21 D-H 0.70 d-r 0.53 l-z 0.38 t-C 0.54 F-H
Mean 0.48 A 0.35 B 0.26 C 0.32 A 0.24 B 0.18 C 0.80 A 0.59 B 0.43 C
Means sharing the same letter are statistically similar while different letters are significantly different at p ≤ 0.05.
27
Figure 4.2. Effect of ACC deaminase containing PGPR on wheat shoot nitrogen concentration under drought stress. Means sharing the
same letter are statistically similar while different letters are significantly different at p ≤ 0.05.
28
Figure 4.3. Effect of ACC deaminase containing PGPR on wheat shoot phosphorus concentration under drought stress. Means sharing
the same letter are statistically similar while different letters are significantly different at p ≤ 0.05.
29
Figure 4.4. Effect of ACC deaminase containing PGPR on wheat shoot potassium concentration under drought stress. Means sharing
the same letter are statistically similar while different letters are significantly different at p ≤ 0.05.
30
4.3.4. PGPR characteristics
Selected drought-tolerant PGPR strains viz. L. adecarboxylata, A. fabrum, B. amyloliquefaciens
and P. aeruginosa were able to produce indole acetic acid (IAA) with and without L-tryptophan
(L-TRP; Sigma, Shanghai, China). The L. adecarboxylata produced 291% higher IAA as
compared to B. amyloliquefaciens in the presence of L-tryptophan. The phosphorus and potassium
solubilizing activities were the maximum in L. adecarboxylata while minimum in A. fabrum. In
the case of ACC deaminase production, the performance of A. fabrum was best among all the
studied drought-tolerant ACC deaminase containing PGPR (Table 4.5).
Table 4.5. Characterization of most efficient ACC deaminase containing PGPR
Source Wheat isolated rhizobacteria
PGPR experiment code AbW1 CbW2 CbW3 AbW5
Closet type strain and its NR_104933.1 NR_074266.1 FN597644.1 CP012001.1
accession number Leclercia Agrobacterium Bacillus Pseudomonas
adecarboxylata fabrum amyloliquefaciens aeruginosa
P-Solubilization (µg/ml) 26.6 ± 1.04 16.2 ± 1.48 20.9 ± 2.48 22.8 ± 1.36
K-Solubilization (µg/ml) 20.1 ± 1.02 26.7 ± 1.49 23.4 ± 1.92 17.9 ± 1.02
IAA (µg/ml) 67.8 ± 2.20 58.8 ± 3.27 17.3 ± 2.34 24.8 ± 1.49
IAA (µg/ml) 3.42 ± 0.27 2.43 ± 0.34 1.12 ± 0.6 3.16 ± 0.21
ACCD activity
(µmol α-ketobutyrate 304.9 ± 24.1 349.6 ± 21.4 313.2 ± 34.3 245.4 ± 19.5
nmol mg protein h-1)
-1
31
4.4. Discussion
In this pot study, PEG (20%) induced osmotic stress was more severe, which substantially
reduced the wheat growth; while seed inoculation with ACC deaminase containing PGPR,
especially L. adecarboxylata and A. fabrum, substantially counteracted the damaging effects of
PEG-induced osmotic stress on wheat growth. PEG-induced osmotic stress impaired the root and
shoots growth of wheat possibly due to impaired cell division and cell elongation. Similar kind of
results regarding the reduction in root and shoot growth under PEG induced osmotic stress were
also noted by Baalbaki et al. (1999) and Hellal et al. (2018). Both cell division and elongation are
the key components of plant growth negatively affected by osmotic stress (Zeiger and Taiz, 2010;
Hussain et al., 2018; Paul et al., 2018) leading to decrease in root and shoot length and dry weights
as was observed in this study. According to Gargallo-Garriga et al. (2014), drought stress
deactivates metabolic processes that result in a reduction of shoot length. Small uptake of NPK
was primarily linked with small root growth and decreased water uptake due to elevated osmotic
stress. Reduction in nutrients uptake is quite common in crop plants subjected to drought stress
due to the impaired root system and PEG accelerated osmotic stress (Izzo et al., 1989; Hussain et
al., 2018). Likewise, decreased wheat seedling dry weight in this pot study was linked with small
root growth, limited nutrients uptake and a decrease in chlorophyll contents. Drought stress results
in elevated production of ethylene.
Stress conditions stimulates the methionin to change into S-adenosyl-Met. Activation of
enzyme ACC synthase converts S-adenosyl-Met into ACC. This ACC is further catalyzed by ACC
oxidase which converts it into stress generating ethylene. Accumulation of stress ethylene in
plants, decreases the roots elongation while increases its thickness (Glick et al., 1998). The
thickness in roots due to stress generating ethylene is mainly characterized by accumulation of
dead cells in the cortex which resulted in lysigenous aerenchyma formation (He et al., 1996).
However, poor roots and shoot elongation are characterized by inhibition of cell divison due to
higher accumulation of ethyelene in hypocotyls (Skirycz et al., 2011).
Ethylene also deteriorates the cell membrane integrity by lipid molecules degradation due to
its direct contact with chloroplast that activates chlorophyllase (chlase) gene. This activation of
chlorophyllase (chlase) gene severely damage chlorophyll in plants (Matile et al., 1997).
Nonetheless, application of PGPR, L. adecarboxylata and A. fabrum in specific, markedly
counteracted the damaging effects of osmotic stress on wheat growth i.e., shoot and root lengths and
32
dry weights. The reasons behind this growth improvement might be the reduction in endogenous
ethylene production due to their higher ACC-deaminase activity and secretion of growth hormone
i.e., IAA that resulted in better roots elongation and intake of nutrients. The proposed mechanism of
ACC deaminase functioning by Glick et al. (1999) under abiotic stress strengthen our argument of
better root elongation and improvement in growth attributes by a reduction in endogenous stress
ethylene through ACC-deaminase producing L. adecarboxylata and A. fabrum inoculation.
According to Glick et al. (1999), enzyme ACC-deaminase initially breakdown ethylene precursor, 1-
aminocyclopropane-1-carboxylic acid into α-ketobutyrate and NH3. Reduction in rhizospheric
ethylene resulted in the movement of roots accumulated ethylene from the inside of roots to outside
in rhizosphere along a concentration gradient. Thus, low accumulation of stress generating ethylene
in roots resulted in significant improvement in root elongation. Similar kind of improvement in plant
growth attributes was also documented by many scientists where ACC-deaminase containing PGPR
were inoculated (Chandra et al., 2018; Zhang et al., 2018). The findings of Xie et al. (1996) also
supported our argument and suggested that growth hormone IAA is an allied factor, which might also
be responsible for an improvement in root elongation. High secretion of IAA by PGPR significantly
enhance root surface area, adventitious and lateral root length (Mohite, 2013). The effective drought
tolerant ACC-deaminase containing PGPR (L. adecarboxylata and A. fabrum) of the current study
were also capable to produce IAA with and without L-tryptophan (Table 1). According to Safronova
et al. (2006) colonization of PGPR increase surface area of roots for nutrients absorption and their
ability to solubilize immobile nutrients (e.g., phosphorus) which might be one of the reason behind
the improvement in shoot nutrients concentration through inoculation of ACC deaminase containing
PGPR. Nonetheless, higher NPK shoot concentration under L. adecarboxylata and A. fabrum
inoculation was linked with higher root growth and their higher P and K solubilizing activity.
Moreover, higher chlorophyll contents and better uptake of NPK in these treatments might have been
translated into higher seedling weight both under normal and PEG-induced osmotic stress conditions.
According to Hassan et al. (2015, 2016) better uptake of N, P and K nutrients play a vital role in the
improvement of the shoot and root dry weight. Stefan et al. (2013) also confirmed our results of
improvement in chlorophyll contents as they observed and suggested that secretion of IAA by PGPR
is a co-factor responsible for the improvement in chlorophyll contents of runner bean (Phaseolus
coccineus L.). However, according to Wu et al. (2006), better intake of N and P stimulates the
synthesis of chlorophyll contents.
33
4.5. Conclusion
The PEG-induced drought stress impaired the early wheat growth while application of newly
reported drought tolerant ACC-deaminase containing PGPR i.e. L. adecarboxylata and A. fabrum
counteracted these damaging effects. The improvement in wheat growth under optimal and sub-
optimal conditions with PGPR application was primarily linked with their higher ACC deaminase
activity, IAA production and NPK uptake. However, further investigations are needed at field level
to introduce L. adecarboxylata and A. fabrum as new drought tolerant ACC-deaminase containing
PGPR to improve growth of cereal crops.
34
CHAPTER 5
Mitigation of drought stress in maize by inoculation of drought tolerant ACC

deaminase containing PGPR under axenic conditions
Abstract
Drought is an abiotic factor that hampers the growth and yield of crops via an elevated level of
ethylene and a limited supply of nutrients. It is suggested that ACC deaminase containing PGPR
can mitigate drought stress in crops by decreasing the synthesis and accumulation of ethylene.
Keeping in mind the significance of maize as a widely used cereal grain and fodder crop, a glass
jar study was conducted for the screening of drought-tolerant ACC deaminase containing PGPR
under axenic condition. It was noted that under various levels (0, 10 and 20%) of polyethylene
glycol (PEG) induced drought stress, some of Pseudomonas aeruginosa (DtM10), Enterobacter
cloacae (DtM16), Achromobacter xylosoxidans (DtM29) and Leclercia adecarboxylata
significantly enhanced shoot and root length, shoot fresh and dry weight, root fresh and dry weight
in maize seedlings. A significant improvement in chlorophyll content, N, P and K concentrations
in maize shoot validated the efficacious functioning of Pseudomonas aeruginosa (DtM10),
Enterobacter cloacae (DtM16), Achromobacter xylosoxidans (DtM29) and L. adecarboxylata
regarding less ethylene accumulation in maize seedlings roots under PEG induced drought. It is
concluded that P. aeruginosa, E. cloacae and A. xylosoxidans are previously documented but L.
adecarboxylata is a new drought tolerant ACC deaminase containing PGPR that might have the
potential to alleviate drought stress by decreasing ethylene.
Keywords: Chlorophyll pigments, Stress, Crops, Morphological attributes, Rhizobacteria
5.1. Introduction
Water is an essential part of every living organism and its deficiency for irrigation is called drought
stress (Aslam et al., 2015). Drought is considered one of the most critical environmental abiotic
stress that can decrease the production of crops (Lambers et al., 2008). It has been predicted by
climatic models, that the ongoing changes in climate are going to increase the frequency and
severity of drought in near future (IPCC, 2007; Farooq et al., 2009). The higher rate of
evapotranspiration and low precipitation leads towards the development of drought condition
35
(Mishra and Cherkauer, 2010). The demand for water is expected to increase by 10% up to 2050’s
for the cultivation of crops (Wada et al., 2013).
Under drought condition, most of the plants are unable to uptake ample water which is required
for normal growth (Manivannan et al., 2008). Less uptake of water resulted in the loss of turgor,
decrease in leaf water potential, enzymes impairment, reduction in cell division and elongation
(Kiani et al., 2007; Farooq et al., 2009; Hussain et al., 2009; Taiz and Zeiger, 2010). Drought
stress also decreases the duration of the growing cycle in crops by disturbing their phenology
(Desclaux and Roumet, 1996). Higher biosynthesis of abscisic acid (ABA) as a defensive
mechanism to mitigate drought stress decrease the conductance of stomata which reduce
evapotranspiration rate in plants (Yamaguchi-Shinozaki and Shinozaki, 2006).
Reduction in the uptake of nitrogen, phosphorus and potassium in shoot and root is a general
phenomenon in crops under drought stress (Subramanian et al., 2006). Due to changes in the
physiological and biochemical processes under drought stress, the productivity of crops can be
reduced up to 50% (Hoekstra et al., 2001; Anjum et al., 2011c; Zafar-ul-Hye et al., 2014). Plants
which are cultivated in the drought stress usually show a low leaf area which decreases the intake
of CO2. This reduction in CO2 impaired the ATP and carboxylation enzymes resulted in the
destruction of the photosynthesis mechanism (Yamane et al., 2003).
Higher biosynthesis and accumulation of ethylene under drought stress is an established fact, that
has been reported by many scientists (Mayak et al., 2004a; Zahir et al., 2008; Zafar-ul-Hye et al.,
2014). Server drought stimulates the ethylene precursor 1 aminocyclopropane-1-carboxylic acid
for higher ethylene synthesis (Wang et al., 2003). Due to the accumulation of ethylene stem
becomes thick and low elongated (Knight and Crocker, 1913). Less supply of energy and limited
water availability at imbibition phase significantly decreased the germination of seeds due to the
biosynthesis of ethylene, (Taiz and Zeiger, 2010; Ricardo, 2012). Stomatal closure, high
transpiration rate, less biological nitrogen fixation, inhibition of abscisic acid activity and evoking
of physiological responses are some of the major drawbacks of higher ethylene accumulation in
the plants (Tamimi and Timko, 2003; Wang et al., 2003; Tanaka et al., 2005).
Most of the plant growth promoting rhizobacteria (PGPR) not only increase the productivity of
crops but also protected them from abiotic stresses (Saleem et al., 2007; Saraf et al., 2010; Ngumbi
and Kloepper, 2016; Vurukonda et al., 2016). However, there are some PGPR that can mitigate
abiotic stresses via the activity of ACC deaminase (Shahzad et al., 2013). The polymeric ACC
36
deaminase enzyme is dependent on pyridoxal 5-phosphate (PLP) (Honma and Shimomura, 1978)
that is efficacious to mitigate drought stress by decreasing the ethylene (Mayak et al., 2004a; Zahir
et al., 2008; Zafar-ul-Hye et al., 2014). This enzyme hydrolyzed ethylene into α-ketobutyrate and
ammonia (Glick et al., 1997) thus, improve the stomatal conductance and photosynthesis (Jiang et
al., 2012).
Maize (Zea mays L.) is considered the third important cereal grain crop cultivated worldwide. The
share of maize in cereal grains production is 62% (Farhad et al., 2011). As nutritional diet grains
of maize are rich in protein (10%), starch (78%), fibre (8.5%), oil (4.8%) and sugar (3.1%) which
also helped to decrease the cholesterol humans blood (Chaudhry, 1983; Chaudhary et al., 2014).
However, the cultivation of maize under drought stress can decrease up to 17% yield (loss of 24
million tons yr-1) as compared to well-watered production (Edmeades et al., 1993). That’s why a
glass jar experiment was conducted under the axenic condition to isolate the ACC deaminase
containing PGPR to mitigate the drought stress in maize. The novelty and aim of the study were
to find some new drought tolerant ACC deaminase containing PGPR for mitigation of drought
stress.

5.2.1. Collection of rhizosphere
See chapter 3 section 3.1 subsection 3.1.1
5.2.2. Isolation, incubation and purification of isolates
See chapter 3 section 3.1 subsection 3.1.2.
5.2.3. Selection of drought-tolerant isolates
For the selection of drought-tolerant ACC deaminase containing PGPR, Polyethylene Glycol 6000
(PEG) was added at the rate of 20% in the DF media. There were 37 isolates which were able to
grow on 20% PEG containing DF media. These isolates were considered as drought tolerant ACC
deaminase containing PGPR.
5.2.4. Design and site of experiment
5.2.5. Seeds sterilization and inoculation
37
5.2.6. Application of Hoagland solution
Hoagland solution (Hoagland and Arnon, 1950) was used for the application of all macro and
micronutrients for culturing of maize hydroponically. Started from sowing, 5ml Hoagland solution
was applied after every 5 days, to meet the macro and micronutrients requirement of maize.
5.2.7. Artificial drought stress
The polyethylene glycol was used at three different rates (control = 0% (-0.08 MPa), 10% (-0.67
MPa) and 20% (-1.85 MPa) PEG) to induced artificial drought stress as described by Piwowarczyk
et al. (2014).
5.2.8. Harvesting and Morphological attributes
After 21 days of sowing the seedlings were harvested by removing filter papers. The
morphological growth attributes were noted soon after harvesting of seedlings. The dry weights
were noted by drying the samples at 70 oC for 48h on analytical grade weight balance.
5.2.9. Nutrients analysis
5.2.11. Molecular identification of effective drought tolerant PGPR
See chapter 3 section 3.1 subsection 3.1.7. The most effective drought tolerant ACC deaminase
containing PGPR were identified as Pseudomonas aeruginosa (DtM10), Enterobacter cloacae
(DtM16), Achromobacter xylosoxidans (DtM29) and Leclercia adecarboxylata (DtM34) (Figure
5.1).
38
5.2.12. Biochemical characterization of most efficient PGPR
See chapter 3 section 3.1 subsection 3.1.6. The characteristics of most efficient drought tolerant
ACC deaminase containing PGPR is provided in Table 5.1.
Table 5.1. Characterization of ACC deaminase containing PGPR

Source Maize isolated rhizobacteria
PGPR experiment code DtM10 DtM16 DtM29 DtM34
No. of nucleotide 6317050 1480 6813182 1527
Closet type strain and its CP012001.1 CP001918.1 LN831029.1 NR_104933.1
accession number Pseudomonas Enterobacter Achromobacter Leclercia
aeruginosa cloacae xylosoxidans adecarboxylata
P-Solubilization (µg/ml) 29.1 ± 1.19 66.3 ± 0.38 77.4 ± 0.98 20.1 ± 1.29
K-Solubilization (mg/ml) 12.6 ± 0.92 19.1 ± 0.82 24.5 ± 0.42 16.4 ± 1.40
IAA (Tryptophan) (µg/ml) 21.3 ± 0.37 78.8 ± 0.35 61.2 ± 0.14 61.6 ± 0.20a
IAA (No Tryptophan) (µg/ml) 2.94 ± 0.49 3.39 ± 0.41 5.52 ± 0.79 2.11 ± 0.17a
ACCD activity 115.2 ± 16.1 402.1 ± 27.3 381.17 ± 11.7 296.1 ± 21.7
(µmol α-ketobutyrate g -1 protein h-1)

