PHOSPHATE SOLUBILIZING
BACTERIA: THEIR ISOLATION,
CHARACTERIZATION AND IMPACT
ON PLANT GROWTH
IQRA MUNIR
JULY, 2018
Department of Microbiology & Molecular Genetics
University of the Punjab, Lahore
Pakistan
PHOSPHATE SOLUBILIZING
BACTERIA: THEIR ISOLATION,
CHARACTERIZATION AND IMPACT
ON PLANT GROWTH
THESIS SUBMITTED TO THE UNIVERSITY OF THE
PUNJAB IN PARTIAL FULFILLMENT OF THE
REQUIREMENT FOR THE DEGREE OF DOCTORATE OF
PHILOSOPHY
BY
IQRA MUNIR
JULY, 2018
Department of Microbiology & Molecular Genetics
University of the Punjab, Lahore
Pakistan
Dedicated to my Father
May Allah grant him best place in Jannah, Ameen
ACKNOWLEDGEMENTS
In the name of ALLAH who is the most merciful and kind. I would express my gratitude to ALLAH
for all blessings upon me and offer millions and millions thanks to my beloved prophet HAZAT
MUHAMMAD (PBUH) from the core of my heart, for showing me the way to the ALLAH almighty
and to be blessed from his blessings.
Firstly, I would like to express my sincere gratitude to my supervisor Prof. Dr. Muhammad Faisal,
Department of Microbiology and Molecular Genetics, University of the Punjab, for his continuous
support during my Ph.D study and related research, for his patience, motivation, and immense
knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not
have imagined having a better advisor and mentor for my Ph.D study.
I would like to thank Prof. Dr. Anjum Nasim Sabri, Chairperson, Department of Microbiology and
Molecular Genetics, University of the Punjab, for providing me the opportunity to conduct this work.
My sincere thanks to Dr. Mehboob Ahmed and Dr. Yasir Rehman who helped me a lot whenever I
needed it. I would like to thank Dr. Abdurehman for his prayers and motivation.
I would like to offer my special thanks to my friends Humaira, Huma, Adeela, Sara, Saher, Kashaf,
Hina, Sana, Mehvish and Sumaira for all your support, encouragement and fun time we had during this
research work. I am also thankful to other lab mates for their help and motivation.
Heartfelt thanks goes to my father, whom love inspired me to take the right path and to complete my
studies. His strong encouragement helped me throughout my research work and its only because of him,
what I am today. No expressions, verbal or written, can express my heartiest feelings for my mother, the
most beautiful gift of Allah for me, who always prayed for my success and blooming. I cannot give any
reward for her deep affection, love and care. I am profoundly grateful to my sisters Nosheen, Maira and
Bareera who always worked to release my tension and made me laugh during the toughest times.
I would like to express my sincerest gratitude and appreciation to Prof. Dr. Mohamed Hijri, Institut de
Recherche en Biologie Végétales (IRBV), Département de sciences Biologiques, Université de Montréal,
Canada for providing me the opportunity to work in his lab under his guidance for six months. I would
also say thanks to my all lab colleagues in Hijri Lab, Soon-jae Lee, Kenza Samlali, Mengxuan Kong,
Bachir Iffis, Fahad Al Otaibi and Désiré Kanku.
My research would have been impossible without the support of Higher Education Commission (HEC)
Pakistan. I would like to thank for the award of Indigenous fellowship and for awarding me six months
scholarship to Université de Montréal, Canada.
Iqra Munir
TABLE OF CONTENT
Serial No. Title Page No.
List of abbreviations i
List of tables ii
List of figures vi
Summary xix
Chapter 01 Introduction 1
Chapter 02 Material and methods 10
Chapter 03 Isolation and characterization of phosphate solubilizing bacteria 54
Chapter 04 Phylogenetic analysis of phosphate solubilizing bacteria 77
Chapter 05 Phosphate solubilization potential of bacterial isolates 102
Chapter 06 Plant growth promoting attributes of phosphate solubilizing
bacteria 134
Chapter 07 Wheat root elongation assay in the presence and absence of
pesticide stress 147
Chapter 08 Impact of phosphate solubilizing bacteria and inorganic
phosphate on wheat (Triticum aestivum) under pesticide stress 163
Chapter 09 Interaction between phosphate solubilizing bacteria and
arbuscular mycorrhizal fungi 203
Chapter 10 Discussion 225
Chapter 11 References 241
Appendix-I Conferences attended
Appendix-II Publication
Appendix-III Table of soil properties
i
List of Abbreviations
Abbreviation Description
P Phosphorous
PGPR Plant growth promoting bacteria
PSB Phosphate solubilizing bacteria
NBRIP National Botanical Research Institute’s Phosphate
OD Optical density
PCR Polymerase chain reaction
MIC Minimum inhibitory concentration
SI Solubilization index
SE Solubilization efficiency
BLAST Basic local alignment search tool
ALP Aluminium phosphate
FP Ferric phosphate
TCP Tricalcium phosphate
AMF Arbuscular mycorrhizal fungi
ii
List of Tables
Table No. Description Page No.
Table 2.1 Pikovskaya agar 10
Table 2.2 Pikovskaya broth 10
Table 2.3 NBRIP agar 10
Table 2.4 NBRIP broth 11
Table 2.5 L-Agar 11
Table 2.6 L- Broth 11
Table 2.7 Crystal violet solution 11
Table 2.8 Gram’s iodine solution 12
Table 2.9 Decolorizer 12
Table 2.10 Safranin solution 12
Table 2.11 3% hydrogen peroxide 12
Table 2.12 1% Oxidase reagent 12
Table 2.13 Simmons citrate agar 13
Table 2.14 MR-VP broth 13
Table 2.15 Methyl red indicator 13
Table 2.16 Barritt’s reagent 13
Table 2.17 Medium for nitrate reduction test 14
Table 2.18 α-Naphthylamine 14
Table 2.19 Sulfanilic acid 14
Table 2.20 Medium for indole production 14
Table 2.21 King’s A medium 14
Table 2.22 King’s B medium 14
Table 2.23 Medium for starch hydrolysis 15
Table 2.24 Medium for lipid hydrolysis 15
Table 2.25 Nutrient gelatin broth 15
Table 2.26 Urea broth 15
Table 2.27 Antibiotics for sensitivity testing 16
Table 2.28 Chloromolybdic acid 16
iii
Table 2.29 Chlorostanous acid 16
Table 2.30 Phosphate standard 16
Table 2.31 Trypticase soy agar 16
Table 2.32 50% Ethanol 17
Table 2.33 0.5% Phenolphthalein 17
Table 2.34 8.4% Ammonium hydroxide 17
Table 2.35 p-nitrophenyl phosphate disodium (PNPP) 0.115M 17
Table 2.36 0.5 M sodium acetate buffer 17
Table 2.37 0.5 M CaCl2 17
Table 2.38 0.5 M NaOH 17
Table 2.39 L-tryptophan stock solution 17
Table 2.40 Solution I: 0.05M Ferric chloride solution 18
Table 2.41 Solution II: Perchloric acid 18
Table 2.42 Salkowski’s reagent 18
Table 2.43 Blue dye Solution 1 18
Table 2.44 Blue dye Solution 2 18
Table 2.45 Blue dye Solution 3 18
Table 2.46 Minimal Media 9 (MM9) Salt Solution Stock 19
Table 2.47 20% Glucose Stock 19
Table 2.48 NaOH stock 19
Table 2.49 Casamino Acid Solution 19
Table 2.50 CAS agar 19
Table 2.51 Growth medium for Hydrogen cyanide production 20
Table 2.52 Picric acid reagent 20
Table 2.53 Peptone water 20
Table 2.54 Nessler’s reagent 20
Table 2.55 Ninhydrin reagent 20
Table 2.56 DF medium 21
Table 2.57 DF-ACC medium 21
Table 2.58 Stock solutions of ACC 21
iv
Table 2.59 0.1% HgCl2 solution for seed sterilization 22
Table 2.60 Pesticides 22
Table 2.61 80 % acetone 22
Table 2.62 3% Sulfosalycylic acid 22
Table 2.63 Orthophosphoric acid (6N) 22
Table 2.64 Acid Ninhydrin reagent 22
Table 2.65 Phosphate buffer (0.1M) 23
Table 2.66 1% Guaiacol solution 23
Table 2.67 H2O2 solution 23
Table 2.68 0.1M Tris HCL buffer 23
Table 2.69 Citrate buffer 24
Table 2.70 Disodium phenyl phosphate 24
Table 2.71 Phenol standard (stock) 24
Table 2.72 Phenol solution (working) 24
Table 2.73 0.5 N Sodium hydroxide solution 24
Table 2.74 0.5N Sodium bicarbonate solution 24
Table 2.75 4-Amino antipyrin 24
Table 2.76 Potassium ferricyanide 25
Table 2.77 Folin’s mixture: Solution A 25
Table 2.78 Folin’s mixture: Solution B 25
Table 2.79 Folin’s mixture: Solution C 25
Table 2.80 Growth medium for proximal compartment 25
Table 2.81 Medium for distal compartment 26
Table 3.1 Isolation of phosphate solubilizing bacteria from different
sampling sites. 59
Table 3.2 Morphological and biochemical characterization of phosphate
solubilizing bacterial isolates. 60
Table 3.3 Extracellular hydrolytic enzyme production ability of isolated
bacterial strains. 64
v
Table 3.4 Determination of antibiotic resistance profiling of phosphate
solubilizing isolates. 66
Table 3.5 Determination of Minimum Inhibitory Concentration (MIC) of
pesticides. 67
Table 4.1 GenBank accession numbers of isolated phosphate solubilizing
bacteria and their % similarity with nearest homologues. 80
Table 5.1 Characteristics of phosphate solubilizing bacteria. 105
Table 6.1 Plant growth promoting activities of isolated phosphate
solubilizing bacterial strains. 137
Table 8.1
Effect of phosphate solubilizing bacteria on plant growth
parameters of bacterial inoculated wheat plants in the presence of
different inorganic phosphate sources in the absence and presence
of pesticide stress. The data shown represents Mean (n=3) and ±
standard deviation. The interaction significance between different
treatments was judged by 2-way ANOVA followed by Duncans’s
analysis at the level of 95% significance.
171
Table 8.2
Effect of phosphate solubilizing bacteria on chlorophyll content
of bacterial inoculated wheat plants in the presence of different
inorganic phosphate sources in the absence and presence of
pesticide stress. The data shown represents Mean (n=3) and ±
standard deviation. The interaction significance between different
treatments was judged by 2-way ANOVA followed by Duncans’s
analysis at the level of 95% significance.
182
vi
List of Figures
Figure No. Description Page No.
Figure 3.1 South Asian map showing location of Pakistan. 57
Figure 3.2 Soil sampling site (highlighted as red circle) from Lahore,
Punjab, Pakistan (31.497658, 74.296866).
58
Figure 3.3 Soil sampling sites (highlighted as red circle) in Chakwal and
Kallar Kahar, Punjab, Pakistan (32.781758, 72.709010 and
32.936859, 72.863817).
58
Figure 3.4 Effect of various pH levels (3, 5, 7, 9, 11) of medium on growth
of phosphate solubilizing bacterial isolates after 24 hours of
incubation at temperature 28oC.
65
Figure 3.5 Crowding pattern of phosphate solubilizing bacteria after
spreading of soil samples on Pikovskaya agar plates after 7 days
of incubation period at 28oC.
68
Figure 3.6 Biochemical characterization of phosphate solubilizing bacterial
isolates. Catalase test (A), oxidase test (B), citrate utilization test
(C) nitrate reduction test (D), and indole production test (E).
69
Figure 3.7 Determination of extracellular hydrolytic enzymatic activities of
isolated bacteria. Starch hydrolysis (A), Gelatin hydrolysis (B),
and Urea hydrolysis (C).