39
5.3. Results
5.3.1. Shoot and root length
Both main and interactive effects of PGPR and various levels of drought (D) were significant (p ≤
0.05) for shoot and root length of maize seedlings. The strains DtM2, DtM3, DtM10, DtM14,
DtM16, DtM25, DtM26, DtM29, DtM32, DtM33, DtM34 and DtM35 performed significantly best
from control at 0% PEG for shoot length. Inoculation of DtM29 performed best and differ
significantly from control at 10% PEG for shoot length (Table 5.2). However, at 20% PEG the
isolates DtM10, DtM16, DtM27, DtM28, DtM29, DtM32 and DtM34 performed significantly
better for shoot length. Maximum increase of 0.84, 1.21 and 3.22-fold in shoot length was noted
from control (No PGPR) at 0, 10 and 20% PEG respectively where DtM29 was applied as an
inocula. In case of root length DtM9, DtM10, DtM16, DtM18, DtM26, DtM28, DtM29 and DtM33
differ significantly from control at 0% PEG for root length. Inoculation of DtM16 and DtM29
remained statistically alike to each other but performed significantly best at 10% PEG for root
length. At 20% PEG, inoculation of DtM16 was significantly best from control for root length.
Maximum increase of 0.94-fold in root length was recorded as compared to the control (No PGPR)
at 0% PEG where DtM29 was inoculated. However, at 10 and 20% PEG maximum increase of
1.10 and 1.35-fold in root length was noted from control where DtM16 was applied as an inocula.
40
Table 5.2. Effect of ACC deaminase containing PGPR on shoot length (cm) and root
length (cm) of maize seedlings under various levels of PEG induced drought
Shoot Length (cm) Root Length (cm)
Various levels of PEG induced drought
PGPR
IE (PGPR × D) IE (PGPR × D)
+ME +ME
0% 10% 20% 0% 10% 20%
Control 24.6 i-v 17.5 r-w 7.70 w 16.6 M 15.5 u-O 13.2 E-U 9.20 Q-V 12.6 MN
DtM1 29.1 b-p 26.4 f-t 25.5 f-u 27.0 E-K 14.0 B-S 12.1 J-V 9.10 R-V 11.7 N
DtM2 35.4 a-h 33.5 b-l 24.5 i-v 31.1 B-H 22.5 d-l 15.3 u-P 14.2 A-R 17.3 F-K
DtM3 35.9 a-f 25.2 g-u 22.5 m-v 27.9 D-K 20.1 g-v 13.1 F-V 9.10 S-V 14.1 L-N
DtM4 35.0 a-j 33.2 b-l 27.0 f-s 31.7 B-G 18.2 k-E 15.7 u-O 15.5 u-O 16.5 H-L
DtM5 29.1 b-p 21.2 o-v 7.50 w 19.3 LM 23.9 c-h 14.5 y-P 8.20 UV 15.5 KL
DtM6 34.6 a-k 30.0 b-p 26.2 f-t 30.3 B-I 17.5 l-I 16.1 t-M 12.1 J-V 15.2 K-M
DtM7 32.2 b-n 30.4 b-o 27.3 f-s 30.0 B-I 21.0 f-t 20.0 g-w 16.1 t-M 19.0 E-H
DtM8 26.9 f-s 25.3 f-u 21.1 o-v 24.4 J-L 15.0 w-P 14.6 y-P 12.9 G-V 14.2 L-N
DtM9 32.2 b-n 29.8 b-p 17.4 r-w 26.5 F-K 26.1 a-f 18.1 k-F 13.2 E-U 19.1 E-G
DtM10 39.1 ab 30.5 b-o 30.0 b-p 33.2 B-D 25.9 a-f 21.6 f-q 20.3 g-u 22.6 BC
DtM11 30.0 b-p 28.2 c-q 25.0 g-u 27.7 D-K 21.4 f-s 20.1 g-v 19.4 h-y 20.3 C-E
DtM12 27.9 d-r 27.1 f-s 24.6 i-v 26.5 F-K 17.5 l-I 13.6 B-T 13.4 C-T 14.8 K-M
DtM13 33.3 b-l 27.6 e-s 27.0 f-s 29.3 B-J 17.2 n-I 16.0 t-M 12.7 H-V 15.3 KL
DtM14 39.1 ab 31.3 b-o 22.1 n-v 30.8 B-H 17.7 k-H 15.8 u-N 14.1 A-S 15.9 KL
DtM15 27.1 f-s 24.2 k-v 23.6 l-v 25.0 I-K 16.6 q-K 14.3 z-Q 13.4 C-T 14.8 K-M
DtM16 38.6 a-c 33.8 b-l 29.1 b-p 33.8 AB 29.3 ab 27.7 ab 21.6 f-q 26.2 A
DtM17 17.2 s-w 17.1 s-w 15.9 t-w 16.7 M 13.7 B-S 11.7 K-V 10.6 O-V 12.0 N
DtM18 27.6 e-s 25.1 g-u 22.3 n-v 25.0 I-K 26.7 a-e 21.6 f-r 10.9 N-V 19.7 D-G
DtM19 32.0 b-n 29.9 b-p 19.5 p-v 27.1 E-K 22.1 e-p 19.3 h-z 10.4 P-V 17.3 G-K
DtM20 34.1 b-l 25.8 f-t 24.4 j-v 28.1 D-K 17.9 k-G 16.5 r-L 14.4 y-P 16.3 I-L
DtM21 26.6 f-s 26.6 h-v 24.8 h-v 26.0 H-K 18.4 j-C 16.0 t-M 11.4 L-V 15.3 KL
DtM22 29.7 b-p 22.1 n-v 17.3 r-w 23.0 KL 24.6 b-g 19.4 h-y 12.5 I-V 18.8 E-I
DtM23 29.7 b-p 27.2 f-s 22.1 n-v 26.3 G-K 22.2 e-n 19.1 h-A 14.8 y-P 18.7 E-J
DtM24 27.2 f-s 26.7 f-s 24.1 k-v 26.0 H-K 21.4 f-s 18.3 k-D 8.60 T-V 16.1 J-L
DtM25 35.1 a-i 31.2 b-o 15.0 u-w 27.1 E-K 22.3 e-m 17.0 p-J 8.10 V 15.8 KL
ab b-n v-w B-J a-d e-m s-M
DtM26 38.9 32.6 14.2 28.6 27.4 22.3 16.4 22.0 B-D
DtM27 35.1 a-i 33.0 b-m 29.2 b-p 32.4 B-E 17.1 o-J 15.7 u-O 13.2 D-U 15.3 KL
DtM28 33.1 b-m 31.5 b-o 31.4 b-o 32.0 B-E 27.1 a-e 24.1 c-h 19.9 g-x 23.7 AB
DtM29 45.2 a 38.7 a-c 32.5 b-n 38.8 A 30.0 a 27.1 a-e 10.6 O-V 22.6 BC
DtM30 27.2 f-s 24.6 i-v 23.5 l-v 25.1 I-K 17.3 m-I 15.7 u-O 15.1 v-P 16.0 KL
DtM31 30.5 b-o 29.2 b-p 25.3 f-u 28.3 C-K 15.8 u-M 14.9 x-P 14.1 A-S 14.9 K-M
DtM32 39.1 ab 30.5 b-o 30.0 b-p 33.2 B-D 24.1 c-h 17.7 k-H 14.5 y-P 18.8 E-I
DtM33 38.0 a-e 32.1 b-n 25.9 f-t 32.0 B-E 25.0 a-g 22.6 d-k 14.9 x-P 20.8 C-E
DtM34 38.3 a-d 33.1 b-m 29.7 b-p 33.7 A-C 23.6 c-i 23.4 c-j 19.9 g-x 22.3 B-D
DtM35 35.6 a-g 33.2 b-l 26.9 f-s 31.9 B-F 21.4 f-s 18.6 i-B 17.8 k-G 19.3 E-G
DtM36 32.4 b-n 31.7 b-o 30.7 b-o 31.6 B-G 21.0 f-t 12.7 H-V 11.4 M-V 15.0 K-M
DtM37 31.2 b-o 28.8 b-p 18.0 q-w 26.0 H-K 23.4 c-j 22.1 e-o 14.2 A-R 19.9 D-F
*ME 32.5 A 28.6 B 23.4 C 21.1 A 17.8 B 13.6 C
*ME = Main Effect of drought; +ME = Main Effect of PGPR; IE = Interactive Effect
41
5.3.2. Shoot fresh and dry weight
The main effect of PGPR and various levels of D were significant but interaction remained non-
significant for shoot fresh weight (Table 5.3). For shoot dry weight both main and interactive
effects of PGPR and various levels of D differ significantly. The strains DtM2, DtM4, DtM6,
DtM9, DtM10, DtM14, DtM16, DtM27, DtM29, DtM33 and DtM34 differ significantly as
compared to the control (No PGPR) for shoot fresh weight. At 0% PEG the shoot fresh weight was
significantly higher as compared to 10% and 20% PEG. Maximum increase of 1.33-fold in shoot
fresh weight was noted from control where DtM16 was inoculated. For shoot dry weight, the
isolate DtM29 performed significantly best at 0, 10 and 20% PEG from control. Maximum
increase of 0.80, 0.93 and 1.25-fold in shoot dry weight was noted at 0, 10 and 20% PEG induced
drought respectively from control where DtM29 was applied as an inoculum.
5.3.3. Root fresh and dry weight
Main effects of PGPR and various level of D were significant but interaction was non-significant
for root fresh weight (Table 5.4). For root dry weight, both main and interactive effects of PGPR
and D were significant. The strains DtM7, DtM10, DtM16, DtM18, DtM26, DtM27, DtM29 and
DtM34 differ significantly different from control for root fresh weight. At 0% PEG the root fresh
weight was significantly higher as compared to 10% and 20% PEG Maximum increase of 3.31-
fold in the root fresh weight was noted as compared to the control where DtM29 was applied as
inoculum. For root dry weight, DtM29 performed significantly best as compared to the control at
0, 10 and 20% PEG. Maximum increase of 0.82, 0.83 and 1.56-fold in the root dry weight was
noted as compared to the control (No PGPR) at 0, 10 and 20% PEG-induced drought respectively
in DtM29.
42
Table 5.3. Effect of ACC deaminase containing PGPR on shoot fresh weight (g) and
shoot dry weight (g) of maize seedlings under various levels of PEG induced drought
Shoot Fresh Weight (g) Shoot Dry Weight (g)
PGPR
+ME +ME
0% 10% 20% 0% 10% 20%
Control 0.41 0.26 0.15 0.27 G 0.050 c-e 0.040 e-g 0.020 h-j 0.037 E-H
DtM1 0.54 0.46 0.31 0.44 A-G 0.050 c-e 0.040 e-g 0.010 j 0.033 F-I
A-F e-g e-g h-j
DtM2 0.61 0.57 0.36 0.51 0.040 0.040 0.020 0.033 F-I
DtM3 0.59 0.41 0.32 0.44 A-G 0.050 c-e 0.030 f-i 0.010 j 0.030 G-J
A-E c-e f-i f-i
DtM4 0.58 0.53 0.52 0.54 0.050 0.030 0.030 0.037 E-H
DtM5 0.58 0.50 0.26 0.45 A-G 0.040 e-g 0.020 h-j 0.010 j 0.023 IJ
A-D bc e-g f-i
DtM6 0.63 0.53 0.52 0.56 0.060 0.040 0.030 0.044 B-E
DtM7 0.53 0.49 0.40 0.47 A-G 0.050 c-e 0.040 e-g 0.030 f-i 0.040 C-G
DtM8 0.37 0.34 0.33 0.35 E-G 0.030 f-i 0.020 h-j 0.015 ij 0.021 J
DtM9 0.72 0.55 0.49 0.59 A-C 0.070 b 0.050 c-e 0.030 f-i 0.051 B
DtM10 0.72 0.55 0.49 0.59 A-C 0.060 b-d 0.050 c-e 0.040 e-g 0.050 BC
DtM11 0.60 0.32 0.25 0.39 C-G 0.040 e-g 0.030 f-i 0.010 j 0.027 H-J
DtM12 0.49 0.50 0.36 0.45 A-G 0.030 f-i 0.030 f-i 0.020 h-j 0.027 H-J
DtM13 0.52 0.52 0.38 0.47 A-G 0.050 c-e 0.020 h-j 0.010 j 0.027 H-J
DtM14 0.61 0.50 0.40 0.50 A-F 0.030 f-i 0.030 f-i 0.010 j 0.023 IJ
G c-e e-g j
DtM15 0.42 0.26 0.17 0.28 0.050 0.040 0.010 0.033 F-I
DtM16 0.70 0.62 0.56 0.63 A 0.070 b 0.050 c-e 0.030 f-i 0.051 B
G
DtM17 0.34 0.31 0.20 0.28 0.050 c-e 0.040 e-g 0.010 j 0.033 F-I
DtM18 0.35 0.30 0.29 0.31 FG 0.070 b 0.040 e-g 0.020 h-j 0.043 B-F
DtM19 0.54 0.52 0.13 0.40 C-G 0.050 c-e 0.050 c-e 0.030 f-i 0.043 B-F
DtM20 0.64 0.38 0.30 0.44 A-G 0.060 bc 0.040 e-g 0.030 f-i 0.044 B-E
DtM21 0.42 0.40 0.29 0.37 D-G 0.040 e-g 0.040 e-g 0.020 h-j 0.033 F-I
DtM22 0.48 0.34 0.21 0.34 E-G 0.050 c-e 0.030 f-i 0.020 h-j 0.033 F-I
DtM23 0.38 0.32 0.31 0.34 E-G 0.030 f-i 0.030 f-i 0.010 j 0.023 IJ
A-G c-e f-i j
DtM24 0.52 0.50 0.23 0.42 0.050 0.030 0.010 0.030 G-J
DtM25 0.68 0.56 0.17 0.47 A-G 0.050 c-e 0.040 e-g 0.020 h-j 0.038 D-G
DtM26 0.58 0.57 0.22 0.46 A-G 0.050 c-e 0.030 f-i 0.020 h-j 0.033 F-I
DtM27 0.69 0.45 0.43 0.52 A-F 0.060 b-d 0.030 f-i 0.030 f-i 0.040 C-G
DtM28 0.55 0.47 0.31 0.44 A-G 0.030 f-i 0.020 h-j 0.010 j 0.020 J
AB a ab c-f
DtM29 0.84 0.59 0.41 0.61 0.090 0.077 0.045 0.071 A
DtM30 0.54 0.52 0.31 0.46 A-G 0.040 e-g 0.030 f-i 0.030 f-i 0.033 F-I
DtM31 0.49 0.47 0.22 0.39 C-G 0.040 e-g 0.040 e-g 0.020 h-j 0.033 F-I
DtM32 0.49 0.39 0.34 0.41 B-G 0.060 bc 0.050 c-e 0.030 f-i 0.048 B-D
DtM33 0.74 0.44 0.40 0.53 A-E 0.070 b 0.040 e-g 0.030 f-i 0.048 B-D
DtM34 0.76 0.55 0.53 0.61 AB 0.070 b 0.043 d-f 0.030 f-i 0.050 B-D
DtM35 0.73 0.53 0.36 0.54 A-E 0.040 e-g 0.040 e-g 0.010 j 0.030 G-J
DtM36 0.55 0.45 0.27 0.42 A-G 0.060 b-d 0.030 f-i 0.030 f-i 0.040 C-G
DtM37 0.46 0.45 0.21 0.37 D-G 0.050 c-e 0.040 e-g 0.020 h-j 0.037 E-H
ME 0.56 A 0.46 B 0.33 C 0.051 A 0.037 B 0.022 C
*ME = Main Effect of drought; +ME = Main Effect of PGPR
43
Table 5.4. Effect of ACC deaminase containing PGPR on root fresh weight (g) and
root dry weight (g) of maize seedlings under various levels of PEG induced drought
Root Fresh Weight (g) Root Dry Weight (g)
PGPR
+ME +ME
0% 10% 20% 0% 10% 20%
Control 0.17 0.14 0.09 0.13 G 0.022 cd 0.018 de 0.009 fg 0.016 E-G
DtM1 0.44 0.23 0.19 0.29 B-G 0.022 cd 0.018 de 0.013 ef 0.018 C-E
DtM2 0.31 0.30 0.19 0.27 B-G 0.018 de 0.018 de 0.009 fg 0.015 E-G
DtM3 0.36 0.13 0.10 0.20 D-G 0.022 cd 0.013 ef 0.004 g 0.013 F-H
DtM4 0.30 0.29 0.29 0.29 B-G 0.022 cd 0.013 ef 0.013 ef 0.016 E-G
DtM5 0.35 0.33 0.10 0.26 B-G 0.018 de 0.009 fg 0.004 g 0.010 HI
DtM6 0.38 0.24 0.24 0.29 B-G 0.026 bc 0.018 de 0.013 ef 0.019 B-D
DtM7 0.48 0.40 0.27 0.38 A-E 0.022 cd 0.018 de 0.013 ef 0.018 C-E
DtM8 0.29 0.25 0.21 0.25 B-G 0.013 ef 0.009 fg 0.004 g 0.009 I
DtM9 0.46 0.27 0.13 0.29 B-G 0.022 cd 0.018 de 0.004 g 0.015 E-G
DtM10 0.56 0.48 0.22 0.42 A-C 0.031 b 0.022 cd 0.013 ef 0.022 B
DtM11 0.31 0.24 0.21 0.25 B-G 0.018 de 0.013 ef 0.004 g 0.012 G-I
DtM12 0.25 0.17 0.16 0.19 E-G 0.013 ef 0.013 ef 0.009 fg 0.012 G-I
DtM13 0.33 0.26 0.20 0.26 B-G 0.022 cd 0.009 fg 0.004 g 0.012 G-I
DtM14 0.32 0.23 0.18 0.24 B-G 0.013 ef 0.013 ef 0.004 g 0.010 HI
DtM15 0.22 0.22 0.16 0.20 D-G 0.022 cd 0.018 de 0.004 g 0.015 E-G
DtM16 0.52 0.44 0.33 0.43 AB 0.026 bc 0.022 cd 0.018 de 0.022 B
DtM17 0.35 0.15 0.09 0.20 D-G 0.022 cd 0.018 de 0.004 g 0.015 E-G
DtM18 0.55 0.31 0.21 0.36 A-F 0.031 b 0.018 de 0.009 fg 0.019 B-D
DtM19 0.35 0.30 0.05 0.23 B-G 0.022 cd 0.022 cd 0.013 ef 0.019 B-D
DtM20 0.33 0.24 0.21 0.26 B-G 0.026 bc 0.018 de 0.013 ef 0.019 B-D
DtM21 0.28 0.14 0.11 0.18 E-G 0.018 de 0.018 de 0.009 fg 0.015 E-G
DtM22 0.25 0.18 0.06 0.16 FG 0.022 cd 0.013 ef 0.009 fg 0.015 E-G
DtM23 0.22 0.18 0.10 0.17 E-G 0.013 ef 0.013 ef 0.004 g 0.010 HI
DtM24 0.38 0.24 0.18 0.27 B-G 0.022 cd 0.013 ef 0.004 g 0.013 F-H
DtM25 0.42 0.26 0.06 0.25 B-G 0.022 cd 0.018 de 0.009 fg 0.016 E-G
DtM26 0.52 0.46 0.33 0.44 AB 0.031 b 0.013 ef 0.013 ef 0.019 B-D
DtM27 0.48 0.40 0.36 0.41 A-D 0.026 bc 0.022 cd 0.013 ef 0.021 BC
DtM28 0.34 0.25 0.16 0.25 B-G 0.022 cd 0.013 ef 0.009 fg 0.015 E-G
DtM29 0.78 0.56 0.34 0.56 A 0.040 a 0.033 ab 0.023 cd 0.032 A
DtM30 0.39 0.33 0.32 0.35 A-G 0.018 de 0.013 ef 0.013 ef 0.015 E-G
DtM31 0.39 0.31 0.22 0.31 B-G 0.018 de 0.018 de 0.009 fg 0.015 E-G
DtM32 0.39 0.31 0.22 0.31 B-G 0.026 bc 0.022 cd 0.013 ef 0.021 BC
DtM33 0.25 0.20 0.17 0.21 C-G 0.018 de 0.018 de 0.004 g 0.013 F-H
DtM34 0.56 0.40 0.32 0.43 AB 0.031 b 0.018 de 0.013 ef 0.021 BC
DtM35 0.33 0.31 0.22 0.29 B-G 0.013 ef 0.009 fg 0.004 g 0.009 I
DtM36 0.53 0.15 0.15 0.28 B-G 0.026 bc 0.013 ef 0.013 ef 0.018 C-E
DtM37 0.25 0.20 0.11 0.19 E-G 0.022 cd 0.018 de 0.009 fg 0.016 BC
*ME 0.38 A 0.28 B 0.19 C 0.022 A 0.016 B 0.009 C
*ME = Main Effect of drought; +ME = Main Effect of PGPR;
44
5.3.4. Chlorophyll content
Both main and interactive effects of PGPR and D were significant for the improvement in the
chlorophyll a and total chlorophyll in maize seedlings leaves. For chlorophyll b, main effect of
PGPR and D were significant but their interaction was non-significant (Table 5.5). The strain
DtM16 and DtM16 were significantly best from control at 0% PEG for chlorophyll a content.
Maximum increase of 1.27-fold in the chlorophyll a was noted as compared to the control at 0%
PEG where DtM16 was inoculated. In case of chlorophyll b content, DtM10, DtM14, DtM16,
DtM19, DtM25, DtM26, DtM27, DtM29, DtM30, DtM31 and DtM34 performed significantly
better from control. Maximum increase of 2.39-fold in the chlorophyll b was noted from control
in DtM29. For total chlorophyll content, DtM16 and DtM29 differ significantly from control at
0% PEG. At 10% PEG-induced drought, DtM29 performed significantly better as compared to the
control for total chlorophyll content. All the treatments remained statistically similar to each other
at 20% PEG for total chlorophyll. Maximum increase of 1.48 and 2.17-fold in total chlorophyll
was noted as compared to the control at 0 and 10% PEG where DtM16 and DtM20 were applied
as inocula respectively.
5.3.5. N, P and K concentration in shoot
Both main and interactive effects of PGPR and D were significant for shoot nitrogen (Figure 5.2),
phosphorus (Figure 5.3) and potassium (Figure 5.4) concentration. For nitrogen and phosphorus
concentration, the strains DtM29 and DtM34 were significantly best as compared to the control at
0% and 10% PEG. Maximum increase of 2.56 and 2.33-fold in the shoot nitrogen and phosphorus
concentrations were noted at 0% PEG respectively from control where DtM29 was inoculated. For
shoot potassium concentration, the strain DtM10 and DtM34 differ significantly at 0 % PEG as
compared to the control. At 10% PEG performance of DtM16 was significantly best from control
for shoot potassium (Figure 5.3). Maximum increase of 0.38-fold in the maize shoot potassium
concentration was noted at 0% PEG from control where DtM10 was inoculated.
45
Table 5.5. Effect of ACC deaminase containing PGPR on cholorophyll a (mg/g), cholorophyll b (mg/g) and total
cholorophyll (mg/g) synthesis in maize seedlings under various levels of PEG induced drought
Chlorophyll a (mg/g) Chlorophyll b (mg/g) Total Chlorophyll (mg/g)
PGPR
IE (PGPR × D) IE (PGPR × D) IE (PGPR × D)
+ME +ME +ME
0% 10% 20% 0% 10% 20% 0% 10% 20%
Control 1.13 b-i 0.67 d-i 0.49 g-i 0.77 DE 0.92 0.47 0.32 0.57 G-J 2.05 c-l 1.15 h-l 0.82 kl 1.34 IJ
a-i b-i d-i B-E D-J
DtM1 1.31 1.02 0.75 1.03 1.16 0.78 0.64 0.86 2.47 c-l 1.80 e-l 1.39 f-l 1.89 E-J
a-i b-i d-i B-E E-J
DtM2 1.42 0.98 0.67 1.02 0.98 0.73 0.60 0.77 2.40 c-l 1.71 f-l 1.27 h-l 1.79 E-J
DtM3 1.39 a-i 0.95 b-i 0.75 d-i 1.03 B-E 0.56 0.38 0.30 0.41 IJ 1.95 d-l 1.34 f-l 1.04 j-l 1.44 IJ
a-i a-i a-i A-E H-J
DtM4 1.37 1.23 1.22 1.27 0.55 0.50 0.41 0.49 1.92 d-l 1.73 f-l 1.63 f-l 1.76 E-J
DtM5 1.37 a-i 1.18 a-i 0.61 f-i 1.05 B-E 0.55 0.48 0.25 0.43 IJ 1.92 d-l 1.66 f-l 0.86 kl 1.48 G-J
a-i a-i d-i A-E H-J
DtM6 1.72 1.25 0.85 1.27 0.70 0.51 0.35 0.52 2.42 c-l 1.76 f-l 1.20 h-l 1.79 E-J
DtM7 1.32 a-i 1.23 a-i 1.16 a-i 1.23 A-E 0.53 0.50 0.46 0.50 H-J 1.85 d-l 1.73 f-l 1.62 f-l 1.73 E-J
c-i d-i d-i C-E J
DtM8 0.87 0.80 0.78 0.82 0.35 0.32 0.31 0.33 1.22 h-l 1.12 h-l 1.09 i-l 1.15 J
DtM9 1.37 a-i 1.34 a-i 0.52 g-i 1.08 B-E 0.55 0.55 0.21 0.44 IJ 1.92 d-l 1.89 d-l 0.73 l 1.51 G-J
a-e a-g a-i AB A-C a-d a-i c-l
DtM10 2.05 1.85 1.20 1.70 2.14 1.58 1.25 1.66 4.19 3.43 2.45 3.36 AB
a-i a-i d-i B-E IJ c-l f-l j-l
DtM11 1.41 1.23 0.75 1.13 0.59 0.35 0.31 0.41 2.00 1.57 1.06 1.54 G-J
DtM12 1.65 a-i 1.46 a-i 1.32 a-i 1.48 A-D 0.69 0.59 0.53 0.60 G-J 2.33 c-l 2.05 c-l 1.85 d-l 2.08 D-J
a-i b-i c-i B-E B-J
DtM13 1.27 1.09 0.89 1.08 1.13 0.90 0.77 0.93 2.40 c-l 1.99 c-l 1.66 f-l 2.01 D-J
DtM14 1.33 a-i 1.22 a-i 0.98 b-i 1.18 A-E 1.55 1.20 0.73 1.16 A-I 2.89 a-l 2.42 c-l 1.71 f-l 2.34 A-J
a-c a-i a-i AB IJ
DtM15 2.28 1.73 1.17 1.73 1.86 1.42 1.01 1.43 4.14 a-e 3.15 a-k 2.17 c-l 3.15 A-D
DtM16 2.57 a 1.66 a-i 1.36 a-i 1.86 A 2.52 1.00 0.89 1.47 A-F 5.09 a 2.66 b-l 2.25 c-l 3.33 A-C
a-i a-i g-i A-E B-J
DtM17 1.75 1.21 0.52 1.16 0.93 0.83 1.17 0.97 2.69 b-l 2.03 c-l 1.68 f-l 2.13 C-J
DtM18 1.82 a-h 1.31 a-i 0.75 d-i 1.29 A-E 0.82 0.91 1.13 0.95 B-J 2.63 b-l 2.23 c-l 1.88 d-l 2.25 B-J
a-f f-i a-i A-E A-H
DtM19 1.97 0.58 1.22 1.26 2.11 0.95 0.71 1.26 4.08 a-f 1.53 f-l 1.94 d-l 2.52 A-I
DtM20 1.83 a-g 1.32 a-i 1.48 a-i 1.54 A-C 1.13 1.05 0.60 0.93 B-J 2.96 a-l 2.37 c-l 2.08 c-l 2.47 A-I
a-i a-i d-i B-E E-J
DtM21 1.20 1.28 0.75 1.08 1.01 0.78 0.50 0.76 2.21 c-l 2.06 c-l 1.25 h-l 1.84 E-J
a-i b-i g-i B-E D-J
DtM22 1.46 1.15 0.51 1.04 0.49 0.87 1.07 0.81 1.94 d-l 2.01 c-l 1.58 f-l 1.85 E-J
DtM23 0.99 b-i 1.44 a-i 0.66 e-i 1.03 B-E 1.10 0.78 0.77 0.88 C-J 2.09 c-l 2.21 c-l 1.43 f-l 1.91 E-J
b-i d-i a-i B-E B-J
DtM24 0.95 0.77 1.42 1.05 0.98 0.74 1.14 0.95 1.93 d-l 1.50 f-l 2.57 b-l 2.00 D-J
DtM25 1.53 a-i 1.16 a-i 1.21 a-i 1.30 A-E 1.93 1.55 0.90 1.46 A-F 3.47 a-h 2.71 b-l 2.11 c-l 2.76 A-F
a-i b-i b-i A-E A-E
DtM26 1.39 1.14 0.96 1.16 1.74 1.48 1.30 1.51 3.13 a-k 2.62 b-l 2.26 c-l 2.67 A-H
DtM27 1.79 a-i 1.55 a-i 0.73 d-i 1.36 A-E 1.00 1.71 1.27 1.32 A-G 2.79 a-l 3.26 a-k 1.99 c-l 2.68 A-G
b-i a-i a-g A-D B-J
DtM28 1.05 1.53 1.83 1.47 1.41 0.63 0.70 0.92 2.46 c-l 2.16 c-l 2.53 b-l 2.38 A-I
DtM29 2.34 ab 1.58 a-i 0.79 d-i 1.57 AB 2.50 2.06 1.22 1.93 A 4.84 ab 3.64 a-g 2.01 c-l 3.49 A
a-i b-i a-i B-E AB
DtM30 1.15 0.95 1.28 1.13 1.87 1.95 1.22 1.68 3.02 a-l 2.90 a-l 2.51 b-l 2.81 A-E
c-i a-i a-i A-E A-H
DtM31 0.87 1.24 1.38 1.16 1.30 1.13 1.36 1.26 2.17 c-l 2.36 c-l 2.74 a-l 2.42 A-I
DtM32 0.77 d-i 0.58 f-i 0.81 d-i 0.72 E 1.71 0.56 0.83 1.03 B-J 2.48 c-l 1.14 h-l 1.64 f-l 1.75 E-J
a-i b-i b-i B-E D-J
DtM33 1.26 1.13 0.97 1.12 1.03 0.87 0.69 0.86 2.29 c-l 2.00 c-l 1.66 f-l 1.98 D-J
DtM34 2.08 a-d 1.34 a-i 1.19 a-i 1.54 A-C 2.23 1.60 0.96 1.60 A-D 4.32 a-c 2.93 a-l 2.16 c-l 3.14 A-D
a-i b-i b-i B-E C-J
DtM35 1.26 0.99 0.92 1.06 0.96 0.77 0.94 0.89 2.23 c-l 1.76 f-l 1.86 d-l 1.95 D-J
DtM36 1.28 a-i 0.40 hi 0.38 i 0.69 E 1.45 0.61 0.55 0.87 C-J 2.73 b-l 1.01 j-l 0.93 j-l 1.56 F-J
b-i d-i g-i DE F-J
DtM37 1.11 0.77 0.47 0.78 0.82 0.69 0.53 0.68 1.93 d-l 1.46 f-l 1.00 j-l 1.46 H-J
*ME 1.47 A 1.17 B 0.94 C 1.21 A 0.91 B 0.76 C 2.67 A 2.08 B 1.70 C
*ME = Main Effect of drought; +ME = Main Effect of PGPR; IE = Interactive Effect
46
Figure 5.2. Effect of ACC deaminase containing PGPR on nitrogen concentration in shoot of maize seedlings under various levels of
PEG induced drought
47
Figure 5.3. Effect of ACC deaminase containing PGPR on phosphorus concentration in shoot of maize seedlings under various levels
of PEG induced drought
48
Figure 5.4. Effect of ACC deaminase containing PGPR on potassium concentration in shoot of maize seedlings under various levels of
PEG induced drought
49
5.4. Discussion
The experiment was conducted to examine the effect of drought-tolerant ACC deaminase
PGPR on growth attributes, pigments synthesis and nutrients concentration in maize seedlings
under drought stress. It was noted that growth attributes, pigments synthesis and nutrient
concentration in maize seedlings were decreased without ACC deaminase containing PGPR under
drought stress. This reduction was might be due to higher biosynthesis of ethylene as suggested
by Mayak et al. (1999).
Stress conditions stimulates the methionin to covert into S-adenosyl-Met. Activation of
enzyme ACC synthase changes into S-adenosyl-Met into ACC. This ACC is then catalyzed by
ACC oxidase, resulting in stress generating ethylene (Glick et al., 1998). Higher biosynthesis and
accumulation of ethylene ultimately induced negative changes in the development phases and
decreased plant growth (Arshad et al., 2008). Higher accumulation of stress ethylene in plant roots,
play an imperative role in decreasing the root elongation. It promotes the thickness of plant roots
via accumulation of dead cell in the root cortex. Such accumulation of death cells in root cortex
results in the formation of lysigenous aerenchyma. In hypocotyls region, stress ethylene also
decreases the cell divison. Low cell divison eventually results in poor elongation of roots and shoot
in plants (He et al., 1996; Skirycz et al., 2011).
In the current experiment, there were four ACC deaminase containing PGPR (DtM10, DtM16,
DtM29 and DtM34) that significantly enhanced shoot and root length of maize seedlings. This
improvement in shoot and root length was might be due to a reduction in ethylene biosynthesis by
the activity of ACC deaminase secreted by Pseudomonas aeruginosa (DtM10), Enterobacter
cloacae (DtM16), Achromobacter xylosoxidans (DtM29) and Leclercia adecarboxylata (DtM34).
The findings of Zafar-ul-Hye et al. (2014) supported our argument regarding improvement in the
growth of crops by inoculation of ACC deaminase containing PGPR. Similarly, Zahir et al. (2009)
also suggested the inhibition of ethylene accumulation by ACC deaminase PGPR as the main trait
that promotes plant growth. According to Glick et al. (1999), the ACC deaminase enzyme breaks
the ethylene into NH3 and α-ketobutyrate. Diffusion of root ethylene in rhizosphere along
concentration gradient significantly decreased the accumulation of ethylene in plants (Glick, 2004;
Siddikee et al., 2011). However, Xie et al. (1996) suggested the improvement in root length as
IAA producing trait of PGPR. Similar kind of improvement in adventitious and lateral root was
also noted by Gamalero and Glick (2011) and Mohite (2013) due to IAA secretion by PGPR. It
50
was noted that the most effective drought tolerant ACC deaminase containing PGPR of current
experiment P. aeruginosa (DtM10), E. cloacae (DtM16), A. xylosoxidans (DtM29) and L.
adecarboxylata (DtM34) were also capable to secret IAA with and without L-tryptophan that might
be an allied factor for improvement in maize growth under drought (Table 5). In the current study
a significant improvement in shoot N, P and K, was might be due to better elongation of roots.
According to Reid and Renquist (1997), the better elongation of roots helps the plants to uptake
relatively more water that improves water use efficiency under drought (Zahir et al., 2008). The
findings of Safronova et al. (2006) regarding better nutrients uptake in pea plants by inoculation
of ACC deaminase containing PGPR P. brassicacearum and P. marginalis also supports out
results. However, chlorophyll a, b and total was significantly decreased where no PGPR was
inoculated at 10 and 20% PEG. Matile et al. (1997) also observed a similar reduction in the
synthesis of chlorophyll in plants as a result of higher biosynthesis of ethylene under stress. They
suggested that the outburst of ethylene under stress condition degrade the lipid which resulted in
the loss of chloroplast cell membrane integrity. In chloroplast, the chlorophyllase (chlase) gene
become stimulated by higher ethylene accumulation which starts degradation when becoming in
contact with chlorophyll (Matile et al., 1997). A significant improvement in the synthesis of
chlorophyll a, chlorophyll b and total chlorophyll is a solid justification of reduction in ethylene
due to ACC deaminase secreted by P. aeruginosa (DtM10), E. cloacae (DtM16), A. xylosoxidans
(DtM29) and L. adecarboxylata that grant resistance to maize against drought (Table 4). In addition
to the above argument, Stefan et al. (2013) also suggested the activity of IAA as an allied factor
which improves the synthesis of chlorophyll.
5.5. Conclusion
It is concluded from results that significant improvement in maize seedlings was might be due to
the reduction in ethylene, better root and shoot elongation and IAA secretion by PGPR
Pseudomonas aeruginosa (DtM10), Enterobacter cloacae (DtM16), Achromobacter xylosoxidans
(DtM29) and Leclercia adecarboxylata under PEG induced drought stress. However, more
investigation is yet suggested to introduce Leclercia adecarboxylata as new drought-tolerant ACC
deaminase containing PGPR.
51
CHAPTER 6
Co-application of ACC-deaminase producing PGPR and timber-waste
biochar improves pigments formation, growth and yield of wheat under
drought stress
Abstract
Besides other deleterious effects, drought elevates ethylene level too in plants. Increased ethylene
concentration reduces root elongation and development and consequently retards plant growth and
yield. There are certain PGPR which produce ACC-deaminase. The ACC-deaminase converts
ACC (an immediate precursor of ethylene biosynthesis in methionine pathway in higher plants)
into ammonia and α-ketobutyrate instead of ethylene. Regularization of ethylene level in plants
mitigate the effects of drought. On the other hand, biochar has been reported to be rich in nutrients
and exhibiting higher water holding capacity. So, a pot study was conducted with the hypothesis
that the combined application of ACC-deaminase producing PGPR and biochar would minimize
the drought effects on wheat growth. The ACC-deaminase producing PGPR were applied on wheat
seeds in combination with two biochar doses. Three moisture levels were maintained throughout
the trial. The data obtained revealed that the B. amyloliquefaciens + 2BC improved the chlorophyll
a, chlorophyll b, photosynthetic rate, transpiration rate, 100-grain weight, and grain N, P and K up
to 114%, 123%, 118%, 73%, 59%, 58%, 18% and 23%, respectively, under drought conditions. It
is concluded that co-application of PGPR and biochar is an effective technique to mitigate the
drought effects.
Keywords: Biochar, Chlorophyll contents, Growth, Rhizobacteria, Wheat, Yield
6.1. Introduction
Various biotic (pests, pathogens) and abiotic (soil compaction, salinity, waterlogging, heavy
metals and poor nutrition) stresses are a big cause of low crops productivity around the globe
(Timmusk et al., 2018). Drought stress is very common in worldwide arid and semi-arid areas.
Moreover, climate change is going to creat a worst situation in this regard (Anjum et al., 2011b;
Griffin et al., 2013; Mehran et al., 2017; Zhang et al., 2017; Saikia et al., 2018). The demand for
irrigation water is expected to increase to 10% by the 2050s (Wada et al., 2013).
52
Under drought stress, growth and yield of crops are usually decreased due to less intake of
nutrients, poor photosynthesis (Fahad et al., 2017) and limited supply of water (Ludlow and
Muchow, 1990). In addition, drought accelerates the biosynthesis of ethylene (Glick et al., 1998;
Johnson and Ecker, 1998) which retards the roots elongation and development (Morgan and Drew,
1997; Mayak et al., 2004a; Zafar-ul-Hye et al., 2014).
Although, traditional breeding, water management and genetic engineering are thought to be much
effective tools to alleviate drought stress but high technicalities are involved to adopt and
implement these approaches (Niu et al., 2018). However, the use of plant growth promoting
rhizobacteria (PGPR) is an alternative technique for mitigation of drought effects (Niu et al.,
2018). A large number of rhizospheric bacteria are well documented that show growth promotion
in plants under stressful conditions (Zafar-ul-Hye et al., 2014). As far as regularization of ethylene
biosynthesis under drought stress is concerned, using 1-aminocyclopropane-1-carboxylate
deaminase (ACC-deaminase), producing PGPR is found to be quite effective (Honma and
Shimomura, 1978; Saleem et al., 2007; Saraf et al., 2010; Ngumbi and Kloepper, 2016; Vurukonda
et al., 2016). The ACC-deaminase cleaves the ACC (1-aminocyclopropane-1-carboxylic acid, an
immediate precursor of ehtylene biosynthesis through methionine pathway in higher plants) into
ammonia and α-ketobutyrate instead of ethylene (Glick et al., 1998, 1999a; Glick, 2004). Besides
ethylene regularization, the PGPR also help in better root development (Belimov et al., 2001a),
secretion of growth hormones (auxins or cytokines) (Glick et al., 1999a) and solubilization of
immobile nutrients (e.g. phosphorus, potassium etc.) (Alam et al., 2008; Basak and Biswas, 2010).
On the other hand, the imperative role of organic amendments in mitigation of drought stress by
improving soil water holding capacity and availability of nutrients cannot be denied (Danish et al.,
2014; Qayyum et al., 2014). Biochar (BC), is a black carbon compound which is a good source of
nutrients. It is produced through pyrolysis at high temperature under low or no supply of oxygen
(Lehmann, 2007; Singh et al., 2010; Qayyum et al., 2014). The physio-chemical properties of BC
depend on the nature of waste material used and temperature of the pyrolysis (Glaser et al., 2002;
Navia and Crowley, 2010). High surface area and pore spaces of BC structure improve its soil
water and nutrients holding capacity. (Gundale and DeLuca, 2006; Hartmann et al., 2006;
Amonette and Joseph, 2009; Warnock, 2009).
Wheat (Triticum aestivum L.) is an important cereal crop and staple food in most parts of the world.
It contains 55% carbohydrates and 8-12% proteins (Bos et al., 2005). It is an important crop due
53
to its worldwide trade too (FAO, 2003). Cultivation of wheat under a limited supply of water
significantly decreases the yield (Singh and Chaudhary, 2006) while its demand is increasing at
the rate of 1.6% / annum (Ortíz-Castro et al., 2008). The need of time is to enhance wheat yield
even under drought stress.
In recent past, the researchers focused on the application of either ACC-deaminase containing
PGPR or BC in separate to mitigate the drought stress. The novelty and aim of the present study
are to examine the combined effect of ACC-deaminase producing PGPR and timber-waste BC for
the alleviation of drought effects. Keeping in mind the importance of wheat, the current study was
conducted with the hypothesis that co-application of drought tolerant ACC deaminase producing
PGPR and timber waste BC could be very effective to alleviate drought effects.

6.2.1. ACC deaminase PGPR
See chapter 3 section 3.1 subsection 3.1.10. The physio-chemical characteristics of biochar is
provided in table 6.1.
Table 6.1. Characteristics of soil and timber waste biochar (BC)

Soil Unit Value Biochar Unit Value
Sand % 55 pH - 7.03
Silt % 30 ECe dS m-1 0.89
Clay % 15 Volatile Matter % 10.19
Texture Sandy Loam Ash Content % 30.26
pHs - 8.43 Fixed Carbon % 59.55
-1
ECe dS m 1.95 Total N % 0.29
Organic Matter % 0.45 Total P % 0.53
Total N % 0.023 Total K % 1.36
-1
Extractable P µg g 8.16 Total Na % 0.28
-1
Extractable K µg g 204
54
6.2.4. Pots dimensions and soil characterization
The plastic bag (30 cm deep × 20 cm in diameter) was used as a pot, having capacity to carry 8 kg
soil. The soil was collected from the plough layer of bank of the Chenab River, Multan, Punjab,
Pakistan. The soil of the selected area was previously characterized as dark yellowish brown,
moderately calcareous, weakly structured and well drained with Cambic subsurface horizon and
an Ochric epipedon (Abid et al., 2017). For soil characterization see chapter 3 section 3.1
subsection 3.1.11.
6.2.5. Pots preparation

In each plastic pot, 8 kg soil was filled. To fulfil macronutrients requirement nitrogen (N),
phosphorus (P) and potassium (K) fertilizers were added at the rate of 120: 90 and 60 kg ha-1
respectively, as recommended dose (Adnan et al., 2014). The urea was added in three split doses.
As far as diammonium phosphate (DAP) and muriate of potash (MOP) fertilizers are concerned,
recommended rates of fertilizers were applied in a single dose at the time of sowing. Timber waster
biochar was added at three different rates including control no biochar (0BC), 0.75% of soil (60g
biochar per 8 kg soil) biochar (1BC) and 1.50% of soil (120g biochar per 8 kg soil) biochar (2BC).
6.2.6. Seeds collection and sterilization
6.2.7. PGPR inoculation
6.2.8. Experiment site and treatments
The pot experiment was conducted in the research area of the Department of Soil Science,
Bahauddin Zakariya University Multan, Pakistan under drought stress on wheat. There were 15
treatments with 3 replications following factorial completely randomized design (CRD). The
treatments included: Control (No PGPR + No BC), L. adecarboxylata, A. fabrum, P. aeruginosa,
B. amyloliquefaciens, 1BC, L. adecarboxylata + 1BC, A. fabrum + 1BC, P. aeruginosa + 1BC, B.
amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa +
2BC, B. amyloliquefaciens + 2BC.
6.2.9. Seeds sowing and drought
In each pot, 10 seeds of wheat were initially sown. In control, the soil normal moisture (NM) was
maintained at the level of 70% of field capacity (FC70) throughout the experiment on weight basis.
55
However, to introduce mild drought (MD) and severe drought (SD) stress as per treatment plan,
the soil moisture was maintained at the level of 50% and 30% of field capacity (FC50 and FC30),
respectively, throughout the trial as suggested by Boutraa et al. (2010). After germination of seeds,
five healthy seedlings were maintained in each pot by thinning.
6.2.10. Reproductive stage harvesting and yield attributes
The wheat plants were harvested after 125 days of sowing for the determination of growth and
yield attributes.
6.2.11. Nutrients analysis
6.2.14. Proline
6.2.15. Statistical analysis
6.3. Results
6.3.1. Shoot length and electrolyte leakage
Both main and interactive effects of treatments (T) and various levels of drought (D) were
significant for shoot length and electrolyte leakage in wheat leaves. At SD, the B.
amyloliquefaciens + 2BC and P. aeruginosa + 2BC remained significantly best as compared to all
other treatments for shoot length. Application of 1BC and 2BC remained statistically alike with
respect to each other but significantly different as compared to control at SD as compared to control
for shoot length (Figure 6.1). However, B. amyloliquefaciens + 2BC also remained significantly
better among all the treatments for shoot length at MD. Maximum increase, 1.53 and 0.79-fold in
shoot length was noted at SD and MD respectively, as compared to control where B.
amyloliquefaciens + 2BC was applied. In case of electrolyte leakage at SD, A. fabrum + 2BC and
B. amyloliquefaciens + 2BC were found to be significantly best as compared to control.
Application of 1BC and 2BC remained statistically alike with each other but significantly different
56
as compared to the control at SD as compared to control for electrolyte leakage. It was noted that
L. adecarboxylata, P. aeruginosa, B. amyloliquefaciens without BC also decreased the electrolyte
leakage as compared to control at SD. Maximum reduction (0.50-fold) in electrolyte leakage was
noted as compared to control where A. fabrum + 2BC and B. amyloliquefaciens + 2BC were
applied at SD.
timber waste biochar (1BC and 2BC) on shoot length (A) electrolyte leakage (B) in wheat leaves
under various levels of drought (D). Means sharing the same letter are statistically similar. Error
bars represent ± standard deviations. NM = Normal Moisture; MD = Mild Drought; SD = Severe
Drought
57
6.3.2. Yield attributes
Both main and interactive effects of T and D were significant for grain yield pot -1 and straw yield pot-1.
In case of 100-grain weight main effect of T and D was significant but their interaction remained non-
significant. It was noted that the PGPR with and without 1BC, as well as 1BC and 2BC treatments, were
statistically alike to each other and to the control at SD for grain yield pot-1. However, the treatments L.
adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC
differed significantly as compared to control for grain yield pot-1 at MD and SD (Table 6.2). The
treatments B. amyloliquefaciens + 1BC also remained significantly better as compared to control and
other PGPR with 1BC at MD for grain yield pot -1. At NM application of B. amyloliquefaciens + 1BC,
2BC, L. adecarboxylata + 2BC, remained significantly different as compared to control for grain yield
pot -1. Maximum increase, 0.40, 1.55 and 2.15-fold in grain yield pot -1 was noted at NM, MD and SD
as compared to control where L. adecarboxylata + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens
+ 2BC were applied respectively. For 100-grains weight, all the treatments (except A. fabrum) remained
significantly better as compared to control. Application of 1BC and 2BC remained statistically alike
with each other for 100-grains weight. Among all treatments, 1BC, A. fabrum + 1BC, B.
amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and
B. amyloliquefaciens + 2BC remained significantly best for 100-grains weight. Maximum increase
(59%) in 100-grains weight was noted as compared to control where B. amyloliquefaciens + 2BC was
applied. In case of straw yield pot-1, the PGPR with and without biochar remained significantly better as
compared to control at NM, MD and SD. However, 1BC and 2BC remained statistically alike to each
other for straw yield pot-1. Among all the treatments, L. adecarboxylata + 1BC, A. fabrum + 1BC, P.
aeruginosa + 1BC, B. amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P.
aeruginosa + 2BC and B. amyloliquefaciens + 2BC remained significantly best for straw yield pot-1.
Maximum increase of 1.81 and 1.78-fold in straw yield pot-1 was noted as compared to control where B.
amyloliquefaciens + 2BC and A. fabrum + 2BC were applied at SD and MD respectively.
58
Table 6.2. Effect of ACC deaminase containing PGPR in combination with various rates of timber waste biochar (0BC, 1BC and 2BC) on grains yield
pot-1 100-grains weight and straw yield under various levels of drought (D)
Grain Yield Pot -1 (g) 100-grains weight (g) Straw Yield Pot-1 (g)
Various levels of drought (D)
Treatments IE (T × D) IE (T × D) IE (T × D) ME (T)
(Means of 3 replicates) ME (T) (Means of 3 replicates) ME (T) (Means of 3 replicates)
NM MD SD NM MD SD NM MD SD
d-m q-u u E E h-n st
Control (No PGPR + No BC) 5.82 2.93 1.84 3.53 2.89 2.01 1.30 2.07 15.3 6.80 4.70 t 8.90 F
L. adecarboxylata 6.03 b-k 4.56 j-r 2.73 r-u 4.44 C-E 2.89 2.80 2.26 2.65 CD 16.1 g-l 12.9 l-q 9.50 q-s 12.9 E
f-o p-u s-u DE DE
A. fabrum 5.53 3.34 2.45 3.77 3.04 2.47 1.95 2.49 16.7 f-k 14.9 h-n 10.6 p-r 14.1 DE
P. aeruginosa 5.74 e-n 3.74 m-u 2.17 tu 3.88 DE 3.00 2.83 1.90 2.58 CD 16.1 g-l 12.2 m-r 9.20 rs 12.5 E
B. amyloliquefaciens 5.90 c-l 3.87 l-u 2.95 q-u 4.24 DE 3.12 2.81 2.19 2.71 B-D 17.4 e-j 14.8 i-n 10.1 p-s 14.1 DE
1BC 6.69 a-i 4.44 j-s 2.87 q-u 4.67 CD 3.13 2.97 2.52 2.87 A-D 18.5 e-h 14.4 j-o 9.10 rs 14.0 DE
L. adecarboxylata + 1BC 6.45 a-j 4.76 i-r 2.73 r-u 4.65 CD 3.07 2.88 2.43 2.79 B-D 20.0 a-f 16.7 f-k 10.4 p-r 15.7 CD
A. fabrum + 1BC 7.21 a-g 5.37 g-p 3.70 n-u 5.43 BC 3.29 3.10 2.54 2.98 A-D 21.1 a-d 15.6 g-m 12.0 n-r 16.2 BC
P. aeruginosa + 1BC 6.28 a-j 4.90 h-q 2.87 q-u 4.68 CD 3.11 2.96 2.14 2.74 B-D 20.8 a-e 16.9 f-j 9.90 p-s 15.9 BC
B. amyloliquefaciens + 1BC 8.06 ab 6.33 a-j 3.81 l-u 6.07 AB 3.28 3.18 2.51 2.99 A-C 21.3 a-c 17.0 f-j 11.9 n-r 16.7 A-C
2BC 7.95 a-c 5.87 c-l 3.62 o-u 5.81 AB 3.35 3.00 2.52 2.96 A-D 21.5 a-e 17.9 c-j 11.1 o-r 16.8 A-C
L. adecarboxylata + 2BC 8.13 a 7.18 a-g 4.00 k-t 6.43 AB 3.48 3.13 2.47 3.03 A-C 22.7 a 18.2 c-i 12.0 n-r 17.6 AB
A. fabrum + 2BC 7.90 a-d 6.92 a-h 4.51 j-s 6.44 AB 3.52 3.19 2.82 3.18 AB 22.8 a 18.9 b-g 12.8 l-q 18.2 A
P. aeruginosa + 2BC 7.73 a-e 7.48 a-f 4.17 k-t 6.46 AB 3.55 3.29 2.23 3.02 A-C 22.4 ab 17.6 d-j 11.9 n-r 17.3 A-C
B. amyloliquefaciens + 2BC 7.58 a-f 7.14 a-g 5.79 e-n 6.84 A 3.58 3.38 2.94 3.30 A 22.9 a 17.9 c-j 13.2 k-p 18.0 A
ME (D) 6.87 A 5.25 B 3.35 C 3.22 A 2.93 B 2.31 C 19.7 A 15.5 B 10.6 C
Means sharing different letters are significantly different (p ≤ 0.05). Non-significant interactive effect (T × D) did not have any letter.
ME indicates main effect; IE indicates interactive effect; NM = Normal Moisture; MD = Mild Drought; SD = Severe Drought
59
6.3.3. N, P and K concentration in grain
Both main and interactive effects of T and D were significant for N, P and K concentration in grains. At
SD, inoculation of L. adecarboxylata, A. fabrum, P. aeruginosa and B. amyloliquefaciens with and
without 1BC and 2BC differed significantly as compared to control for grain nitrogen. The treatments
B. amyloliquefaciens, 1BC, L. adecarboxylata + 1BC, A. fabrum + 1BC, P. aeruginosa + 1BC and B.
amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and
B. amyloliquefaciens + 2BC remained significantly better as compared to control for grain nitrogen at
MD (Table 6.3). Application of A. fabrum + 2BC performed significantly best as compared to control
for grain nitrogen at NM. For grain phosphorus, all the treatments (except P. aeruginosa) were
significantly different as compared to control at SD. However, at MD and NM all the treatments were
statistically alike with control for grain P. In case of grain K, all the treatments were significantly
different as compared to control at SD. Inoculation of A. fabrum and P. aeruginosa at 2BC remained
significantly better than at 1BC for grain potassium at SD. At MD, inoculation of PGPR with 1BC and
2BC proved significantly better than control for grain K. The performance of A. fabrum was significantly
better at 2BC than 1BC for grain K at MD. Among all the treatments A. fabrum + 1BC, B.
amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC, B.
amyloliquefaciens + 2BC performed significantly best at NM. Application of 2BC performed
significantly better than 1BC for grain potassium at NM, MD and SD. Maximum increase in grain N
(58%), P (18%) and K (23%) were noted by B. amyloliquefaciens + 2BC as compared to control at SD.
Main effects of T and D were significant but their interaction was non-significant for N and P
concentration in shoot. For K concentration in shoot, both main and interactive effects of T and D were
significant. The treatments L. adecarboxylata, A. fabrum, P. aeruginosa and B. amyloliquefaciens
differed significantly as compared to the control for shoot N concentration. Among 1BC and 2BC,
application of 2BC remained significantly better as compared to 1BC for N concentration in shoot (Table
6.4). It was noted that all the PGPR (L. adecarboxylata, A. fabrum, P. aeruginosa and B.
amyloliquefaciens) with 2BC performed significantly better as compared to 1BC (except B.
amyloliquefaciens + 1BC) for N concentration in shoot. For phosphorus concentration in shoot,
However, 1BC and 2BC significantly differed as compared to control. In case of P concentration in
shoot, L. adecarboxylata, A. fabrum and B. amyloliquefaciens were statistically alike to each other but
significantly different as compared to control. Among 1BC and 2BC, application of 2BC significantly
60
increased (28%) P concentration in shoot as compared to 1BC. Statistical analysis confirmed that among
all the treatments A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC proved
significantly best for P concentration in shoot. Maximum increase in N (0.73-fold) and P (1.50-fold)
concentration in shoot was noted as compared to control where B. amyloliquefaciens + 2BC was applied.
In case of K concentration in shoot, all the treatments remained significantly better as compared to
control at SD. The treatments 1BC and 2BC remained statistically alike with each other without PGPR
for K concentration in shoot at MD and SD. Among all the treatments results of B. amyloliquefaciens +
1BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC were significantly best
for K concentration in shoot at MD. Maximum increase of 0.38, 0.85 and 1.38-fold in K concentration
in shoot was noted where B. amyloliquefaciens + 2BC was applied as compared to control at NM, MD
and SD respectively.
61
Table 6.3. Effect of ACC deaminase containing PGPR in combination with various rates of timber waste biochar (0BC, 1BC and 2BC) on grains nitrogen,
phosphorus and potassium concentration under various levels of drought (D)
Grain Nitrogen (%) Grain Phosphorus (%) Grain Potassium (%)
Treatments IE (T × D) IE (T × D) IE (T × D)
(Means of 3 replicates) ME (T) (Means of 3 replicates) ME (T) (Means of 3 replicates) ME (T)
b-k no p G a-i i-n o D k-m mn
Control (No PGPR + No BC) 1.78 1.56 1.11 1.48 0.305 0.283 0.247 0.278 0.465 0.453 0.422 n 0.447 H
h-m h-m
L. adecarboxylata 1.77 c-m 1.67 h-n 1.49 o 1.64 F 0.303 a-j 0.287 e-n 0.273 mn 0.288 B-D 0.481 0.474 0.460 1-m 0.472 G
A. fabrum 1.79 b-k 1.69 g-n 1.56 no 1.68 EF 0.306 a-g 0.299 b-l 0.275 mn 0.293 A-C 0.491 f-l 0.481 h-m 0.460 l-m 0.478 FG
P. aeruginosa 1.80 b-j 1.67 h-n 1.56 no 1.68 EF 0.301 a-k 0.280 j-n 0.268 no 0.283 CD 0.482 h-m 0.471 j-m 0.455 m 0.469 G
B. amyloliquefaciens 1.79 b-j 1.74 e-m 1.60 m-o 1.71 D-F 0.309 a-g 0.279 k-n 0.274 mn 0.287 B-D 0.493 f-k 0.484 g-m 0.461l-m 0.479 E-G
1BC 1.82 a-i 1.78 b-l 1.61 l-o 1.74 C-E 0.307 a-g 0.280 j-n 0.280 j-n 0.289 B-D 0.505 d-h 0.489 f-l 0.470 j-m 0.488 EF
L. adecarboxylata + 1BC 1.87 a-f 1.77 c-m 1.62 k-o 1.75 B-E 0.310 a-e 0.283 i-n 0.277 l-n 0.290 BC 0.506 d-h 0.497 f-j 0.481 h-m 0.495 DE
A. fabrum + 1BC 1.86 a-g 1.78 c-l 1.61 l-o 1.75 B-E 0.307 a-h 0.283 i-n 0.280 k-n 0.290 BC 0.535 a-d 0.503 d-i 0.473 i-m 0.504 CD
P. aeruginosa + 1BC 1.86 a-f 1.78 b-l 1.64 j-o 1.76 B-E 0.309 a-f 0.283 i-n 0.274 mn 0.289 B-D 0.520 b-f 0.497 f-j 0.465 k-m 0.494 DE
B. amyloliquefaciens + 1BC 1.91 a-d 1.82 a-i 1.66 i-o 1.80 A-C 0.312 a-d 0.287 e-n 0.279 k-n 0.293 BC 0.542 a-c 0.519 b-f 0.498 f-j 0.520 B
2BC 1.92 a-d 1.81 a-i 1.66 i-o 1.80 A-D 0.321 ab 0.286 g-n 0.276 mn 0.294 A-C 0.543 a-c 0.532 a-e 0.502 e-i 0.526 AB
L. adecarboxylata + 2BC 1.94 a-c 1.84 a-h 1.67 h-n 1.82 A-C 0.315 a-c 0.290 d-n 0.287 f-n 0.297 AB 0.550 ab 0.519 b-f 0.484 g-m 0.517 BC
A. fabrum + 2BC 1.97 a 1.88 a-f 1.71 f-n 1.85 A 0.318 ab 0.293 c-m 0.279 k-n 0.296 AB 0.560 a 0.541 a-c 0.515 c-g 0.539 A
P. aeruginosa + 2BC 1.90 a-e 1.82 a-i 1.67 h-n 1.80 A-C 0.309 a-g 0.290 d-n 0.285 h-n 0.295 AB 0.543 a-c 0.531 a-e 0.515 c-g 0.530 AB
B. amyloliquefaciens + 2BC 1.95 ab 1.78 c-l 1.75 d-m 1.82 AB 0.322 a 0.299 a-l 0.291 d-n 0.304 A 0.559 a 0.541 a-c 0.518 c-f 0.539 A
ME (D) 1.86 A 1.76 B 1.59 C 0.310 A 0.287 B 0.276 C 0.518 A 0.502 B 0.479 C
62
Table 6.4. Effect of ACC deaminase containing PGPR in combination with various rates of timber waste biochar (0BC, 1BC and 2BC) on shoot N, P
and K concentration under various levels of drought (D)
Shoot Nitrogen (%) Shoot Phosphorus (%) Shoot Potassium (%)
Control (No PGPR + No BC) 1.66 1.43 1.28 1.45 F 0.29 0.20 0.12 0.20 H 2.06 f-o 1.44 r 0.99 s 1.50 I
L. adecarboxylata 2.01 1.93 1.52 1.82 E 0.37 0.24 0.21 0.27 FG 2.11 e-o 1.94 i-q 1.62 p-r 1.89 GH
A. fabrum 2.08 1.95 1.64 1.89 DE 0.37 0.25 0.22 0.28 FG 2.13 e-o 2.01 g-p 1.57 qr 1.90 F-H
P. aeruginosa 2.06 1.93 1.56 1.85 E 0.33 0.21 0.19 0.24 GH 2.09 e-o 1.88 k-q 1.52 qr 1.83 H
B. amyloliquefaciens 2.05 1.97 1.63 1.88 DE 0.39 0.26 0.18 0.27 FG 2.15 e-n 2.02 g-p 1.71 o-r 1.96 F-H
1BC 2.24 2.13 1.85 2.07 CD 0.45 0.30 0.22 0.32 EF 2.29 c-l 1.93 i-q 1.73 n-r 1.99 E-H
L. adecarboxylata + 1BC 2.37 2.23 2.00 2.20 BC 0.49 0.33 0.25 0.35 C-E 2.37 b-i 2.14 e-o 1.77 n-r 2.10 D-G
A. fabrum + 1BC 2.47 2.26 1.94 2.22 BC 0.52 0.36 0.30 0.39 B-D 2.42 a-g 2.32 c-k 1.82 n-r 2.19 C-E
P. aeruginosa + 1BC 2.42 2.26 1.97 2.22 BC 0.46 0.35 0.20 0.34 D-F 2.34 c-j 2.27 d-m 1.72 n-r 2.11 D-F
B. amyloliquefaciens + 1BC 2.52 2.35 2.06 2.31 AB 0.54 0.41 0.31 0.42 B 2.50 a-f 2.43 a-g 1.84 m-r 2.25 B-D
2BC 2.59 2.48 1.90 2.32 AB 0.55 0.39 0.28 0.41 BC 2.45 a-g 2.36 b-i 1.92 j-q 2.24 B-D
L. adecarboxylata + 2BC 2.67 2.51 2.15 2.44 A 0.53 0.42 0.31 0.42 BC 2.68 a-d 2.38 b-h 1.86 l-r 2.30 B-D
A. fabrum + 2BC 2.72 2.55 2.06 2.44 A 0.60 0.48 0.39 0.49 A 2.79 ab 2.61 a-d 1.94 i-q 2.44 AB
P. aeruginosa + 2BC 2.77 2.57 2.13 2.49 A 0.55 0.42 0.37 0.45 AB 2.71 a-c 2.53 a-e 1.94 h-q 2.39 BC
B. amyloliquefaciens + 2BC 2.79 2.57 2.17 2.51 A 0.61 0.49 0.40 0.50 A 2.85 a 2.67 a-d 2.35 b-j 2.63 A
ME (D) 2.36 A 2.21 B 1.86 C 0.47 A 0.34 B 0.26 C 2.40 A 2.20 B 1.75 C
63
Main effect of T and D was significant, while their interaction (T × D) remained non-significant
for the rate of photosynthesis and transpiration. For stomatal conductance, both main and
interactive effects of T and D were significant. It was noted that the photosynthetic rate was
significantly improved as compared to the control where P. aeruginosa and B. amyloliquefaciens
were applied as inocula. Biochar application without PGPR significantly enhanced photosynthetic
rate as compared to the control but application rate 2BC was more effective than 1BC for the
improvement in photosynthetic rate (Table 6.5). However, among all the treatments P. aeruginosa
+ 2BC and B. amyloliquefaciens + 2BC remained the best for a significant increase in
photosynthetic rate. For transpiration rate, a significant improvement was noted as compared to
control in all the treatments. Application of 1BC and 2BC without PGPR gave statistically similar
results regarding transpiration rate. However, P. aeruginosa + 2BC and B. amyloliquefaciens +
2BC remained significantly best for transpiration rate as compared to L. adecarboxylata, A.
fabrum, P. aeruginosa, B. amyloliquefaciens, 1BC and the control. Maximum increase in
photosynthetic rate (1.18-fold) and transpiration (0.73-fold) rate was noted as compared to control
where B. amyloliquefaciens + 2BC was applied. Application of 1BC and 2BC remained significant
at SD as compared to control for stomatal conductance. However, at MD application of 2BC
remained significantly better than 1BC and control regarding stomatal conductance. Statistical
analysis confirmed that B. amyloliquefaciens + 2BC at SD while 2BC, P. aeruginosa + 2BC and
B. amyloliquefaciens + 2BC at MD performed significantly best among all the treatments for
stomatal conductance. However, stomatal conductance was maximum as compared to control in
2BC (0.56-fold), B. amyloliquefaciens + 2BC (1.46-fold) and B. amyloliquefaciens + 2BC (5.62-
fold) at NM, MD and SD, respectively.
Main effects of T and D were significant but their interactions remained non-significant for
chlorophyll a, chlorophyll b and total chlorophyll contents in wheat leaves. The treatments L.
adecarboxylata, A. fabrum, P. aeruginosa and B. amyloliquefaciens were statistically alike with
each other but differed significantly as compared to control for cholorophyll a content (Table 6.6).
With and without PGPR, application of 1BC and 2BC gave statistically similar results but
remained significantly different as compared to control for cholorophyll a content. For
cholorophyll b content, with and without 1BC all the PGPR treatments (except B.
64
amyloliquefaciens + 1BC) were statistically alike with each other but differed significantly as
compared to control. Among 1BC and 2BC without PGPR the 2BC was significantly better as
compared to control for cholorophyll b content. However, A. fabrum + 2BC and B.
amyloliquefaciens + 2BC proved significantly best as compared to all other treatments regarding
cholorophyll b content. In case of total cholorophyll content, the treatments L. adecarboxylata, A.
fabrum, P. aeruginosa and B. amyloliquefaciens differed significantly as compared to control.
Statistically, 2BC was significantly better than 1BC for total cholorophyll. However, both 1BC
and 2BC signifcantly increased total chlorophyll as compared to control. It was noted that 2BC, L.
were statistically alike and performed the best among all the treatments for total cholorophyll
content. Maximum increase in cholorophyll a (1.14-fold), cholorophyll b (1.23-fold) and total
cholorophyll (1.15-fold) was noted in B. amyloliquefaciens + 2BC as compared to control.
6.3.7. Carotenoids and proline
Both main and interactive effects of T and D were significant for carotenoids and proline. In case
of carotenoids, the treatments L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC
and B. amyloliquefaciens + 2BC were statistically alike to each other but differ significantly as
compared to control for carotenoids at SD (Figure 6.2). The result of 2BC was significantly better
as compared to 1BC and control for carotenoids at SD. It was noted that 1BC, L. adecarboxylata
+ 1BC, A. fabrum + 1BC, P. aeruginosa + 1BC, B. amyloliquefaciens + 1BC, 2BC, L.
were statistically alike with each other but significantly different as compared to control for
carotenoids at MD. Maximum increase, 0.42, 0.48 and 2.20-fold in carotenoids was noted at NM,
MD and SD respectively as compared to control where B. amyloliquefaciens + 2BC was applied.
For proline reduction, A. fabrum, P. aeruginosa and B. amyloliquefaciens differed significantly
as compared to control at SD. Application of 1BC also significantly decreased proline as compared
to control but result of 2BC was statistically better for proline reduction as compared to 1BC at
SD. All PGPR strains remained significantly better with 2BC for proline reduction as compared to
1BC at SD. However, application of B. amyloliquefaciens + 2BC showed a maximum decrease of
0.34-fold proline at SD as compared to control.
65
Table 6.5. Effect of ACC deaminase containing PGPR in combination with various rates of timber waste biochar (0BC, 1BC and 2BC) on gas
exchange attributes under various levels of drought (D)
Photosynthetic Rate Transpiration Rate Stomatal Conductance
[µmol (CO2) m-2 s-1] (mmol (H2O) m s ) -2 -1 mol (CO2) m-2 s-1
Treatments
IE (T × D) IE (T × D) IE (T × D)
G D e-l m-p
Control (No PGPR + No BC) 35.9 22.0 16.0 24.6 1.84 1.12 0.70 1.22 35.7 21.7 6.70 r 21.4 H
FG CD d-j l-p qr
L. adecarboxylata 44.1 32.6 17.7 31.5 1.98 1.40 0.89 1.42 39.8 24.5 8.71 24.3 GH
A. fabrum 43.4 31.4 14.1 29.6 FG 2.07 1.79 1.02 1.63 BC 41.4 b-h 28.9 h-n 8.38 qr 26.2 GH
P. aeruginosa 46.7 38.0 21.2 35.3 D-F 2.12 1.76 1.03 1.64 BC 36.4 e-l 26.0 k-n 12.4 p-r 24.9 GH
B. amyloliquefaciens 46.9 38.2 27.4 37.5 C-F 2.11 1.80 1.08 1.66 BC 37.5 d-k 29.0 h-n 13.1 o-r 26.5 F-H
1BC 46.7 34.6 19.1 33.5 EF 2.24 1.87 0.84 1.65 BC 37.9 d-k 32.2 e-n 28.1 i-n 32.7 EF
L. adecarboxylata + 1BC 52.3 46.5 33.9 44.2 BC 2.36 1.84 1.33 1.84 AB 36.2 e-l 28.5 i-n 25.8 k-o 30.1 E-G
A. fabrum + 1BC 50.6 43.7 35.4 43.2 B-D 2.43 1.80 1.31 1.85 AB 37.4 d-l 27.6 j-n 20.1 n-q 28.4 FG
P. aeruginosa + 1BC 49.0 41.8 36.8 42.5 B-D 2.41 1.79 1.32 1.84 AB 39.2 d-j 35.8 e-l 32.2 e-n 35.7 DE
B. amyloliquefaciens + 1BC 48.3 43.3 33.6 41.7 B-E 2.42 1.91 1.36 1.90 AB 41.6 b-h 35.2 e-l 29.5 g-n 35.4 DE
2BC 52.5 45.2 37.9 45.2 BC 2.40 1.89 1.39 1.89 AB 55.8 a 44.9 a-e 39.9 d-j 46.9 A-C
L. adecarboxylata + 2BC 47.6 43.8 36.2 42.5 B-D 2.40 2.03 1.32 1.92 AB 55.7 a 40.5 c-i 33.8 e-m 43.4 BC
A. fabrum + 2BC 50.5 49.9 34.1 44.8 BC 2.34 2.08 1.24 1.88 AB 53.9 ab 37.2 d-l 31.8 f-n 41.0 CD
P. aeruginosa + 2BC 54.3 49.5 39.3 47.7 AB 2.40 2.19 1.54 2.04 A 55.0 a 49.6 a-d 41.9 b-g 48.8 AB
B. amyloliquefaciens + 2BC 62.9 58.6 39.4 53.6 A 2.32 2.27 1.73 2.11 A 55.3 a 53.3 a-c 44.2 a-f 50.9 A
ME (D) 48.8 A 41.3 B 29.5 C 2.26 A 1.84 B 1.21 C 43.9 A 34.3 B 25.1 C
66
Table 6.6. Effect of ACC deaminase containing PGPR in combination with various rates of timber waste biochar (0BC, 1BC and 2BC) on
chlorophyll content under various levels of drought (D)
Chlorophyll a (mg g-1) Chlorophyll b (mg g-1) Total Chlorophyll (mg g-1)
E G
Control (No PGPR + No BC) 1.04 0.67 0.39 0.70 0.29 0.26 0.10 0.22 1.33 0.93 0.50 0.92 H
D EF
L. adecarboxylata 1.21 0.92 0.78 0.97 0.43 0.34 0.15 0.31 1.64 1.26 0.93 1.28 G
D EF
A. fabrum 1.14 0.93 0.83 0.96 0.40 0.38 0.12 0.30 1.54 1.31 0.95 1.27 G
D F
P. aeruginosa 1.09 0.92 0.78 0.93 0.36 0.31 0.14 0.27 1.45 1.24 0.93 1.20 G
CD EF
B. amyloliquefaciens 1.12 1.04 1.04 1.07 0.42 0.39 0.17 0.32 1.54 1.42 1.21 1.39 FG
BC EF
1BC 1.43 1.31 1.07 1.27 0.40 0.40 0.17 0.32 1.82 1.71 1.24 1.59 EF
AB E
L. adecarboxylata + 1BC 1.58 1.36 1.17 1.37 0.43 0.38 0.18 0.33 2.02 1.74 1.36 1.70 C-E
AB DE
A. fabrum + 1BC 1.42 1.38 1.29 1.36 0.42 0.40 0.22 0.35 1.84 1.78 1.52 1.71 C-E
AB E
P. aeruginosa + 1BC 1.58 1.26 1.15 1.33 0.45 0.38 0.18 0.34 2.03 1.64 1.33 1.67 DE
AB CD
B. amyloliquefaciens + 1BC 1.53 1.35 1.24 1.37 0.48 0.41 0.29 0.40 2.01 1.76 1.54 1.77 B-E
AB BC
2BC 1.60 1.42 1.17 1.40 0.51 0.47 0.26 0.41 2.10 1.89 1.44 1.81 A-D
AB BC
L. adecarboxylata + 2BC 1.64 1.37 1.30 1.44 0.51 0.44 0.28 0.41 2.14 1.81 1.58 1.85 A-D
AB AB
A. fabrum + 2BC 1.59 1.44 1.35 1.46 0.55 0.52 0.31 0.46 2.15 1.96 1.66 1.92 AB
AB BC
P. aeruginosa + 2BC 1.61 1.51 1.29 1.47 0.52 0.49 0.29 0.43 2.12 2.00 1.58 1.90 A-C
A A
B. amyloliquefaciens + 2BC 1.65 1.49 1.35 1.50 0.56 0.51 0.38 0.49 2.21 2.00 1.74 1.98 A
A B C A B C A B
ME (D) 1.41 1.22 1.08 0.45 0.41 0.22 1.86 1.63 1.30 C
67
A
timber waste biochar (1BC and 2BC) on carotenoids (A) proline (B) in wheat leaves under various
levels of drought (D). Means sharing the same letter are statistically similar. Error bars represent
± standard deviations. NM = Normal Moisture; MD = Mild Drought; SD = Severe Drought
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6.4. Discussion
In current study, reduction in shoot length of wheat at MD and SD in control as compared to co-
application of PGPR and 2BC might be due to competition for water and nutrients between roots
and shoot. According to Gargallo-Garriga et al. (2014) stated that drought stress deactivated shoot
metabolic activity to conserve water and food which facilitated roots elongation. The reduction in
water and nutrients movement in shoot, enhanced the up-regulation of ethylene precursor 1‐
aminocyclopropane‐1‐carboxylic acid (ACC) from root to shoot (Sobeih et al., 2004) while
significant amount of ACC generated ethylene restricted the root elongation (Mayak et al., 2004).
A significant reduction in stress ethylene by activity of ACC-deaminase is well documented (Glick
et al., 1998). According to Glick et al. (1998), the synthesis of indole acetic acid (IAA) by PGPR
stimulates the elongation of plants cells and activates ACC synthase which convert S-
adenosylmethionine to ACC. A significant amount of ACC is exuded by plants roots and seeds in
rhizosphere which is hydrolyzed by ACC deaminse into NH3 and α-ketobutyrate that resulted in
better roots elongation. Secretion of roots exudates (phytosiderophores, sugars, organic acids,
amino acids, vitamins, nucleosides and mucilage) also attracts PGPR of rhizo-microbiome that
colonize roots and facilitates better uptake of water and nutrients via PGPR colonization (Shukla
et al., 2011; Drogue et al., 2013). Besides imperative role of PGPR, high surface area, ion
exchange capacity, nutrients and water holding capacity of BC make it an effective amendment
for better intake of nutrients and water in plants (Lehmann et al., 2006; Shenbagavalli and
Mahimairaja, 2012; Paetsch et al., 2018). A significant improvement in photosynthetic rate,
tranpiration rate and stomatal conductance under MD and SD, signified the effectiveness of co-
application of L. adecarboxylata, A. fabrum, P. aeruginosa, B. amyloliquefaciens with 2BC,
comparative to their sole application and control. This increase in gas exchange attributes was
might be due better uptake of water and nutrients, improvement in soil water holding capacity
(WHC), ACC deaminase contaning PGPR (Zafar-ul-Hye et al., 2014; Akhtar et al., 2015b),
redcution in ethylene and better PGPR colonization due to co-application of 2BC (Novak et al.,
2009; Lehmann et al., 2011).
Hydrophobicity of biochar surface is considered as one of the major cause of greater rhizobacteria
diversity, proliferation and activity. The strong adhesion affinity of rhizobacteria with biochar is
mainly characterized by the presence of divalent ions in biochar. The micropores of biochar also
69
serve as a shelter for rhizobacteria. These micropores increase the survival of rhizpbacteria and
decrease their competition for food, shelter and space in the rhizosphere. Although biochar is
resistant against mineralization, slow degradation of biochar also facilitates in privision of
nutrients to the rhizobacteria that plays an imperative role in better microbial proliferation
(Lehmann and Rondon, 2002).
According to Zheng et al. (2003) and Borch et al. (1999) the deficiency of nitrogen and phosphorus
significantly decreased the growth of crops. Less stomatal conductance is one of main cause of
reduction in rate of transpiration under drought (Siddique et al., 1999). Drought stress decreased
the intake of CO2 due to less stomatal conductance (Tholen et al., 2008) which rectrict
carboxylation and reduced rate of photosynthesis (Chaves et al., 2009). However, A significant
improvement in chlorophyll a, chlorophyll b, total chlorophyll, shoot and grains nutrients
concentration validated the efficacious functioning of co-application of ACC deaminase producing
PGPR and 2BC that also significantly increase yield attributes (100-grains weight, straw yield and
grains yield pot-1) of wheat plants at MD and SD. In different experiments conducted by
Richardson et al. (2009) and Zahir et al. (2011), observed that better roots elongation and secretion
of organic acids by PGPR for P and K solubilization are key factors responsible for better nutrients
uptake, improvement in dry weight and yield of crops. However, biochar ability to sorp nutrients
also reduced the losses of N and improved its uptake in plants (Decai et al., 2014; Younis et al.,
2014a). According to Chan et al. (2008) the high surface area of biochar is basic reason for
improved cation exchange sites in soil which resulted in better bioavailibility of nutrients. In
addition, geometry, size, distribution and number of microspores in biochar play an efficacious
role in the sorption of nutrients and water. Application of biochar in soil also makes rapid cycling
of nutrients. Higher retention of nutrients and diversity of rhizobacteria improve the fertility level
of soil and nutrients availability to the plants (Lehmann and Rondon, 2002) It also releases a
significant amount of nutrients in soil solution that become part of the biochar structure during
pyrolysis The concentration of nutrients present in biochar is dependent on the type of waste
feedstock which is used to develop the biochar (Novak et al., 2009).
Results of the current study also showed that electrolyte leakage in the wheat plants leaves was
decreased where L. adecarboxylata, A. fabrum, P. aeruginosa, B. amyloliquefaciens with 2BC was
applied comparative to control under MD and SD. The reduction in electrolyte leakage was might
70
be due to activity of ACC deaminase, better availability of water and nutrients by co-application
of PGPR and 2BC. In an experiment Senaratna and McKersie (1983), also observed a significant
increase in electrolyte leakage due to cell membrane damage caused by drought stress which makes
it more permeable. According to Matile et al. (1997), cell usually lost its membrane integrity as a
result of lipid degradation by ethylene. When lipid in cell membrane becomes degraded then
ethylene comes in contact with the chloroplast and activates the chlorophyllase (chlase) gene
which causes severe damage to the cholorophyll (Matile et al., 1997). However, the addition of
2BC and PGPR in combination significantly decreased electrolyte leakage which was might be
due to activity of ACC deaminase, better availability of water and nutrients intake.
6.5. Conclusion
It is concluded that co-application of drought tolerant ACC deaminase producing PGPR and 2BC
is comparatively better approach than sole application to mitigate drought stress in wheat.
Inoculation of L. adecarboxylata and P. aeruginosa were also effective but A. fabrum and B.
amyloliquefaciens with 2BC gave maximum increase in gas exchange attributes, nutrients
concentration in shoot and grains, photosynthetic pigments and yield of wheat. More investigation
is needed at field level to introduce A. fabrum and B. amyloliquefaciens with 2BC to improve
growth and yield of wheat under drought stress.
71
CHAPTER 7
PGPR capable to produce ACC deaminase and biochar mitigate drought
effects in maize
Abstract
Availability of good quality irrigation water is a big challenge in arid and semi-arid regions of the
world. Drought results in poor plant growth and low yield. Accumulation of stress ethylene has
also been reported to be one of the reasons of stunted plant growth under drought conditions. The
ACC-deaminase can alleviate stress ethylene level in plants. So, the rhizobacteria, capable of
producing ACC-deaminase are likely to improve crop growth and productivity under drought
stress. Similarly, biochar is a nutrients rich organic amendment with high water holding capacity.
So, a pot experiment was carried out with the view to evaluate the role of PGPR capable to produce
ACC deaminase in separate and combinations with timber-waste biochar (BC) in improving maize
growth under drought stress. ACC-deaminase producing rhizobacteria, Pseudomonas aeruginosa,
Enterobacter cloacae, Achromobacter xylosoxidans and Leclercia adecarboxylata were studied
along with two rates (0.75 and 1.50 % of the soil weight) of biochar under three moisture levels.
The Enterobacter cloacae in conjunction with the higher rate of biochar produced a significant
improvement i.e., up to 60, 73, 43, 69, 76 and 42 % respectively, in grain yield pot-1, photosynthetic
rate, stomatal conductance chlorophyll a, total chlorophyll and carotenoids contents of maize over
control under mild drought condition. Similarly, Achromobacter xylosoxidans along with the
higher rate of biochar also significantly enhanced the grain yield pot-1, photosynthetic rate,
stomatal conductance, chlorophyll a, total chlorophyll and carotenoids contents of maize up to
200, 213, 113, 152, 148 and 284 % respectively over control under severe drought condition.
Hence, combined use of ACC-deaminase containing PGPR and BC proved to be an effective
technique to improve maize growth and productivity under drought conditions.
Keywords: Biofertilizers, Biochar, Drought, Morphological attributes, Nutrients, Chlorophyll

content, Maize, Yield.
7.1. Introduction
Drought would be a big threat to sustainable crop production, in future, around the globe (Timmusk
et al., 2018). Climatic models reveal that the degree of drought severity is increasing day by day
72
(Farooq et al., 2009; Griffin et al., 2013; IPCC 2007; Mehran et al., 2017; Saikia et al., 2018;
Zhang et al., 2017). The demand of irrigation water is expected to be raised by 10% up to 2050
(Wada et al., 2013). Higher evapotranspiration rate and less precipitation would also be the reasons
of elevation in drought intensity, if visualized in connection with the present climate change trend
(Mishra and Cherkauer, 2010).
Drought results in less uptake of nutrients by plants, poor root growth and photosynthesis (Fahad
et al., 2017). Drought like other abiotic stresses also stimulates ethylene production referred as
stress ethylene via raised level of 1-aminocyclopropane-1-carboxylic acid (ACC) in higher plants,
a precursor of ethylene in methionine pathway (Wang et al., 2003). Accumulation of stress
ethylene restricts the elongation of roots and consequently growth of shoots (Knight and Crocker,
1913).
Although, water management, traditional breeding and genetic engineering are supposed to be
quite effective to mitigate osmotic stress but high expertise made them less adoptable approaches
than plant growth promoting rhizobacteria (PGPR) inoculation that is effective and can be
implemented easily (Niu et al., 2018; Zafar-ul-Hye et al., 2018). Inoculation of PGPR improves
the growth of plant through better root elongation (Belimov et al., 2001), phosphorus (P) and
potassium (K) solubilization (Alam et al., 2008; Basak and Biswas, 2010) and secretion of growth
hormones i.e. auxin, cytokinins (Glick et al., 1999). ACC-deaminase containing PGPR can
regulate ethylene level via its break down into α-ketobutyrate and ammonia (Glick et al., 1997)
under abiotic stresses (Mayak et al., 2004; Zafar-ul-Hye et al., 2014; Zahir et al., 2008). The plants
treated with PGPR capable to produce ACC deaminase have shown a significant improvement in
stomatal conductance and photosynthesis (Jiang et al., 2012).
Activated black carbon biochar (BC) is another organic amendment which can reduce the osmotic
stress by increasing soil water retention (Verheijen et al., 2010). It is manufactured by the process
of pyrolysis at high temperature and under limited or no supply of oxygen using waste feedstock
(Lehmann, 2007; Liang et al., 2006; Qayyum et al., 2014; Younis et al., 2015). Low surface area,
high porosity and resistance against decomposition (Atkinson et al., 2010) make biochar one of
most useful amendment for the improvement in physio-chemical characteristics of soil (Abid et
al., 2017; Younis et al., 2015) and mitigation of drought stress (Danish et al., 2014; Fiaz et al.,
2014; Keshavarz et al., 2016).
73
In recent past, most of the scientists concentrated on either inoculation of PGPR capable to produce
ACC deaminase or application of BC solely to alleviate drought stress. But aim of the current
study is to inspect the effectiveness of combined application of PGPR capable to produce ACC
deaminase and timber-waste BC to grant resistant to maize against drought. Keeping in view the
importance of maize for its nutritional value (i.e., 78% starch, 10% proteins, 8.5% fibre, 4.8% oil
and 3.1% sugars) (Chaudhary et al., 2014) current pot study was carried out with hypothesis that
combined use of ACC deaminase PGPR and timber waste BC would be much efficacious to
mitigate drought stress.
7.2. Materials and methods