70
Figure 3.8 Antibiotic resistance profiling of phosphate solubilizing bacteria
after 24 hours of incubation at 28oC.
71
Figure 4.1 Neighbor joining phylogenetic tree of S1 strain, constructed from
16S rRNA gene of isolate and its nearest homologues obtained
from NCBI nucleotide data base.
82
Figure 4.2 Neighbor joining phylogenetic tree of S2 strain, constructed from
16S rRNA gene of isolate and its nearest homologues obtained
from NCBI nucleotide data base.
82
vii
Figure 4.3 Neighbor joining phylogenetic tree of Rad1 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
83
Figure 4.4 Neighbor joining phylogenetic tree of Rad2 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
83
Figure 4.5 Neighbor joining phylogenetic tree of Ros1 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
84
Figure 4.6 Neighbor joining phylogenetic tree of Ros2 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
84
Figure 4.7 Neighbor joining phylogenetic tree of JA10 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
85
Figure 4.8 Neighbor joining phylogenetic tree of R12 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
85
Figure 4.9 Neighbor joining phylogenetic tree of R14 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
86
Figure 4.10 Neighbor joining phylogenetic tree of R15 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
86
Figure 4.11 Neighbor joining phylogenetic tree of SL8 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
87
Figure 4.12 Neighbor joining phylogenetic tree of M6 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base
87
viii
Figure 4.13 Neighbor joining phylogenetic tree of L6 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
88
Figure 4.14 Neighbor joining phylogenetic tree of L19 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
88
Figure 4.15 Neighbor joining phylogenetic tree of L20 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
89
Figure 4.16 Neighbor joining phylogenetic tree of L22 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
89
Figure 4.17 Neighbor joining phylogenetic tree of SF strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
90
Figure 4.18 Neighbor joining phylogenetic tree of SpA strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
90
Figure 4.19 Neighbor joining phylogenetic tree of CS1 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
91
Figure 4.20 Neighbor joining phylogenetic tree of R2 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
91
Figure 4.21 Neighbor joining phylogenetic tree of S62 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
92
Figure 4.22 Neighbor joining phylogenetic tree of W94 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
92
ix
Figure 4.23 Neighbor joining phylogenetic tree of W95 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
93
Figure 4.24 Neighbor joining phylogenetic tree of W96 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
93
Figure 4.25 Neighbor joining phylogenetic tree of P1 strain, constructed from
16S rRNA gene of isolate and its nearest homologues obtained
from NCBI nucleotide data base.
94
Figure 4.26 Neighbor joining phylogenetic tree of U.P strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
94
Figure 4.27 Neighbor joining phylogenetic tree of C14 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
95
Figure 4.28 Neighbor joining phylogenetic tree of C50 strain, constructed
from 16S rRNA gene of isolate and its nearest homologues
obtained from NCBI nucleotide data base.
95
Figure 4.29 Neighbor joining phylogenetic tree of all isolated phosphate
bacterial strains constructed from 16S rRNA gene sequeces.
101
Figure 5.1 Phosphate solubilization on Pikovskaya agar medium after seven
days of incubation at 28 oC.
106
Figure 5.2 Phosphate solubilization on NBRIP agar medium after seven
days of incubation at 28 oC.
106
Figure 5.3 Phosphatases detection on Tryptic Soy Agar (TSA)
supplemented with phenolphthalein indicator. Pink coloration
shows the production of phosphatases after 48 hours of
incubation at 28 oC.
107
Figure 5.4 Determination of Solubilization Index (SI) by bacterial isolates
on Pikovskaya agar and NBRIP agar after 7 days of incubation
at 28 oC.
108
x
Figure 5.5 Determination of percentage solubilization Efficiency (SE) by
bacterial isolates on Pikovskaya agar and NBRIP agar after 7
days of incubation at 28 oC.
108
Figure 5.6 Solubilization of aluminium phosphate by phosphate
solubilizing bacteria after 7 days of incubation at 28 oC.
115
Figure 5.7 Effect of aluminium phosphate solubilization on pH and titrable
acidity of culture supernatant after 7 days of incubation at 28 oC.
115
Figure 5.8 Solubilization of ferric phosphate by phosphate solubilizing
bacteria after 7 days of incubation at 28 oC.
116
Figure 5.9 Effect of ferric phosphate solubilization on pH and titrable
acidity of culture supernatant after 7 days of incubation at 28 oC.
116
Figure 5.10 Solubilization of tricalcium phosphate by phosphate solubilizing
bacteria after 7 days of incubation at 28 oC.
117
Figure 5.11 Effect of tricalcium phosphate solubilization on pH and titrable
acidity of culture supernatant after 7 days of incubation at 28 oC.
117
Figure 5.12 Effect of different carbon sources on phosphate solubilization
ability of isolated phosphate solubilizing bacteria after 7 days of
incubation at 28 oC
118
Figure 5.13 Effect of phosphate solubilization on pH and titrable acidity in
the presence of different carbon sources after 7 days of
incubation at 28 oC.
119
Figure 5.14 Acid phosphatase production by phosphate solubilizing bacteria
in the presence of different carbon sources.
120
Figure 5.15 Alkaline phosphatase production by phosphate solubilizing
bacteria in the presence of different carbon sources.
121
Figure 5.16 Effect of pesticide stress on phosphate solubilization ability of
isolated phosphate solubilizing bacteria after 7 days of
incubation at 28 oC.
122
xi
Figure 5.17 Effect of phosphate solubilization on pH and titrable acidity in
the presence of different pesticides after 7 days of incubation at
28 oC.
123
Figure 5.18 Acid phosphatase production by phosphate solubilizing bacteria
in the presence of pesticide stress.
124
Figure 5.19 Alkaline phosphatase production by phosphate solubilizing
bacteria in the presence of pesticide stress.
125
Figure 6.1 Hydrogen cyanide production by isolated phosphate solubilizing
bacteria after four days of incubation at 28oC.
138
Figure 6.2 Qualitative determination of Indole Acetic Acid (IAA) by
isolated phosphate solubilizing bacterial strains. T- represents
IAA production in the absence of L-tryptophan, T+ represents
IAA production in the presence of L-tryptophan.
138
Figure 6.3 Ammonia production by isolated phosphate solubilizing
bacterial strains after incubation of three days at 28oC.
139
Figure 6.4 Siderophore production by isolated phosphate solubilizing
bacterial strains on Chrom Azurol S (CAS) agar after four days
of incubation at 28oC.
139
Figure 6.5 Quantitative determination of Indole Acetic Acid (IAA) by
isolated phosphate solubilizing bacterial strains in the absence
and presence of L-tryptophan. Error bars Mean ± standard error
(n=3).
140
Figure 6.6 ACC deaminase production by isolated phosphate solubilizing
bacteria measured after 24 hours of incubation in DF-ACC
medium. Error bars Mean ± standard error (n=3).
141
Figure 7.1 Effect of bacterial inoculated wheat seeds on percentage
germination in the presence of Chlorpyrifos (0.5 µg mL-1) and
Pyriproxyfen (1.3 µg mL-1). Error bars Mean ± standard error
(n=3), ANOVA followed by Duncan (P<0.05).
153
Figure 7.2 Effect of bacterial inoculated wheat seeds on shoot length in the
presence of Chlorpyrifos (0.5 µg mL-1) and Pyriproxyfen (1.3 µg
154
xii
mL-1). Error bars Mean ± standard error (n=3), ANOVA
followed by Duncan (P<0.05).
Figure 7.3 Effect of bacterial inoculated wheat seeds on root length in the
presence of Chlorpyrifos (0.5 µg mL-1) and Pyriproxyfen (1.3 µg
mL-1). Error bars Mean ± standard error (n=3), ANOVA
followed by Duncan (P<0.05).
155
Figure 7.4 Effect of bacterial inoculated wheat seeds on number of root in
the presence of Chlorpyrifos (0.5 µg mL-1) and Pyriproxyfen (1.3
µg mL-1). Error bars Mean ± standard error (n=3), ANOVA
followed by Duncan (P<0.05).
156
Figure 7.5 Effect of Pseudomonas putida-Rad2 inoculation on root length
in the presence of Pyriproxyfen (1.3 µg mL-1) on wheat
compared to uninoculated control in gnotobiotic root elongation
assay.
161
Figure 8.1 Effect of phosphate solubilizing bacterial inoculation on proline
content in wheat plants as compared to uninoculated control in
the absence and presence of pesticide in natural soil. The graph
shows the mean ± standard deviation (n=3). Data judged from 2-
way ANOVA followed by Duncan’s (p<0.05).
186
Figure 8.2 Effect of phosphate solubilizing bacterial inoculation on proline
content in wheat plants as compared to uninoculated control in
the absence and presence of pesticide in soil amended with
aluminium phosphate. The graph shows the mean ± standard
deviation (n=3). Data judged from 2-way ANOVA followed by
Duncan’s (p<0.05).
186
Figure 8.3 Effect of phosphate solubilizing bacterial inoculation on proline
content in wheat plants as compared to uninoculated control in
the absence and presence of pesticide in soil amended with ferric
phosphate. The graph shows the mean ± standard deviation
(n=3). Data judged from 2-way ANOVA followed by Duncan’s
(p<0.05).
187
xiii
Figure 8.4 Effect of phosphate solubilizing bacterial inoculation on proline
content in wheat plants as compared to uninoculated control in
the absence and presence of pesticide in soil amended with
tricalcium phosphate. The graph shows the mean ± standard
deviation (n=3). Data judged from 2-way ANOVA followed by
Duncan’s (p<0.05).
187
Figure 8.5 Effect of phosphate solubilizing bacterial inoculation on
peroxidase content in wheat plants as compared to uninoculated
control in the absence and presence of pesticide in natural soil.
The graph shows the mean ± standard deviation (n=3). Data
judged from 2-way ANOVA followed by Duncan’s (p<0.05).
188
Figure 8.6 Effect of phosphate solubilizing bacterial inoculation on
peroxidase content in wheat plants as compared to uninoculated
control in the absence and presence of pesticide in soil amended
with aluminium phosphate. The graph shows the mean ±
standard deviation (n=3). Data judged from 2-way ANOVA
followed by Duncan’s (p<0.05).
188
Figure 8.7 Effect of phosphate solubilizing bacterial inoculation on
peroxidase content in wheat plants as compared to uninoculated
control in the absence and presence of pesticide in soil amended
with ferric phosphate. The graph shows the mean ± standard
deviation (n=3). Data judged from 2-way ANOVA followed by
Duncan’s (p<0.05).
189
Figure 8.8 Effect of phosphate solubilizing bacterial inoculation on
peroxidase content in wheat plants as compared to uninoculated
control in the absence and presence of pesticide in soil amended
with tricalcium phosphate. The graph shows the mean ± standard
deviation (n=3). Data judged from 2-way ANOVA followed by
Duncan’s (p<0.05).
189
Figure 8.9 Effect of phosphate solubilizing bacterial inoculation on acid
phosphatase content in wheat plants as compared to uninoculated
190
xiv
control in the absence and presence of pesticide in natural soil.
The graph shows the mean ± standard deviation (n=3). Data
judged from 2-way ANOVA followed by Duncan’s (p<0.05).
Figure 8.10 Effect of phosphate solubilizing bacterial inoculation on proline
content in wheat plants as compared to uninoculated control in
the absence and presence of pesticide in soil amended with
aluminium phosphate. The graph shows the mean ± standard
deviation (n=3). Data judged from 2-way ANOVA followed by
Duncan’s (p<0.05).
190
Figure 8.11 Effect of phosphate solubilizing bacterial inoculation on acid
phosphatase content in wheat plants as compared to uninoculated
control in the absence and presence of pesticide in soil amended
with ferric phosphate. The graph shows the mean ± standard
deviation (n=3). Data judged from 2-way ANOVA followed by
Duncan’s (p<0.05).
191
Figure 8.12 Effect of phosphate solubilizing bacterial inoculation on acid
phosphatase content in wheat plants as compared to uninoculated
control in the absence and presence of pesticide in soil amended
with tricalcium phosphate. The graph shows the mean ± standard
deviation (n=3). Data judged from 2-way ANOVA followed by
Duncan’s (p<0.05).
191
Figure 8.13 Effect of phosphate solubilizing bacterial inoculation on protein
content in wheat plants as compared to uninoculated control in
the absence and presence of pesticide in natural soil. The graph
shows the mean ± standard deviation (n=3). Data judged from 2-
way ANOVA followed by Duncan’s (p<0.05).
192
Figure 8.14 Effect of phosphate solubilizing bacterial inoculation on protein
content in wheat plants as compared to uninoculated control in
the absence and presence of pesticide in soil amended with
aluminium phosphate. The graph shows the mean ± standard
192
xv
deviation (n=3). Data judged from 2-way ANOVA followed by
Duncan’s (p<0.05).
Figure 8.15 Effect of phosphate solubilizing bacterial inoculation on protein
content in wheat plants as compared to uninoculated control in
the absence and presence of pesticide in soil amended with ferric
phosphate. The graph shows the mean ± standard deviation
(n=3). Data judged from 2-way ANOVA followed by Duncan’s
(p<0.05).
193
Figure 8.16 Effect of phosphate solubilizing bacterial inoculation on protein
content in wheat plants as compared to uninoculated control in
the absence and presence of pesticide in soil amended with
tricalcium phosphate. The graph shows the mean ± standard
deviation (n=3). Data judged from 2-way ANOVA followed by
Duncan’s (p<0.05).
193
Figure 8.17 Effect of phosphate solubilizing Pseudomonas plecoglossicida-
R14 inoculation and aluminium phosphate on vegetative growth
of the wheat plant.
197
Figure 8.18 Effect of phosphate solubilizing Pseudomonas aeruginosa-SpA
inoculation and aluminium phosphate on vegetative growth of
the wheat plant under pesticide stress.
197
Figure 8.19 Effect of phosphate solubilizing Enterobacter aerogenes-W96
inoculation and ferric phosphate on vegetative growth of the
wheat plant.
198
Figure 8.20 Effect of phosphate solubilizing Enterobacter cloacae-W95
inoculation and tricalcium phosphate on vegetative growth of
wheat plant under pesticide stress.
198
Figure 9.1 Bi-compartment petri plate having mychorrized chicory roots
with Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198,
grown in proximal compartment for 21 days at 28oC. The distal
208
xvi
compartment containing minimal growth medium inoculated
with Acinetobacter baumanii- JA10 followed by incubation for
6 weeks at 28oC.
Figure 9.2 Bi-compartment petri plate having mychorrized chicory roots
with Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198,
grown in proximal compartment for 21 days at 28oC. The distal
compartment containing minimal growth medium supplemented
with tricalcium phosphate inoculated with Pseudomonas putida-
Rad2 followed by incubation for 6 weeks at 28oC.
208
Figure 9.3 Effect of phosphate solubilizing bacterial isolates on pH of
minimal growth medium in the absence of Arbuscular
Mycorrhizal Fungi (AMF), RiDAOM 19198. Plates were
incubated for six weeks after bacterial inoculation at 28oC. Error
bars Mean ± standard error (n=5). Different letters on bars
indicate significant difference between treatments using
Duncan’s multiple range test (P<0.05).
209
Figure 9.4 Effect of phosphate solubilizing bacterial isolates on pH of
minimal growth medium supplemented with tricalcium
phosphate in the absence of Arbuscular Mycorrhizal Fungi
(AMF), RiDAOM 19198. Plates were incubated for six weeks
after bacterial inoculation at 28oC. Error bars Mean ± standard
error (n=5). Different letters on bars indicate significant
difference between treatments using Duncan’s multiple range
test (P<0.05).
210
Figure 9.5 Effect of interaction between phosphate solubilizing bacterial
isolates and Arbuscular Mycorrhizal Fungi (AMF), RiDAOM
19198 on pH of minimal growth medium. Plates were incubated
for six weeks after bacterial inoculation at 28oC. Error bars
Mean ± standard error (n=5). Different letters on bars indicate
significant difference between treatments using Duncan’s
multiple range test (P<0.05).
211
xvii
Figure 9.6 Effect of interaction between phosphate solubilizing bacterial
isolates and Arbuscular Mycorrhizal Fungi (AMF), RiDAOM
19198 on pH of minimal growth medium supplemented with
tricalcium phosphate. Plates were incubated for six weeks after
bacterial inoculation at 28oC. Error bars Mean ± standard error
(n=5). Similar letter on bars indicate non-significant difference
between treatments using Duncan’s multiple range test (P<0.05).
212
Figure 9.7 Effect of phosphate solubilizing bacterial isolates on P
solubilization in minimal growth medium in the absence of
Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198. Plates
were incubated for six weeks after bacterial inoculation at 28oC.
Error bars Mean ± standard error (n=5). Different letters on bars
indicate significant difference between treatments using
Duncan’s multiple range test (P<0.05).
213
Figure 9.8 Effect of phosphate solubilizing bacterial isolates on P
solubilization in minimal growth medium supplemented with
tricalcium phosphate in the absence of Arbuscular Mycorrhizal
Fungi (AMF), RiDAOM 19198. Plates were incubated for six
weeks after bacterial inoculation at 28oC. Error bars Mean ±
standard error (n=5). Different letters on bars indicate significant
difference between treatments using Duncan’s multiple range
test (P<0.05).
214
Figure 9.9 Effect of interaction between phosphate solubilizing bacterial
isolates and Arbuscular Mycorrhizal Fungi (AMF), RiDAOM
19198 on P solubilization in minimal growth medium. Plates
were incubated for six weeks after bacterial inoculation at 28oC.
Error bars Mean ± standard error (n=5). Different letters on bars
indicate significant difference between treatments using
Duncan’s multiple range test (P<0.05).
215
Figure 9.10 Effect of interaction between phosphate solubilizing bacterial
isolates and Arbuscular Mycorrhizal Fungi (AMF), RiDAOM
216
xviii
19198 on P solubilization in minimal growth medium
supplemented with tricalcium phosphate. Plates were incubated
for six weeks after bacterial inoculation at 28oC. Error bars
Mean ± standard error (n=5). Similar letter on bars indicate non-
significant difference between treatments using Duncan’s
multiple range test (P<0.05).
Figure 9.11 Interaction between phosphate solubilizing Ochrobactrum
pseudogrignonense (S1) with Arbuscular Mycorrhizal Fungi
(AMF), RiDAOM 19198 on minimal growth medium. Plates
were incubated for six weeks after bacterial inoculation at 28oC.
The interaction was analyzed using stereo microscope.
220
Figure 9.12 Positive interaction between phosphate solubilizing
Pseudomonas putida (Rad2) with Arbuscular Mycorrhizal Fungi
(AMF), RiDAOM 19198 on minimal growth medium. Plates
were incubated for six weeks after bacterial inoculation at 28oC.
The interaction was analyzed using stereo microscope.
221
Figure 9.13 Positive interaction between phosphate solubilizing
Acinetobacter baumanii (JA10) with Arbuscular Mycorrhizal
Fungi (AMF), RiDAOM 19198 on minimal growth medium.
Plates were incubated for six weeks after bacterial inoculation at
28oC. The interaction was analyzed using stereo microscope.
222
Figure 9.14 Positive interaction between phosphate solubilizing
Pseudomonas aeruginosa (SpA) with Arbuscular Mycorrhizal
Fungi (AMF), RiDAOM 19198 on minimal growth medium.
Plates were incubated for six weeks after bacterial inoculation at
28oC. The interaction was analyzed using stereo microscope.
223
Figure 9.15 Positive interaction between phosphate solubilizing
Enterobacter aerogenes (W96) with Arbuscular Mycorrhizal
Fungi (AMF), RiDAOM 19198 on minimal growth medium.
Plates were incubated for six weeks after bacterial inoculation at
28oC. The interaction was analyzed using stereo microscope.
224
xix
Thesis Summary
Phosphorous is an important macronutrient required by plants for fundamental processes.
Phosphate exist in very high quantities in soil, but the plant available form of phosphate is
a limiting factor. The available quantity of phosphate ranges from 0.01 milligrams to 0.2
milligrams per kilogram of soil. In soil, phosphorous usually remains adsorbed by
aluminium, ferrous, calcium and magnesium and their oxides. It also lead to their gradual
conversion towards more complexity. The adsorption of phosphorous is greatly influenced
by pH of soil. Bacteria present in plant rhizosphere are versatile in transformation,
mobilization and solubilization of nutrients as compared to other bacterial species of soil.
Phosphate solubilizing bacteria can improve the availability of nutrients to plants which
ultimately improves nutrient uptake by plants and as a result, crop yield improves.
The present investigation deals with the isolation and characterization of 28 phosphate
solubilizing bacterial strains (S1, S2, Rad1, Rad2, Ros1, Ros2, JA10, R12, R14, R15, SL8,
M6, L6, L19, L20, L22, SF, SpA, CS1, R2, S62, W94, W95, W96, P1, UP, C14 and C50)
isolated from rhizosphere of different plants and from barren soil. The major aim of the
present study was to check the phosphate solubilization potential of the isolates. All the
isolates were gram negative rods and were characterized in genus Pseudomonas,
Acinetobacter, Klebsiella, Enterobacter and Ochrobactrum on the basis of their
morphological, biochemical and genetic characteristics. The isolates were genetically
identified by 16S rRNA gene sequencing as Ochrobactrum pseudogrignonense-S1,
Acinetobacter olivorans-S2, Pseudomonas putida-Rad1, Pseudomonas putida-Rad2,
Pseudomonas parafulva-Ros1, Pseudomonas sp-Ros2, Acinetobacter baumanii-JA10,
Klebsiella pneumoniae-R12, Pseudomonas plecoglossicida-R14, Pseudomonas
xx
aeruginosa-R15, Pseudomonas japonica-SL8, Ochrobactrum sp-M6, Acinetobacter pittii-
L6, Acinetobacter pittii-L19, Pseudomonas koreensis-L20, Pseudomonas
frederiksbergensis-L22, Pseudomonas oryzihabitans-SF, Pseudomonas aeruginosa-SpA,
Acinetobacter pittii-CS1, Acinetobacter calcoaceticus-R2, Acinetobacter calcoaceticus-
S62, Acinetobacter sp.-W94, Enterobacter cloacae-W95, Enterobacter aerogenes-W96,
Pseudomonas fluorescens-P1, Pseudomonas reinekei-UP, Acinetobacter calcoaceticus-
C14 and Acinetobacter sp.-C50.
The optimum pH for bacterial growth was 7 however, the isolates showed notable growth
at pH range from 5 to 9. Several phosphate solubilizing bacterial isolates showed starch,
lipid and gelatin hydrolysis. Majority of phosphate solubilizing strains showed resistance
against antibiotics including Amoxicillin, Cloxacillin, and Ceftazidime while sensitivity
was observed against Imipenem. Majority of the isolated strains showed resistance toward
commonly used pesticides. The isolates showed tolerance to Chlorpyrifos and
Pyriproxyfen for up to 80 mg mL-1.
All the isolated bacterial strains exhibited phosphate solubilizion on agar media and also
showed phosphatase production on tryptic soya agar. The strains showed highest
solubilization index and solubilization efficiency on NBRIP agar as compared to
Pikovskaya agar. In liquid media supplemented with aluminium phosphate and ferric
phosphate as an inorganic source, strain L22 exhibited highest solubilization potential and
solubilized 118 µg mL-1 phosphate. In ferric phosphate solubilization, highest
solubilization was shown by strain SpA. Remarkable results were observed by isolated
strains for tricalcium phosphate solubilization, the strains exhibited solubilization range
from 650 µg mL-1 to 980 µg mL-1. High titrable acidity and decreased pH was recorded as
xxi
a result of phosphate solubilization. Moreover, for majority of the isolates, the most
suitable carbon source for phosphate solubilization was glucose. All phosphate solubilizing
bacterial isolates showed acid and alkaline phosphatase activities. The phosphate
solubilization activities of all isolates were affected in the presence of pesticides.