7.2.1. Drought-tolerant PGPR
Out of 37 PGPR isolated from maize rhizosphere, four most efficacious drought-tolerant ACC
deaminase containing PGPR identified as Pseudomonas aeruginosa, Enterobacter cloacae,
Achromobacter xylosoxidans and Leclercia adecarboxylata were screened out previously. These
strains were capable to propagate at osmotic potential of –0.85MPa produced via 20%
polyethylene Glycol 6000 (PEG). The DF minimal salt medium was used to grow the ACC
deaminase producing PGPR (Dworkin and Foster, 1958). The strains were grown on DF minimal
salt medium (Dworkin and Foster, 1958).
7.2.2. Characteristics of PGPR

For confirmation of AcdS gene NCBI gene bank was consulted that confirmed that Enterobacter
cloacae (https://www.ncbi.nlm.nih.gov/nuccore/KM501058.2), Pseudomonas aeruginosa
(https://www.ncbi.nlm.nih.gov/nuccore/CP014948.1/) and Achromobacter xylosoxidans
(https://www.ncbi.nlm.nih.gov/nuccore/AY604540.1),(https://www.ncbi.nlm.nih.gov/nuccore/A
Y604539.1) have AcdS. Work on L. adecarboxylata is continued. For determination of ACC
deaminase activity, synthesized by PGPR (Enterobacter cloacae = 402.1 ± 27.29, Achromobacter
xylosoxidans = 381.17 ± 11.69, Pseudomonas aeruginosa = 115.2 ± 16.14 and Leclercia
adecarboxylata = 296.1 ± 21.69 µmol α-ketobutyrate g-1 protein h-1) methodologies of El-Tarabily
(2008) and Honma and Shimomura (1978) were followed. Indole acetic acid without
(Enterobacter cloacae = 3.39 ± 0.41, Achromobacter xylosoxidans = 5.52 ± 0.79, Pseudomonas
aeruginosa = 2.94 ± 0.49 and Leclercia adecarboxylata = 2.11 ± 0.17µg/ml) and with L-
74
tryptophan (Enterobacter cloacae = 78.79 ± 0.35, Achromobacter xylosoxidans = 61.19 ± 0.14,
Pseudomonas aeruginosa = 21.3 ± 0.37 and Leclercia adecarboxylata = 61.59 ± 0.20µg/ml) was
determined by using Salkowski reagent (Glickmann and Dessaux, 1995). Pikovskaya’s medium
was utilized to determine phosphorus solubilizing activity of PGPR (Enterobacter cloacae = 66.3
± 0.38, Achromobacter xylosoxidans = 77.4 ± 0.98, Pseudomonas aeruginosa = 29.1 ± 1.19 and
Leclercia adecarboxylata = 20.1 ± 1.29 µg/ml) (Vazquez et al., 2000). Potassium solubilizing
activity of PGPR (Enterobacter cloacae = 19.1 ± 0.82, Achromobacter xylosoxidans = 24.5 ± 0.42,
Pseudomonas aeruginosa = 12.6 ± 0.92 and Leclercia adecarboxylata = 16.4 ± 1.40 µg/ml) was
examined according to Setiawati and Mutmainnah (2016).
7.2.3. Production of Timber-waste biochar

7.2.4. Characterization of timber-waste biochar
See chapter 3 section 3.1 subsection 3.1.10. The characteristics of biochar are given in Table 6.1.
7.2.5. Soil characteristic
The plastic bags having dimensions of 75 cm deep × 45 cm diameter were used as a pot. Each bag
has the capacity to carry 15kg soil. A bulk sample of soil was collected from the plough layer near
Chenab River (30° 18′ 22.4″ N; 71° 26′ 16.3″ E). The soil of the selected area was previously
characterized as dark yellowish brown, moderately calcareous, weakly structured and well-drained
with Cambic subsurface horizon and an Ochric epipedon (Abid et al., 2017). For soil
characterization see chapter 3 section 3.1 subsection 3.1.11. The pre-experimental soil
characteristics are provided in Table 6.1.
75
Table 7.1. Pre-experimental characteristics of soil and timber waste biochar (BC)
Sand % 60 pH - 7.26
ECe dS m-1 1.69 Total N % 0.21
Extractable P µg g-1 9.26 Total K % 1.61
Extractable K µg g-1 229 Total Na % 0.19
7.2.6. Polythene bags preparation

In each polythene bag, 12 kg of soil was filled. The N, P and K were applied at the rate of
200:150:100 kg ha-1 (Zafar-ul-Hye et al., 2015). Three splits of urea were applied during the
growth period while diammonium phospahate (DAP) and muriate of potash (MOP) were at the
time of sowing in a single dose.
7.2.7. Seed inoculation
The seeds of maize (cv. Kenzo-123 Hybrid) were purchased from certified (Government of Punjab,
Pakistan) dealer of seeds. Weak and damaged seeds were initially screened out manually. For seeds
sterilization see chapter 3 section 3.1 subsection 3.1.3.
7.2.8. Seeds Sowing and Drought stress
In each pot, four seeds of maize were sown. Normal moisture (NM) was applied in control to
maintain 70% field capacity (FC70). Mild drought (MD) was maintained at 50% FC (FC50) and
severe drought (SD) at 30% FC (FC30) throughout the study as suggested by Boutraa et al. (2010).
After seeds germination, two healthy seedlings were maintained by thinning in each pot.
7.2.9. Experiment site
The experiment was carried out in the research area of the Department of Soil Science, Bahauddin
Zakariya University Multan, Pakistan. There were fifteen treatments with three replicates
following factorial completely randomized design (CRD).
76
7.2.10. Application rate of biochar and treatments
The application rates of BC were as follows; control having no biochar (BC0), 0.75% w/w biochar
(BC0.75) and 1.50% w/w biochar (BC1.50) of total soil contained in a pot. The treatments (T)
included: control (No PGPR + No BC), P. aeruginosa, E. cloacae, A. xylosoxidans, L.
adecarboxylata, BC0.75, P. aeruginosa + BC0.75, E. cloacae + BC0.75, A. xylosoxidans + BC0.75, L.
adecarboxylata + BC0.75, BC1.50, P. aeruginosa + BC1.50, E. cloacae + BC1.50, A. xylosoxidans +
BC1.50 and L. adecarboxylata + BC1.50.
7.2.11. Harvesting at vegetative stage
For shoot length, electrolyte leakage, photosynthetic pigments, proline contents and nutrients
concentration in the shoot vegetative stage harvesting (one plant from each pot) was done after 50
days of sowing. The shoot length of maize was measured using the measuring tape.
7.2.12. Harvesting for yield attributes
The maize plants were harvested at maturity for the determination of shoot dry weight, 100-grains
weight and grains yield pot-1.
7.2.13. Nitrogen, phosphorus and potassium concentration in grain and shoot
7.2.14. Electrolyte leakage
7.2.16. Proline and Chlorophyll contents
See chapter 3 section 3.1 subsection 3.1.5 and 3.1.4.

7.3. Results
7.3.1. Shoot length and shoot dry weight
Effects of treatments (T) and various levels of drought (D) were significant for the shoot length
and shoot dry weight. The PGPR without BC remained statistically at par with each other and with
the control for shoot length at MD. At SD, the E. cloacae without BC showed statistically better
77
results over control. Application of BC0.75 also remained not significant at MD but was
significantly at SD for shoot length over control (Table 6.2). However, the E. cloacae + BC0.75 and
A. xylosoxidans + BC0.75 remained significant from BC0.75 alone and the control for shoot length
at MD and SD. No significant difference in shoot length was noted due to BC 0.75 and BC1.50
application rates with respect to each other at MD and SD. Maximum increase of 0.67-fold in shoot
length due to A. xylosoxidans + BC1.50 at MD while that of 1.35-fold in case of E. cloacae + BC1.50
at SD were observed over control. The BC0.75 alone remained non-significantly different over
control for shoot dry weight at SD. From BC0.75, no significant difference was noted where PGPR
were applied along with BC0.75 for shoot dry weight (except with L. adecarboxylata + BC0.75) at
NM, MD and SD. Maximum increase of 0.40-fold in shoot dry weight as a result of E. cloacae +
BC1.50 at MD and that of 0.92-fold due to A. xylosoxidans + BC1.50 at SD were noted.
7.3.2. 100-grains weight and grain yield pot-1
Effects of T and D were significant for the 100-grain weight and grain yield pot-1. In case of 100-
grain weight, application of E. cloacae + BC1.50 and A. xylosoxidans + BC1.50 remained significant
from E. cloacae + BC0.75 and A. xylosoxidans + BC0.75 at SD (Table 6.3). From BC0.75, no
significant difference was noted where PGPR were applied with BC0.75 as far as 100-grains weight
(except L. adecarboxylata + BC0.75) and grain yield pot-1 at NM, MD and SD. The application of
BC0.75 was significantly over control for 100-grains weight and grain yield pot-1. In case of 100-
grain weight, E. cloacae + BC0.75 gave a maximum increase of 0.45-fold at MD while A.
xylosoxidans + BC1.50 exhibited 1.07-fold improvement at SD over control. For gran yield pot-1, E.
cloacae + BC1.50 gave a maximum increase of 0.61-fold at MD while A. xylosoxidans + BC1.50
showed 2.0-fold increase at SD.
78
waste BC biochar (BC0, BC0.75 and BC1.50) on shoot length and shoot dry weight under various levels
of drought
Shoot Length (cm) Shoot dry weight (g)
Drought levels
Treatments
IE (T × D) IE (T × D)
ME (T) ME (T)
NM MD SD NM MD SD
Control (No PGPR + No BC) 125.2a-h 88.1j-m 55.4o 89.6E 68.8 a-g 48.4 h-m 32.2 m 49.8 G
P. aeruginosa 117.5c-j 97.0h-l 56.5no 90.3E 75.1 a-d 48.7 h-m 36.1lm 53.3 FG
E. cloacae 136.3a-c 115.2d-k 85.7k-n 112.4CD 72.1 a-f 57.3 e-j 38.1 k-m 55.9 E-G
A. xylosoxidans 142.1a-e 116.6c-j 83.6l-o 114.1CD 73.6 a-e 61.3 c-j 44.7 i-m 59.9 C-F
L. adecarboxylata 119.9b-i 93.9i-l 59.2m-o 91.0E 73.3 a-e 48.4 h-m 38.7 k-m 53.5 FG
BC0.75 126.0a-h 114.1d-k 89.8i-l 110.0D 75.1 a-d 57.4 e-j 46.5 i-m 59.6 C-F
P. aeruginosa + BC0.75 138.2a-e 117.5c-j 86.0k-n 113.9CD 73.5 a-e 53.1 g-k 44.5 j-m 57.0 D-G
E. cloacae + BC0.75 153.0a 141.2a-e 127.5a-g 140.6A 74.4 a-d 68.4 a-g 56.3 f-j 66.4 A-C
A. xylosoxidans + BC0.75 151.7a 144.1a-d 119.5b-i 138.4A 75.6 a-d 65.1 b-h 53.7 g-k 64.8 B-D
L. adecarboxylata + BC0.75 155.4a 112.4e-l 89.7i-l 119.2B-D 78.4 ab 56.2 f-j 44.9 i-m 59.9 C-F
BC1.50 153.2a 133.7a-f 103.9f-l 130.3AB 77.6 a-c 61.5 c-i 50.6 h-l 63.2 C-E
P. aeruginosa + BC1.50 155.1a 127.0a-h 97.1h-l 126.4A-C 84.8 a 60.2 d-j 49.2 h-l 64.7 B-D
E. cloacae + BC1.50 153.7a 140.7a-e 128.6a-g 141.0A 84.6 a 73.0 a-f 60.3 d-j 72.6 AB
A. xylosoxidans + BC1.50 143.0a-d 146.8a-c 118.9c-i 136.2A 84.5 a 71.5 a-f 64.9 b-h 73.7 A
L. adecarboxylata + BC1.50 149.7ab 127.6a-g 102.8g-l 126.7A-C 83.7 a 63.8 b-h 50.7 h-l 66.0 A-C
ºME (D) 141.3A 121.1B 93.6C 77.0 A 59.6 B 47.4 C
Different letters on means showing significant difference (p ≤ 0.05).
ME = main effect; IE = interactive effect; BC0.75 = 0.75% Biochar, BC1.50 = 1.50% Biochar
79
waste BC (BC0, BC0.75 and BC1.50) on 100-grains weight and grains yield pot-1 under various
levels of drought
100-grains weight (g) Grains yield pot -1 (g)
Drought levels
Treatments
IE (T × D) IE (T × D)
ME (T) ME (T)
NM MD SD NM MD SD
Control (No PGPR + No BC) 16.30 a-f 10.68 k-p 6.96 r 11.31 F 125 a-g 79 h-k 34 mn 79 G
P. aeruginosa 16.80 a-e 10.90 i-p 7.42 qr 11.71 EF 137 a-d 77 h-k 35 l-n 83 FG
E. cloacae 16.80 a-e 13.80 e-j 9.02 n-r 13.21 B-E 146 ab 97 f-i 56 k-n 100 D-F
A. xylosoxidans 16.37 a-f 13.83 e-j 8.78 o-r 12.99 C-E 139 a-c 92 f-k 58 j-n 96 E-G
L. adecarboxylata 16.83 a-e 12.12 h-m 8.11 p-r 12.35 D-F 135 a-e 74 h-k 26 n 78 G
BC0.75 17.43 a 13.97 c-h 10.24 l-q 13.88 BC 142 ab 96 f-j 73 h-l 104 B-E
P. aeruginosa + BC0.75 17.13 ab 13.21 g-l 10.02 m-q 13.46 B-D 137 a-d 91 f-k 77 h-k 102 C-F
E. cloacae + BC0.75 17.10 ab 15.51 a-g 10.88 i-p 14.49 AB 138 a-c 120 a-g 95 f-j 118 A-D
A. xylosoxidans + BC0.75 17.30 a 15.47 a-g 10.81 j-p 14.53 AB 141 ab 125 a-g 90 f-k 119 A-C
L. adecarboxylata + BC0.75 16.90 a-d 11.92 h-n 9.85 m-r 12.89 C-E 143 ab 88 g-k 69 i-m 100 C-F
BC1.50 17.13 ab 13.90 d-i 12.60 g-m 14.54 AB 137 a-d 108 b-h 77 h-k 107 A-E
P. aeruginosa + BC1.50 17.10 ab 13.80 e-j 12.80 g-m 14.57AB 146 a 99 d-i 72 h-l 106 A-E
E. cloacae + BC1.50 17.00 a-c 15.35 a-g 14.14 b-h 15.50 A 150 a 127 a-f 97 f-i 125 A
A. xylosoxidans + BC1.50 17.27 a 15.29 a-g 14.44 a-h 15.67 A 145 ab 120 a-g 102 c-i 122 AB
L. adecarboxylata + BC1.50 16.80 a-e 13.35 f-k 11.46 h-o 13.87 BC 140 a-c 98 e-i 77 h-k 105 B-E
ºME (D) 16.95 A 13.54 B 10.50 C 140 A 99 B 69 C
Different letters on means showing significant difference (p ≤ 0.05).
ME = main effect; IE = interactive effect; BC0.75 = 0.75% Biochar, BC1.50 = 1.50% Biochar
80
Effects of T and D were significant for N, P and K concentration in the grains. The PGPR without
BC did not vary significantly from control for N, P and K concentration in grains at SD. However,
the A. xylosoxidans without BC remained significant for N concentration in grains from control at
MD (Table 6.4). Both BC0.75 and BC1.50 were significantly at SD but remained statistically alike at
MD for N concentration in grains. However, the BC0.75 and BC1.50 remained significant over
control for N concentration in grains at SD and MD. For grain N, the P. aeruginosa, A.
xylosoxidans and L. adecarboxylata gave significant results along with BC1.50 from BC0.75 alone at
SD. Maximum increase of 0.51 and 1.51-fold in grain N concentration was noted as a result of E.
cloacae + BC1.50 over control at MD and SD respectively. Regarding grain P concentration, the E.
cloacae + BC1.50 and A. xylosoxidans + BC1.50 remained significant from BC1.50 alone at SD. From
E. cloacae + BC0.75, A. xylosoxidans + BC0.75 and L. adecarboxylata + BC0.75, the E. cloacae +
BC1.50, A. xylosoxidans + BC1.50 and L. adecarboxylata + BC1.50 remained statistically better for
grain P concentration at SD. Maximum increase of 0.93-fold in grain P concentration using E.
cloacae + BC1.50 at SD while that of 0.40-fold through E. cloacae + BC1.50 and A. xylosoxidans +
BC1.50 at MD were recorded over control. For grain K concentration no significant difference was
noted among PGPR when applied with BC0.75 and BC1.50 at MD and SD.
81
Table 7.4. Effect of ACC deaminase producing PGPR with and without different rates of timber waste BC (BC0, BC0.75 and BC1.50) on grains N, P and K
concentration under various levels of drought
Grain Nitrogen (%) Grain Phosphorus (%) Grain Potassium (%)
Drought levels
Treatments
IE (T × D) IE (T × D) IE (T × D)
ME (T) ME (T) ME (T)
Control (No PGPR + No BC) 1.34 b-g 0.97 k-p 0.63 qr 0.98 F 0.22 a-g 0.16 j-o 0.10 r 0.16 F 0.35 a-e
0.25 g-k
0.18 l 0.26 HI
P. aeruginosa 1.38 a-f 0.94 l-p 0.62 qr 0.98 F 0.22 a-f 0.16 j-p 0.11 r 0.16 EF 0.35 a-d 0.24 h-k 0.18 l 0.26 HI
E. cloacae 1.42 a-e 1.19 f-k 0.78 p-r 1.13 E 0.22 a-f 0.18 i-n 0.13o-r 0.18 D-F 0.36 ab 0.28 f-h 0.21 j-l 0.29 E-H
A. xylosoxidans 1.42 a-e 1.20 e-j 0.82 o-q 1.15 E 0.23 a-d 0.18 h-m 0.13 p-r 0.18 DE 0.36 a-c 0.29 e-h 0.22 i-l 0.29 D-G
L. adecarboxylata 1.40 a-f 1.04 j-o 0.59 r 1.01 F 0.23 a-d 0.16 j-p 0.11 r 0.16 EF 0.36 a-c 0.23 i-l 0.18 l 0.26 I
BC0.75 1.47 a-d 1.28 c-j 0.91 m-p 1.22 C-E 0.23 a-e 0.18 h-n 0.13 o-r 0.18 D-F 0.36 ab 0.28 f-i 0.21 j-l 0.28 F-H
P. aeruginosa + BC0.75 1.48 a-d 1.22 e-j 0.87 n-p 1.19 DE 0.23 a-d 0.18 g-l 0.13 o-r 0.18 CD 0.36 ab 0.29 f-h 0.21 k-l 0.29 F-H
E. cloacae + BC0.75 1.48 a-d 1.37 a-f 1.07 i-n 1.31 BC 0.24 a-d 0.21 b-h 0.15 l-q 0.20 BC 0.37 b-g 0.31 b-g 0.26 g-j 0.31 B-E
A. xylosoxidans + BC0.75 1.47 a-d 1.34 b-g 1.04 j-n 1.29 B-D 0.24 a-c 0.21 b-h 0.15 n-q 0.20 BC 0.37 c-g 0.30 c-g 0.26 g-k 0.31 C-F
L. adecarboxylata + BC0.75 1.49 a-c 1.21 e-j 0.87 n-p 1.19 DE 0.24 a-d 0.18 h-m 0.12 qr 0.18 D-F 0.37 a 0.26 g-k 0.22 j-l 0.28 G-H
BC1.50 1.54 ab 1.42 a-e 1.14 g-l 1.36 AB 0.24 a-c 0.21 c-i 0.15 m-q 0.20 BC 0.37 a 0.32 a-f 0.25 g-k 0.31 A-D
P. aeruginosa + BC1.50 1.57 a 1.33 b-h 1.10 i-m 1.33 AB 0.25 ab 0.21 d-i 0.16 k-q 0.20 BC 0.37 a 0.32 a-f 0.26 g-k 0.32 A-C
E. cloacae + BC1.50 1.54 ab 1.46 c-i 1.28 c-i 1.42A 0.25 ab 0.23 a-e 0.19 e-j 0.22 A 0.38 a 0.35 a-e 0.30 d-h 0.34 AB
A. xylosoxidans + BC1.50 1.51 ab 1.41 a-f 1.27 d-i 1.39 AB 0.25 a 0.22 a-e 0.19 f-k 0.22 A 0.37 a 0.35 a-d 0.30 c-g 0.34 A
a a-f h-m AB
L. adecarboxylata + BC1.50 1.59 1.40 1.12 1.37 0.25 a 0.21 d-i 0.16 j-o 0.21 AB 0.38 a 0.32 a-f 0.26 g-k 0.32 A-C
ME (D) 1.47 A 1.25 B 0.94 C 0.24 A 0.19 B 0.14 C 0.36 A 0.29 B 0.24 C
Different letters on means showing significant difference (p ≤ 0.05). ME = main effect; IE = interactive effect
BC0.75 = 0.75% Biochar, BC1.50 = 1.50% Biochar
82
Effects of T and D were significant for N, P and K concentration in the shoot of maize. Application
of BC0.75 and BC1.50 remained statistically but significantly different over control for N
concentration in the shoot at MD and SD. The PGPR with BC0.75 and BC1.50 also remained
statistically alike at MD and SD for N concentration in the shoot. Maximum increase of 0.44 and
0.92-fold in shoot N concentration was noted where A. xylosoxidans + BC1.50 was applied over
control at MD and SD respectively (Table 6.5). For P concentration inoculation the E. cloacae and
A. xylosoxidans without BC remained significant over control at MD and SD. Application of BC0.75
did not vary significantly from control for P concentration in maize shoot at MD and SD. However,
application of BC1.50 remained significant for P concentration at SD but non-significant at MD
from applying BC0.75 and the control. No significant improvement in the P concentration was noted
among PGPR when applied with BC0.75 and BC1.50. Maximum increase of 0.37 and 0.87-fold in P
concentration was noted where E. cloacae + BC1.50 and A. xylosoxidans + BC1.50 were applied over
control at MD and SD respectively. In case of shoot K concentration, the PGPR and BC 0.75
remained statistically alike. The BC1.50 was significant over control at SD. However, the E. cloacae
+ BC0.75 and A. xylosoxidans + BC0.75 remained significant over control at MD and SD for K
concentration in the shoot. The E. cloacae and A. xylosoxidans with BC0.75 and BC1.50 remained
statistically alike for shoot K concentration. Maximum increase of 1.50-fold using A. xylosoxidans
at SD while that of 0.57-fold through use of E. cloacae + BC1.50 at MD were observed over control.
7.3.5. Gas Exchange Attributes
Effects of T and D were significant for photosynthetic rate, transpiration rate and stomatal
conductance in maize leaves. The PGPR without BC did not vary significantly for photosynthetic
rate, transpiration rate and stomatal conductance at NM, MD and SD. The BC0.75 proved significant
for photosynthetic and transpiration rate but did not vary for stomatal conductance over control at
SD (Table 6.6). Similarly, application of BC0.75 and BC1.50 were also statistically similar to each
other at NM, MD and SD for photosynthetic rate, transpiration rate and stomatal conductance.
Maximum increase of 0.73 and 2.13-fold in photosynthetic rate, was noted over control where E.
cloacae + BC1.50 and A. xylosoxidans + BC1.50 were applied at MD and SD respectively.
Application of A. xylosoxidans + BC1.50 gave a maximum increase of 0.83 and 2.46-fold in
transpiration rate at MD and SD respectively. However, E. cloacae + BC1.50 gave a maximum
83
increase of 0.42-fold in stomatal conductance at MD while A. xylosoxidans + BC1.50 showed 1.10-
fold improvement in stomatal conductance at SD over control.
Effects of T and D were significant in case of chlorophyll a and total chlorophyll. For chlorophyll
b the main effect of T and D varied significant but interaction was similar. Inoculation of P.
aeruginosa, E. cloacae, A. xylosoxidans, L. adecarboxylata without BC were similar for
chlorophyll a and total chlorophyll at NM, MD and SD. Application of BC 0.75 and BC1.50 were
statistically alike but showed significant improvement over control in chlorophyll a at MD and SD
(Table 6.7). However, the BC1.50 was significant for total chlorophyll over control for total
chlorophyll. Maximum increase (0.22-fold) in chlorophyll a was noted at NM where P. aeruginosa
+ BC1.50 and E. cloacae + BC1.50 were applied. However, at MD and SD, the application of E.
cloacae + BC1.50 and A. xylosoxidans + BC1.50 gave a maximum increase of 0.69 and 1.52-fold in
chlorophyll a content. For total chlorophyll, application of E. cloacae + BC1.50 and A. xylosoxidans
+ BC1.50 gave a maximum increase of 0.76 and 1.48 at MD and SD respectively. In the case of
chlorophyll b, application of BC0.75 and BC1.50 vary significantly from control but BC1.50 was
significant than BC0.75. Inoculation of P. aeruginosa, E. cloacae, A. xylosoxidans and L.
adecarboxylata along with BC1.50 proved to be relatively better than BC0.75 for production of
chlorophyll b. The application of + BC1.50, P. aeruginosa + BC1.50, E. cloacae + BC1.50, A.
xylosoxidans + BC1.50 and L. adecarboxylata + BC1.50 were found to be significant for chlorophyll
b production in maize from all other treatments. Maximum increase of 0.74-fold in chlorophyll b
was noted where L. adecarboxylata + BC1.50 was applied over control.
84
Table 7.5. Effect of ACC deaminase producing PGPR with and without different rates of timber waste BC (BC0, BC0.75 and BC1.50) on shoot N, P and K
concentration under various levels of drought
Drought levels
Treatments
IE (T × D) IE (T × D) IE (T × D)
Control (No PGPR + No BC) 1.01 a-g 0.70i-n 0.49 n 0.73 G 0.14 a-f 0.11i-m 0.07 o 0.11 F 0.47 d-h
0.37 h-k
0.20 l 0.35 G
P. aeruginosa 1.07 a-f 0.68 j-n 0.49 n 0.74 G 0.14 a-f 0.11 j-m 0.08 m-o 0.11 EF 0.51 c-f 0.35 h-k 0.20 l 0.35 G
E. cloacae 1.07 a-d 0.81 g-l 0.63 l-n 0.84 D-G 0.15 a-f 0.13 c-j 0.11i-m 0.13 CD 0.51 b-f 0.43 f-i 0.27 kl 0.40 FG
A. xylosoxidans 1.02 a-g 0.81 g-l 0.65 k-n 0.83 E-G 0.14 a-g 0.13 d-k 0.10 k-n 0.12 C-E 0.51 c-f 0.41 f-j 0.29 j-l 0.40 FG
L. adecarboxylata 1.07 a-e 0.68i-n 0.57mn 0.77 FG 0.15 a-e 0.11i-m 0.07 n-o 0.11 EF 0.52 b-f 0.35 h-k 0.20 l 0.36 G
BC0.75 1.09 a-c 0.86 d-k 0.75 h-m 0.90 B-E 0.15 a-e 0.12 f-l 0.09 l-o 0.12 DE 0.59 a-d 0.42 f-i 0.31i-l 0.44 EF
P. aeruginosa + BC0.75 1.11 ab 0.87 d-j 0.79 h-l 0.92 A-E 0.16 a-c 0.12 e-l 0.09 l-o 0.12 CD 0.59 a-d 0.45 e-h 0.31i-l 0.45 D-F
E. cloacae + BC0.75 1.11 ab 1.03 a-f 0.88 c-j 1.01 AB 0.15 a-d 0.14 a-h 0.12 g-l 0.13 A-D 0.61 a-c 0.50 c-g 0.39 f-k 0.50 C-E
A. xylosoxidans + BC0.75 1.11 ab 1.01 a-g 0.86 d-k 0.99 AB 0.15 a-f 0.13 b-i 0.12 f-l 0.13 B-D 0.57 a-e 0.51 b-f 0.38 g-k 0.49 C-E
L. adecarboxylata + BC0.75 1.11 ab 0.85 f-k 0.67 j-n 0.88 C-F 0.15 a-d 0.12 g-l 0.09 l-o 0.12 DE 0.58 a-d 0.45 e-h 0.30i-l 0.45 EF
BC1.50 1.12 a 0.96 a-h 0.85 e-k 0.98 A-C 0.16 a-c 0.13 c-j 0.11 h-l 0.13 B-D 0.64 a 0.51 c-f 0.42 f-i 0.52 A-C
P. aeruginosa + BC1.50 1.13 a 0.87 d-k 0.81 g-l 0.94 A-D 0.15 a-d 0.13 c-j 0.11 h-l 0.13 B-D 0.60 a-c 0.52b-f 0.40 f-j 0.51 B-D
E. cloacae + BC1.50 1.07 a-e 0.97 a-h 0.90 b-i 0.98 A-C 0.16 a 0.15 a-e 0.13 b-i 0.15 A 0.65 a 0.58 a-d 0.47 d-h 0.56 AB
A. xylosoxidans + BC1.50 1.13 a 1.01 a-g 0.94 a-h 1.03 A 0.16 a-d 0.14 a-g 0.13 c-j 0.14 AB 0.64 ab 0.57 a-e 0.50 c-g 0.57 A
a c-j g-l A-D ab b-i g-l
L. adecarboxylata + BC1.50 1.14 0.87 0.81 0.94 0.16 0.13 0.12 0.14 A-C 0.62 a-c 0.51 b-f 0.40 f-j 0.51 A-C
ME (D) 1.09 A 0.87 B 0.74 C 0.15 A 0.13 B 0.10 C 0.57 A 0.46 B 0.34 C
Different letters on means showing significant difference (p ≤ 0.05). ME = main effect; IE = interactive effect
85
Table 7.6. Effect of ACC deaminase producing PGPR with and without different rates of timber waste BC (BC0, BC0.75 and BC1.50) on gas exchange
attributes under various levels of drought
[µmol (CO2) m-2 s-1] (mmol (H2O) m s ) -2 -1 mol (CO2) m-2 s-1
Treatments Drought levels
IE (T × D) IE (T × D) IE (T × D)
Control (No PGPR + No BC) 24.6 a-h 13.1 m-q 6.30 q 14.7 G 3.50 a-g 2.13i-m 1.01 n 2.21I 0.19 a-h 0.14 h-l 0.08 m 0.14 G
P. aeruginosa 25.0 a-g 13.3 l-q 7.20 p-q 15.2 FG 3.75 a-e 2.07i-m 1.05 n 2.29 HI 0.21 a-d 0.14 h-l 0.09 m 0.15 FG
E. cloacae 27.0 a-c 16.4i-o 9.40 o-q 17.6 D-G 3.87 a-d 2.50 h-l 1.72 l-n 2.70 GH 0.21 a-d 0.17 d-j 0.12 k-m 0.17 D-F
A. xylosoxidans 26.3 a-d 19.0 e-m 10.6 n-q 18.6 C-F 3.58 a-g 2.76 f-k 1.86 k-n 2.73 F-H 0.21 a-d 0.17 c-i 0.12 j-m 0.17 D-F
L. adecarboxylata 27.5 a 13.8 k-p 7.40 p-q 16.2 E-G 3.76 a-e 2.15i-m 1.34mn 2.42 HI 0.22 a-d 0.14 h-l 0.10lm 0.15E-G
BC0.75 27.3 ab 18.6 e-m 13.9 k-p 19.9 A-D 4.14 a-c 2.88 e-j 2.04 j-m 3.02 E-G 0.22 ab 0.17 c-i 0.13i-m 0.17B-E
P. aeruginosa + BC0.75 26.2 a-d 18.2 f-m 13.5 l-p 19.3 B-E 4.16 a-c 2.94 d-j 2.16i-m 3.09 D-G 0.21 a-d 0.17 c-i 0.13i-m 0.17 C-E
E. cloacae + BC0.75 25.3 a-e 20.9 a-k 17.7 h-n 21.3 A-C 4.30 ab 3.40 b-h 2.64 g-l 3.45 B-E 0.22 a 0.19 a-h 0.17 d-j 0.19 A-C
A. xylosoxidans + BC0.75 24.5 a-h 20.2 b-l 18.1 g-m 20.9 A-D 4.04 a-c 3.29 c-h 2.65 g-l 3.33 C-E 0.21 a-d 0.19 a-h 0.16 e-k 0.19 A-D
L. adecarboxylata + BC0.75 27.5 a 18.0 g-m 14.1 k-p 19.8 A-D 4.42 a 2.98 d-j 2.20 i-m 3.20 C-F 0.22 a-c 0.17 c-i 0.12 j-m 0.17 C-E
BC1.50 26.3 a-d 21.9 a-j 17.1i-n 21.7 A-C 4.40 a 3.68 a-f 2.92 d-j 3.67 A-C 0.22 a-d 0.19 a-g 0.15 f-k 0.19 A-D
P. aeruginosa + BC1.50 27.1 ab 19.9 c-m 15.3 j-o 20.8 A-D 4.40 a 3.43b-h 3.03 d-i 3.62 A-C 0.23 a 0.20 a-e 0.14 g-l 0.19 A-D
E. cloacae + BC1.50 25.2 a-f 22.6 a-i 19.4 d-m 22.4 AB 4.34 ab 3.78 a-e 3.37 b-h 3.83 AB 0.22 a-d 0.20 a-e 0.17 c-i 0.20 AB
ab a-j d-m A a a-d a-g A
A. xylosoxidans + BC1.50 27.2 22.4 19.7 23.1 4.40 3.89 3.54 3.94 0.23 a 0.20 a-f 0.17b-i 0.20 A
L. adecarboxylata + BC1.50 27.5 a 20.3 b-l 16.7i-n 21.5 A-C 4.32 ab 3.40 b-h 2.87 e-j 3.53 A-D 0.22 a-c 0.18 a-h 0.14 h-l 0.18 A-D
ME (D) 26.3 A 18.6B 13.7C 4.09A 3.02B 2.29C 0.22 A 0.17B 0.13C
Different letters on means showing significant difference (p ≤ 0.05). ME = indicates main effect; IE = interactive effect
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Table 7.7. Effect of ACC deaminase producing PGPR with and without different rates of timber waste BC (BC0, BC0.75 and BC1.50) on chlorophyll
content under various levels of drought
Chlorophyll a (mg g-1 F.W.) Chlorophyll b (mg g-1 F.W.) Total Chlorophyll (mg g-1 F.W.)
Drought levels
Treatments
IE (T × D) IE (T × D) IE (T × D)
b-i k-n o-p G F d-l p-t
Control (No PGPR + No BC) 0.76 0.49 0.29 0.51 0.39 0.26 0.15 0.27 1.15 0.75 0.45 u 0.78 G
P. aeruginosa 0.83 a-e 0.55 j-n 0.25 p 0.54 FG 0.43 0.30 0.15 0.29 EF 1.26 b-h 0.84 n-s 0.41 u 0.84 FG
E. cloacae 0.83 a-e 0.62 g-m 0.39 n-p 0.61 EF 0.40 0.29 0.21 0.30 D-F 1.22 c-i 0.91 l-r 0.60tu 0.91 F
A. xylosoxidans 0.84 a-e 0.61 h-m 0.44 m-p 0.63 D-F 0.43 0.31 0.21 0.32 C-F 1.27 b-h 0.92 k-r 0.65 s-u 0.95 EF
L. adecarboxylata 0.86 a-e 0.55 j-n 0.29 p 0.57 FG 0.42 0.27 0.16 0.28 EF 1.28b-h 0.83 o-t 0.45 u 0.85 FG
BC0.75 0.91 a-c 0.72 d-j 0.49 k-n 0.71 C-E 0.43 0.34 0.26 0.34 C-E 1.34 a-e 1.05 h-o 0.74 q-t 1.05 DE
P. aeruginosa + BC0.75 0.89 a-d 0.72 c-j 0.48 l-o 0.70 C-E 0.43 0.35 0.24 0.34 C-E 1.33 a-f 1.07 h-o 0.73 r-t 1.04 DE
E. cloacae + BC0.75 0.90 a-d 0.80 a-h 0.58i-m 0.76 A-C 0.42 0.36 0.30 0.36 CD 1.32 a-f 1.16 d-k 0.89 m-s 1.12 CD
A. xylosoxidans + BC0.75 0.89 a-d 0.79 a-h 0.63 f-l 0.77 A-C 0.45 0.38 0.31 0.38 BC 1.34 a-e 1.17 d-j 0.94 j-r 1.15 B-D
L. adecarboxylata + BC0.75 0.89 a-d 0.72 d-j 0.51 k-n 0.71C-E 0.48 0.35 0.26 0.36 CD 1.37 a-d 1.07 g-n 0.76 p-t 1.07 CD
BC1.50 0.81 a-f 0.74 b-j 0.61 g-m 0.72 B-D 0.51 0.43 0.38 0.44 AB 1.32 a-f 1.16 d-k 0.99 i-p 1.16 B-D
P. aeruginosa + BC1.50 0.93 ab 0.73 c-j 0.62 g-m 0.76 A-C 0.51 0.42 0.36 0.43 AB 1.44 a-c 1.15 d-l 0.98 j-q 1.19 A-C
E. cloacae + BC1.50 0.93 ab 0.83 a-e 0.72 d-j 0.82 A 0.55 0.49 0.34 0.46 A 1.48 ab 1.32 a-g 1.06 h-o 1.28 A
A. xylosoxidans + BC1.50 0.88 a-d 0.80 a-g 0.73 c-j 0.80 AB 0.52 0.45 0.39 0.45 A 1.40 a-d 1.26 b-h 1.12 e-m 1.26 AB
L. adecarboxylata + BC1.50 0.97 a 0.76 b-i 0.68 e-k 0.80 AB 0.56 0.42 0.42 0.47 A 1.53 a 1.18 d-j 1.09 f-m 1.27 AB
*ME (D) 0.88A 0.69 B 0.51 C 0.46 A 0.36 B 0.28 C 1.34 A 1.06 B 0.79 C
Different letters on means showing significant difference (p ≤ 0.05). Non-significant interactive effect (T × D) did not have any letter.
ME = indicates main effect; IE = interactive effect F.W.= Fresh weight
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Effects of T and D were significant for electrolyte leakage in maize. The BC1.50 significantly
decreased the electrolyte leakage over control at MD. However, all the PGPR without BC did not
vary significantly over control for electrolyte leakage at MD. However, at SD co-application of P.
aeruginosa + BC0.75 proved significant from P. aeruginosa sole for electrolyte leakage (Figure
6.1). Application of BC0.75 and BC1.50 did not vary significantly for the reduction in electrolyte
leakage of maize at MD and SD with respect to each other. Maximum reduction of 0.22, 0.86 and
0.81-fold in the electrolyte leakage was noted over control at NM, MD and SD where A.
xylosoxidans + BC1.50 was applied.
7.3.8. Carotenoids and Proline
Effects of T and D were observed to be significant different for carotenoids and proline contents
in maize leaves. The BC0.75 with and without PGPR was significant over control for carotenoids at
SD. Application of BC0.75 and BC1.50 were statistically alike for carotenoids contents at NM, MD
and SD. Maximum increase of 0.18, 0.42 and 2.84-fold in carotenoids was noted over control
where P. aeruginosa + BC1.50, E. cloacae + BC1.50 and A. xylosoxidans + BC1.50 were applied at
NM, MD and SD respectively. The A. xylosoxidans without BC was significant from P.
aeruginosa, E. cloacae, L. adecarboxylata and control at NM (Fig. 6.2-6.3). However, the BC0.75
significantly decreased the proline from control, while remained statistically at par with BC1.50 at
NM, MD and SD. Maximum reduction of 0.42 and 0.50-fold in proline was noted in A.
xylosoxidans + BC1.50 at MD and SD respectively.
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waste BC (BC0, BC0.75 and BC1.50) on electrolyte leakage under various levels of moisture (Normal
Moisture (NM), Mild Drought (MD) and Severe Drought (SD)). BC0.75 = 0.75% Biochar, BC1.50
= 1.50% Biochar
waste BC (BC0, BC0.75 and BC1.50) on carotenoids contents under various levels of moisture
(Normal Moisture (NM), Mild Drought (MD) and Severe Drought (SD)). BC0.75 = 0.75%
Biochar, BC1.50 = 1.50% Biochar
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waste BC (BC0, BC0.75 and BC1.50) on proline content under various levels of moisture (Normal
Moisture (NM), Mild Drought (MD) and Severe Drought (SD)). BC0.75 = 0.75% Biochar, BC1.50
= 1.50% Biochar
7.4. Discussion
In the current study, the co-application of P. aeruginosa, E.cloacae, A. xylosoxidans, L.
adecarboxylata and timber-waste biochar significantly enhanced maize shoot growth, nutrients
uptake and yield under various levels of drought (NM, MD and SD). A significant improvement
in shoot growth shows efficacy of co-application of E. cloacae and A. xylosoxidans along with
BC0.75 from BC0.75 alone and/or control under drought. The improvement in shoot growth might be
due to reduction in stress ethylene level by E. cloacae and A. xylosoxidans. Higher synthesis of
ACC deaminase by E. cloacae and A. xylosoxidans might be the major cause of their better
performance from P. aeruginosa and L. adecarboxylata regarding maize growth and yield under
drought. In one of experiment conducted by Mayak et al. (2004) studied that elevated level of 1‐
aminocyclopropane‐ 1‐ carboxylic acid (ACC) in plants, especially under limited availability of
water and nutrients, increases ethylene concentration in root and shoot of plants. Less elongation
and radial swelling of the stem is an indications of stress ethylene generation (Abeles et al., 1992)
that decreases the supply of energy and water at the imbibition phase (Taiz and Zeiger, 2010;
Aroca, 2012). The plant roots and seeds exude the ACC into rhizosphere that is converted by PGPR
secreted ACC deaminase into NH3 and α-ketobutyrate and ultimately ethylene level reduces.
Reduction in ethylene results in better roots elongation that facilitates plant to intake water and
nutrients by increasing rhizosphere area (Glick et al., 1999).
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The E. cloacae and A. xylosoxidans in specific, distinctly counteracted the adverse effects of
drought in maize in terms of improvement in grain yield, photosynthetic rate, stomatal conductance
chlorophyll a, total chlorophyll and carotenoids contents of maize. In addition to ACC-deaminase
activity, the improvement in maize growth might also be favoured due to more production of
growth hormone i.e., IAA by E. cloacae and A. xylosoxidans as from P. aeruginosa and L.
Adecarboxylata. According to Xie et al. (1996) IAA as an allied factor, playing an important role
in crops growth improvement. High IAA synthesis by PGPR increases surface area and length of
adventitious and lateral roots in plants that play an important role in nutrients uptake (Mohite,
2013). In addition to roots exudates, other organic metabolities i..e., organic acids,
phytosiderophores, sugars, vitamins, amino acids, nucleosides and mucilage positivly attract and
stimulates the PGPR for better proliferation and colonization in rhizosphere. This improvement
also facilitates the uptake of water and solubilization of immobilized (P and K) nutrients (Alam et
al., 2008; Basak and Biswas, 2010; Shukla et al., 2011; Drogue et al., 2013).
Furthermore, application of biochar might also be one of the probable reasons for the increase in
the rate of photosynthesis, rate of transpiration, stomatal conductance, nutrients uptake and
reduction in electrolyte leakage under drought stress. Better water and nutrients holding capacity
of biochar due to its high surface area are directly linked with improvement in crops growth
parameters (Novak et al., 2009; Lehmann et al., 2011; Younis et al., 2014; Keshavarz et al., 2016).
Majority of biochar types across a wide range of pH shows negative zeta potential which indicates
most of the negative charges at surface of the biochar. These negatively charged surfaces attaract
the cation nutrients, thus improve nutrients retension in soil. Furthermore, hydraulic cations when
become adsorbed in biochar surface, the associated water molecules also become attached or
stored in micropores of biochar (Glaser et al., 2002). Development of funtional groups (phenolic
and carboxylic) on the surface of biochar during pyrolysis are composed of carbon, hydrogen and
oxygen. Presence of aromatic carbon rings faciliates the redox reaction during binding of nutrients
with functional groups of biochar. At the same time, shuttle electrons attached at biochar surface
triger the metaboliasm of microbial community in rhiospehre. Such conditions improve the cycling
of nutrients and their phytoavailibility in soil (Almeida et al., 2015).
In an experiment, Chan et al. (2008) observed that an improvement in soil cation exchange
capacity by application of biochar played a vital role in the availability of N. Same kind of results
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were also observed by Younis et al. (2014) regarding uptake of P by addition of cotton sticks
biochar. Better uptake of K in maize at MD and SD through co-application of E. cloacae / A.
xylosoxidans along with BC might be another favourable factor responsible for the mitigation of
drought stress. According to Singh et al. (2016) it is the higher amount of K in biochar ash that
contributes in the improvement of K concentration in plants. Better uptake of K might have
maintained the turgor of cells and stomatal conductance by osmoregulation (Wilkinson and
Davies, 2002; Shabala, 2003). Similarly, an increase in shoot P (E. cloacae and A. xylosoxidans)
and K (E. cloacae + BC0.75 and A. xylosoxidans + BC0.75) concentration was observed through co-
application of PGPR and BC. This increase in P and K might also be due to better proliferation
and activity of the PGPR in the presence of biochar at MD and SD. Findings of Singh et al. (2016)
supported our argument that the presence of organic carbon in biochar increases the growth of
PGPR (Singh et al., 2016).
7.5. Conclusion
It is concluded that the application of BC with E. cloacae / A. xylosoxidans would be very effective
for the promotion of growth and yield of maize under drought. Though from an economical point
of view, BC0.75 is suitable rate, however, in specific, for grain weight improvement of maize the
combined application of E. cloacae / A. xylosoxidans with BC1.50 would be better practice from
BC0.75.
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CHAPTER 8
ACC deaminase producing PGPR Bacillus amyloliquefaciens and