Majority of the phosphate solubilizing bacterial isolates exhibited in vitro plant growth
promoting activities. All the isolated strains exhibited indole acetic acid, ammonia and
ACC deaminase production, however, some of these isolates showed hydrogen cyanide
and siderophores production. In root elongation assay with wheat plant, twelve individual
phosphate solubilizing bacterial strains (S1, S2, Rad1, Rad2, Ros2, JA10, R14, SL8, SpA,
W95, W96 and UP) showed different results in the absence and presence of pesticide
compounds. The percentage seed germination generally increased in the absence of
pesticide stress and decrease in its presence. The shoot lengths were decreased in both
conditions, however, strain Rad2 inoculation showed significantly increased root length in
the presence of Pyriproxyfen. Moreover, the number of roots were significantly increased
with majority of bacterial inoculations with and without stress.
Phosphate solubilizing bacterial inoculation to wheat plant in field conditions resulted in
significantly increased shoot length in different treatments. Considerable increase in shoot
dry weight (54%) was exhibited in the supplemented inorganic phosphates (aluminium
phosphate, ferric phosphate and tricalcium phosphate). Majority of the strain inoculations
resulted in remarkable increase in spike length. Spike and seed weights were increased
significantly by all bacterial inoculated wheat plants in natural soil as well as with the
supplementation of aluminium phosphate to soil. However, the presence of pesticide
showed toxic effects and resulted in reduced weight of seeds and spikes per plant. The
xxii
effect on number of tillers as a result of bacterial inoculations resulted in either no effect
or resulted in its reduction.
The chlorophyll content in leaf was generally increased due to phosphate solubilizing
bacterial inoculation in wheat plants, whereas, the supplementation of pesticide stress
resulted in significant decrease in chlorophyll content. In contrast, leaf proline content was
increased in the stress condition. The peroxidase content in leaf was considerably increased
due to bacterial inoculations. The acid phosphatase activity and protein content were
negatively affected due the toxic effects of pesticide compounds.
The pH of growth media as a result of interaction between phosphate solubilizing bacteria
and arbuscular mycorrhizal fungi remain unaffected. Whereas, the solubilized phosphate
content was significantly increased due to arbuscular mycorrhizal fungi in the presence of
tricalcium phosphate as an inorganic phosphate source. The interaction between arbuscular
mycorrhizal fungi and phosphate solubilizing bacteria under microscope were also
observed by majority of the inoculated strains. Present study shows the phosphate
solubilization potential of phosphate solubilizing bacteria, plant growth promoting
potential and their interaction with wheat plants and arbuscular mychorrhizal fungi.
1
Chapter 01
Introduction
Phosphorous is an important macronutrient required by plants for fundamental processes.
It is a vital component of Adenosine Tri-Phosphate, involved in metabolic activities of
plants (Arfarita et al., 2017). It is a major component for growth and development of plants
and is generally used as fertilizers to enhance plant growth (Wei et al., 2015; Wei et al.,
2017). Phosphorous, potassium and nitrogen are the primary nutrients for crops that are
required for enhanced growth and production (Ayub et al., 2010; Kumar et al., 2015).
Phosphorous has been found to have importance in several metabolic activities in plants.
These activities include photosynthesis, energy transfer, respiration, biosynthesis and
signal transduction (Ahemad and Khan, 2012a). Phosphorous is a main component of soil
but it remains sequestered by different elements present in soil which are responsible for
its un-availability to plants. This deficiency leads to low productivity of plants (Zhang et
al., 2017).
Composition and mineral status of soil is very important. In addition to Phosphorous
quantities present in soil, its availability equally depends on microbial activity for its
solubility. Phosphate is present in very high quantities in soil, but the plant available form
of phosphate is a limiting factor. The available quantity of phosphate ranges from 0.01
milligrams to 0.2 milligrams per kilogram of soil (Arfarita et al., 2017). The P levels in soil
are further classified as its organic and inorganic forms. Phosphorus in its organic forms is
present as phytate which is synthesized by plants and microorganisms. The other organic
2
forms present in soil includes phosphotriesters, nucleic acids, phospholipids and
phosphomonoesters (Paul and Clark, 1988; Behera et al., 2014).
In soil, phosphorous usually remains adsorbed by aluminium, ferrous, calcium and
magnesium and their oxides. It also lead to their gradual conversion towards more
complexity. The adsorption of phosphorous is greatly influenced by pH of soil. Calcium
bound phosphorous occur predominantly in alkaline soils while aluminium and ferric
bound forms usually occur in acidic environments (Banerjea and Gosh, 1970; Maitra et al.,
2015). According to an estimate, around eight to eighty two percent of total phosphorous
is present in bound form. Out of which around 50% is bound to calcium (Qian et al., 2010;
Rzepechi, 2010; Renjith et al., 2011; Maitra et al., 2015).
Soil contains a huge variety of microorganisms and in rhizospheric zone there are different
microorganisms which are involved in direct or indirect mechanisms of plant growth
promotion. Phosphate solubilizing bacteria are known to promote plant growth. The
bacterial diversity in plant rhizosphere can be related to root system as well as the nature
of root exudates (Rajapaksha and Senanayake, 2011). The phosphate solubilizing bacteria
have the ability to solubilize the fixed or insoluble form of phosphorous in soil from
different bound forms (aluminium phosphate, ferric phosphate, and tricalcium phosphate)
(Sharma et al., 2013). The microbial activities in soil helps to overcome the lower quantities
of available phosphate. They are helpful in conversion of un-available phosphate to
available forms and also help plant roots to reach towards available phosphate (Arfarita et
al., 2017).
Rhizosphere is the area of soil adjacent to plant roots. However, rhizobacteria are the group
of species living in close proximity to plant roots (Kloepper et al., 1991; Ahemad and
3
Kibert, 2014). Bacteria present in plant rhizosphere are versatile in transformation,
mobilization and solubilization of nutrients as compared to other soil bacterial species
(Hayat et al., 2010; Ahemad and Kibert, 2014). The rhizospheric region of crops is a very
important environment of soil ecology and play an important role in the interaction of
microorganism, plant and soil. A number of indigenous microbes colonize and surround
the root system and as a result associative, symbiotic, parasitic and neutralistic relationship
develops in the plant soil system. These associations are based on microbial type, nutrient
status in soil, environment of soil and defence system of plant (Kumar et al., 2015).
In addition to providing nutritional benefits to plants, rhizobacteria also protect them from
phytopathogens by different mechanisms. They are responsible for soil structure
improvement and bioremediation in soils polluted from different pollutants by
sequestration of toxic classes of heavy metals and degradation of xenobiotics such as
pesticides (Braud et al., 2009; Rajkumar et al., 2010; Hayat et al., 2010; Ahemad and Malik,
2011; Ahemad, 2012).
Soil present around plant roots contain large number of active bacterial species. These
bacteria are also called as plant growth promoting rhizobacteria (PGPR) (Kloepper et al.,
1980; Reetha et al., 2014). It is estimated that above 95% of bacteria exist in the rhizosphere
of plants are responsible to help plants in obtaining nutrients from soil. According to
current approaches, researchers are trying to isolate and study bacteria having plant growth
promoting (PGP) abilities (Ullah and Bano, 2015). Plant growth enhancement by bacteria
can be due to direct mechanisms as well as it can be due to some indirect mechanisms.
Bacteria present in plant rhizosphere also help plants to survive in stress conditions either
biotic or abiotic (Park et al., 2016). The indirect mechanisms include the production of
4
phytohormones specifically related to stress conditions. These stress associated
phytohormones include ethylene or jasmonic acid. The other indirect mechanisms include
the induction of systemic resistance in plants and the production of antibiotics to compete
in rhizosphere. The direct mechanisms responsible for enhanced plant growth include
phosphate solubilization, fixation of atmospheric nitrogen, siderophore production and
phytohormone production including auxins, cytokinins, gibberallins and nitric oxide
(Cassan et al., 2014). These rhizospheric bacterial population mostly include Pseudomonas
spp. Enterobacter spp. Bacillus spp. and Rhizobium spp. Their most common plant growth
promoting abilities include solubilization of phosphate, zinc and potassium, auxin
production, and biocontrol activities such as antibiotic production, hydrolytic enzyme
production and hydrogen cyanide production (Singh et al., 2015; Zhang et al., 2013;
Yadegari and Mosadeghzad, 2012; Phua et al., 2012; Verma et al., 2012).
The development and growth of plants is a changing process that favor in adapting the
environments where the plants are restricted. Plants conform their growth according to the
external and internal stimulus by the hormonal activities. Plant growth depends on the key
phytohormones which include ethylene, auxin and abscisic acid (Vanstraelen and
Benekova, 2012; Thole at al., 2014). Abscisic acid responds to many stress conditions
(Culter et al., 2010; Thole et al., 2014).
Bacterial isolates involved in plant growth promotion have been isolated from different
plants (Fernandes et al., 2013; Zhao et al., 2015; Afzal et al., 2017). Native environment
friendly microorganisms play an important role in sustainability of plants, soil and
environment. Microorganisms promote plant growth by activating different enzymes and
provide several benefits such as nutrient uptake, disease resistance towards diseases,
5
transportation of starch and sugars, improved photosynthesis, maintenance of turgor
pressure and protein synthesis. The use of renewable input that have the ability to provide
environmental benefits and reduces ecological hazards is very important in the
sustainability of agriculture (Verma et al., 2013; Kumar et al., 2015).
The solubilization of inorganic phosphate have been explained by different reports. The
main mechanism for solubilization includes production of carbon dioxide, hydroxyl ion,
protons, siderophores and most importantly the production of organic acids (Sharma et al.,
2013; Alori et al., 2017). The production of organic acids along with hydroxyl and carboxyl
ions caused the chelation of cations or cause reduction in pH which releases the P from
bound phosphate (Seshachala and Tallapragada, 2012). The production of organic acid take
place in periplasmic space as a result of oxidation (Zhao et al., 2014). Due to decrease in
pH, the organic acids excrete out which acidify the bacterial cell and its environment. As a
result, phosphorous ion released due to substitution of proton for calcium ion (Goldstein,
1994; Alori et al., 2017).
A vast majority of bacterial species have been reported for mobilization of unavailable
phosphorous through solubilizing and mineralizing them. The species include
Bacillus, Agrobacterium, Pseudomonas, (Babalola and Glick, 2012),
Burkholderia (Mamta et al., 2010; Zhao et al., 2014; Istina et al., 2015), Rhizobium,
Ralstonia (Tajini et al., 2012), Azotobacter (Kumar et al., 2014), Kushneria (Zhu et al.,
2011), Bacillus (Jahan et al., 2013; David et al., 2014), Paenibacillus (Fernández et al.,
2011), Erwinia, Enterobacter (Chakraborty et al., 2009), Thiobacillus, Sinomonas,
Salmonella, Bradyrhizobium, Serratia and Rhodococcus (David et al., 2014; Alori et al.,
2017).
6
Phosphate solubilizing microorganisms improve the availability of nutrients to plants
which ultimately improves nutrient uptake by plants and as a result, yield and production
of crops increases (Verma et al., 2010). The solubilization of phosphorous in rhizosphere
is the best way that is exhibited by plants growth promoting rhizobacteria. By this mean,
these rhizobacteria augment plants by providing nutrients in available forms (Richardson,
2001; Kumar et al., 2015). Microorganisms in soil play a crucial role in conversion or
transformation of nutrients from one form to another (Maitra et al., 2015). There are several
reports of isolation of phosphate solubilizing bacteria from rhizospheric region of different
plants (Singh et al., 2013; Panda et al., 2016; Tomer et al., 2017).