Agrobacterium fabrum along with biochar improve wheat productivity under
drought stress
Abstract
Drought stress retards wheat plant’s vegetative growth and physiological processes and results in
low productivity. Stressed plant synthesizes ethylene which inhibits root elongation; however, the
enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase catabolizes ethylene produced
under water stress. Therefore, the ACC deaminase producing plant growth promoting rhizobacteria
(PGPR) can be used to enhance crop productivity under drought stress. Biochar (BC) is an
organically active and potentially nutrient-rich amendment that when applied to the soil, can
increase pore volume, cation exchange capacity and nutrient retention and bioavailability. We
conducted a field experiment to study the effect of drought tolerant, ACC deaminase producing
PGPR (with and without timber waste BC) on plant growth and yield parameters under drought
stress. Two PGPR strains, Agrobacterium fabrum or Bacillus amyloliquefaciens were applied
individually and in combination with 30 Mg ha-1 BC under three levels of irrigation, i.e.,
recommended four irrigations (4I), three irrigations (3I) and two irrigations (2I). Combined
application of B. amyloliquefaciens and 30 Mg ha-1 BC under 3I, significantly increased growth
and yield traits of wheat: grain yield (36%), straw yield (50%), biological yield (40%). Same soil
application under 2I resulted in greater increases in several of the growth and yield traits: grain
yield (77%), straw yield (75%), above- and below-ground biomasses (77%), as compared to
control; however, no significant increases in chlorophyll a, b or total, and photosynthetic rate and
stomatal conductance in response to individual application of PGPR strains (without BC) were
observed. Therefore, we suggest that the combined soil application of B. amyloliquefaciens and
BC more effectively mitigates drought stress and improves wheat productivity as compared to any
of the individual soil applications tested in this study.
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8.1. Introduction
Wheat is a staple and cash crop globally recognized for its nutritional and economic
importance (Bos et al., 2005; Shiferaw et al., 2013). Wheat grain (flour) constitutes 20% of daily
human diet and contains protein (8-12%) and a high amount of carbohydrates (55%). Drought is a
worldwide most critical abiotic factor due to which sustainable wheat crop productivity is at risk
(Kilic et al., 2010; Waraich, 2011; Ahmad et al., 2018). Drought severity is predicted to
successively increase under climate change scenarios of atmospheric and soil warmings and
altered precipitation patterns (Griffin et al., 2013; Munir et al., 2015; Zhang et al., 2017; Saikia et
al., 2018; Bechtold et al., 2019; Strack et al., 2019). Consistent and prolonged warming and
drought conditions combined with associated abiotic and biotic changes (Preston et al., 2008) may
drastically retard crop productivity and risk food security (Mishra and Cherkauer, 2010; Allen et
al., 2014).
Drought stress reduces nutrient uptake, which can cause poor development of roots, low
transpiration and photosynthetic rates, closure of leaf stomata and desiccation resulting in wilting
of plants (Reddy et al., 2014; Fahad et al., 2017; Munir et al., 2017). Like other abiotic stresses,
the drought also stimulates stress ethylene synthesis through an elevated level of 1-
Aminocyclopropane-1-carboxylic acid (ACC; an ethylene precursor) via the methionine pathway,
in higher plants (Wang et al., 2013; Zafar-ul-Hye et al., 2018). Accumulation of stress ethylene
in-turn inhibits roots elongation and consequently, shoot growth in plants (Sharp et al., 2002).
Water management strategies and genetic engineering are useful tools to adapt to or
mitigate drought stress. While irrigation water is being managed in irrigation-dependent cropping
systems, genetic engineering to cope with water stress remains limited. However, a vital biological
approach to combat drought impacts is the soil inoculation of plant growth promoting rhizobacteria
(PGPR). The PGPR are frequently reported to efficiently elongate plant roots in the pot (Danish
and Zafar-ul-Hye, 2019) and mitigate drought impacts in field or greenhouse conditions
(Stromberger et al., 2017; Salem el al., 2018), and mobilize the immobile nutrients that lead to
significant increases in plant vegetative growth (Kumputa et al., 2019) and crop yield (Basak et
al., 2010; Mwajita et al., 2013). PGPR produces ACC deaminase enzyme, which catabolizes stress
ethylene through cleavage of ACC into α-ketobutyrate and ammonium ion (NH4+) under drought
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stress (Belimov et al., 2001), thus reducing the level of stress ethylene (Mayak et al., 2004; Zahir
et al., 2008).
Biochar (BC) is an organically active soil amendment with very high soil pore volume and
cation exchange capacity and has been reported to reduce drought stress in plants (Gundale et al.,
2006; Hartmann et al., 2006; De Jesus Duarte et al., 2019; Wacal et al., 2019). Biochar is a
nutrient-rich, black carbon soil amendment (De la Rosa et al., 2018) that is produced through
pyrolysis of waste feedstock at high temperature (Paneque et al., 2017) under anaerobic or partially
anaerobic condition (Lehmann, 2007; Singh et al., 2010; Hagemann et al., 2017).
While individual soil application of ACC-deaminase containing PGPR or BC has been
frequently investigated for combating drought effects in pot experiments, controlled field
experimentation for evaluation of cumulative mitigating effects remains limited. Therefore, the
objective of this research was to observe the efficiency of combined application of ACC-
deaminase producing PGPR and timber waste BC in granting resistance to field-scale wheat crop
against drought impacts. We hypothesized that soil inoculation of drought-tolerant ACC-
deaminase containing PGPR along with timber waste BC amendment would be a more efficient
technique to mitigate adverse drought effects on wheat growth and yield traits.
We conducted this experiment in the research area of the Department of Soil Science,
Bahauddin Zakariya University, Multan, Pakistan, in November 2016. A total of 54 same size
plots (9 m2) were prepared and randomly divided into six triplicate treatments (T) (6×3=18) with
each applied at three levels of irrigation (I), (i.e., 4I, 3I and 2I) following a randomized complete
block design (RCBD; 18×3=54 plots). The experimental area was cropped with wheat and maize
(rotation) during the last five years.
Recommended nitrogen (N), phosphorus (P) and potassium (K) fertilizers (RNPKF) were
applied at the rates of 120, 60 and 60 kg ha-1 (Sarfraz et al., 2008; Ahmad et al., 2018). Full doses
of P (as diammonium phosphate) and K (as sulphate of potash), and a 1/3rd dose of N were
incorporated to topsoil at the seedbed preparation stage, and the remaining two splits of N were
top-dressed after 30 and 60 days of seeding. We used standard crop management practices such as
irrigation, fertilization, weeding, hoeing and plant protection to grow wheat crop during the study
season. Timber-waster biochar (BC) was applied at a rate of 1.5%, i.e., 30 Mg ha-1. Treatments
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included: Control (No PGPR + No BC + RNPKF), A. fabrum, B. amyloliquefaciens, 30 Mg ha-1
BC, A. fabrum + 30 Mg ha-1 BC and B. amyloliquefaciens + 30 Mg ha-1 BC.
Two most competent drought-tolerant ACC-deaminase producing PGPR strains,
Agrobacterium fabrum (NR_074266.1) and Bacillus amyloliquefaciens (FN597644.1) as
documented by (Danish and Zafar-ul-Hye, 2019) were provided from the collection of Soil and
Environmental Microbiology Laboratory, Bahauddin Zakariya University Multan, Pakistan. Both
strains were initially tested and found eligible to grow in DF minimal salt medium at -0.87 Mpa
osmotic potential, generated by 20% polyethylene glycol 6000 (PEG). For experimental purpose,
DF minimal salt medium without agar was used to prepare inoculum of desired PGPR strains
(Dworkin and Foster, 1958). For measuring ACC-deaminase produced by PGPR strains (A. fabrum
= 349.6 ± 21.4 and B. amyloliquefaciens = 313.2 ± 34.3 µmol α-ketobutyrate mg-1 protein h-1), we
followed El-Tarabily (2008). Glickmann and Dessaux methods (1995) was used for assessment of
indole acetic acid with (A. fabrum = 58.8 ± 3.27 and B. amyloliquefaciens = 17.3 ± 2.34 µg/ml)
and without 0.5 gL-1 L-tryptophan (A. fabrum = 2.43 ± 0.34 and B. amyloliquefaciens = 1.12 ±
0.60 µg/ml) using Salkowski reagent. Vazquez et al. (2006) and Sheng and He (2006)
methodologies were followed for determination of P (A. fabrum = 16.2 ± 1.48 and B.
amyloliquefaciens = 20.9 ± 2.48 µg/ml) and K solubilizing activities (A. fabrum = 26.7 ± 1.49 and
B. amyloliquefaciens = 23.4 ± 1.92 µg/ml) (Danish and Zafar-ul-Hye, 2019). For biochar
production and characterization see chapter 3 section 3.1 subsection 3.1.9 and 3.1.10. For soil
characterization see chapter 3 section 3.1 subsection 3.1.11. The physiochemical characteristics of
soil are provided in Table 8.1.
Table 8.1. Characteristics of soil and timber waste biochar (BC)
Sand % 55 pH - 7.26
Texture Sandy Clay Loam Ash Content % 28.9
ECe dS m-1 3.69 Total N % 0.21
Total N % 0.02 Total K % 1.61
Extractable P µg g-1 5.26 Total Na % 0.19
-1
Extractable K µg g 170
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Wheat seeds (Glaxay-2013) were purchased from the Government of Punjab certified seed dealer.
Weak seeds were initially screened out manually. For seeds sterilization see chapter 3 section 3.1
subsection 3.1.3.
In each of the 18 plots (9 m2), six rows of seeds were sown using the drill method. Four irrigations
were applied according to the production technology of wheat recommended and published by the
Directorate of Agricultural Information Punjab (Sheikh et al., 2003). There was no precipitation
event during the study period, therefore, no precipitation-induced soil moisture variations were
monitored. To create a mild drought, 3 irrigations were applied (1 irrigation was skipped at the
tillering stage). However, severer drought stress was induced by using 2 irrigations (2 irrigations
were skipped; one at the tillering stage and other at the milky stage). The irrigation schedule was:
1st = 25 days after sowing (Crown root Initiation)
2nd = 55 days after sowing (Tillering stage)
3rd = 80 days after sowing (Heading stage)
4th = 110 days after sowing (Milky stage / soft dough)
After 65 days of sowing (vegetative phase), we collected vegetative samples from four random
spots in each plot for the determination of chlorophyll contents, gas exchange attributes, electrolyte
leakage and nutrient concentrations in the shoot. At the vegetative phase, samples were collected
only from 4I (control) and 3I (mild drought) treatments (no 2I (severe drought) treatment was
available at this point of time). Skipping one irrigation created mild drought treatment as compared
to skipping two irrigations (2nd and 4th) which created severe drought treatment. The drought and
control treatments were sampled at maturity point of time for estimating yield attributes.
We followed Kumar et al. (1993) for root sampling and Newman (1966) for root length
measurement at 120 days after seeding. Briefly, an augar of 10 cm internal diameter was used and
the core samples were taken at 10 cm depth intervals to a total depth of 90 cm. Random sampling
locations within each plot included sampling at row and midway between rows for collecting four,
90 cm depth samples. Soil/root cores were placed on a 32 cm mesh screen and gently washed in
water (Kumar et al., 1993). Root length was measured by the line intercept technique of Newman
(Newman, 1966; Kumar et al., 1993). For yield attributes and grain analyses, harvesting was done
at the time of maturity when soil and plants were fully dried. The plant height, spike length, grains
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spike-1, spikelets spike-1, 1000-grains weight, grains yield, straw yield and biological yield
(aboveground + root biomass) data were collected at the time of maturity (approx. 140 days).
For nutrients analyses of plants parts see chapter 3 section 3.1 subsection 3.1.4. Regarding gas
exchange attributes see chapter 3 section 3.1 subsection 3.1.14. For the determination of
chlorophyll contents see chapter 3 section 3.1 subsection 3.1.5. Regarding electrolyte leakage
please see chapter 3 section 3.1 subsection 3.1.12.
Maximum increase (%) was calculated by using the formula:
Maximum Increase (%) = (Highest Value – Control value / Control value) × 100 (6)
Statistical analysis was performed using standard statistical procedures as described by Steel and
Torrie (1980). Two factorial ANOVA was applied on Statistix 8.1 software for determination of
treatments significance under various levels of irrigations. Tukey's test at p ≤ 0.05 was applied for
comparison of treatments.
8.3. Results
8.3.1. Plant height, root length and spike length
Both the individual and interactive effects of T and I were significant on plant height and root
length. For spike length, main effects were significantly different while interactive effects (T × I)
remained nonsignificant. Application of BC, A. fabrum + BC and B. amyloliquefaciens + BC
significantly improved plant height compared to control, with 4I and 2I. The treatments A. fabrum,
B. amyloliquefaciens, BC, A. fabrum + BC and B. amyloliquefaciens + BC differed significantly
from control at 3I for plant height (Table 8.2). A maximum increase of 0.31-fold in plant height
was observed in A. fabrum + BC at 4I while 0.81-fold in B. amyloliquefaciens + BC with 2I from
control. However, plant height was the maximum (0.42-fold) from control, in responses to A.
fabrum + BC and B. amyloliquefaciens + BC treatments. For root length, the BC, A. fabrum + BC
and B. amyloliquefaciens + BC differed significantly from control at 4I and 3I. The B.
amyloliquefaciens, BC, A. fabrum + BC and B. amyloliquefaciens + BC were significantly better
from control for root length with 2I. Maximum increases, i.e., 0.49, 1.11 and 0.90-fold in root
length were noted over control in B. amyloliquefaciens + BC with 4I, 3I and 2I, respectively. In
the case of spike length, all the treatments were statistically alike but different from control.
98
8.3.2. Grain, straw and biological yield
Both the individual and interactive effects of T and I were significantly different for grain, straw
and biological yield of wheat (Figure 8.1-8.3). The A. fabrum + BC and B. amyloliquefaciens +
BC differed significantly from control for grain yield with 4I. Applications of A. fabrum, B.
amyloliquefaciens, BC, A. fabrum + BC and B. amyloliquefaciens + BC differed significantly from
control for grain yield at 3I. However, the BC, A. fabrum + BC and B. amyloliquefaciens + BC
showed significantly better results over control for grain yield at 2I. The maximum increases, i.e.,
0.29, 0.36 and 0.77-fold in grain yield were noted from control in B. amyloliquefaciens + BC with
4I, 3I and 2I, respectively. For straw yield, the B. amyloliquefaciens, BC, A. fabrum + BC and B.
amyloliquefaciens + BC differed significantly from control with 3I and 2I. Maximum increases of
0.25, 0.50 and 0.75-fold in straw yield were noted from control in B. amyloliquefaciens + BC. In
case of biological yield, the B. amyloliquefaciens, BC, A. fabrum + BC and B. amyloliquefaciens
+ BC differed significantly from control with 4I and 2I. From control, the A. fabrum, B.
amyloliquefaciens, BC, A. fabrum + BC and B. amyloliquefaciens + BC differed significantly at
3I for biological yield. The maximum increases of 0.28, 0.40 and 0.77-fold in biological yield were
noted from control in B. amyloliquefaciens + BC with 4I, 3I and 2I, respectively.
8.3.3. Spikelets spike-1, grains spike-1 and 1000 grain weight

Main effects of T and I differed significantly for spikelets spike-1, grains spike-1 and 1000 grain
weight but the interaction (T × I) was significantly different only for 1000 grain weight (Table
8.3). From control, the applications of B. amyloliquefaciens, BC, A. fabrum + BC and B.
amyloliquefaciens + BC differed significantly from control for spikelets spike-1. The treatment B.
amyloliquefaciens + BC differed significantly over BC and B. amyloliquefaciens for spikelets
spike-1. Similarly, A. fabrum + BC differed significantly as compared to A. fabrum but did not
differ significantly as compared to BC for spikelets spike-1. A maximum increase of 0.24-fold in
spikelets spike-1 was noted from control in B. amyloliquefaciens + BC. In the case of grains spike-
1
, the BC, A. fabrum + BC and B. amyloliquefaciens + BC were statistically alike but differed
significantly from control. Inoculation of B. amyloliquefaciens also differed significantly from
control for grains spike-1. A maximum increase of 0.51-fold in grains spike-1 was noted from
control in B. amyloliquefaciens + BC. For 1000 grain weight, the A. fabrum + BC differed
significantly from control with 4I. It was noted that A. fabrum + BC and B. amyloliquefaciens +
99
BC differed significantly from control at 3I for 1000 grain weight. However, the applications of
B. amyloliquefaciens, BC, A. fabrum + BC and B. amyloliquefaciens + BC differed significantly
from control with 2I for 1000 grain weight. A maximum increase of 0.20-fold in 1000 grain weight
was noted as compared to control in A. fabrum + BC with 4I. With 3I, the application of B.
amyloliquefaciens + BC gave a maximum increase of 0.29-fold as compared to control in 1000
grains weight. However, the BC and A. fabrum + BC gave a maximum rise of 0.46-fold as
compared to control in 1000 grain weight with 2I.
100
Table 8.2. Effect of Agrobacterium fabrum, Bacillus amyloliquefaciens with/without biochar (30 Mg ha-1) on plant height, root length
and spike length of wheat cultivated in field conditions
Plant Height (cm) Root Length (cm) Spike Length (cm)
No. of Irrigations (I)
Treatments
IE (T × I) ME IE (T × I) ME IE (T × I) ME
4I 3I 2I (T) 4I 3I 2I (T) 4I 3I 2I (T)
d-f gh i D d-f hi i D
Control 59.0 48.1 33.0 46.7 8.82 5.65 4.46 6.31 5.75 4.86 4.34 4.98 B
c-f ef hi C b-d f-h g-i C
A. fabrum 65.5 58.3 40.1 54.6 10.1 7.08 6.22 7.79 6.68 6.22 4.87 5.92 A
b-e d-f hi BC b-d f-h gh C
B. amyloliquefaciens 66.2 60.0 39.6 55.3 10.3 7.46 6.36 8.03 6.69 6.27 4.78 5.91 A
a-c d-f h B a-c c-e e-g B
BC 71.2 60.9 45.6 59.2 11.5 9.67 7.81 9.67 6.67 6.46 4.96 6.03 A
a a-d fg A a a-c d-f A
A. fabrum + BC 76.8 68.3 56.5 67.2 12.8 11.2 8.46 10.8 7.13 6.33 5.10 6.19 A
ab a-d d-f A a ab d-f A
B. amyloliquefaciens + BC 75.5 68.3 59.7 67.8 13.1 11.9 8.49 11.2 7.12 6.55 5.05 6.24 A
A B C A B C A B
ME (I) 69.0 60.7 45.8 11.1 8.83 6.97 6.67 6.12 4.85 C
Different letters on means showing significant difference (p ≤ 0.05). Non-significant interactive effect (T × I) did not have any letter.
ME = indicates main effect; IE = interactive effect; 4I = Normal Moisture; 3I = Mild Drought; 2I = Severe Drought
101
amyloliquefaciens and biochar (30 Mg ha-1) on grains yield (tons acre-1) in wheat grains cultivated
in field conditions.
amyloliquefaciens and biochar (30 Mg ha-1) on straw yield (tons acre-1) in wheat grains cultivated
in field conditions.
102
amyloliquefaciens and biochar (30 Mg ha-1) on biological yield (tons acre-1) in wheat grains
cultivated in field conditions.
103
Table 8.3. Effect of Agrobacterium fabrum, Bacillus amyloliquefaciens with/without biochar (30 Mg ha-1) on spikelets spike-1, grains
spike-1 and 1000 grains weight of wheat cultivated in field conditions
Spikelets spike-1 Grains Spike-1 1000 Grains Weight (g)
Treatments
4I 3I 2I (T) 4I 3I 2I (T) 4I 3I 2I (T)
D C b-d fg h
Control 15.3 13.0 10.7 13.0 37.7 27.7 22.0 29.1 35.3 28.2 20.2 27.9 C
A. fabrum 16.0 13.7 13.0 14.2 CD 38.3 33.7 26.7 32.9 BC 34.2 c-f 30.7 c-g 25.5 gh 30.2 BC
B. amyloliquefaciens 16.0 13.3 12.7 14.0 C 39.3 35.0 29.7 34.7 B 35.2 b-e 31.0 c-g 26.8 g 31.0 B
BC 16.3 14.3 13.0 14.6 BC 47.3 42.0 32.7 40.7 A 36.3 a-c 31.1 c-g 29.4 d-f 32.3 B
A. fabrum + BC 17.0 15.3 14.3 15.6 AB 45.3 40.7 39.0 41.7 A 42.5 a 34.7 b-e 29.4 d-f 35.5 A
B. amyloliquefaciens + BC 17.3 15.7 15.3 16.1 A 49.0 43.0 39.3 43.8 A 40.8 ab 36.5 a-c 28.9 e-f 35.4 A
ME (I) 16.3 A 14.2 B 13.2 C 42.8 A 37.0 B 31.6 C 37.4 A 32.0 B 26.7 C
ME = indicates main effect; IE = interactive effect; 4I = Normal Moisture; 3I = Mild Drought; 2I = Severe Drought
104
8.3.4. N, P and K concentration in grains
Both the main and interactive effects of T and I were significant for N, P and K concentrations in
wheat grains (Table 8.4). All the treatments were statistically alike with 4I for grains N
concentration. Applications of BC, A. fabrum + BC and B. amyloliquefaciens + BC performed
significantly better from control with 3I and 2I for grains N concentration. The maximum increases
of 0.13, 0.37 and 0.57-fold in grains N concentration were noted in B. amyloliquefaciens + BC
with 4I, 3I and 2I respectively. In case of grains P concentration, B. amyloliquefaciens, BC, A.
fabrum + BC and B. amyloliquefaciens + BC remained statistically alike but only A. fabrum + BC
and B. amyloliquefaciens + BC differed significantly from control with 4I. The A. fabrum, B.
amyloliquefaciens, BC, A. fabrum + BC and B. amyloliquefaciens + BC were significantly better
from control with 3I and 2I for grains P concentration. Both the A. fabrum + BC and B.
amyloliquefaciens + BC showed a maximum increase of 0.32-fold in grains P concentration from
control with 4I. However, with 3I and 2I, the B. amyloliquefaciens + BC gave the maximum
increases of 0.91 and 1.64-fold in grains P concentration from control, respectively. For grains K
concentration, the A. fabrum, B. amyloliquefaciens, BC, A. fabrum + BC and B. amyloliquefaciens
+ BC were statistically similar to each other while, the BC, A. fabrum + BC and B.
amyloliquefaciens + BC differed significantly from control with 4I. The A. fabrum + BC and B.
amyloliquefaciens + BC differed significantly with 3I from control for grains K concentration.
However, the A. fabrum, B. amyloliquefaciens, BC, A. fabrum + BC and B. amyloliquefaciens +
BC differed significantly over control for grains K concentration with 2I. The maximum increases
of 0.22, 0.27 and 0.61-fold in grains K concentration were noted in B. amyloliquefaciens + BC
with 4I, 3I and 2I, respectively.

Both the individual and interactive effects of T and I differed significantly for shoot nitrogen
compared to only individual effects of T and I were significant for P and K concentrations in
wheat (Table 8.5). All treatments were statistically alike with 4I for shoot nitrogen concentration.
The A. fabrum + BC, B. amyloliquefaciens + BC and BC differed significantly from control at 3I
for shoot nitrogen concentration. A. fabrum and B. amyloliquefaciens were non-significant over
control for shoot nitrogen concentration. A maximum increase of 0.32-fold in shoot nitrogen
concentration was noted from control with 3I in both the B. amyloliquefaciens + BC and A. fabrum
105
+ BC treatments. For P concentration in the shoot, B. amyloliquefaciens + BC differed significantly
from control. A. fabrum and B. amyloliquefaciens and BC also differed substantially from control.
Maximum increases of 0.44-fold in shoot P concentration were noted from control in B.
amyloliquefaciens + BC treatment. However, wheat cultivation with 4I gave 0.44-fold higher P
shoot concentration from 3I. For shoot K concentration, A. fabrum + BC and B. amyloliquefaciens
+ BC remained statistically alike but significantly better from control. Both the A. fabrum and B.
amyloliquefaciens inoculations also differed significantly from control for shoot K concentration.
We observed that BC was significantly different from A. fabrum, B. amyloliquefaciens and control
treatments for shoot K concentration. Maximum increases of 0.51-fold in shoot K concentration
were noted from control in A. fabrum + BC. However, wheat cultivation with 4I gave 0.13-fold
higher K shoot concentration from 3I treatment.

Main effects of T and I differed significantly but interactive effect (T × I) was non-significant for
photosynthetic rate and stomatal conductance (Table 8.6). For photosynthetic rate, the BC, A.
fabrum + BC and B. amyloliquefaciens + BC were statistically alike but differed significantly from
control. Applications of A. fabrum and B. amyloliquefaciens were non-significant from control for
photosynthetic rate. A maximum increase of 0.48-fold in photosynthetic rate was observed from
control in B. amyloliquefaciens + BC treatment. However, wheat cultivation with 4I showed 0.35-
fold higher photosynthetic rate from 3I. In case of transpiration rate, A. fabrum + BC and B.
amyloliquefaciens + BC were statistically similar to each other but differed significantly from
control. A. fabrum, B. amyloliquefaciens and BC proved significantly better treatments from
control for transpiration rate. A maximum increase of 0.81-fold in the rate of transpiration was
noted from control in B. amyloliquefaciens + BC. However, wheat cultivation with 4I showed
0.32-fold higher transpiration rate from 3I. For stomatal conductance, the BC, A. fabrum + BC and
B. amyloliquefaciens + BC treatments remained statistically alike but were significantly different
from A. fabrum and control. The A. fabrum and B. amyloliquefaciens were statistically similar to
control for stomatal conductance. The maximum increases of 0.42-fold in stomatal conductance
were noted from control in B. amyloliquefaciens + BC treatment. However, wheat cultivation with
4I showed 0.24-fold higher stomatal conductance from 3I.
106
Table 8.4. Effect of Agrobacterium fabrum, Bacillus amyloliquefaciens with/without biochar (30 Mg ha-1) on nitrogen, phosphorus
and potassium concentration in wheat grains cultivated in field conditions
Grains Nitrogen (%) Grains Phosphorus (%) Grains Potassium (%)
Treatments
4I 3I 2I (T) 4I 3I 2I (T) 4I 3I 2I (T)
a-e h-j k D d-g i j D b-e e f
Control 2.58 1.99 1.54 2.04 0.66 0.43 0.25 0.44 0.50 0.45 0.31 0.42 D
A. fabrum 2.78 ab 2.21 f-i 1.79 jk 2.26 C 0.71 c-f 0.60 f-h 0.45 i 0.59 C 0.55 a-c 0.47 de 0.43 e 0.48 C
B. amyloliquefaciens 2.73 a-c 2.33 e-h 1.86 i-k 2.31 C 0.75 a-e 0.61 e-h 0.48 hi 0.61 BC 0.56 a-c 0.48 c-e 0.46 e 0.50 C
BC 2.74 a-c 2.55 b-f 2.13 g-j 2.47 B 0.78 a-d 0.68 c-g 0.54 g-i 0.67 B 0.58 a 0.50 b-e 0.46 e 0.51 BC
A. fabrum + BC 2.87 ab 2.68 a-d 2.33 d-h 2.63 AB 0.87 ab 0.73 b-f 0.62 e-h 0.74 A 0.60 a 0.54 a-d 0.49 c-e 0.55 AB
B. amyloliquefaciens + BC 2.92 a 2.76 a-c 2.42 c-g 2.70 A 0.87 a 0.82 a-c 0.66 d-g 0.78 A 0.61 a 0.57 ab 0.50 b-e 0.56 A
ME (I) 2.77 A 2.42 B 2.01 C 0.77 A 0.65 B 0.50 C 0.57 A 0.50 B 0.44 C
ME = indicates main effect; IE = interactive effect 4I = Normal Moisture; 3I = Mild Drought; 2I = Severe Drought
107
Table 8.5. Effect of Agrobacterium fabrum, Bacillus amyloliquefaciens with/without biochar (30 Mg ha-1)
on nitrogen, phosphorus and potassium concentration in wheat shoot cultivated in drought-stressed field
conditions
Treatments
IE (T × I) IE (T × I) IE (T × I)
4I 3I 4I 3I 4I 3I
Control 1.88 a 1.33 c 1.60 B 0.42 0.26 0.34 C 1.85 1.56 1.71 D
A. fabrum 1.85 a 1.58 bc 1.71 AB 0.46 0.36 0.41 B 2.20 1.97 2.09 C
B. amyloliquefaciens 1.85 a 1.57 bc 1.71 AB 0.45 0.38 0.42 B 2.20 2.01 2.11 C
BC 1.88 a 1.59 b 1.73 AB 0.48 0.42 0.45 AB 2.56 2.17 2.37 B
A. fabrum + BC 1.93 a 1.75 ab 1.84 A 0.51 0.43 0.47 A 2.69 2.47 2.58 A
B. amyloliquefaciens + BC 1.94 a 1.75 ab 1.85 A 0.53 0.45 0.49 A 2.68 2.44 2.56 A
ME (I) 1.89 A 1.59 B 0.47 A 0.39 B 2.37 A 2.10 B
Different letters on means showing significant difference (p ≤ 0.05). Non-significant interactive effect (T × I)
did not have any letter. ME = indicates main effect; IE = interactive effect; 4I = Normal Moisture; 3I = Mild
Drought
108
Table 8.6. Effect of Agrobacterium fabrum, Bacillus amyloliquefaciens with/without biochar (30 Mg ha-1) on
gas exchange attributes of wheat cultivated in drought-stressed field conditions
(µmol (CO2) m-2 s-1) (mmol (H2O) m s )-2 -1 (µmol (CO2) m-2 s-1)
Treatments No. of Irrigations (I)
IE (T × I) IE (T × I) IE (T × I)
4I 3I 4I 3I 4I 3I
Control 14.5 9.07 11.8 C 4.35 2.97 3.66 D 150.7 105.3 128.0 C
A. fabrum 16.1 10.7 13.4 C 4.90 4.23 4.56 C 148.3 125.7 137.0 C
B. amyloliquefaciens 15.9 10.1 13.0 BC 5.41 4.17 4.79 BC 166.7 127.7 147.2 BC
BC 17.4 13.5 15.5 AB 6.27 4.64 5.46 B 181.3 145.0 163.2 AB
A. fabrum + BC 18.6 15.6 17.1 A 7.35 5.85 6.60 A 193.0 156.3 174.7 A
B. amyloliquefaciens + BC 19.1 15.8 17.5 A 7.86 5.43 6.64 A 193.3 172.3 182.8 A
ME (I) 16.9 A 12.5 B 6.02 A 4.55 B 172.2 A 138.7 B
Different letters on means showing significant difference (p ≤ 0.05). Non-significant interactive effect (T × I)
did not have any letter. ME = indicates main effect; IE = interactive effect; 4I = Normal Moisture; 3I = Mild
Drought
109
Main effects of T and I were significantly different but interaction (T × I) was non-significant for
chlorophyll a, chlorophyll b and total chlorophyll contents in wheat leaves (Table 8.7). In the case
of chlorophyll a, the A. fabrum + BC and B. amyloliquefaciens + BC were statistically similar,
while both differed significantly from control. The BC also differed significantly from control for
chlorophyll a content. The A. fabrum and B. amyloliquefaciens did not vary significantly from
control for chlorophyll a content. A maximum increase of 0.40-fold in chlorophyll a was noted in
B. amyloliquefaciens + BC treatment over control. However, wheat cultivation with 4I showed
0.15-fold higher chlorophyll a content from 3I. For chlorophyll b, the B. amyloliquefaciens + BC
and A. fabrum + BC treatments differed significantly from control. The BC also differed
significantly from control for chlorophyll b. While, A. fabrum and B. amyloliquefaciens, did not
differ significantly from control for chlorophyll b, maximum increase of 0.42-fold in chlorophyll
b was noted from control in B. amyloliquefaciens + BC treatment. However, wheat cultivation
with 4I showed 0.20-fold higher chlorophyll b content from 3I. In case of total chlorophyll, A.
fabrum + BC and B. amyloliquefaciens + BC differed significantly from control. Inoculation of A.
fabrum and B. amyloliquefaciens did not vary significantly but BC was significant from control
for total chlorophyll. A maximum increase of 0.41-fold in total chlorophyll was noted over control
due to B. amyloliquefaciens + BC application. However, wheat cultivation with 4I showed 0.17-
fold higher total chlorophyll content from 3I.

Main effects of T and I were significantly different from control for electrolyte leakage. The A.
fabrum + BC and B. amyloliquefaciens + BC treatments differed significantly from control for
electrolyte leakage. The A. fabrum, B. amyloliquefaciens and BC were statistically similar to
control for electrolyte leakage. The B. amyloliquefaciens + BC exhibited significant reduction, i.e.,
0.21-fold in electrolyte leakage compared to control. However, with 4I application wheat plants
showed significant reduction (0.20-fold) in electrolyte leakage from 3I.
110
Table 8.7. Effect of Agrobacterium fabrum, Bacillus amyloliquefaciens with/without biochar (30 Mg ha-1) on photosynthetic
pigments synthesis and electrolyte leakage in wheat leaves cultivated in drought-stressed field conditions.
Chlorophyll a Chlorophyll b Total Chlorophyll Electrolyte Leakage
(mg g-1) -1
(mg g ) (mg g )-1 (%)
Treatments No. of Irrigations (I)
IE (T × I) IE (T × I) IE (T × I) IE (T × I) ME (T)
4I 3I 4I 3I 4I 3I 4I 3I
C C C
Control 0.87 0.68 0.77 0.42 0.34 0.38 1.29 1.02 1.15 41.0 59.3 50.2 A
A. fabrum 0.91 0.78 0.85 BC 0.47 0.39 0.43 BC 1.38 1.17 1.28 BC 40.3 55.3 47.8 AB
B. amyloliquefaciens 0.90 0.78 0.84 BC 0.48 0.38 0.43 BC 1.37 1.16 1.27 BC 41.3 54.0 47.7 AB
BC 0.99 0.85 0.92 B 0.48 0.42 0.45 B 1.47 1.27 1.37 B 41.0 47.0 44.0 AB
A. fabrum + BC 1.16 0.98 1.07 A 0.53 0.45 0.49 AB 1.68 1.44 1.56 A 39.0 41.0 40.0 B
B. amyloliquefaciens + BC 1.10 1.06 1.08 A 0.59 0.49 0.54 A 1.69 1.55 1.62 A 37.0 42.3 39.7 B
ME (I) 0.99 A 0.86 B 0.49 A 0.41 B 1.48 A 1.27 B 39.9 B 49.8 A
Different letters on means showing significant difference (p ≤ 0.05). Non-significant interactive effect (T × I) did not have any
letter. ME = indicates main effect; IE = interactive effect; 4I = Normal Moisture; 3I = Mild Drought
111
8.4. Discussion
Sole application of BC under 2I significantly improved the root length and grain yield of
wheat as compared to control. Biochar is frequently reported to have very high pore volume, water
holding and cation exchange capacities (Horel et al., 2019), which properties stimulate root growth
and facilitate better water and nutrient uptakes resulting in improved vegetative and reproductive
growth (Sánchez-Monedero et al., 2019). Significantly greater K concentrations in shoot and grain
and improved plant yield in this field and an earlier pot study (Danish and Zafar-ul-Hye, 2019)
have validated the reportedly productive characteristics of BC. However, the specific objective of
this study was to investigate and present the cumulative role of drought tolerant ACC-deaminase
producing PGPR and BC in mitigating drought stress in wheat crop under field conditions.
Combined application of ACC-deaminase producing PGPR Agrobacterium fabrum or
Bacillus amyloliquefaciens and timber-waste BC significantly improved the growth and yield of
field grown wheat under mild (3I) and severe drought (2I) conditions. Our field study results
validate earlier pot study results of improved growth and yield in response to comparable drought
conditions (Salem et al., 2018; Danish and Zafar-ul-Hye, 2019). Both the PGPR strains, A. fabrum
and B. amyloliquefaciens along with BC significantly enhanced root length and plant height
compared to those under control condition. Similar results were also observed in a previous pot
study where A. fabrum and B. amyloliquefaciens significantly improved morphological growth
attributes in wheat under drought stress (Danish and Zafar-ul-Hye, 2019). As the A. fabrum and B.
amyloliquefaciens were capable of producing ACC-deaminase, improvement in root length and
plant height might be due to a reduction in ethylene level.
According to Mayak et al. (2004), raised level of 1‐ aminocyclopropane‐ 1‐ carboxylic
acid (ACC) in plants exposed to drought, raises ethylene concentration in root and shoot of plants.
Roots secrete accumulated ACC into rhizosphere which is cleaved by PGPR secreted ACC-
deaminase into NH3 and α-ketobutyrate, and ultimately ethylene level decreases. The decrease in
ethylene concentration results in better root coverage, which results in improvements in the uptake
of water and nutrients due to the enhanced rhizospheric area (Glick et al., 1997).
Significant improvements in grain yield, photosynthetic rate, transpiration rate, stomatal
conductance chlorophyll a, chlorophyll b and total chlorophyll validated the enhanced functioning
of the A. fabrum and B. amyloliquefaciens when applied in combination with BC, as compared to
using the same rhizobacteria without BC (Kumputa et al., 2019). Secretion of growth hormone,
112
i.e., IAA by the A. fabrum and B. amyloliquefaciens and greater water holding capacity of BC in
addition to ACC-deaminase production are the allied factors responsible for the improvement in
wheat growth. Findings of previous pot studies also support this argument (Salem et al., 2018;
Danish and Zafar-ul-Hye, 2019). Xie et al. (1996) described IAA as a co-factor, playing a crucial
role in crop growth enhancement. Moreover, increases in surface area and length of lateral and
adventitious roots due to high IAA secretion by PGPR play a vital role in better nutrient uptake
(Mohite, 2013).
This study finds that both the A. fabrum and B. amyloliquefaciens were solubilizing P and
K, which may explain why grain and shoot P and K concentrations were significantly improved
(Pérez-Fernández et al., 2017; Ma et al., 2019) with and without BC. Also, the increases in N, P
and K contents in shoot and grain in responses to BC (without rhizobacteria) might be due to the
retention of N and presences of P and K in BC. Improvement in cation exchange sites through BC
addition also increases the retentions of mobile nutrients like N (Rosa et al., 2016), thus, enhancing
its bioavailability by decreasing leaching and volatilization losses (Chan et al., 2008). Significant
improvements in total chlorophyll, chlorophyll a, and chlorophyll b in the current study were
probably due to better uptake of N.
According to Singh et al. (2016), greater K concentration in BC ash also contributed to
better K uptake. Improvement in K concentration might have maintained the cell turgor pressure
and regulated the stomatal conductance by osmoregulation (Shabala, 2003). Similarly, Novak et
al. (2009) and Lehmann et al. (2011) also observed a significant improvement in water holding
capacity of soil where BC was applied. The greater surface area and pore spaces of BC facilitate
the retention of water when used in soil (Abbas et al., 2018; De Jesus Duarte et al., 2019). Polar
and dispersive surface of biochar along with solid surface enerygy is directly associated with the
retension of water molecules. The negative zeta potential of biochar in majority cases shows the
prsecence of negative charges at biochar surface. Electrostatic force of attraction between
negatively charged biochar surface and cations in soil solution facilitates the adsorption of
nutrients at biochar surface (Glaser et al., 2002). Progressive degradation of cellulose and lignin
in waste feedstock make the amorphous surface of biochar. This amorphous surface of biochar has
micropores. Emission of volatile compound during pyrolysis creats the spaces which plays a role
in absorption of water when biochar is applied in soil as an amendment (Zhao et al., 2017)
113
The organic carbon in BC significantly facilitates PGPR for improvement in their growth
(Singh et al., 2016). Danish and Zafar-ul-Hye (2019) also documented the synergistic effects of
PGPR and BC against drought. They argued that root elongation and retention of water and
nutrients by PGPR and BC respectively create a favourable environment in rhizosphere for plants
to perform better under drought. Specifically, significant increases in growth and yield of wheat
through co-application of both ACC-deaminase PGPR (A. fabrum and B. amyloliquefaciens) along
with BC might be due to better survivability, activity and proliferation of PGPR in combination
with water and nutrients holding potentials of BC under 3I and 2I.
8.5. Conclusion
Combined application of PGPR and biochar more effectively mitigates drought impacts as
compared to individual PGPR inoculation or BC application, in field-grown wheat crop.
Specifically, soil application of drought-tolerant ACC-deaminase producing PGPR Agrobacterium
fabrum or Bacillus amyloliquefaciens in addition to timber waste BC (30 Mg ha-1), significantly
promotes growth and yield traits of wheat under field drought condition
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CHAPTER 9
Sole and combined application A. xylosoxidans and E. cloacae with timber-

waste biochar mitigated the drought induced stress in maize under field
condition
Abstract
Anthropogenic activities in conjoint with climate change are leading towards scarcity of good
quality irrigation. Drought is going to be a big risk to sustainable cultivation of crops especially in
arid and semi-arid areas around the globe. On the other hand, food demand is continuously
increasing due to exponential increase in human population. It is need of the time to introduce such
environmental friendly technologies that can improve crops productivity in the areas subjected to
drought conditions. ACC deaminase producing plant growth promoting rhizobacteria (PGPR) can
perform an imperative role in this regard by decreasing stress ethylene in plants. Biochar (BC) due
to its special physio-chemical attributes can also alleviate drought stress. So, a field study was
carried out to examine the role of drought tolerant ACC deaminase producing PGPR (i.e.,
Achromobacter xylosoxidans and Enterobacter cloacae) with/without timber waste (15 Mg ha-1)
under varying number of irrigations i.e., normal (12 irrigations) and drought conditions (10 and 8
irrigations). A significant improvement in shoot dry weight (28%), 1000-grain weight (19%), grain
yield (27%), grain N (43%), P (92%) and K (71%) concentration, photosynthetic rate (33%),
transpiration rate (55%), stomatal conductance (104%), chlorophyll a (33%), chlorophyll b (62%)
and total chlorophyll (45%) of maize was noted under drought stress where E. cloacae + BC was
applied. In conclusion, drought tolerant ACC-deaminase containing PGPR E. cloacae and A.
xylosoxidans proved very effective with 15 Mg ha-1 timber waste BC to improve maize growth and
yield under drought stress due to higher ACC-deaminase synthesis, better nutrients availability
and IAA production.
9.1. Introduction
Changing climatic conditions and scarcity of water has made the situation adversely severe
for the cultivation of crops (Anjum et al., 2011). Increasing temperature of earth due to global
warming is playing a significant part in the expansion of the drought area over cultivatable land
(Mir et al., 2012). Under drought stress, most of the plants are unable to uptake ample water which
115
is required for regular growth (Manivannan et al., 2008) due to which drought is considered, most
crucial abiotic stress among all stresses (Anjum et al., 2011; Zafar-ul-Hye et al., 2014).
When plants are cultivated in a limited supply of water, they produced a higher level of
stress generating ethylene (Mayak et al., 2004a; Zahir et al., 2008; Zafar-ul-Hye et al., 2014).
Server drought stimulates 1-aminocyclopropane-1-carboxylic acid (ACC) that increases ethylene
accumulation (Wang et al., 2003). Stomatal closure, high transpiration rate, less biological
nitrogen fixation, inhibition of abscisic acid activity and evoking of physiological responses are
some other major drawbacks of higher ethylene accumulation in plants (Tamimi and Timko, 2003;
Wang et al., 2003; Tanaka et al., 2005).
To overcome the problem of drought produced stress ethylene, certain plant growth
promoting rhizobacteria (PGPR) have been reported and studied that are capable to produce ACC
deaminase (1-aminocyclopropane-1-carboxylate deaminase) (Glick, 2004). The polymeric ACC
deaminase enzyme is dependent on pyridoxal 5-phosphate (PLP) (Honma and Shimomura, 1978)
that serves as a sink for ACC (ethylene precursor) (Shah et al., 1998). The decrease in ACC by its
deamination via ACC deaminase resulted in less biosynthesis of ethylene that is an important and
beneficial trait of ACC deaminase producing PGPR for plants under stress environment (Glick,
2004).
In recent years, use of activated black carbon named biochar (BC) has also become
debatable among scientists of the world (Lehmann, 2007; Spokas et al., 2010). Research conducted
on Amazonian dark earth called terra preta provided the basis for the application of BC as a soil
amendment (Glaser et al., 2001). As compared to other soils, higher cation exchange capacity
(Glaser et al., 2001; Steiner et al., 2008), improved soil fertility status, high concentration of
phosphorus and organic contents (Glaser et al., 2001) were such attributes of terra preta soil that
captured the attention of scientists to use BC as a soil amendment (Ippolito et al., 2012; Tian et
al., 2016). Biochar is produced by the process of pyrolysis that is an effective carbon sequestration
technique which can be used for recycling of agricultural and industrial wastes (Chen et al., 2010).
Application of BC in soil significantly increased water holding in sandy soil due to its high capacity
for sorption of water and nutrients (Yu et al., 2013), thus can be effective to mitigate drought
effects (Fiaz et al., 2014; Keshavarz et al., 2016). It also has potential to improve the microbial
population in rhizosphere, depending upon taxa and production of specific biochar. Thus, biochar
plays an imperative role in the cycling of nutrients (Ventura et al., 2013).
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Among cereals, maize is 3rd vital grain crop which is cultivated worldwide. The share of
maize in cereal grains production is 62% (Farhad et al., 2011). As a nutritional diet, grains of maize
have approximately 7.8% starch, 10% protein, 8.5% fibre, 4.8% oil and 3.1% sugar which also
help to decrease the cholesterol of humans blood (Chaudhary et al., 2014). However, cultivation
of maize under drought stress can decrease yield over well-watered production (Edmeades et al.,
1993).
A lot of work on sole application of BC and drought tolerant ACC deaminase containing
PGPR have been done by many scientists (Abid et al., 2017; Danish et al., 2019, 2015; Younis et
al., 2014a) but no investigation has been made regarding cumulative use of drought-tolerant ACC
deaminase containing PGPR with timber waste BC to mitigate the drought effects in maize. The
current study was conducted with the hypothesized that the use of ACC deaminase containing
PGPR and timber waste BC might be an effective and environment-friendly approach to mitigate
drought effects in maize.
9.2. Materials and methods

9.2.1. PGPR strains
Two most effective drought-tolerant ACC deaminase containing PGPR strains Enterobacter
cloacae and Achromobacter xylosoxidans which were able to grow at -0.78 MPa osmotic potential
in DF minimal salt medium (4g KH2PO4, 6g Na2HPO4, 2g glucose, 2g gluconic acid, 2g citric acid
and 0.2g MgSO4.7H2O with trace elements: 124.6 mg ZnSO4.7H2O, 11.2 mg MnSO4.H2O, 78.22
mg CuSO4.5H2O, 10 mg H3BO3, 10 mg MoO3, 1mg FeSO4.7H2O and 0.5M ACC, as a nitrogen
source in one litre of autoclaved deionized water with 7.2 pH) (Dworkin and Foster, 1958) .
Inoculum broth of the desired PGPR was made using the medium without agar.
9.2.2. PGPR characterization
For ACC deaminase activity (E. cloacae = 402.1 ± 27.29, A. xylosoxidans = 381.17 ± 11.69 µmol
α-ketobutyrate mg-1 protein h-1) Honma and Shimomura (1978) and El-Tarabily (2008)
methodologies were adopted. Glickmann and Dessaux (1995) method was used for indole acetic
acid determination using Salkowski reagent with (E. cloacae = 78.79 ± 0.35 and A. xylosoxidans
= 61.19 ± 0.14 µg/ml) and without L-tryptophan (E. cloacae = 3.39 ± 0.41 and A. xylosoxidans =
5.52 ± 0.79µg/ml). Vazquez et al. (2000) and Setiawati and Mutmainnah (2016) methods were
adopted for phosphorus (E. cloacae = 66.3 ± 0.38 and A. xylosoxidans = 77.4 ± 0.98 µg/ml) and
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potassium solubilizing activities (E. cloacae = 19.1 ± 0.82 and A. xylosoxidans = 24.5 ± 0.42
µg/ml) of both strains.
See chapter 3 section 3.1 subsection 3.1.10. Physio-chemical characteristics of BC are provided
in Table 9.1.
Table 9.1. Pre-experimental characteristics of soil and timber waste BC
Sand % 60 pH - 7.26
-1
ECe dS m 3.84 Total N % 0.21
-1
Extractable P µg g 4.31 Total K % 1.61
-1
Extractable K µg g 110 Total Na % 0.19
9.2.5. Experimental site and soil characteristic
The experiment was conducted in the research area of the Department of Soil Science, Bahauddin
Zakariya University Multan. For soil characterization see chapter 3 section 3.1 subsection 3.1.11.
The physio-chemical characteristics of soil is provided in Table 8.1.
9.2.6. Seeds collection and inoculation
The seeds of maize (cv. Kenzo Hybride-123) were purchased from certified seed dealer of the
Government of Punjab, Pakistan. Weak seeds were initially screened out manually by hand. For
seeds sterilization see chapter 3 section 3.1 subsection 3.1.3.
9.2.7. Field preparation, nutrients and biochar application
To provide macronutrients for maize cultivation 200, 150, 100 kg ha-1 N, P and K were applied
respectively as recommended NPK fertilizer (RNPKF) respectively (Zafar-ul-Hye et al., 2015).
All P and K fertilizers were applied at seeds sowing in a single dose. Nitrogen was applied in 4
splits (1st = 5-6 leaf stage, 2nd 8-10 leaf stage, 3rd 14-16 leaf stage and 4th tasseling stage). For
application of NPK fertilizers, urea, DAP and SOP were used. Timber waster biochar (BC) was
applied at the rate of 15 Mg ha-1 as selected best application rate in pot experiment.
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9.2.8. Experimental design and treatment plan
There were 6 treatments applied at 3 levels of irrigation (12, 10 and 8) with 3 replications following
randomized complete block design (RCBD) design. The treatments included; control (No PGPR
and No BC), E. cloacae, A. xylosoxidans, 15Mg ha-1 BC, E. cloacae + 15Mg ha-1 BC and A.
xylosoxidans + 15Mg ha-1 BC.
9.2.9. Seeds sowing and Drought stress
In each plots (3m length × 4m width = 12 m2) sown of seeds was done by hand. The seed rate was
8 kg ha-1. For handling of PGPR inoculated seeds, sterilized gloves were used to avoid any
contamination. In control, 12 irrigations were provided according to the production technology of
maize 2017 issued by Directorate of Agricultural Information Punjab (GOP, 2017) to fulfill the
requirement of water (600 mm) for maize (Reddy, 2006). Weather data (Figure 8.1) of experiment
was collected from the Central Cotton Research Institute, Multan, during the study years (2017).
Maximum Temperature Minimum Temperature Humidity
100 45
90 40
80 35
Temperature °C
Humidity (%)
70 30
60
25
50
20
40
30 15
20 10
10 5
0 0
February March April May June
Months of year 2017
Figure 8.1. Weathering data 2017

To introduce drought, 10 irrigations (1 skipped at tasseling, 2nd at flowering) and 8 irrigations (1
skipped at after end of juvenile, 2nd at tasseling, 3rd at flowering and 4th at milking) were provided.
9.2.10. Harvesting
After 45 days of sowing, vegetative harvesting from 4 random spots of each plot was done for
determination of photosynthetic pigments, electrolyte leakage and nutrients concentration in the
shoot.
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9.2.11. Yield attributes
The maize plants cob length, grains weight cob-1, number of grains cob-1, grains yield, 1000-grains
weight and biological yield were noted at the time of maturity (115 days after sowing).
9.2.12. Nitrogen, phosphorus and potassium concentration in grain and shoot
9.2.13. Nutrients use efficiency (NUE)
For nutrients (N, P and K) use efficiency equation of Fageria et al. (1997) was used.
Total Nutrients uptake in treatments(kg ha−1 ) − Total Nutrients uptake incontrol(kg ha−1 )
NUE (%) = × 100
Nutrients Applied (kg ha−1 )
Nutrient uptake (kg ha−1 ) = Nutrients concentration (%) × yield (Mg ha−1 ) × 1000 /100
Total nutrients uptake (kg ha−1 ) = Grains nutrients uptake + Shoot nutrients uptake
9.2.14. Gas exchange parameters

9.3. Results
9.3.1. Plant height, cob length and number of grains cob-1
Both main and interactive effects of treatments (T) and irrigations (I) differed significantly for
plant height. However, main effects of T and I remained significantly different for cob length and
number of grains cob-1. For plant height, application of BC, A. xylosoxidans + BC and E. cloacae
+ BC differed significantly over control for plant height (Table 9.2). However, A. xylosoxidans
and BC did not differ significantly with each other for plant height. Highest increase of 11% in
plant height of maize was noted over control where A. xylosoxidans + BC and E. cloacae + BC
were applied. For cob length, application of BC and A. xylosoxidans + BC remained significantly
better over control (Table 9.2). Highest increase of 9% in cob length was noted over control where
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A. xylosoxidans + BC was applied. In case of number of grains cob-1, A. xylosoxidans + BC and E.
cloacae + BC proved significantly better over control (Table 9.2). Application of A. xylosoxidans
+ BC and E. cloacae + BC found to be significantly better over BC, A. xylosoxidans and E. cloacae
for number of grains cob-1. The E. cloacae and BC were significantly different over control for
number of grains cob-1. Highest increase of 21% in number of grains cob-1 was noted over control
where E. cloacae + BC was applied.
9.3.2. 1000-grain weight, grain yield and biological yield
Main effects of T and I were significantly different for 1000-grain weight, biological and grain
yield of maize (Table 9.2). For 1000-grain weight, application of E. cloacae + BC remained
significantly different over control. Highest increase of 19% in 1000-grain weight was noted over
control where E. cloacae + BC was applied. In case of grain yield, BC, E. cloacae + BC and A.
xylosoxidans + BC remained significantly better over control. Both E. cloacae + BC and A.
xylosoxidans + BC remained significantly better over E. cloacae and A. xylosoxidans respectively,
for grains yield. Inoculation of E. cloacae and A. xylosoxidans gave significantly better results
over control for grain yield. Highest increase of 27% in grain yield was noted over control where
E. cloacae + BC was applied. For biological yield, E. cloacae + BC and A. xylosoxidans + BC
remained significantly better over control. E. cloacae + BC and BC were statistically alike but A.
xylosoxidans + BC differed significantly over BC for biological yield (Table 9.2). Both E. cloacae
+ BC and A. xylosoxidans + BC remained significantly better over E. cloacae and A. xylosoxidans
for biological yield. Highest increase of 30% in biological yield was noted over control where A.
xylosoxidans + BC was applied.

Main effects of T and I were significantly different for N, P and K concentration in maize grain
(Table 9.3). In case of grain N concentration, BC, A. xylosoxidans + BC and E. cloacae + BC
proved significantly better over control. No significant change was observed in grains N
concentration where A. xylosoxidans and E. cloacae was used over control. Inoculation of A.
xylosoxidans and E. cloacae with BC remained significantly better over without BC for grain N
concentration. Highest increase of 43% in grains N concentration was noted over control where E.
cloacae + BC was applied. For grain P concentration, A. xylosoxidans + BC and E. cloacae + BC
remained significantly better over control (Table 9.3). Application of A. xylosoxidans + BC was
statistically similar to BC, A. xylosoxidans and E. cloacae for grain P concentration. The BC, A.
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xylosoxidans and E. cloacae remained significantly better over control for grains P concentration.
Inoculation of E. cloacae with BC remained significantly better over without BC for grains P
concentration. Highest increase of 92% in grains P concentration was noted over control where E.
cloacae + BC was applied. In case of grains K concentration, A. xylosoxidans + BC and E. cloacae
+ BC differed significantly over control (Table 9.3). Inoculation of E. cloacae with BC remained
significantly better over without BC for grain K concentration. It was noted that BC, A.
xylosoxidans and E. cloacae differed significantly over control for grain K concentration. Highest
increase of 71% in grain K concentration was noted over control where E. cloacae + BC was
applied.