Increasing population demands increased amount of food production. The urbanization has
limited the land for agricultural use (Hamuda and Patko, 2013; Namli et al., 2017). Due to
this reason, the production of damage free food with good quality is of great concern.
Chemical fertilizers are used increasingly but their increased use is raising so many
concerns. The limitation of phosphorous is compensated by the application of different
phosphate fertilizers. Excessive farming practices with the help of mineral fertilizers are
maximizing the crop yield, are expensive as well as they are creating problems to
environment (Kumar et al., 2015). The application of these chemical fertilizers are posing
risks to the environment. Soil supplementation with chemical fertilizers to fulfill the
phosphorous requirements and the manufacture of chemical fertilizers require enormous
cost. Due to these factors, researchers are finding cost effective and ecofriendly approaches
(Zhang et al., 2017).
The occurrence of phosphate solubilizing microorganisms in soil suggests that they can be
a good option to be study and to be used as biofertilizers (Majeed et al., 2015). A number
7
of studies has been conducted on phosphate solubilizing bacteria and it has been found that
they also showed other plant growth promoting abilities including siderophore production,
secondary metabolite and antibiotic production, ACC deaminase enzyme, gibberellins and
auxin production (Taurian et al., 2010; Namli et al., 2017). The enzymatic activity of
bacteria is greater in the rhizospheric region of soil (Gianfreda, 2015).
A good alternative to chemical fertilizers is the use of plant growth promoting bacteria. To
fulfil the needs of deficient phosphorous in agricultural land, phosphate solubilizing
bacteria can be used (Hamuda and Patko, 2013; Namli et al., 2017). In different agricultural
soils, phosphorus is an important limiting nutrient and its deficiency affects plant growth.
Phosphate solubilizing isolates have been reported to be used as bio-inoculants for a
number of crops. The use of microbial inoculants helps to increase the microbial population
in plant rhizosphere (Rajapaksha and Senanayake, 2011). The crop or soil inoculation with
phosphate solubilizing bacteria is a likely approach to improve phosphorous absorption by
plants thereby causing reduction in the usage of chemical fertilizers that adversely affect
the environment (Alori et al., 2017). The application of biofertilizers can help increasing P
availability from the accumulated P in the soil. Among symbiotic rhizobacterial species
Mesorhizobium, Bradyrhizobium and Rhizobium are most prominent while among non-
symbionts Azomonas, Pseudomonas, Azospirillum, Bacillus, Azotobacter and Klebsiella
are of great importance and are used as biofertilizers worldwide (Gianfreda, 2015).
Presently, improvement of crops using biological approaches have attained prime
importance among crop producers, and agronomists. In this regard a large number of
researches are going on worldwide. Their common interest is to explore a wide variety of
rhizobacterial species having unique characteristics such as detoxification of heavy metals
8
(Ma et al., 2011), tolerance or degradation of pesticides (Ahemad and Khan, 2012a),
tolerance towards salinity (Tank and Saraf, 2010), plant protection from phytopathogens
(Hynes et al., 2008; Russo et al., 2008) as well as other plant growth improvements
including P solubilization, nitrogenase, ammonia (Glick, 2012), hydrogen cyanide, 1-
amino cyclopropane 1-carboxylate, siderophores (Jahanian et al., 2012) and other
phytohormone production (Ahemad and Khan, 2012b).
In soil, another challenging condition for microbial survival is the increased applications
of large quantities of pesticides which are used to prevent plants and crops from different
infections. These pesticides are harmful for environment as well as for the microbial
communities in soil. Some microorganisms somehow manage to survive in the presence of
these harmful chemicals either by developing resistance mechanisms or by developing
mechanisms for their degradation (Nuraini et al., 2015). It is reported that in terms of
sustainability, the indigenous microbes are more viable than the other induced
microorganisms applied as biofertilizers or bioremediators (Arfarita et al., 2016).
The presence of pesticide causes toxic effects on microbial population in soil and they also
affect their characteristics. Therefore the identification of phosphate solubilizing bacteria
having plant growth promoting abilities as well as tolerance towards pesticides can be
helpful to optimize the productivity of crops in pesticide stress conditions (Ahemad and
Khan, 2011c).
The application of pesticides leads to the long term persistence of these toxic compounds
in the soil which ultimately affects the microbial communities and is also affects their
functionality (Eliason et al., 2004; Ahemad and Khan, 2012a). To reduce or to overcome
the harmful effects of pesticides on plants, a good alternative is to treat the seeds with the
9
pesticide resistant strains having plant growth promoting abilities (Wani et al., 2005;
Ahemad and Khan 2012a).
Objective
The main objective of the proposed study is to exploit the potential of phosphate
solubilizing bacteria to enhance the growth of plants by different ways. As phosphorous is
the second most important nutrient and is required by plants. Large quantities of chemical
fertilizers are needed to fulfill phosphorous requirements but this is costly and also causing
problems to the environment. The use of native phosphate solubilizing bacterial strains as
bio-fertilizers will help in reducing the use of chemical fertilizers and also they will be
effective in reducing the cost of cultivation and maintaining the natural fertility of soil. Use
of these phosphate solubilizing bacteria as bio-inoculants will increase the available P in
soil, and will promote the sustainable agriculture. The pesticides are xenobiotic compounds
that are deliberately spread into the environment to control the pest that affects crop
production. On application into the soil, it may harm the native microbial population,
affects bacterial diversity and influence the soil biochemical processes including
degradation of organic matter, nitrogen fixation, nitrification, denitrification,
ammonification and P solubilization. The pesticide-tolerance in these phosphate
solubilizing rhizobacteria may be important in the decontamination of agricultural soils
polluted with pesticides. In addition, a great deal of functional diversity can be found
among phosphate solubilizing rhizobacteria isolated from different sites. Furthermore, the
interaction of these isolated bacteria with fungi will be studied.
10
Chapter 02
Materials and Methods
All media, solutions and buffers were prepared using glass distilled water. Media, solutions
and glass apparatus were sterilized by autoclaving.
Table 2.1: Pikovskaya agar (Pikovskaya, 1948)
S. No. Component Quantity (g L-1)
1 Glucose 10.0
2 Yeast extract 0.5
3 FeSO4.H2O 0.006
4 MnSO4.7H2O 0.006
5 KCl 0.2
6 MgSO4.7H2O 0.1
7 Ca3(PO4)2 5.0
8 (NH4)SO4 0.5
9 Agar 15.0
pH adjusted to 7.0
Table 2.2: Pikovskaya broth (Pikovskaya, 1948)
S. No. Component Quantity (g L-1)
1 Glucose 10.0
2 Yeast extract 0.5
3 FeSO4.H2O 0.006
4 MnSO4.7H2O 0.006
5 KCl 0.2
6 MgSO4.7H2O 0.1
7 Ca3(PO4)2 5.0
8 (NH4)SO4 0.5
pH adjusted to 7.0
Table 2.3: NBRIP agar (Nautiyal, 1999)
S. No. Component Quantity (g L-1)
1 Glucose 10.0
2 (NH4)SO4 0.1
3 MgCl2.6H2O 5.0
4 Ca3(PO4)2 5.0
11
5 KCl 0.2
6 MgSO4.7H2O 0.25
7 Agar 15.0
pH adjusted to 7.0
Table 2.4: NBRIP broth (Nautiyal, 1999)
S. No. Component Quantity (g L-1)
1 Glucose 10.0
2 (NH4)SO4 0.1
3 MgCl2.6H2O 5.0
4 Ca3(PO4)2 5.0
5 KCl 0.2
6 MgSO4.7H2O 0.25
Table 2.5: L-Agar (Gerhardt et al., 1994)
S. No. Component Quantity (g L-1)
1 Tryptone 10.0
2 NaCl 5.0
3 Yeast extract 5.0
4 Agar 15.0
pH adjusted to 7.0
Table 2.6: L- Broth (Gerhardt et al., 1994)
S. No. Component Quantity (g L-1)
1 Tryptone 10.0
2 NaCl 5.0
3 Yeast extract 5.0
pH adjusted to 7.0
Solutions for Gram Staining
Table 2.7: Crystal violet solution
Solution A:
S. No. Component Quantity
1 Ethyl alcohol (95%) 20 mL
12
2 Crystal violet 2.0 g
Solution B:
S. No. Components Quantity
1 Distilled water 80 mL
2 Ammonium oxalate 0.8 g
Table 2.8: Gram’s iodine solution
S. No. Components Quantity
1 Potassium iodide 2.0 g
2 Iodine 1.0 g
3 Distilled water 300 mL
Table 2.9: Decolorizer
S.
No.
Components Quantity (100 mL-1)
1 Ethyl alcohol 95 mL
2 Distilled water 5 mL
Table 2.10: Safranin solution
S. No. Components Quantity (100 mL-1)
1 Ethyl alcohol (95%) 10 mL
2 Safranin O 0.25 g
3 Distilled water 90 mL
Reagent for Catalase Test
Table 2.11: 3% hydrogen peroxide (H2O2) (Cappuccino and Sherman, 2005)
S. No. Components Quantity (10 mL-1)
1 Hydrogen Peroxide 0.3 mL
2 Distilled water 10 mL
Reagent for Cytochrome Oxidase Test
Table 2.12: 1% Oxidase reagent (Tetramethyl-para-phenylenediamine dihydrochloride)
S. No. Components Quantity (10 mL-1)
1 Oxidase Reagent 0.1 g
2 Distilled water 10 mL
13
Table 2.13: Simmons citrate agar (Cappuccino and Sharman, 2005)
S. No. Components Quantity (g L-1)
1 Dipotassium phosphate 1.0
2 Sodium citrate 2.0
3 Sodium chloride 5.0
4 Bromothymol blue 0.08
5 Ammonium dihydrogen phosphate 1 .0
6
Agar 15.0
7 Magnesium sulfate 0.2
pH adjusted to 7.2
Table 2.14: MR-VP broth (Cappuccino and Sharman, 2005)
S. No. Components Quantity (g L-1)
1 Dextrose 5
2 Potassium phosphate 5
3 Peptone 7
pH adjusted to 6.9
Table 2.15: Methyl red indicator (Gerhardt et al., 1994)
S. No. Components Quantity (10 mL-1)
1 95% ethanol 10 mL
2 Methyl red 0.003 g
Table 2.16: Barritt’s reagent for Voges Proskauer test
Solution A:
S. No. Components Quantity (100 mL-1)
1 Absolute ethanol 95 mL
2 α-naphthol 5 g
Solution B:
S. No. Components Quantity (100 mL-1)
1 Potassium hydroxide 40 g
2 Distilled water 100 mL
14
Table 2.17: Medium for nitrate reduction test (Gerhard et al., 1994)
S. No. Components Quantity ( g L-1 )
1 KNO3 1.0
2 Beef extract 3.0
3 Peptone 5.0
pH adjusted to 7.1
Table 2.18: α-Naphthylamine
S. No. Components Quantity (100 mL-1)
100 ml-1 1 N-(1-Naphthyl)-ethylenediamine
Dihydrochloride
0.6 g
2 Acetic Acid (5 N) 100 mL
Table 2.19: Sulfanilic acid
S. No. Components Quantity (100 mL-1)
1 Sulfanilic acid 0.8 g
2 Acetic Acid (5 N) 100 mL
Table 2.20: Medium for indole production (Cappuccino and Sherman, 2005)
S. No. Components Quantity (L-1)
1 Peptone/Tryptone 1 g
2 Distilled water 1000 mL
Medium for Pigment Production (King et al., 1954)
Table 2.21: King’s A medium
S. No. Components Quantity (L-1)
1 Peptone 20.0
2 MgCl2(anhydrous) 3.5
3 Glycerol 10.0
4 K2SO4(anhydrous) 10.0
5 Agar 12.0
pH adjusted to 7.2-7.4
Table 2.22: King’s B medium
S. No. Components Quantity (L-1)
1 Peptone 20.0
2 K2HPO4 1.5
3 MgSO4.H2O 1.5
15
4 Glycerol 10.0
5 Agar 12.0
pH adjusted to 7.2-7.4
Extracellular Enzyme Tests
Table 2.23: Medium for starch hydrolysis (Gerhardt et al., 1994)
S. No. Components Quantity (g L-1)
1 Tryptone 10.0
2 Starch 2.0
3 Yeast extract 5.0
4 NaCl 5.0
5 Agar 20.0
Table 2.24: Medium for lipid hydrolysis (Cappuccino and Sharman, 2005)
S. No. Components Quantity (g L-1)
1 Beef extract 3
2 Peptone 5.0
3 Tributyrin 10
4 Agar 15.0
pH adjusted to 7.2
Table 2.25: Nutrient gelatin broth (Cappuccino and Sharman, 2005)
S. No. Components Quantity (g L-1)
1 Peptone 5.0
2 Beef extract 3.0
3 Gelatin 120.0
pH adjusted to 6.8
Table 2.26: Urea broth (Cappuccino and Sharman, 2005)
S. No. Components Quantity (L-1)
1 Urea 10 g
2 Urea broth base 24 g
3 Distilled water Up to 1000 mL
pH adjusted to 6.9
Urea broth base was added to water and autoclaved separately, cooled to 45 to 50oC, 1%
filter sterilized urea solution was added in tubes aseptically.
16
Table 2.27: Antibiotics for sensitivity testing
S. No. Components Concentration (µg)
1 Amoxicillin (Amc 30)
30
2 Cloxacillin (Cx1) 1
3 Imipenem (Ipm 10) 10
4 Ceftazidime (Caz 30) 30
Reagents for Phosphate Estimation
Table 2.28: Chloromolybdic acid
S. No. Components Quantity (L-1)
1 Ammonium molybdate 7.5 g
2 Concentrated H2SO4 162.0 mL
Table 2.29: Chlorostanous acid
S. No. Components Quantity (L-1)
1 Stannous chloride 25.0 g
2 Concentrated H2SO4 100.0 mL
Table 2.30: Phosphate standard
S. No. Components Quantity (10 mL-1)
1 KH2PO4 100 mg
2 Distilled water 10 mL
Reagents for Detection of Phosphatase
Table 2.31: Trypticase soy agar
S. No. Components Quantity (g 100 mL-1)
1 Sodium chloride 5.0
2 Phytane 5.0
3 Trypticase 15.0
4 Agar 15.0
pH adjusted to 7.3
Table 2.32: 50% Ethanol
S. No. Components Quantity (100 mL-1)
1 Ethanol 50 mL
2 Distilled water 50 mL
17
Table 2.33: 0.5% Phenolphthalein
S. No. Components Quantity (100 mL-1)
1 Phenolphthalein 0.5 g
2 50% ethanol 100 mL
Table 2.34: 8.4% Ammonium hydroxide
S. No. Components Quantity (L-1)
1 Ammonium hydroxide 8.4 g
Acid and Alkaline Phosphatase Estimation (Naseby and Lynch, 1997;
Tabatabai and Bremmer, 1969)
Table 2.35: p-nitrophenyl phosphate disodium (PNPP) 0.115M
S. No. Components Quantity (L-1)
1 p-nitrophenyl phosphate disodium (PNPP) 42.68 g
Table 2.36: 0.5 M sodium acetate buffer
S. No. Components Quantity (L-1)
1 Sodium acetate 41.01 g
pH adjusted to 6.5 for acid phosphatase and 11 for alkaline phosphatase
Table 2.37: 0.5 M CaCl2
S. No. Components Quantity (L-1)
1 CaCl2 55.49 g
Table 2.38: 0.5 M NaOH
S. No. Components Quantity (L-1)
1 NaOH 19.99 g
PLANT GROWTH PROMOTING ACTIVITIES
Solutions for Auxin Estimation (Brick et al., 1991)
Table 2.39: L-tryptophan stock solution
S. No. Components Quantity
1 L-Tryptophan 1 g
18
2 Autoclaved distilled water 40 mL
Table 2.40: Solution I: 0.05M ferric chloride solution
S. No. Components Quantity (10 mL-1)
1 FeCl3 0.08125 g
Table 2.41: Solution II: Perchloric acid
S. No. Components Quantity (100 mL-1)
1 HClO4 50 mL
Table 2.42: Salkowski’s reagent
S. No. Components Quantity
1 0.05M FeCl3 1 mL
2 35% HClO4 50 mL
Siderophores Production (Louden et al., 2011)
Table 2.43: Blue dye solution 1
S. No. Components Quantity (50 mL-1)
1 Chrome Azurol S (CAS) 0.06 g
2 Double distilled water 50 mL
Table 2.44: Blue dye solution 2
S. No. Components Quantity (10 mL-1)
1 FeCl3-6H2O 0.0027 g
2 10 mM HCl 10 mL
Table 2.45: Blue dye solution 3
S. No. Components Quantity (40 mL-1)
1 Hexadecyltrimethylammonium bromide
(HDTMA)
0.073 g
2 Double distilled water 40 mL
Solution 1 was mixed with 9 mL of solution 2 followed by the addition of solution 3.
Resulting blue mixture was autoclaved and stored in plastic bottle.
19
Table 2.46: Minimal Media 9 (MM9) salt solution stock
S. No. Components Quantity (w/v)
1 KH2PO4 15 g
2 NaCl 25 g
3 NH4Cl 50 g
4 Double distilled water 500 mL
Table 2.47: 20% Glucose stock
S. No. Components Quantity (w/v)
1 Glucose 20 g
2 Double distilled water 100 mL
Table 2.48: NaOH stock
S. No. Components Quantity (w/v)
1 NaOH 25 g
2 Double distilled water 150 mL
pH adjusted to ~12
Table 2.49: Casamino acid solution
S. No. Components Quantity (w/v)
1 Casamino acid 3 g
2 Double distilled water 27 mL
Extracted with 3% 8-hydroxyquinoline in chloroform to remove any trace iron and
filter sterilized
Table 2.50: CAS agar
S. No. Components Quantity (L-1)
- 1 Minimal Media 9 100 mL
2 piperazine-N,N′-bis(2- ethanesulfonic acid)
PIPES
32.24 g
3 Agar 15 g
4 Casamino acid solution 30 mL
5
6
20% glucose solution 10 mL
6 Blue dye solution 100 mL
Minimal Media 9, piperazine-N,N′-bis (2- ethanesulfonic acid) PIPES and Agar were
mixed, autoclaved, cooled and mixed with sterile solutions of Casamino acid, 20% glucose
solution, and Blue dye solution. The mixture was gently agitated to thoroughly mix the
solutions and poured into petri dishes aseptically.
20
Medium for Hydrogen Cyanide Production
Table 2.51: Growth medium for Hydrogen cyanide production (Lorck, 1948)
S. No. Components Quantity (g L-1)
1 Glycine 4.4
2 Peptone 3.0
3 Beef extract 5.0
4 Agar 15.0
Table 2.52: Picric acid reagent
S. No. Components Quantity (g 100 mL-1)
1 Sodium carbonate 2.0
2 Picric acid 0.5
Medium for Ammonia Production
Table 2.53: Peptone water
S. No. Components Quantity (100 mL-1)
1 Peptone 4 g
2 Distilled water 100 mL
Table 2.54: Nessler’s reagent (for ammonia detection)
S. No. Components Quantity (L-1)
1 Potassium iodide 50 g
2 Mercuric chloride solution Until saturation
3 Potassium hydroxide solution (50%) 400 mL
4 Ammonia free distilled water Up to 1L
ACC Deaminase Production Test
Table 2.55: Ninhydrin reagent
S. No. Components Quantity (mL-1)
- 1 Ascorbic acid 15 mg
2 Ethylene glycol 60 mL
3 Ninhydrin 500 mg
4 Citrate buffer 1 M (pH 6.0) 60 mL
Citrate buffer was added just before use.
21
Table 2.56: DF medium (Penrose and Glick, 2003)
S. No. Components Quantity (L-1)
- 1 KH2PO4 4 g
2 Glucose 2 g
3 Gluconic acid 2 g
4 NaHPO4 6 g
5 Citric acid 2 g
6 MgSO4.7H2O 0.2 g
7 ZnSO4 70 µg
8 FeSO4.7H2O 1 mg
9 MoO3 10 µg
10 H3BO3 10 µg
11 MnSO4 10 µg
Table 2.57: DF-ACC medium (Penrose and Glick, 2003)
S. No. Components Quantity (L-1)
- 1 KH2PO4 4 g
2 Glucose 2 g
3 Gluconic acid 2 g
4 NaHPO4 6 g
5 Citric acid 2 g
6 MgSO4.7H2O 0.2 g
7 ZnSO4 70 µg
8 FeSO4.7H2O 1 mg
9 MoO3 10 µg
10 H3BO3 10 µg
11 MnSO4 10 µg
12 1-aminocyclopropane-1-carboxylate (ACC) 0.303 g
Table 2.58: Stock solutions of ACC (0.5 M)
S. No. Components Quantity (100 mL-1)
- 1 ACC 10.1 g
2 Distilled water 100 mL
22
REAGENTS FOR PLANT MICROBE INTERACTION EXPERIMENT
Table 2.59: 0.1% HgCl2 solution for seed sterilization
S. No. Components Quantity (100 mL-1)
1 HgCl2 0.1 g
2 Autoclaved distilled water 100 mL
Table 2.60: Pesticides
S. No. Common
name
Grade
(purity)
Chemical name Chemical family
1 Chlorpyrifos 40% O,O-diethyl O-3,5,6-
trichloropyridin-2-yl
phosphorothioate
organophosphate
2 Pyriproxyfen 10.8% 4-phenoxyphenyl (RS)-2-
(2-pyridyloxy)propyl ether
pyridine
PLANT BIOCHEMICAL TESTS
Chlorophyll Estimation
Table 2.61: 80% acetone
S. No. Components Quantity (100 mL-1)
1 Acetone 80 mL
2 Distilled water 20 mL
Proline Estimation
Table 2.62: 3% Sulfosalycylic acid
S. No. Components Quantity (100 mL-1)
1 Sulphosalycylic acid 3 g
Table 2.63: Orthophosphoric acid (6N)
S. No. Components Quantity (100 mL-1)
1 Orthophosphoric acid 38.1 mL
2 Distilled water 61.9 mL
Table 2.64: Acid ninhydrin reagent
S. No. Components Quantity (w/v)
23
1 Ninhydrin 1.25 g
2 Glacial acetic acid 30 mL
3 Orthophosphoric acid (6N) 20 mL
Other Chemicals for Proline Estimation
Glacial acetic acid
Toluene
Proline for standard
Solutions for Peroxidases Activity (David and Murray, 1965)
Table 2.65: Phosphate buffer (0.1M)
S. No. Components Quantity (g L-1)
1 K2HPO4 17.4
2 KH2PO4 13.6
pH adjusted to 7.0
Table 2.66: 1% Guaiacol solution
S. No. Components Quantity ( 100 mL-1)
1 Guaiacol 1 mL
2 Distilled water 100 mL
Table 2.67: H2O2 solution
S. No. Components Quantity ( 100 mL-1)
1 H2O2 (stock 35%) 0.85 mL
Solutions for Acid Phosphatase Activity (Iqbal and Rafique, 1987)
Table 2.68: 0.1M Tris HCL buffer
S. No Components Quantity (300 mL-1)
1 C4H11NO3 [Tris (hydroxymethyl)
aminomethane]
6.057 g
pH was adjusted to 6.5 using concentrated HCL
24
Table 2.69: Citrate buffer
S. No. Components Quantity (g L-1)
1 C6H8O7.H2O (Citric acid) 42.0
2 NaOH (1N) 376 mL
pH was adjusted to 4.9
Table 2.70: Disodium phenyl phosphate
S. No. Components Quantity (L-1)
1 C6H5PO4Na2 (Di-sodium Phenyl
Orthophosphate)
2.54 g
Solution was cooled immediately after boiling.