Main effect of T and I were significantly different for N, P and potassium K concentration in shoot
of maize (Table 9.3). For N and P concentration, A. xylosoxidans + BC and E. cloacae + BC proved
significantly better over control. However, for P concentration in shoot inoculation of A.
xylosoxidans and E. cloacae remained significantly better over control. Both A. xylosoxidans + BC
and E. cloacae + BC differed significantly over A. xylosoxidans and E. cloacae for N and P
concentration in shoot. Highest increase of 45 and 73% was noted in N and P concentration in
shoot respectively over control in E. cloacae + BC. In case of K concentration in shoot, BC, A.
xylosoxidans + BC and E. cloacae + BC differed significantly over control. However, for K
concentration in shoot, inoculation of A. xylosoxidans and E. cloacae proved significantly better
over control (Table 9.3). The A. xylosoxidans + BC and E. cloacae + BC remained significantly
better over A. xylosoxidans and E. cloacae for K concentration in shoot. Highest increase of 71%
in K concentration was noted over control where A. xylosoxidans + BC was applied.
9.3.5. Nutrients use efficiency

Main effects of T and I were significant for nutrients use efficiency in maize. For N, P and K use
efficiency, application of E. cloacae + BC and BC remained significantly best. No significant
change was noted among A. xylosoxidans + BC, E. cloacae + BC and BC in case of N use
efficiency (Table 9.3). However, E. cloacae + BC and BC differed significantly better over A.
xylosoxidans + BC for P and K use efficiency in maize. Highest increase in N (72.8%), P (70.4%)
and K (69.5%) use efficiencies was noted where A. xylosoxidans + BC was applied over sole
inoculation of A. xylosoxidans.
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Table 9.2. Effect of sole and combined application of E. cloacae and A. xylosoxidans with biochar (15 Mg ha-1) on growth and yield
of maize cultivated under different level of irrigation in field conditions
Plant Cob Number of 1000-grains Grain Biological
Treatment Height length grains weight yield yield
(cm) (cm) cob-1 (g) (Mg ha-1) (Mg ha-1)
12 Irrigations 176 A 17.0 A 381 A 258.3 A 3.60 A 12.8 A
10 Irrigations 162 B 15.1 B 338 B 212.8 B 3.20 B 11.2 B
8 Irrigations 149 C 13.7 C 300 C 168.1 C 2.38 C 8.65 C
Drought tolerant ACC deaminase producing PGPR and Timber Waste Biochar
Control 154 C 14.5 B 307 C 194.2 B 2.66 C 9.51 C
E. cloacae 155 C 15.2 AB 328 B 210.2 AB 2.92 B 10.2 C
A. xylosoxidans 159 BC 14.9 AB 324 BC 196.6 B 2.94 B 10.2 C
BC 165 AB 15.6 A 341 B 222.7 AB 3.13 AB 11.2 B
E. cloacae + BC 171 A 15.5 AB 373 A 232.9 A 3.37 A 11.9 AB
A. xylosoxidans + BC 171 A 15.8 A 364 A 221.9 AB 3.34 A 12.4 A
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Table 9.3. Effect of sole and combined application of E. cloacae and A. xylosoxidans with biochar (15 Mg ha-1) on grains and shoot
nutrients concentration of maize cultivated under different level of irrigation in field conditions
Grains Grains Grains Shoot Shoot Shoot Nitrogen use Phosphorus Potassium
Treatment Nitrogen Phosphorus Potassium Nitrogen Phosphorus Potassium efficiency use efficiency use efficiency
(%) (%) (%) (%) (%) (%) (%) (%) (%)
A A
12 Irrigations 1.89 A 0.23 A
1.15 A
2.07 A
0.26 A
1.25 A
33.2 4.21 20.41 A
10 Irrigations 1.56 B 0.18 B 0.92 B 1.76 B
0.22 B 1.07 B 23.9 B 3.00 B 14.74 B
8 Irrigations 1.26 C 0.14 C 0.68 C 1.32 C
0.17 C 0.77 C 15.9 C 1.72 C 8.99 C
Control 1.30 B 0.12 C 0.66 D 1.42 D 0.15 D 0.73 C - - -
E. cloacae 1.43 B 0.17 B 0.86 C 1.63 CD 0.21 C 0.95 B 25.1 BC 3.42 AB 16.7 B
A. xylosoxidans 1.42 B 0.17 B 0.88 BC 1.53 CD 0.20 C 0.94 B 21.7 C 2.84 B 13.8 B
BC 1.68 A 0.19 B 0.98 BC 1.72 BC 0.22 BC 1.10 AB 33.1 AB 3.66 AB 18.5 AB
E. cloacae + BC 1.86 A 0.23 A 1.13 A 2.06 A 0.26 A 1.21 A 37.5 A 4.84 A 23.4 A
A. xylosoxidans + BC 1.75 A 0.20 AB 1.01 AB 1.93 AB 0.25 AB 1.25 A 28.3 A-C 3.10 B 15.9 B
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9.3.6. Electrolyte leakage and gas exchange attributes
Main effect of T and I were significantly different for electrolyte leakage, photosynthetic rate,
respiration rate and stomatal conductance of maize (Table 9.4). For electrolyte leakage, A.
xylosoxidans, BC, A. xylosoxidans + BC and E. cloacae + BC showed significantly better results
over control. Both A. xylosoxidans + BC and E. cloacae + BC did not differ significantly over A.
xylosoxidans and E. cloacae for electrolyte leakage. A significant decrease of 35% in electrolyte
leakage was noted over control where A. xylosoxidans + BC was applied. For photosynthetic rate,
BC, A. xylosoxidans + BC and E. cloacae + BC were statistically alike but remained significantly
better over control. It was noted that E. cloacae and BC were statistically similar to each other for
photosynthetic rate. Both A. xylosoxidans + BC and E. cloacae + BC remained significantly better
over A. xylosoxidans and E. cloacae for photosynthetic rate. Highest increase of 33% in
photosynthetic rate was noted over control where E. cloacae + BC was applied. In case of
transpiration rate, A. xylosoxidans + BC and E. cloacae + BC differed significantly over control
for transpiration rate (Table 9.4). However, E. cloacae + BC remained significantly better over E.
cloacae and BC for transpiration rate. Highest increase of 55% in transpiration rate was noted over
control where E. cloacae + BC was applied. Application of A. xylosoxidans + BC and E. cloacae
+ BC were significantly better over control for stomatal conductance (Table 9.4). Application of
E. cloacae + BC remained significantly better over E. cloacae and BC for stomatal conductance.
Highest increase of 104% was noted over control where E. cloacae + BC was applied.

Main effect of T and I differed significantly for chlorophyll contents of maize. Application of A.
xylosoxidans + BC and E. cloacae + BC were significantly better over control for chlorophyll a
content. The E. cloacae + BC and A. xylosoxidans + BC remained significantly better over E.
cloacae and A. xylosoxidans for chlorophyll a (Table 9.4). For chlorophyll a content, inoculation
of E. cloacae and BC remained significantly better over control. Highest increase of 33% in
chlorophyll a content was noted over control where E. cloacae + BC was applied. For chlorophyll
b, A. xylosoxidans + BC and E. cloacae + BC differed significantly over control. Application of
BC remained statistically similar with A. xylosoxidans + BC for chlorophyll b content (Table 9.4).
The E. cloacae + BC remained significantly better over BC and E. cloacae for chlorophyll b.
Similarly, A. xylosoxidans + BC differed significantly over A. xylosoxidans. The E. cloacae and
A. xylosoxidans remained statistically alike but differed significantly over control for chlorophyll
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b content. Highest increase of 62% in chlorophyll b content was noted over control where E.
cloacae + BC was applied. In case of total chlorophyll, A. xylosoxidans + BC and E. cloacae + BC
differed significantly over control. Both A. xylosoxidans + BC and E. cloacae + BC remained
significantly better over BC, A. xylosoxidans and E. cloacae for total chlorophyll (Table 9.4).
Inoculation of A. xylosoxidans and E. cloacae differed significantly over control for total
chlorophyll content. Highest increase of 45% in total chlorophyll content was noted over control
where E. cloacae + BC was applied.
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Table 9.4. Effect of sole and combined application of E. cloacae and A. xylosoxidans with biochar (15 Mg ha-1) on electrolyte
leakage, gas exchange attributes and chlorophyll contents of maize cultivated under different level of irrigation in field conditions
Electrolyte Photosynthetic Transpiration Stomatal Cholorophyll Cholorophyll Total
Treatment leakage rate rate conductance a b chlorophyll
(%) (µmol m-1 s-1) (mmol m-1 s-1) (µmol m-1 s-1) (mg g-1) (mg g-1) (mg g-1)
12 Irrigations 29.0 C 17.0 A 381 A 98.4 A 0.75 A 0.54 A 1.29 A
10 Irrigations 45.7 B 15.1 B 338 B 72.1 B 0.64 B 0.46 B 1.10 B
8 Irrigations 56.9 A 13.7 C 300 C 50.9 C 0.54 C 0.35 C 0.89 C
Control 54.6 A 18.2 C 2.49 C 0.19 C 0.55 E 0.34 D 0.89 D
E. cloacae 47.1 AB 19.7 BC 2.86 BC 0.21 C 0.61 CD 0.42 C 1.03 C
A. xylosoxidans 43.9 BC 18.3 C 2.76 BC 0.22 C 0.59 DE 0.41 C 1.00 C
BC 43.1 BC 21.5 AB 3.11 BC 0.30 B 0.66 BC 0.48 B 1.14 B
E. cloacae + BC 39.1 BC 24.2 A 3.86 A 0.38 A 0.73 A 0.55 A 1.29 A
A. xylosoxidans + BC 35.4 C 23.0 A 3.37 AB 0.33 AB 0.71 AB 0.51 AB 1.23 A
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9.4. Discussion
In the current study, the application of drought tolerent ACC-deaminase containing PGPR, E.
cloacae and A. xylosoxidans with timber-waste BC significantly enhanced maize growth and yield
cultivated with varying number of irrigations (12I, 10I and 8I). Under 8I stress, co-application of
ACC-deaminase containing PGPR E. cloacae and A. xylosoxidans with 15 Mg ha-1 BC produced
significantly better results as compred to sole inoculation of E. cloacae and A. xylosoxidans
regarding plant height and shoot dry weight of maize. Better PGPR colonization, ACC deaminase
activity and improvement in water holding capacity by co-application of PGPR and BC might be
responsible for improvement in plant height and shoot dry weight of maize. According to Mayak
et al. (2004) the upregulation of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) from root to shoot
under drought stress and limited availibility of nutrients enhances the synthesis of stress ethylene
in root and shoot of plants. Less elongation and radial swelling of the stem are indications of higher
ethylene accumulation (Abeles et al., 1992). At early stages of crop growth, higher level of
ethylene decreases the supply of energy and water at the imbibition phase (Taiz and Zeiger, 2010;
Aroca, 2012). Similarly, Glick et al. (1998) proposed that the synthesis of indole acetic acid (IAA)
by PGPR stimulates ACC synthase enzyme that converts S-adenosylmethionine to ACC. Plants
roots and seeds exude ACC in rhizosphere which is cleaved into NH3 and α-ketobutyrate by PGPR
secreted ACC deaminase. Decrease in ethylene due to its cleavage of ACC resulted in better
elongation of roots. This improvement in root elongation facilitated plant to uptake water and
nutrients by increasing rhizosphere area (Glick et al., 1999).
In additon to reasoning given above, higher surface area and pore spaces are such characteristics
that make BC an effective soil amendment for improvement in bioavailibility of water to plants
under drought stress (Fiaz et al., 2014). Hydrophobicity of biochar surface is thought to be one of
the cause of rhizobacteria diversity, proliferation and activity. The adhesion affinity of
rhizobacteria with biochar is mainly characterized by the presence of divalent ions in biochar. The
micropores of biochar also serve as a shelter for rhizobacteria. These micropores increase the
survival of rhizpbacteria and decrease their competition for food, shelter and space in the
rhizosphere. Slow mineralization of biochar also facilitates in provision of nutrients to the
rhizobacteria that play an imperative role in better microbial proliferation (Lehmann and Rondon,
2002). Polar and dispersive surface of biochar with surface enerygy is connected with the
possession of water molecules. The negative zeta potential of biochar in majority cases reflects the
128
presence of negative charges at the biochar surface. Electrostatic force of attraction between
negative charges of biochar surface and cations in soil solution facilitate the adsorption of nutrients
at biochar surface (Glaser et al., 2002).
A significant improvement in plant height signified the imperative role of co-application of E.
cloacae and A. xylosoxidans with BC over sole application of BC and the control. Application of
BC with PGPR in the current study also significantly enhanced photosynthetic rate, transpiration
rate and stomatal conductance especially at 10I and 8I. The improvement in photosynthetic rate,
transpiration rate and stomatal conductance might be due to high water holding capacity (WHC)
of BC and decrease in ethylene biosysnthesis. Roots secrete organic acids, sugars, vitamins,
phytosiderophores,nucleosides amino acids and mucilage that attracts PGPR which results in
better colonization of PGPR, uptake of water and nutrients (Danish and Zafar-ul-Hye, 2019).
However, Akhtar et al. (2015) documented that the colonization of PGPR is also improved when
PGPR are inoculated with biochar. In current study inoculation of E. cloacae and A. xylosoxidans
BC significantly decreased electrolyte leakage over control which is another solid evidence of less
ethylene accumulation. The findings of Nadeem et al. (2017) in cucumber under drought stress
justified our results regarding less electrolyte leakage. According to Shi et al. (2012), the
application of 1-aminocyclopropane-1-carboxylic acid (ACC) which is a precursor of ethylene,
increased the electrolyte leakage in plants. Cell membrane mostly loses its integrity by degradation
of lipid molecules as a result of higher accumulation of ethylene. Direct contact of ethylene with
chloroplast by degradation of lipids in cell membrane activated chlorophyllase (chlase) gene that
severely damaged cholorophyll (Matile et al., 1997). A significant improvement in chlorophyll
pigments synthesis at 10I and 8I shows the role of low ethylene accumulation due to deamination
of ACC and improvement in soil water holding capacity by BC. Imrpovement in root elongation
by cell division and better uptake of nutrients are an indicator of enhancement in plant growth
under drought stress (Zeiger and Taiz, 2010; Hussain et al., 2018; Paul et al., 2018). According to
Zheng et al. (2003) and Borch et al. (1999) the limited availability of N and P are allied factor that
significantly contributed in higher biosynthesis and accumulation of ethylene. The improvement
in shoot P (E. cloacae and A. xylosoxidans) and K (E. cloacae + BC and A. xylosoxidans + BC)
concentration at 10I and 8I signified an imperative effect of PGPR and BC for significant
improvement in the yield attributes (1000-grain weight, grain yield and biological yield) of maize
plants. In an experiment conducted by Younis et al. (2014b) also noted similar results for the
129
improvement in the uptake of nutrients by the addition of BC. A significant increase in the fresh
and dry weight of plants by the better uptake of P was also documented by Richardson et al. (2009).
Application of BC significantly increased the P uptake in the plants (Younis et al., 2014a).
According to Chan et al. (2008), a significant increase in the bioavailability of N is due to
improvement in the soil cation exchange capacity when BC is applied as an amendment. In the
current study, better uptake of K in maize at 10I and 8I by co-application of PGPR and BC might
be another allied factor responsible for the mitigation of drought stress.
9.5. Conclusion
From results, it is concluded that the application of BC with E. cloacae and A. xylosoxidans, is a
better approach for mitigation of drought stress, promotion of growth, gas exchange attributes,
nutrients concentration in shoot and grains, and yield in maize. Combine addition of PGPR E.
cloacae and A. xylosoxidans and BC can mitigate drought stress more effectively over sole
inoculation of PGPR.
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SUMMARY
Drought stress retards wheat and maize plant’s vegetative growth and physiological processes that
results in low productivity. Plants under drought stress synthesizes ethylene which inhibits root
elongation. Low stomatal conductivity, transpiration rate and photosynthetic rate are other major
drawbacks of higher ethylene accumulation in plants under limited supply of water. Cultivation of
wheat under a limited supply of water significantly decreases the yield while its demand is
increasing at the rate of 1.6% / annum. Wheat (Triticum aestivum L.) and maize (Zea mays L.) are
important cereal crops and used as a staple food in most parts of the world. Wheat contains 55%
carbohydrates and 8-12% proteins. It is also an important crop due to its worldwide trade too.
Maize also contains 78 % starch, 10 % proteins, 8.5 % fibre, 4.8 % oil and 3.1 % sugars. These
nutrition help to decrease the cholesterol of human’s blood. However, cultivation of maize under
drought stress also decreases its yield drastically i.e., loss of 24 million tons yr-1 over well-watered
production. On the other hand, food demand is continuously increasing day by day due to an
exponential increase in human’s population. That’s why it is need of time to introduce such
technologies that can mitigate drought stress in the agriculture sector and are environment friendly.
Inoculation of ACC deaminase producing plant growth promoting rhizobacteria (PGPR) can play
an imperative role to some extent in that regard. The polymeric enzyme, 1-aminocyclopropane-1-
carboxylate (ACC) deaminase is dependent on pyridoxal 5-phosphate (PLP) that catabolizes
ethylene produced under drought stress. It converts ACC (an immediate precursor of ethylene
biosynthesis in the methionine pathway in higher plants) into ammonia and α-ketobutyrate instead
of ethylene. Regularization of ethylene level in plants mitigate the effects of drought. In addition,
biochar (BC) has been reported to be rich in nutrients that can mitigate drought stress. Biochar is
an organically active amendment that when applied to the soil, can increase pore volume, cation
exchange capacity and nutrient and water retention and bioavailability. It is produced through the
process of pyrolysis that is an effective carbon sequestration technique which can be used for
recycling of agricultural and industrial wastes. This modern technique of pyrolysis is also very
effective for organic production of syngas (CO, H2 and CH4) which is considered as a source of
energy via rapid technical development. Biochar has a large surface area, porosity and cation π-
131
bonding mechanisms, on graphene-like structures or either with –C=O functional groups. The
pyrolysis temperature and nature of waste feedstock are important key factors that decide the pH
and surface area of the BC produced. So far, a lot of work is documented on the sole application
of both amendments but very little information is documented on combined addition of drought
tolerant ACC deaminase producing PGPR and timber waste biochar. The current research project
comprised of six separate experiments to explore the comparative analysis of wheat and maize
growth and productivity with special reference to the combined role of ACC-deaminase producing
PGPR and timber waste biochar under drought stress both in pot culture and field conditions. In
first and second studies, isolation and screening of drought tolerant ACC deaminase producing
PGPR was done. Rhizospheric soils were collected from wheat and maize. It was noted that under
various levels (0, 10 and 20%) of polyethylene glycol (PEG) induced drought stress, some of ACC
deaminase producing PGPR i.e., Pseudomonas aeruginosa, Enterobacter cloacae, Achromobacter
xylosoxidans and Leclercia adecarboxylata improved growth attributes, chlorophyll content and
NPK concentration in maize. Similarly Leclercia adecarboxylata, Agrobacterium fabrum, Bacillus
amyloliquefaciens and Pseudomonas aeruginosa also remained significantly better for
improvement in growth attributes, chlorophyll content and NPK concentration in wheat. In third
pot study, B. amyloliquefaciens proved significantly better as compared to Leclercia
adecarboxylata, Agrobacterium fabrum and Pseudomonas aeruginosa with 1.50% timber waste
biochar for the improvement in growth and yield attributes in wheat. In fourth pot study, both
Enterobacter cloacae and Achromobacter xylosoxidans remained significantly better as compared
to Leclercia adecarboxylata and Pseudomonas aeruginosa with 0.75 and 1.50% timber waste
biochar for the improvement in growth and yield attributes in maize. The B. amyloliquefaciens and
Agrobacterium fabrum were inoculated separately and in combinations with 30 Mg ha-1 in wheat,
under various levels of drought the study 5. Combined application of B. amyloliquefaciens and 30
Mg ha-1 timber waste biochar under 3I significantly increased growth and yield traits of wheat i.e.,
grain yield (36 %), straw yield (50 %) and biological yield (40 %). The same under 2I resulted in
greater increases in several of the growth and yield traits: grain yield (77 %), straw yield (75 %)
and biological yield (77 %), as compared to control. The A. xylosoxidans and E. cloacae were
132
inoculated separately and in combinations with 15 Mg ha-1 in maize, under various levels of
drought in the study 6. The results confirmed that combined addition of A. xylosoxidans with
timber waste biochar significantly enhanced plant height (11 %), cob length (9 %) and decreased
electrolyte leakage (35 %), in maize under drought stress. A significant improvement in shoot dry
weight (28 %), 1000-grain weight (19 %), number of grains cob-1 (21 %), grain weight cob-1 (44
%), grain N (43 %), grain P (43 %), grain N (92 %) and grain yield (71 %) validated the
effectiveness of E. cloacae with timber waste biochar to grant resistance against drought in maize.
133
CONLSUION
In connection with results of less electrolyte leakage, better intake of nutrients and significant
improvement in grain and biological yield it is confirmed that combine addition of both A.
xylosoxidans and E. cloacae with 15 Mg ha-1 timber waste biochar is a better approach to mitigate
drought stress in maize. Similarly, the combined soil application of B. amyloliquefaciens and 30
Mg ha-1 timber waste biochar more effectively mitigates drought stress and improves wheat
productivity as compared to any of the individual soil applications tested in this study.
134
REFERENCES
Abid, M., S. Danish, M. Zafar-ul-Hye, M. Shaaban, M.M. Iqbal, et al. 2017. Biochar increased
photosynthetic and accessory pigments in tomato (Solanum lycopersicum L.) plants by
reducing cadmium concentration under various irrigation waters. Environ. Sci. Pollut. Res.
24(27): 22111–22118.
Abideen, Z., H.W. Koyro, B. Huchzermeyer, R. Ansari, F. Zulfiqar, et al. 2020. Ameliorating
effects of biochar on photosynthetic efficiency and antioxidant defence of Phragmites karka
under drought stress. Plant Biol. 22(2): 259–266.
Administrator. 2011. Crowley Nanjing Biochar inoculants 2011 - Crowley Nanjing Biochar
inoculants 2011.pdf. : 1–45. http://www.biochar-international.org/sites/default/files/Crowley
Nanjing Biochar inoculants 2011.pdf%5Cnpapers2://publication/uuid/F0275312-030D-
4FCB-8B52-EB27E44E436C.
Adnan, M., Z. Shah, A. Khan, M. Shah, G.A. Khan, et al. 2014. Integrated effects of rhizobial
inoculum and inorganic fertilizers on wheat yield and yield components. Am. J. Plant Sci.
05(13): 2066–2073.
Ahmad, I., M.J. Akhtar, Z.A. Zahir, M. Naveed, B. Mitter, et al. 2014. Cadmium-tolerant bacteria
induce metal stress tolerance in cereals. Environ. Sci. Pollut. Res. 21(18): 11054–11065.
Ahmad, M.T., N. Asghar, M. Saleem, M.Y. Khan, and Z.A. Zahir. 2015. Synergistic Effect of
Rhizobia and Biochar on Growth and Physiology of Maize. Agron. J. 107(6): 2327–2334.
Akhtar, S.S., M.N. Andersen, M. Naveed, Z.A. Zahir, and F. Liu. 2015. Interactive effect of
biochar and plant growth-promoting bacterial endophytes on ameliorating salinity stress in
maize. Funct. Plant Biol. 42(8): 770–781.
Akhtar, S.S., G. Li, M.N. Andersen, and F. Liu. 2014. Biochar enhances yield and quality of tomato
under reduced irrigation. Agric. Water Manag. 138: 37–44.
Alam, M.S., N.M. Talukder, M.T. Islam, A. Sarkar, and M.M. Hossain. 2008. Phosphate
135
solubilizing rhizoplane bacteria on growth and yield of transplant aman rice. J. Agrofor.
Environ. 2(1): 11–14.
Ali, S., T.C. Charles, and B.R. Glick. 2012. Delay of flower senescence by bacterial endophytes
expressing 1-aminocyclopropane-1-carboxylate deaminase. J. Appl. Microbiol. 113(5):
1139–1144.
Almeida, V., B. Szpoganicz, and S. Bonneville. 2015. Potentiometric titration and out-of-
equilibrium pH response of the biotite water system. J. Braz. Chem. Soc. 26: 1848–1860.
Amonette, J., and S. Joseph. 2009. Characteristics of Biochar - Micro-chemical Properties. In:
Amonette, J. and Joseph, S., editors, Biochar for Environmental Management: Science and
Technology. Earthscan, London, UK. p. 33–52
Anjum, S.A., M. Farooq, L.C. Wang, L.L. Xue, S.G. Wang, et al. 2011a. Gas exchange and
chlorophyll synthesis of maize cultivars are enhanced by exogenously-applied glycinebetaine
under drought conditions. Plant, Soil Environ. 57(7): 326–331.
Anjum, S.A., L. Wang, M. Farooq, L. Xue, and S. Ali. 2011b. Fulvic Acid Application Improves
the Maize Performance under Well-watered and Drought Conditions. J. Agron. Crop Sci.
197(6): 409–417.
Arnon, D.I. 1949. Copper Enzymes in Isolated Chloroplasts. Polyphenoloxidase in Beta vulgaris.
Plant Physiol. 24(1): 1–15.
Aroca, R. 2012. Plant responses to drought stress from Morphological to Molecular Features (A.
Ricardo, editor). Springer, New York.
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(5): 611–620.
Aslam, M., M.A. Maqbool, and R. Cengiz. 2015. Effects of Drought on Maize. Drought Stress in
Maize (Zea mays L.). SpringerBriefs in Agriculture. Springer, Cham. p. 5–17.
136
Atkinson, C.J., J.D. Fitzgerald, and N.A. Hipps. 2010. Potential mechanisms for achieving
agricultural benefits from biochar application to temperate soils: A review. Plant Soil 337(1):
1–18.
Awasthi, R., N. Kaushal, V. Vadez, N.C. Turner, J. Berger, et al. 2014. Individual and combined
effects of transient drought and heat stress on carbon assimilation and seed filling in chickpea.
Funct. Plant Biol. 41: 1148–1167.
Baalbaki, R.Z., R.A. Zurayk, M.M. Bleik, and S.N. Talhouk. 1999. Germination and seedling
development of drought tolerant and susceptible wheat under moisture stress. Seed Sci.
Technol. 27: 291–302.
Basak, B.B., and D.R. Biswas. 2010. Co-inoculation of potassium solubilizing and nitrogen fixing
bacteria on solubilization of waste mica and their effect on growth promotion and nutrient
acquisition by a forage crop. Biol. Fertil. Soils 46(6): 641–648.
Bates, L.S., R.P. Waldren, and I.D. Teare. 1973. Rapid determination of free proline for water-
stress studies. Plant Soil 39(1): 205–207.
Belimov, A.A., V.I. Safronova, T.A. Sergeyeva, T.N. Egorova, V.A. Matveyeva, et al. 2001a.
Characterization of plant growth promoting rhizobacteria isolated from polluted soils and
containing 1-aminocyclopropane-1-carboxylate deaminase. Can. J. Microbiol. 47(7): 642–
652.
Belimov, A.A., V.I. Safronova, T.A. Sergeyeva, T.N. Egorova, V.A. Matveyeva, et al. 2001b.
Characterization of plant growth promoting rhizobacteria isolated from polluted soils and
containing 1-aminocyclopropane-1-carboxylate deaminase. Can J Microbiol 652: 642–652.
Blackwell, P., E. Krull, G. Butler, A. Herbert, and Z. Solaiman. 2010. Effect of banded biochar on
dryland wheat production and fertiliser use in south-western Australia: An agronomic and
economic perspective. Australian Journal of Soil Research. p. 531–545.
Borch, K., T.J. Bouma, J.P. Lynch, and K.M. Brown. 1999. Ethylene: A regulator of root
137
architectural responses to soil phosphorus availability. Plant, Cell Environ. 22(4): 425–431.
Bos, C., B. Juillet, H. Fouillet, L. Turlan, S. Daré, et al. 2005. Postprandial metabolic utilization
of wheat protein in humans. Am. J. Clin. Nutr. 81(1): 87–94.
Boutraa, T., A. Akhkha, A.A. Al-Shoaibi, and A.M. Alhejeli. 2010. Effect of water stress on
growth and water use efficiency (WUE) of some wheat cultivars (Triticum durum) grown in
Saudi Arabia. J. Taibah Univ. Sci. 3: 39–48.
Chan, K.Y., L. Van Zwieten, I. Meszaros, A. Downie, and S. Joseph. 2008. Using poultry litter
biochars as soil amendments. Aust. J. Soil Res. 46(5): 437–444.
Chandra, D., R. Srivastava, and A.K. Sharma. 2018. Influence of IAA and ACC Deaminase
Producing Fluorescent Pseudomonads in Alleviating Drought Stress in Wheat (Triticum
aestivum). Agric. Res. 7: 290–299.
Chapman, H.D., and P.F. Pratt. 1961. Methods of analysis for soils, plants and water. University
of California, Division of Agricultural Sciences, Berkeley, CA, USA.
Chaudhary, D.P., S. Kumar, and O.P. Yadav. 2014. Nutritive Value of Maize: Improvements,
Applications and Constraints. Maize: Nutrition Dynamics and Novel Uses. Springer India,
New Delhi. p. 3–17.
Chaudhry, A. 1983. Agronomy in “Maize in Pakistan” Punjab Agriculture Coordination board

Univ. Agri. Faisalabad, Pakistan.
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(4): 551–560.
Chen, Y., Y. Shinogi, and M. Taira. 2010. Influence of biochar use on sugarcane growth, soil
parameters, and groundwater quality. Aust. J. Soil Res. 48(6-7): 526–530.
Danish, S., A. Ameer, T.I. Qureshi, U. Younis, H. Manzoor, et al. 2014. Influence of biochar on
growth and photosynthetic attributes of Triticum aestivum L. under half and full irrigation.
138
Int. J. Biosci. 5(7): 101–108.
Danish, S., U. Younis, N. Akhtar, A. Ameer, M. Ijaz, et al. 2015. Phosphorus solubilizing bacteria
and rice straw biochar consequence on maize pigments synthesis. Int. J. Biosci. 5(12): 31–
39.
Danish, S., and M. Zafar-ul-Hye. 2019. Co-application of ACC-deaminase producing PGPR and
timber-waste biochar improves pigments formation, growth and yield of wheat under drought
stress. Sci. Rep. 9: 5999.
Danish, S., M. Zafar-ul-Hye, M. Hussain, M. Shaaban, and A. Núñez-delgado. 2019.

Rhizobacteria with acc-deaminase activity improve nutrient uptake, chlorophyll contents and
early seedling growth of wheat under peg- induced osmotic stress. Int. J. Agric. Biol. 21(6):
1212–1220.
Decai, G., Z. Lei, L. Qiang, R. Xiangmin, Z. Yuping, et al. 2014. Application of biochar in dryland
soil decreasing loss of nitrogen and improving nitrogen using rate. Trans. Chinese Soc. Agric.
Eng. 30(6): 54–61.
Deng, X.P., L. Shan, S. Inanaga, and M. Inoue. 2005. Water-saving approaches for improving
wheat production. J. Sci. Food Agric. 85(8): 1379–1388.
Desclaux, D., and P. Roumet. 1996. Impact of drought stress on the phenology of two soybean
(Glycine max L. Merr) cultivars. F. Crop. Res. 46(1): 61–70.
Drogue, B., E. Combes-Meynet, Y. Moënne-Loccoz, F. Wisniewski-Dyé, and C. Prigent-

Combaret. 2013. Control of the Cooperation Between Plant Growth-Promoting Rhizobacteria
and Crops by Rhizosphere Signals. In: Bruijn, F.J. de, editor, Molecular Microbial Ecology
of the Rhizosphere. Wiley-Blackwell. p. 279–293
Dworkin, M., and J.W. Foster. 1958. Experiments with some microorganisms which utilize ethane
and hydrogen. J. Bacteriol. 75(5): 592–603.
Edmeades, G.O., J. Bolanos, M. Hernandez, and S. Bello. 1993. Causes for silk delay in a lowland
139
tropical maize population. Crop Sci. 33(5): 1029–1035.
El-Tarabily, K.A. 2008. Promotion of tomato (Lycopersicon esculentum Mill.) plant growth by
rhizosphere competent 1-aminocyclopropane-1-carboxylic acid deaminase-producing
streptomycete actinomycetes. Plant Soil 308(1-2): 161–174.
Fahad, S., A.A. Bajwa, U. Nazir, S.A. Anjum, A. Farooq, et al. 2017. Crop Production under
Drought and Heat Stress: Plant Responses and Management Options. Front. Pant Sci. 8: 1147.
file:///C:/Users/Delphine/Downloads/fpls-08-01147 (1).pdf.
FAO. 2003. Basic facts of the world cereal situation, Food Outlook No.4. Food and Agriculture
Organization (FAO) of the United Nations. Rome.
Farhad, W., M. Cheema, M. Saleem, and M. Saqib. 2011. Evaluation of drought tolerance in maize
hybrids. Int. J Agric. Biol 13: 523–528.
Farooq, M., S.M.A. Basra, A. Wahid, N. Ahmad, and B.A. Saleem. 2009. Improving the drought
tolerance in rice (Oryza sativa L.) by exogenous application of salicylic acid. J. Agron. Crop
Sci. 195(4): 237–246.
Fiaz, K., S. Danish, U. Younis, S.A. Malik, M.H. Raza Shah, et al. 2014. Drought impact on Pb/Cd
toxicity remediated by biochar in Brassica campestris. J. soil Sci. plant Nutr. 14(4): 845–854.
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(3): 269–
279.
Gamalero, E., and B.R. Glick. 2011. Mechanisms used by plant growth-promoting bacteria.
Bacteria in Agrobiology: Plant Nutrient Management. p. 17–47.
Gargallo-Garriga, A., J. Sardans, M. Pérez-Trujillo, A. Rivas-Ubach, M. Oravec, et al. 2014.

Opposite metabolic responses of shoots and roots to drought. Sci. Rep. 4: 6829.
Gee, G.W., and J.W. Bauder. 1986. Particle-size analysis. Methods of soil analysis. Part 1. Physical
140
and mineralogical methods. 2nd ed. Madison. p. 383–411.
Glaser, B., L. Haumaier, G. Guggenberger, and W. Zech. 2001. The “Terra Preta” phenomenon:
A model for sustainable agriculture in the humid tropics. Naturwissenschaften 88(1): 37–41.
Glaser, B., J. Lehmann, and W. Zech. 2002. Ameliorating physical and chemical properties of
highly weathered soils in the tropics with charcoal - a review. Biol. Fertil. Soils 35(4): 219–
230.
Glick, B.R. 2004. Bacterial ACC deaminase and the alleviation of plant stress. Adv. Appl.
Microbiol. 56: 291–312.
Glick, B.R., J. Li, S. Shah, D.M. Penrose, B.A. Moffatt, et al. 1999a. ACC deaminase is central to
the functioning of plant growth promoting rhizobacteria. In: Kanellis, A.K., Chang, C., H.,
K., A.B., B., J.C., P., et al., editors, Biology and Biotechnology of the Plant Hormone
Ethylene II. Springer, Dordrecht. p. 293–298
Glick, B.R., C. Liu, S. Ghosh, and E.B. Dumbroff. 1997. Early development of canola seedlings
in the presence of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2.
Soil Biol. Biochem. 29(8): 1233–1239.
Glick, B.R., C.L. Patten, G. Holguin, and D.M. Penrose. 1999b. Biochemical and Genetic
Mechanisms Used by Plant Growth Promoting Bacteria. Imperial College Press, London,
UK.
Glick, B., D. Penrose, and J. Li. 1998. A Model For the Lowering of Plant Ethylene Concentrations
by Plant Growth-promoting Bacteria. J. Theor. Biol. 190(1): 63–68.
Glickmann, E., and Y. Dessaux. 1995. A critical examination of the specificity of the Salkowski
reagent for indolic compounds produced by phytopathogenic bacteria. Appl. Environ.
Microbiol. 61(2): 793–796.
GOP. 2017. Wheat Production Technology. : 9–10. http://dai.agripunjab.gov.pk/croptechnologies.
141
Griffin, M.T., B.E. Montz, and J. S. Arrigo. 2013. Evaluating Climate Change Induced Water
Stress: A Case Study of the Lower Cape Fear basin, NC. Appl. Geogr. 40: 115–128.
Gundale, M.J., and T.H. DeLuca. 2006. Charcoal effects on soil solution chemistry and growth of
Koeleria macrantha in the ponderosa pine/Douglas-fir ecosystem. Biol. Fertil. Soils 43(3):
303–311.
Hartmann, M., A. Fliessbach, H.-R. Oberholzer, and F. Widmer. 2006. Ranking the magnitude of
crop and farming system effects on soil microbial biomass and genetic structure of bacterial
communities. FEMS Microbiol. Ecol. 57(3): 378–88.
Hassan, W., S. Bashir, F. Ali, M. Ijaz, M. Hussain, et al. 2016. Role of ACC-deaminase and/or
nitrogen fixing rhizobacteria in growth promotion of wheat (Triticum aestivum L.) under
cadmium pollution. Environ. Earth Sci. 75(3): 267.
Hassan, W., M. Hussain, S. Bashir, A.N. Shah, R. Bano, et al. 2015. ACC-deaminase and / or
nitrogen fixing rhizobacteria and growth of wheat (Triticum aestivum L.). J. Soil Sci. Plant
Nutr. 15(1): 232–248.
He, C.J., P.W. Morgan, M.C. Drew. 1996. Transduction of an ethylene signal is required for cell
death and lysis in the root cortex of maize during aerenchyma formation induced by hypoxia,
Plant Physiol. 112: 463-472.
Hellal, F.A., H.M. El-Shabrawi, M.A. El-Hady, I.A. Khatab, S.A.A. El-Sayed, et al. 2018.
Influence of PEG induced drought stress on molecular and biochemical constituents and
seedling growth of Egyptian barley cultivars. J. Genet. Eng. Biotechnol. 16(1): 203–212.
Hewitt, K. 1997. Regions of Risk  : A Geographical Introduction to Disasters. Themes in resource

management. p. 389.
Hoagland, D.R., and D.I. Arnon. 1950. The water culture method for growing plants without soil.
University of California, College of Agriculture, Agricultural Experiment Station. 2nd ed.
Berkeley, CA. Circular. p. 374.
142
Hoekstra, F.A., E.A. Golovina, and J. Buitink. 2001. Mechanisms of plant desiccation tolerance.
Trends Plant Sci. 6(9): 431–438.
Honma, M., and T. Shimomura. 1978. Metabolism of 1-aminocyclopropane-1-carboxylic acid.