Table 2.71: Phenol standard (stock)
S. No. Components Quantity (L-1)
1 Phenol (pure crystalline) 1.0 g
2 0.1 N HCl 1000 mL
Table 2.72: Phenol solution (working)
S. No Components Quantity (100 mL-1)
1 Phenol standard (stock) 1 mL
Table 2.73: 0.5 N Sodium hydroxide solution
S. No. Components Quantity (L-1)
1 NaOH 20.0 g
Table 2.74: 0.5N Sodium bicarbonate solution
S. No. Components Quantity (L-1)
1 NaHCO3 42.0 g
Table 2.75: 4-Amino antipyrin
S. No. Components Quantity (L-1)
1 4-amino antipyrin 6.0 g
25
Table 2.76: Potassium ferricyanide
S. No. Components Quantity (L-1)
1 Potassium ferricyanide 24.0 g
Solutions for Soluble Protein Estimation (Lowry et al., 1951)
Folin’s mixture
Table 2.77: Solution A
S. No. Components Quantity (L-1)
1 NaOH 4.0 g
2 Na2CO3 20.0 g
Table 2.78: Solution B
S. No. Components Quantity (L-1)
1 C4H4KNaO6.4H2O 4.0 g
Table 2.79: Solution C
S. No. Components Quantity (L-1)
1 CuSO4 1.0 g
To prepare Folin’s mixture, solution A, B and C were mixed in 100:10:10 ratio.
Folin and ciocalteu’s phenol reagent
Commercially prepared reagent (Sigma) was used.
Interaction between arbuscular mycorrhizal fungi and phosphate
solubilizing bacteria
Table 2.80: Growth medium for proximal compartment
S. No. Components Quantity (mg L-1)
1 KNO3 80
2 MgSO4.7H2O 731
3 KCl 65
4 KH2PO4 4.8
5 Ca(NO3)2.4H2O 288
6 NaFeEDTA 8
26
7 KI 0.75
8 MnCl2.4H2O 6
9 ZnSO4.7H2O 2.65
10 H2BO3 1.5
11 CuSO4.5H2O 0.13
12 Na2MoO4.2H2O 0.0024
13 Glycine 3
14 Thiamine hydrochloride 0.1
15 Pyridoxine hydrochloride 0.1
16 Nicotinic acid 0.5
17 Myo-inositol 50
18 Sucrose 10000
19 phytagel 4000
pH adjusted to 5.5
Table 2.81: Medium for distal compartment
S. No. Components Quantity (mg L-1)
1 KNO3 80
2 MgSO4.7H2O 731
3 KCl 65
4 KH2PO4 4.8
5 Ca(NO3)2.4H2O 288
6 NaFeEDTA 8
7 KI 0.75
8 MnCl2.4H2O 6
9 ZnSO4.7H2O 2.65
10 H2BO3 1.5
11 CuSO4.5H2O 0.13
12 Na2MoO4.2H2O 0.0024
13 Tricalcium phosphate 2500
14 Phytagel 4000
pH adjusted to 5.5
27
METHODS
Soil sampling
For the isolation of phosphate solubilizing bacteria, sampling was done from Lahore,
Chakwal and Kalar Kahar, Punjab, Pakistan. Soil samples were collected from rhizosphere
of different plants including, vegetables, cereals, fruits and ornamental plants as well as
from barren soil. Upper layer of soil was removed to avoid environmental contaminants
and soil was collected with the help of sterile spatula and transported to laboratory in sterile
sampling bags. The pH of samples was determined using digital pH meter and samples
were proceeded for screening of bacteria having ability to solubilize inorganic phosphate.
Isolation of phosphate solubilizing bacteria
Soil suspensions were prepared by adding one gram of soil sample into 100 mL of
autoclaved distilled water and thoroughly mixed by placing on shaker for 30 minutes.
Serially diluted soil suspensions (up to 10-5) were spread on Pikovskaya agar medium
having tricalcium phosphate as an insoluble phosphate source. The plates were incubated
at 28oC for 5-7 days. Twenty eight bacterial strains having different morphology and a
clear zone around their colonies were selected and further purified by streak plate method
on Pikovskaya agar medium. Purified strains were maintained on L-agar medium.
Morphological characterization of phosphate solubilizing bacterial
strains
Colony and cell morphology of isolated bacterial strains was checked by growing them on
agar medium. Morphological characteristic and diverse appearances were studied for
characterization and identification.
28
Colony and cell morphology
To study colony morphological characteristics, 24 hours fresh bacterial cultures were
observed for shape, size, color, elevation and margin with the help of magnifying glass.
Arrangement and shape of cells were studied under light microscope with 100X lens.
Gram’s staining
Gram’s stain is commonly used staining to determine the morphology of cells. This
staining is generally used in the first step to identify bacterial cells as it significantly
differentiate bacteria into two major groups as Gram-negative or Gram-positive.
The basic principle behind Gram staining is the retention of crystal violet in the cell wall
of bacterial cell. The cells which have the ability to retain this primary dye even after
solvent treatment are gram positive but the cells which are unable to retain crystal violet
but retain the counter stain (safranin) are gram negative. The difference in the reaction to
Gram’s stain is because of difference in cell wall of the bacterial cells. Gram positive
microorganisms have thick layer of peptidoglycan and thin layer of lipids in their walls
whereas, in case of gram negative bacterial cells, they have thin peptidoglycan layer and
higher lipid content. The thicker cell wall of gram positive bacteria becomes dehydrated
upon alcohol treatment which closes the pores in cell wall which ultimately blocks the
formation of complex between iodine and crystal violet, as a result cells remain stained.
Whereas, the gram negative cell wall becomes permeable due to solubility of lipid in
alcohol which ultimately enhance crystal violet leaching from cells.
Gram staining was performed by making a smear of 24 hours fresh bacterial culture with
the help of a sterile loop in sterile water drop on a glass slide. The smear was air dried and
heat fixed. Crystal violet was applied for 1 minute, after washing with sterile distilled
29
water, iodine solution was added for 45 seconds and again washed with distilled water.
Extra stain was removed by treating with 95% alcohol. Counter stained with safranin for
60 seconds, the smear was air dried after washing with distilled water and observed with
the help of microscope under 100X lens.
Motility testing
Microorganisms were tested for motility with the help of SIM agar, the media was sterilized
and tubes were prepared. Fresh bacterial cultures were inoculated by stabbing and tubes
were incubated for 24 hours at 28oC. After the completion of incubation time, the growth
pattern of bacteria was observed that either it was restricted to the inoculation line or not.
Bacteria growing along with the inoculation line were non motile whereas the diffused
growth pattern indicated the motility of bacteria.
Biochemical characterization of phosphate solubilizing bacteria
Catalase test
Some bacterial species produce reactive oxygen species as a result of aerobic respiration,
which are harmful to cell and also produce oxidative stress. Aerobic bacteria produce
catalase enzyme which can covert H2O2 into oxygen and water molecule. To check the
bacteria that can produce this enzyme, 3% H2O2 was added on a clean glass slide and fresh
bacterial culture was added to it with the help of sterile wooden tooth pick and was checked
for O2 bubbles evolution which indicated a positive result.
Cytochrome oxidase test
Different bacteria can be identified as aerobic or anaerobic on the basis of oxidase enzyme
production. Cytochromes are iron containing electron carriers in electron transport chain.
30
To test the presence of this enzyme, tetra-methyl-phenylenediamine dihydrochloride is
used as a reagent which acts as a substrate that can contribute electron as a result of
oxidation and produce a black color compound.
To determine the presence of oxidase enzyme in phosphate solubilizing bacteria, spot test
was performed. Bacterial culture was transferred to reagent adsorbed filter papers with the
help of sterile toothpick. Appearance of purple to black color within 1 minute was noticed
as a positive result.
Citrate utilization test
Certain bacteria have the ability to use citrate as the only source for carbon, in the absence
of lactose and glucose. Citrase enzyme metabolizes citrate and as a result an alkaline end
product is formed. Simmon citrate agar medium contains ammonium ions as the only
source for nitrogen and sodium citrate as the only source for carbon. Bacteria are able to
metabolize citrate salt to organic acids and carbon dioxide. Due to the combination of
sodium with sodium carbonate and carbon dioxide, an alkaline salt formation takes place.
The pH indicator in this medium indicates the increase in pH by color change from green
(neutral) to Prussian blue (alkaline). To test the isolated bacterial strains for citrate
utilization ability, Simmon citrate agar medium was prepared and slants were prepared.
Fresh bacterial culture was streaked and tubes were incubated at 28oC. After 24 hours of
incubation, results were observed for change in color of medium.
Methyl Red (MR) test
Several microorganisms have the ability to utilize glucose by converting it into acidic
product and this end product depends on the metabolic pathway present in organism. In
some microorganisms, the acid products remains stable while others can convert them into
31
alkaline forms for example, 2-3, butanediol and acetoin, which ultimately change the pH
of medium. Medium for MR/VP test is a combination medium consisting of peptone,
phosphate buffer and glucose. Bacterial organisms having ability to ferment glucose by
mixed acid fermentation, convert it to larger quantities of stable acids which results in
decline of pH. The pH indicator added to this medium determines the change in pH, for
decreased pH it turns to red while as a result of increase in pH it changes to yellow.
MR/VP broth was prepared and added to test tubes before autoclaving. Fresh bacterial
cultures were inoculated to tubes while control remained un-inoculated. All tubes were
placed in incubator for 24 hours at 28oC. After incubation, the culture content of each tube
was divided into two halves by transferring to a new set of sterile tubes. One set was added
with 5-6 drops of methyl red solution. Color change to bright red was observed and results
were recorded.
Voges Proskauer (VP) test
This test helps to determine the production of alkaline end products as a result of
conversion of acids during glucose fermentation. The main end product of glucose
fermentation in these bacteria are acetoin and 2-3 butandiol. This test determines acetoin
production by using Barritt’s reagent. One set of tubes with remaining half of culture
medium was incubated for another 24 hours. After total 48 hours of incubation, 0.6 mL of
Barritt’s reagent A (5% α napthol) along with 0.2 mL of Barritt’s reagent B (40% KOH)
was added to culture tubes. After mixing, tubes were incubated for 15-60 minutes at 28oC.
Appearance of red color determined the positive result for this test.
32
Nitrate reduction test
Nitrate reductase is an enzyme produced by some aerobes and facultative anaerobes, it can
convert nitrate to nitrites in molecular oxygen deficient environment. These nitrites are
further reduced to ammonia and nitrogen. To check the bacterial ability to reduce nitrate,
bacteria (24 hours fresh culture) were grown in tubes containing 5 mL of nitrate broth. The
tubes were incubated for 2-3 days at 28oC, after the completion of incubation time, 0.5 mL
of solution A (1% sulphanilic acid) and 0.5 mL of solution B (α- naphthylamine) were
added to the tubes. Tube having red color showed that organism was able to reduce nitrate
to nitrite whereas absence of red color after adding reagents showed negative results. Zinc
dust was added to the tubes which showed negative reaction, presence of red color after
adding zinc dust showed that bacteria was not able to reduce nitrate. In case of no color
change, the results indicated that nitrate was reduced to some other compound.
Indole test
Several bacteria produce tryptophanase enzyme which helps in the oxidation of tryptophan.
Tryptophan utilization can be determined by growing bacteria in SIM medium
supplemented with tryptophan. Break down of tryptophan results into the production of
pyruvic acid, ammonia and indole. Kovac’s reagent is used for indole’s presence in
medium by the appearance of cherry red color. Bacterial inoculum was given in SIM
medium tubes and incubated for 24 hours at 28oC. Few drops of reagent were added to
culture tubes, agitated gently and the appearance of cherry red color on top layer indicated
a positive result.