Agric. Biol. Chem. 42(10): 1825–1831.
Huang, L., M. Li, Y. Shao, T. Sun, C. Li, et al. 2018. Ammonium uptake increases in response to
PEG-induced drought stress in Malus hupehensis Rehd. Environ. Exp. Bot. 151: 32–42.
Hunter, A. 2016. The effect of population growth on efficiency in food production. Food Beverage.
https://foodmag.com.au/the-effect-of-population-growth-on-efficiency-in-food-production/.
Hussain, M., S. Farooq, W. Hasan, S. Ul-Allah, M. Tanveer, et al. 2018. Drought stress in
sunflower: Physiological effects and its management through breeding and agronomic
alternatives. Agric. Water Manag. 201: 152–167.
Hussain, M., M.A. Malik, M. Farooq, M.B. Khan, M. Akram, et al. 2009. Exogenous
glycinebetaine and salicylic acid application improves water relations, allometry and quality
of hybrid sunflower under water deficit conditions. J. Agron. Crop Sci. 195(2): 98–109.
IPCC. 2007. Climate Change 2007: The Physical Science Basis.Vol 1009. Cambridge University
Press, Cambridge.
Ippolito, J.A., D.A. Laird, and W.J. Busscher. 2012. Environmental benefits of biochar. J. Environ.
Qual. 41(4): 967–72.
Iqbal, H., M. Garcia-Perez, and M. Flury. 2015. Effect of biochar on leaching of organic carbon,
nitrogen, and phosphorus from compost in bioretention systems. Sci. Total Environ. 521-522:
37–45.
Iqbal, N., R. Nazar, S. Syeed, A. Masood, and N.A. Khan. 2011. Exogenously-sourced ethylene
increases stomatal conductance, photosynthesis, and growth under optimal and deficient
nitrogen fertilization in mustard. J. Exp. Bot. 62(14): 4955–4963.
143
Izzo, R., F. Navari‐ Izzo, and M.F. Quartacci. 1989. Growth and mineral content of roots and
shoots of maize seedlings in response to increasing water deficits induced by PEG solutions.
J. Plant Nutr. 12(10): 1175–1193.
Jalili, F., K. Khavazi, E. Pazira, A. Nejati, H.A. Rahmani, et al. 2009. Isolation and characterization
of ACC deaminase-producing fluorescent pseudomonads, to alleviate salinity stress on canola
(Brassica napus L.) growth. J. Plant Physiol. 166(6): 667–674.
Jahan, S., S. Iqbal, F. Rasul, and K. Jabeen. 2020. Efficacy of biochar as soil amendments for
soybean (Glycine max L.) morphology, physiology, and yield regulation under drought. Arab.
J. Geosci. 13 (356): https://doi.org/10.1007/s12517–020–05318–6.
Jiang, F., L. Chen, A.A. Belimov, A.I. Shaposhnikov, F. Gong, et al. 2012. Multiple impacts of
the plant growth-promoting rhizobacterium Variovorax paradoxus 5C-2 on nutrient and ABA
relations of Pisum sativum. J. Exp. Bot. 63(18): 6421–6430.
Johnson, P.R., and J.R. Ecker. 1998. The ethylene gas signal transduction pathway: a molecular
perspective. Annu. Rev. Genet. 32: 227–254.
Jones, J.B., B. WolfH, and H.A. Mills. 1991. Plant Analysis Handbook: A Practical Sampling,
Preparation, Analysis, and Interpretation Guide. Micro-Macro Publishing Inc., Athens, GA,
USA.
Kammann, C.I., S. Linsel, J.W. Gößling, and H.W. Koyro. 2011. Influence of biochar on drought
tolerance of Chenopodium quinoa Willd and on soil-plant relations. Plant Soil 345(1): 195–
210.
Keshavarz, A.R., M. Hashemi, M. DaCosta, J. Spargo, and A. Sadeghpour. 2016. Biochar

Application and Drought Stress Effects on Physiological Characteristics of Silybum
marianum. Commun. Soil Sci. Plant Anal. 47(6): 743–752.
Kiani, S.P., P. Talia, P. Maury, P. Grieu, R. Heinz, et al. 2007. Genetic analysis of plant water
status and osmotic adjustment in recombinant inbred lines of sunflower under two water
144
treatments. Plant Sci. 172(4): 773–787.
Knight, L.I., and W. Crocker. 1913. Toxicity of Smoke. Bot. Gaz. 55(5): 337–371.
Kogan, F.N. 1997. Global Drought Watch from Space. Bull. Am. Meteorol. Soc. 78(4): 621–636.
Lambers, H., F.S. Chapin, and T.L. Pons. 2008. Plant Physiological Ecology. 2nd ed. Springer,
New York.
Lehmann, J., and M. Rondon. 2002. Bio-Char Soil Management on Highly Weathered Soils in the
Humid Tropics. In: Uphoff, N., editor, Biological Approaches to Sustainable Soil Systems.
CRC Press, Boca Raton, FL. p. 517–530.
Lehmann, J. 2007. Bio-energy in the black. Front. Ecol. Environ. 5(7): 381–387.
Lehmann, J., J. Gaunt, and M. Rondon. 2006. Bio-char Sequestration in Terrestrial Ecosystems –
A Review. Mitig. Adapt. Strateg. Glob. Chang. 11(2): 395–419.
Lehmann, J., M.C. Rillig, J. Thies, C. a. Masiello, W.C. Hockaday, et al. 2011. Biochar effects on
soil biota – A review. Soil Biol. Biochem. 43(9): 1812–1836.
Liang, B., J. Lehmann, D. Solomon, J. Kinyangi, J. Grossman, et al. 2006. Black Carbon Increases
Cation Exchange Capacity in Soils. Soil Sci. Soc. Am. J. 70(5): 1719–1730.
Ludlow, M.M., and R.C. Muchow. 1990. A Critical Evaluation of Traits for Improving Crop
Yields in Water-Limited Environments. Adv. Agron. 43(C): 107–153.
Lutts, S., J.M. Kinet, and J. Bouharmont. 1996. NaCl-induced Senescence in Leaves of Rice
(Oryza sativa L.) Cultivars Differing in Salinity Resistance. Ann. Bot. 78(3): 389–398.
Manivannan, P., C.A. Jaleel, R. Somasundaram, and R. Panneerselvam. 2008. Osmoregulation

and antioxidant metabolism in drought-stressed Helianthus annuus under triadimefon
drenching. Comptes Rendus - Biol. 331(6): 418–425.
Marris, E. 2006. Putting the carbon back: Black is the new green. Nature Publishing Group.
145
Matile, P., M. Schellenberg, and F. Vicentini. 1997. Planta Localization of chlorophyllase in the
chloroplast envelope. Planta 201: 96–99.
Mayak, S., T. Tirosh, and B.R. Glick. 1999. Effect of Wild-Type and Mutant Plant Growth-
Promoting Rhizobacteria on the Rooting of Mung Bean Cuttings. J. Plant Growth Regul.
18(2): 49–53.
Mayak, S., T. Tirosh, and B.R. Glick. 2004. Plant growth-promoting bacteria that confer resistance
to water stress in tomatoes and peppers. Plant Sci. 166(2): 525–530.
Mehran, A., A. AghaKouchak, N. Nakhjiri, M.J. Stewardson, M.C. Peel, et al. 2017. Compounding
Impacts of Human-Induced Water Stress and Climate Change on Water Availability. Sci.
Rep. 7(1): 6282.
Mir, R.R., M. Zaman-Allah, N. Sreenivasulu, R. Trethowan, and R.K. Varshney. 2012. Integrated
genomics, physiology and breeding approaches for improving drought tolerance in crops.
Theor. Appl. Genet. 125(4): 625–645.
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. Agric. For. Meteorol.
150(7-8): 1030–1045.
Mohite, B. 2013. Isolation and characterization of indole acetic acid (IAA) producing bacteria
from rhizospheric soil and its effect on plant growth. J. Sci. Plant Nutr. 13(3): 638–649.
Morgan, P.W., and M.C. Drew. 1997. Ethylene and plant responses to stress. Physiol. Plant.
100(3): 620–630.
Mulcahy, D.N., D.L. Mulcahy, and D. Dietz. 2013. Biochar soil amendment increases tomato
seedling resistance to drought in sandy soils. J. Arid Environ. 88: 222–225.
Nadeem, F., R. Ahmad, M.I.A. Rehmani, A. Ali, M. Ahmad, et al. 2013. Qualitative and Chemical
Analysis of Rice Kernel To Time of Application of Phosphorus in Combination With Zinc
Under Anaerobic Conditions. Asian J. Agric. Biol. 1(2): 67–75.
146
Nadeem, S.M., M. Imran, M. Naveed, M.Y. Khan, M. Ahmad, et al. 2017. Synergistic use of
biochar, compost and plant growth-promoting rhizobacteria for enhancing cucumber growth
under water deficit conditions. J. Sci. Food Agric. 97(15): 5139–5145.
Nadeem, S.M., Z.A. Zahir, M. Naveed, and M. Arshad. 2009. Rhizobacteria containing ACC-
deaminase confer salt tolerance in maize grown on salt-affected fields. Can. J. Microbiol.
55(11): 1302–1309.
Navia, R., and D.E. Crowley. 2010. Closing the loop on organic waste management: biochar for
agricultural land application and climate change mitigation. Waste Manag. Res. 28: 479–480.
Naz, I., A. Rehim, M. Zafar-ul-Hye, Z.A. Zahir, M. Abid, et al. 2013. Effectiveness of ACC-
deaminase containing Pseudomonas strains to induce salinity tolerance in maize under
fertilized and unfertilized field conditions. Soil Environ. 32(2): 167–172.
Nazar, R., M.I.R. Khan, N. Iqbal, A. Masood, and N.A. Khan. 2014. Involvement of ethylene in
reversal of salt-inhibited photosynthesis by sulfur in mustard. Physiol. Plant. 152(2): 331–
344.
Ngumbi, E., and J. Kloepper. 2016. Bacterial-mediated drought tolerance: Current and future
prospects. Appl. Soil Ecol. 105: 109–125.
Niu, X., L. Song, Y. Xiao, and W. Ge. 2018. Drought-tolerant plant growth-promoting
rhizobacteria associated with foxtail millet in a semi-arid and their potential in alleviating
drought stress. Front. Microbiol. 8(JAN). doi: 10.3389/fmicb.2017.02580.
Noor, N.M., A. Shariff, and N. Abdullah. 2012. Slow Pyrolysis of Cassava Wastes for Biochar
Production and Characterization. Iran. J. Energy Environ. 3: 60–65.
Novak, J.M., I. Lima, B. Xing, J.W. Gaskin, C. Steiner, et al. 2009. Characterization of designer
biochar produced at different temperatures and their effects on a loamy sand. Ann. Environ.
Sci. 3(843): 195–206.
Olsen, S.R., and L.E. Sommers. 1982. Phosphorus. In: Page, A.L., editor, Method of soil analysis,
147
Agron. No. 9, part 2: Chemical and microbiological properties. 2nd ed. American Society of
Agronomy, Madison, WI, USA. p. 403–430
Ortíz-Castro, R., E. Valencia-Cantero, and J. López-Bucio. 2008. Plant growth promotion by

Bacillus megaterium involves cytokinin signaling. Plant Signal. Behav. 3(April): 263–265.
Ouji, A., S. El-Bok, M. Mouelhi, M. Ben Younes, and M. Kharrat. 2016. Yield and Yield
Components of Chickpea (Cicer arietinum L.) as Influenced by Supplemental Irrigation
under Semi-arid Region of Tunisia. World J. Agric. Res. 4(5): 153–157.
Paetsch, L., C.W. Mueller, I. Kögel-Knabner, M. Von Lützow, C. Girardin, et al. 2018. Effect of
in-situ aged and fresh biochar on soil hydraulic conditions and microbial C use under drought
conditions. Sci. Rep. 8(1). 6852.
Paul, S., C. Aggarwal, B.. Manjunatha, and M.S. Rathi. 2018. Characterization of osmotolerant
rhizobacteria for plant growth promoting activities in vitro and during plant-microbe
association under osmotic stress. Indian J. Exp. Biol. 56(8): 582–589.
Peters, G.P., G. Marland, C. Le Quéré, T. Boden, J.G. Canadell, et al. 2012. Rapid growth in CO2
emissions after the 2008-2009 global financial crisis. Nat. Clim. Chang. 2(1): 2–4.
Pikovskaya, R.I. 1948. Mobilization of phosphorus in soil in connection with vital activity of some
microbial species. Microbiology 17: 362–370.
Piwowarczyk, B., I. Kamińska, and W. Rybiński. 2014. Influence of PEG generated osmotic stress
on shoot regeneration and some biochemical parameters in Lathyrus culture. Czech J. Genet.
Plant Breed. 50(2): 77–83.
Qayyum, M.F., M. Abid, S. Danish, M.K. Saeed, and M.A. Ali. 2014. Effects of various biochars
on seed germination and carbon mineralization in an alkaline soil. Pakistan J. Agric. Sci.
51(4): 977–982.
Qayyum, M.F., D. Steffens, H.P. Reisenauer, and S. Schubert. 2012. Kinetics of Carbon
Mineralization of Biochars Compared with Wheat Straw in Three Soils. J. Environ. Qual.
148
41(4): 1210.
Reid, J.B., and A.R. Renquist. 1997. Enhanced root production as a feed-forward response to soil
water deficit in field-grown tomatoes. Aust. J. Plant Physiol. 24(5): 685–692.
Richards, L.A. 1954. Diagnosis and improvement of saline and alkali soils. Washington DC, USA.
Richardson, A.E., P.J. Hocking, R.J. Simpson, and T.S. George. 2009. Plant mechanisms to
optimise access to soil phosphorus. Crop Pasture Sci. 60(2): 124–143.
Sadiq, A., and B. Ali. 2013. Growth and yield enhancement of Triticum aestivum L. by
rhizobacteria isolated from agronomic plants. Aust. J. Crop Sci. 7(10): 1544–1550.
Safronova, V.I., V. V. Stepanok, G.L. Engqvist, Y. V. Alekseyev, and A.A. Belimov. 2006. Root-
associated bacteria containing 1-aminocyclopropane-1-carboxylate deaminase improve
growth and nutrient uptake by pea genotypes cultivated in cadmium supplemented soil. Biol.
Fertil. Soils 42(3): 267–272.
Saikia, J., R.K. Sarma, R. Dhandia, A. Yadav, R. Bharali, et al. 2018. Alleviation of drought stress
in pulse crops with ACC deaminase producing rhizobacteria isolated from acidic soil of
Northeast India. Sci. Rep. 8(1): 3560.
Saleem, M., M. Arshad, S. Hussain, and A.S. Bhatti. 2007. Perspective of plant growth promoting
rhizobacteria (PGPR) containing ACC deaminase in stress agriculture. J. Ind. Microbiol.
Biotechnol. 34(10): 635–648.
Saraf, M., S. Vurukonda, S.S.K.P. Vardharajula, M. Shrivastava, A. SkZ, C.K. Jha, et al. 2010.
The Role of ACC Deaminase Producing PGPR in Sustainable Agriculture. In: Maheshwari,
D., editor, Plant Growth and Health Promoting Bacteria. Springer, Berlin, Heidelberg. p. 365–
385.
Sattar, A., A. Sher, M. Ijaz, S. Ul-Allah, M. Butt, et al. 2020. Interactive Effect of Biochar and
Silicon on Improving Morpho-Physiological and Biochemical Attributes of Maize by
Reducing Drought Hazards. J. Soil Sci. Plant Nutr.: https://doi.org/10.1007/s42729–020–
149
00253–7.
Schachtman, D.P., and J.Q.D. Goodger. 2008. Chemical root to shoot signaling under drought.
Trends Plant Sci. 13(6): 281–287.
Schmidt, R., M. Köberl, A. Mostafa, E.M. Ramadan, M. Monschein, K.B. Jensen, R. Bauer, and
G. Berg. 2014. Effects of bacterial inoculants on the indigenous microbiome and secondary
metabolites of chamomile plants. Front. Microbiol. 5: 64.
Senaratna, T., and B.D. McKersie. 1983. Characterization of Solute Efflux from Dehydration
Injured Soybean (Glycine max L. Merr) Seeds. Plant Physiol. 72(4): 911–4.
Setiawati, T.C., and L. Mutmainnah. 2016. Solubilization of potassium containing mineral by

microorganisms from sugarcane rhizosphere. Ital. Oral Surg. 9: 108–117.
Shabala, S. 2003. Regulation of potassium transport in leaves: from molecular to tissue level. Ann.
Bot. 95(5): 627–634.
Shafie, S.T., M.A. Mohd, L.L. Hang, W. Azlina, W. Abdul, et al. 2012. Effect of pyrolysis
temperature on the biochar nutrient and water retention capacity. J. Purity, Util. React.
Environ. 1(6): 323–337.
Shah, S., J. Li, B. a Moffatt, and B.R. Glick. 1998. Isolation and characterization of ACC
deaminase genes from two different plant growth-promoting rhizobacteria. Can. J. Microbiol.
44(9): 833–843.
Shaharoona, B., M. Arshad, and Z.A. Zahir. 2006. Effect of plant growth promoting rhizobacteria
containing ACC-deaminase on maize (Zea mays L.) growth under axenic conditions and on
nodulation in mung bean (Vigna radiata L.). Lett. Appl. Microbiol. 42(2): 155–159.
Shahzad, S.M., A. Khalid, M. Arshad, and Kalil-ur-Rehman. 2013. Screening rhizobacteria

containing ACC-deaminase for growth promotion of wheat under water stress. Pakistan J.
Bot. 45(SPL.ISS): 91–96.
150
Shenbagavalli, S., and S. Mahimairaja. 2012. Production and characterization of biochar from
different biological wastes. Int. J. Plant, Anim. Environ. Sci. 2(1): 197–201.
Shukla, K.P., S. Sharma, N.K. Singh, V. Singh, and K. Tiwari. 2011. Nature and role of root
exudates  : Efficacy in bioremediation. African J. Biotechnol. 10(48): 9717–9724.
Siddikee, M.A., P.S. Chauhan, R. Anandham, G.H. Han, and T. Sa. 2010. Isolation,
characterization, and use for plant growth promotion under salt stress, of ACC deaminase-
producing halotolerant bacteria derived from coastal soil. J. Microbiol. Biotechnol. 20(11):
1577–1584.
Siddikee, M.A., B.R. Glick, P.S. Chauhan, W. jong Yim, and T. Sa. 2011. Enhancement of growth
and salt tolerance of red pepper seedlings (Capsicum annuum L.) by regulating stress ethylene
synthesis with halotolerant bacteria containing 1-aminocyclopropane-1-carboxylic acid
deaminase activity. Plant Physiol. Biochem. 49(4): 427–434.
Siddique, M.R.B., A. Hamid, and Islam. 1999. Drought stress effects on photosynthetic rate and
leaf gas exchange of wheat. Bot. Bull. Acad. Sin. 40: 141–145.
Singh, G.P., and H.B. Chaudhary. 2006. Selection parameters and yield enhancement of wheat
(Triticum aestivum L.) under different moisture stress conditions. Asian J. Plant Sci. 5: 894–
898.
Singh, B., B.P. Singh, and A.L. Cowie. 2010. Characterisation and evaluation of biochars for their
application as a soil amendment. Aust. J. Soil Res. 48(6-7): 516–525.
Singh, A., A.P. Singh, S.K. Singh, S. Rai, and D. Kumar. 2016. Impact of addition of biochar
along with pgpr on rice yield, availability of nutrients and their uptake in alluvial soil. J. Pure
Appl. Microbiol. 10(3): 2181–2188.
Skirycz, A,, H. Claeys, S. De Bodt, A. Oikawa, S. Shinoda, M. Andriankaja, K. Maleux, N.B.

Eloy, F. Coppens , S.D.Yoo, et al. 2011. Pause-and-stop: the effects of osmotic stress on cell
proliferation during early leaf development in Arabidopsis and a role for ethylene signaling
151
in cell cycle arrest. Plant Cell 23: 1876–1888.
Sobeih, W.Y., I.C. Dodd, M.A. Bacon, D. Grierson, and W.J. Davies. 2004. Long-distance signals
regulating stomatal conductance and leaf growth in tomato (Lycopersicon esculentum) plants
subjected to partial root-zone drying. J. Exp. Bot. 55(407): 2353–2363.
Spokas, K.A., J.M. Baker, and D.C. Reicosky. 2010. Ethylene: Potential key for biochar
amendment impacts. Plant Soil 333(1): 443–452.
Stearns, J.C., O.Z. Woody, B.J. McConkey, and B.R. Glick. 2012. Effects of bacterial ACC
deaminase on Brassica napus gene expression. Mol. Plant. Microbe. Interact. 25(5): 668–
676.
Steel, R.G., J.H. Torrie, and D.A. Dickey. 1997. Principles and Procedures of Statistics: A
Biometrical Approach. 3rd ed. McGraw Hill Book International Co., Singapore.
Stefan, M., N. Munteanu, V. Stoleru, and M. Mihasan. 2013. Effects of inoculation with plant
growth promoting rhizobacteria on photosynthesis, antioxidant status and yield of runner
bean. Rom. Biotechnol. Lett. 18(2): 8132–8143.
Steiner, C., B. Glaser, W.G. Teixeira, J. Lehmann, W.E.H. Blum, et al. 2008. Nitrogen retention
and plant uptake on a highly weathered central Amazonian Ferralsol amended with compost
and charcoal. J. Plant Nutr. Soil Sci. 171(6): 893–899.
Subramanian, K.S., P. Santhanakrishnan, and P. Balasubramanian. 2006. Responses of field grown

tomato plants to arbuscular mycorrhizal fungal colonization under varying intensities of
drought stress. Sci. Hortic. (Amsterdam). 107(3): 245–253.
Taiz, L., and E. Zeiger. 2010. Plant physiology. 5th ed. Sinauer Associates Inc., Publishers, MA,
USA.
Tamimi, S.M., and M.P. Timko. 2003. Effects of ethylene and inhibitors of ethylene synthesis and
action on nodulation in common bean (Phaseolus vulgaris L.). Plant Soil 257(1): 125–131.
152
Tanaka, Y., T. Sano, M. Tamaoki, N. Nakajima, N. Kondo, et al. 2005. Ethylene inhibits abscisic
acid-induced stomatal closure in Arabidopsis. Plant Physiol. 138(4): 2337–2343.
Tholen, D., T.L. Pons, L.A. Voesenek, and H. Poorter. 2008. The role of ethylene perception in
the control of photosynthesis. Plant Signal. Behav. 3(2): 108–109.
Tian, J., J. Wang, M. Dippold, Y. Gao, E. Blagodatskaya, et al. 2016. Biochar affects soil organic
matter cycling and microbial functions but does not alter microbial community structure in a
paddy soil. Sci. Total Environ. 556: 89–97.
Timmusk, S., G. Seisenbaeva, and L. Behers. 2018. Titania (TiO2) nanoparticles enhance the
performance of growth-promoting rhizobacteria. Sci. Rep. 8(1): 617.
Vazquez, P., G. Holguin, M.E. Puente, A. Lopez-Cortes, and Y. Bashan. 2000. Phosphate-
solubilizing microorganisms associated with the rhizosphere of mangroves in a semiarid
coastal lagoon. Biol. Fertil. Soils 30(5-6): 460–468.
Van Schouwenberg, J.C., and I. Walinge. 1973. Methods of Analysis for Plant Material.
Agricultural University Wageningen, Wageningen, The Netherlands.
Verheijen, F., S. Jeffery, A.C. Bastos, M. Van Der Velde, and I. Diafas. 2010. Biochar Application
to Soils. A Critical Scientific Review of Effects on Soil Properties, Processes and Functions.
Environment 8: 149.
Vurukonda, S.S.K.P., S. Vardharajula, M. Shrivastava, and A. SkZ. 2016. Enhancement of drought

stress tolerance in crops by plant growth promoting rhizobacteria. Microbiol. Res. 184: 13–
24.
Wada, Y., D. Wisser, S. Eisner, M. Flörke, D. Gerten, et al. 2013. Multimodel Projections and
Uncertainties of Irrigation Water Demand Under Climate Change. Geophys. Res. Lett.
40(17): 4626–4632.
Walkley, A. 1935. An Examination of Methods for Determining Organic Carbon and Nitrogen in
Soils. J. Agric. Sci. 25: 598.
153
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): 1–14.
Warnock, D.D. 2009. Arbuscular mycorrhizal responses to biochars in soils - potential

mechanisms of interaction and observed responses in controlled environments. Graduate
Student Theses, Dissertations, & Professional Papers. 1310. University of Montana.
https://scholarworks.umt.edu/etd/1310/.
Wilhite, D. 2000. Drought:a global assessment. In: D.A. Wilhite, editor, Drought as a natural
hazard. Routledge, London. p. 3–18.
Wilkinson, S., and W.J. Davies. 2002. ABA‐ based chemical signalling: the co‐ ordination of
responses to stress in plants. Plant. Cell Environ. 25(2): 195–210.
Wu, C., Z. Wang, H. Sun, and S. Guo. 2006. Effects of different concentrations of nitrogen and
phosphorus on chlorophyll biosynthesis, chlorophyll a fluorescence, and photosynthesis in
Larix olgensis seedlings. Front. For. China 1(2): 170–175.
Xie, H., J.J. Pasternak, and B.R. Glick. 1996. Isolation and characterization of mutants of the plant
growth-promoting rhizobacterium Pseudomonas putida GR12-2 that overproduce
indoleacetic acid. Curr. Microbiol. 32(2): 67–71.
Yamaguchi-Shinozaki, K., and K. Shinozaki. 2006. Transcriptional regulatory networks in cellular

responses and tolerance to dehydration and cold stresses. Annu. Rev. Plant Biol. 57(1): 781–
803.
Yamane, K., K. Hayakawa, M. Kawasaki, M. Taniguchi, and H. Miyake. 2003. Bundle sheath
chloroplasts of rice are more sensitive to drought stress than mesophyll chloroplasts. J. Plant
Physiol. 160(11): 1319–27.
Younis, U., S. Danish, M.H.R. Shah, and S.A. Malik. 2014a. Nutrient shifts modeling in Spinacea
oleracea L. and Trigonella corniculata L. in contaminated soil amended with biochar. Int. J.
Biosci. 5(9): 89–98.
154
Younis, U., M.F. Qayyum, M.H.R. Shah, S. Danish, A.N. Shahzad, et al. 2015. Growth, survival,
and heavy metal (Cd and Ni) uptake of spinach (Spinacia oleracea) and fenugreek (Trigonella
corniculata) in a biochar-amended sewage-irrigated contaminated soil. J. Plant Nutr. Soil Sci.
178(2): 209–217.
Younis, U., M.H.R. Shah, S. Danish, S.A. Malik, and A. Ameer. 2014b. Biochar role in improving
biometric and growth attributes of S. oleracea and T. corniculata under cadmium stress. Int.
J. Biosci. 5(8): 84–90.
Yu, O.Y., B. Raichle, and S. Sink. 2013. Impact of biochar on the water holding capacity of loamy
sand soil. Int. J. Energy Environ. Eng. 4(1): 44. doi: 10.1186/2251-6832-4-44.
Zafar-ul-Hye, M., M.H. Farooq, and M. Hussain. 2015. Bacteria in combination with fertilizers
promote root and shoot growth of maize in saline-sodic soil. Brazilian J. Microbiol. 46(1):
97–102.
Zafar-ul-Hye, M., H.M. Farooq, Z.A. Zahir, M. Hussain, and A. Hussain. 2014. Application of
ACC-deaminase containing rhizobacteria with fertilizer improves maize production under
drought and salinity stress. Int. J. Agric. Biol. 16(3): 591–596.
Zafar-ul-Hye, M., A. Shahjahan, S. Danish, M. Abid, and M.F. Qayyum. 2018. Mitigation of
cadmium toxicity induced stress in wheat by ACC-deaminase containing PGPR isolated from
cadmium polluted wheat rhizosphere. Pakistan J. Bot. 50(5): 1727–1734.
Zafar-ul-Hye, M., Z.A. Zahir, S.M. Shahzad, M. Naveed, M. Arshad, et al. 2007. Preliminary
screening of rhizobacteria containing ACC-deaminase for promoting growth of lentil
seedlings under axenic condition. Pakistan J. Bot. 39(5): 1725–1738.
Zahir, Z.A., U. Ghani, M. Naveed, S.M. Nadeem, and H.N. Asghar. 2009. Comparative
effectiveness of Pseudomonas and Serratia sp. containing ACC-deaminase for improving
growth and yield of wheat (Triticum aestivum L.) under salt-stressed conditions. Arch.
Microbiol. 191(5): 415–424.
155
Zahir, Z.A., A. Munir, H.N. Asghar, B. Shaharoona, and M. Arshad. 2008. Effectiveness of
rhizobacteria containing ACC deaminase for growth promotion of peas (Pisum sativum)
under drought conditions. J. Microbiol. Biotechnol. 18(5): 958–963.
Zahir, Z.A., M. Zafar-ul-Hye, S. Sajjad, and M. Naveed. 2011. Comparative effectiveness of

Pseudomonas and Serratia sp. containing ACC-deaminase for coinoculation with Rhizobium
leguminosarum to improve growth, nodulation, and yield of lentil. Biol. Fertil. Soils 47(4):
457–465.
Zandalinas, S.I., R. Mittler, D. Balfagón, V. Arbona, and A. Gómez-Cadenas. 2018. Plant

adaptations to the combination of drought and high temperatures. Physiol. Plant. 162(1): 2–
12.
Zeiger, E., and L. Taiz. 2010. Plant Physiology. Sinauer Associates Inc., Publishers, Sunderland,
MA, USA.
Zhang, S., H. Kang, and W. Yang. 2017. Climate change-induced water stress suppresses the
regeneration of the critically endangered forest tree Nyssa yunnanensis. PLoS One 12(8):
e0182012.
Zhang, G., Y. Sun, H. Sheng, H. Li, and X. Liu. 2018. Effects of the inoculations using bacteria
producing ACC deaminase on ethylene metabolism and growth of wheat grown under
different soil water contents. Plant Physiol. 125: 178–184.
Zheng, Z., L.E. Parent, and J.A. MacLeod. 2003. Influence of soil texture on fertilizer and soil
phosphorus transformations in Gleysolic soils. Can. J. Soil Sci. 83(4): 395–403.
Zhao, S.X., T. Na , and X.D. Wang. 2017. Effect of temperature on the structural and
physicochemical properties of biochar with apple tree branches as feedstock material. Energ.
10: 1293.
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EFFECTS OF ACC-DEAMINASE CONTAINING

RHIZOBACTERIA AND BIOCHAR ON WHEAT AND

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE

DOCTOR OF PHILOSOPHY IN AGRICULTURE

DEPARTMENT OF SOIL SCIENCE

FACULTY OF AGRICULTURAL SCIENCES & TECHNOLOGY

BAHAUDDIN ZAKARIYA UNIVERSITY MULTAN

THE MOST BENEFICENT

THE MOST MERCIFUL”

I would like to acknowledge to my mother (Shabana Hameed) and especially my Father

THEY GIVE ME THE DIRECTION TOWARDS SUCCESS AND PRAYS FOR ME IN

LIST OF TABLES ..................................................................................................................... xvi

LIST OF FIGURES ................................................................................................................... xix

LIST OF ABBREVIATIONS ................................................................................................... xxi

REVIEW OF LITERATURE ......................................................................................................... 6

2.1. Climatic changes and food demand .................................................................................... 6

2.2. Drought ............................................................................................................................... 6

2.4. Plant growth promoting rhizobacteria ................................................................................ 8

2.5. Biochar ................................................................................................................................ 9

MATERIALS AND METHODS ............................................................................................... 12

3.1.1. Collection of Rhizosphere Soils ........................................................................................ 12

3.1.2. Isolation, Purification and Selection of Drought Tolerant Isolates ................................. 12

3.1.3. Experimental Details ........................................................................................................ 12

3.1.4. Growth and Chemical Analysis of Plants ......................................................................... 13

3.1.5. Chlorophyll Content.......................................................................................................... 14

3.1.6. Biochemical Characterization of Effective PGPR ............................................................ 14

3.1.7. Identification of PGPR ...................................................................................................... 14

3.1.8. ACC deaminase PGPR ..................................................................................................... 15

3.1.9. Biochar production ........................................................................................................... 15

3.1.11. Soil characterization ......................................................................................................... 16

3.1.12. Electrolyte Leakage .......................................................................................................... 16

3.1.13. Determination of proline................................................................................................... 16

3.1.14. Gas exchange attributes .................................................................................................... 17

3.1.15. Statistical Analysis ............................................................................................................ 17

4.1. Introduction ....................................................................................................................... 19

4.2. Materials and Methods ...................................................................................................... 21

4.3. Results ............................................................................................................................... 21

4.3.1. Growth attributes .............................................................................................................. 21

4.3.2. Chlorophyll contents ......................................................................................................... 26

4.3.3. Nutrients in shoot .............................................................................................................. 26

4.3.4. PGPR characteristics ........................................................................................................ 31

4.4. Discussion ......................................................................................................................... 32

4.5. Conclusion ........................................................................................................................ 34

5.1. Introduction ....................................................................................................................... 35

5.2. Materials and Methods ...................................................................................................... 37

5.2.2. Isolation, incubation and purification of isolates ............................................................. 37

5.2.3. Selection of drought-tolerant isolates ............................................................................... 37

5.2.4. Design and site of experiment ........................................................................................... 37

5.2.5. Seeds sterilization and inoculation ................................................................................... 37

5.2.6. Application of Hoagland solution ..................................................................................... 38

5.2.7. Artificial drought stress .................................................................................................... 38

5.2.8. Harvesting and Morphological attributes ........................................................................ 38

5.2.9. Nutrients analysis.............................................................................................................. 38

5.2.10. Chlorophyll contents ......................................................................................................... 38

5.2.11. Molecular identification of effective drought tolerant PGPR........................................... 38

5.2.12. Biochemical characterization of most efficient PGPR ..................................................... 39

5.2.13. Statistical Analysis ............................................................................................................ 39

5.3. Results ............................................................................................................................... 40

5.3.1. Shoot and root length ........................................................................................................ 40

5.3.2. Shoot fresh and dry weight ............................................................................................... 42

5.3.3. Root fresh and dry weight ................................................................................................. 42

5.3.4. Chlorophyll content .......................................................................................................... 45