33
Pigment production test
Some bacteria especially species belonging to Pseudomonadaceae have the ability to
produce pigments. To check the pigment production by bacterial cultures, King’s A and
King’s B medium were used. Agar slants of each media were prepared and bacterial
cultures were streak inoculated. The slants were incubated for 24 to 48 hours at 28oC,
pigment production was noted after completion of incubation.
Extracellular enzyme production by phosphate solubilizing bacteria
Starch hydrolysis test
Starch is a polysaccharide consisting of two components, unbranched polymer of glucose
called amylose and a branched polymer called as amylopectin. Certain bacteria are capable
of hydrolyzing both amylose and amylopectin into glucose, maltose and dextrins. These
molecules are smaller in size, and can move into the cells and help in energy production
via glycolysis. To check the degrading capability of phosphate solubilizing bacteria, starch
supplemented L-agar plates were prepared. Bacteria were inoculated and plates were
incubated at 28oC for 48 hours. Gram’s iodine was added to the surface of plates for 30
seconds. Starch hydrolysis was indicated by the appearance of clear zone around the
bacterial growth.
Lipid hydrolysis test
Tributyrin agar is usually used for the assessment of bacterial isolates to breakdown lipids
as a result of lipase enzyme production. Lipase enzyme hydrolyze triglyceride lipids to
shorter fragments which are used by the cell in energy production as well as in other
processes. Tributyrin oil makes medium opaque, bacterial strains able to produce lipase
enzyme produce a halo around growth. To check lipase enzyme production by phosphate
34
solubilizing bacteria, tributyrin agar plates were prepared, streak inoculated and incubated
at 28oC. After 48 hours of incubation, results were observed for the halo formation around
bacterial growth.
Gelatin hydrolysis
Different microorganisms are capable of producing gelatinase enzyme and metabolize
gelatin protein into amino acids. Nutrient gelatin medium contains gelatin, which have the
properties to remain in the form of gel at temperatures below 25 oC. If gelatin hydrolysis
occur by the gelatinase activity of bacteria, it loses its gel property and remains liquid even
at low temperature (4 oC). To perform this test, nutrient gelatin medium was prepared in
test tubes and fresh bacterial cultures were inoculated. The tubes were incubated at 28 oC
for 48 hours. After incubation, tubes were placed at 4 oC for 30 minutes and results were
recorded. Cultures that remained liquid even at low temperature showed positive test for
gelatinase enzyme activity.
Urea hydrolysis
Urea hydrolysis is a test to differentiate bacteria particularly the Enterobacteriaceae by their
ability for the production of urease enzyme. Test medium contains urea which upon
hydrolysis by bacteria is converted into ammonia. As a result of ammonia production, pH
of medium changes to alkaline and in the presence of phenol red indicator, the color of
medium changes to pink. To perform this test, urease broth was prepared and urea solution
was added after filter sterilization. Tubes were inoculated with fresh bacterial cultures
whereas control tubes remained un-inoculated. Tubes were incubated at 28oC for 7 days.
Results were recorded after incubation for color change.
35
Physiological characterization of phosphate solubilizing bacteria
Antibiotic resistance test
Bacterial ability to resist different antibiotics or susceptibility towards them was checked
for some commonly used antibiotics including amoxicillin (AMC 30), cloxacillin (CX 1),
imipenem (IPM 10) and ceftazidime (CAZ 30). Optical density of bacterial culture was set
to1.0 at 600 nm to get uniform number of cells. Bacterial culture was spread on L-agar
medium with the help of sterile cotton swab followed by placement of antibiotic discs of
various concentrations onto the agar surface. Inoculated plates were incubated for 48 hours
at 28oC and results were noted for resistance or susceptibility towards antibiotics.
pH range of phosphate solubilizing bacteria
The pH range for bacterial growth can be described as the levels of pH as minimum,
optimal and maximum. Bacteria are not able to grow above and below the maximum and
minimum pH levels. The optimum pH is a point between these extremes at which they
show best growth rate. This decreasing or increasing order of growth indicates direct effect
of hydrogen ions on enzymatic reaction rate.
To study the effect of pH on bacterial growth, L-broth was prepared and different pH levels
(3, 5, 7, 9 and 11) were adjusted separately and medium was autoclaved in conical flasks.
Standardized bacterial culture (50 µl) was inoculated to each flask and incubated on an
orbital shaker at 200 rpm for 24 hours at 28oC. Optical density of cultures was measured
against blank (un-inoculated broth) at 600 nm with the help of spectrophotometer (Cecil
Aquarius CE 7200). The experiment was repeated thrice for each strain and pH.
36
Phosphate solubilization potential of bacterial isolates
Solubilization of inorganic phosphate in solid medium
Inorganic phosphate solubilizing ability of bacterial isolates was also investigated. For this
purpose two different phosphate solubilization media, Pikovskaya agar medium and
National Botanical Research Institute’s Phosphate (NBRIP) (Nautiyal, 1999) medium were
prepared and their pH was adjusted to 7.0 before sterilization. Standardized fresh bacterial
cultures were used for spot inoculation. Plates were incubated at 28oC for seven days.
Solubility index for phosphate was determined by following the method of Edi-Promero et
al. (1996) by measuring diameter of both halozone as well as of colony with the help of
following formula:
𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 𝑠𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑧𝑎𝑡𝑖𝑜𝑛 𝑖𝑛𝑑𝑒𝑥 (𝑆𝐼) =𝐶𝑜𝑙𝑜𝑛𝑦 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 + 𝐻𝑎𝑙𝑜𝑧𝑜𝑛𝑒 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟
𝐶𝑜𝑙𝑜𝑛𝑦 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟
Growth of phosphate solubilizing bacteria in liquid medium
To estimate the solubilized phosphate quantities by isolated bacteria, bacterial isolates were
grown in Pikovskaya broth and NBRIP broth media. Fresh bacterial culture was inoculated
in flasks having sterile culture medium with tricalcium phosphate as a sole phosphate
source. Control received autoclaved distilled water instead of bacterial culture. Experiment
was replicated three times. After inoculation, cultures were incubated for 7 days at 28oC
on an orbital shaker at 150 rpm.
Quantitative estimation of solubilized phosphate
To assess the bacterial ability for phosphate solubilization, method described by King
(1932) was followed. Bacterial cultures were grown in liquid medium and centrifuged at
50000 rpm for 30 minutes. One mL of culture supernatant was taken in conical flask
37
followed by the addition of 10 mL chlormolybdic acid and volume was raised up to 45 mL
with distilled water. Mixture was stirred and 250 µL of chlorostanous acid reagent was
added and final volume of the reaction mixture was raised up to 50 mL with water. As a
result of reaction, blue coloration developed which was read with the help of
spectrophotometer by measuring absorbance at 600 nm. The quantity of solubilized
phosphate was calculated by preparing standard curve of KH2PO4.
Effect on pH and titrable acidity
To determine the influence of phosphate solubilization on pH change, pH of culture
medium was adjusted to 7. After inoculation and completion of incubation time, pH was
recorded with the help of digital pH meter. In order to determine the influence of phosphate
solubilization on titratable acidity, bacterial cultures were centrifuged for 10 minutes at
1000 rpm. Culture supernatant (5 mL) was taken in a conical flask and few drops of
phenolphthalein indicator were added followed by titration against 0.01 N NaOH (Takao,
1965). Titrable acidity was calculated as volume of sodium hydroxide consumed per 5 mL
of supernatant.
Extracellular protein estimation
For the estimation of extracellular protein content, bacterial cultures were centrifuged and
supernatant was used. Experiment was performed in test tubes following Lowry’s method
(1951). Tubes were added with 200 µL of culture supernatant and 2 mL of alkaline copper
sulfate reagent. Tube content was mixed and incubated at room temperature for 10 minutes.
200 µL of 1 N Folin Ciocalteu reagent was added followed by incubation for another 30
minutes. Absorbance of samples was measured against blank at 660 nm (Hartree, 1972).
38
Protein concentration of unknown samples was calculated by preparing standard plot of
known concentrations of protein using Bovine Serum Albumin (BSA).
Phosphatases production test
To detect the production of phosphatases by phosphate solubilizing bacteria, a solution of
phenolphthalein diphosphate (0.5%) was prepared and sterilized with the help of 0.22 µm
membrane filter. Tryptic soya agar medium was prepared and cooled to 50oC and two mL
of sterile phenolphthalein solution was added per 100 mL of medium before pouring into
petri plates. Bacterial cultures were inoculated and incubated for 48 hours at 28oC.
Phosphatase production was determined by adding few drops of ammonium hydroxide
solution (8.4%) onto the lid of petri dish and results were observed for development of pink
coloration after 15 minutes. Pink color development indicated the production of
phosphatases by bacterial isolates.
Acid and alkaline phosphatase biosynthesis
To estimate the production of alkaline or acid phosphatase involved in phosphate
solubilization by phosphate solubilizing bacteria, method of Naseby and Lynch (1997) was
followed. Sodium acetate buffer with pH 6.5 was used for acid phosphatase estimation
while sodium acetate buffer with pH 11 was used for alkaline phosphatase estimation.
Bacterial cultures grown in Pikovskaya broth medium were centrifuged to get supernatant
which was used as enzyme for assay. Reaction mixture containing 0.5 M sodium acetate
buffer (2 mL), p-Nitrophenyl Phosphate (PNPP) substrate (0.5 mL) and supernatant (0.5
mL) was mixed and incubated for 90 minutes at 37oC. Reaction tubes were placed at 2 oC
for 15 minutes to terminate the reaction followed by the addition of 0.5 M CaCl2 (0.5 mL)
and 0.5 M NaOH (2 mL). Centrifugation was done to remove any possible precipitation.
39
Formation of p-Nitrophenol (PNP) was determined by measuring optical density at 398 nm
(Tabatabai and Bremmer, 1969). The concentration of PNP was calculated by preparing
standard curve.
Genetic characterization of phosphate solubilizing bacteria
Identification of phosphate solubilizing bacteria by 16S rRNA gene sequencing
Isolated bacterial strains exhibiting inorganic phosphate solubilization activity were
sequenced using sequencing facility provided by Macrogen (Korea). Breifly, pure colonies
were picked and added to 500 µL saline solution in ependorf (1.5 mL). Tubes were
centrifuged at 10000 rpm for 10 minutes. Pellets were resuspended in 500 µL InstaGen
Matrix (Bio-Rad, USA). Tubes were incubated for 30 minutes at 56oC followed by heating
for 10 minutes at 100oC. PCR was performed for amplification of 16S rRNA gene.
Amplified DNA product was analyzed by gel electrophoresis. Amplified DNA product was
sequenced using Big Dye terminator cycle sequencing kit (Applied, BioSystems, USA).
Product after sequencing was resolved using Applied BioSystems (3730 XL) automated
sequencing system for DNA (Applied, BioSystems, USA).
Phylogenetic analysis
Phylogenetic relationship of bacterial isolates was performed after sequencing. Obtained
nucleotide sequences were analyzed for base calling using Finch TV software version 1.4.0
(Geospiza). For molecular and phylogenetic study, obtained sequences of nucleotides were
compared with the sequences in NCBI database through BlastN. Closest match for
similarity of known affiliating phylogeny were used for assigning specific group for
taxonomy. Sequences with highly similar scoring were retrieved from database and
sequence alignment was performed with the help of ClastalW and phylogenetic tree was
40
inferred through the method of neighbor joining using MEGA7 software (Version 7.0.14)
(Tamura et al., 2011). For construction of tree, boot-strap value was set to 100 replicates
for enhanced reliability (Felsenstein, 1985).
Accession numbers
To obtain a unique identification numbers for each sequence, the obtained nucleotide
sequences were analyzed and submitted to NCBI GenBank through Bankit submission
tool.
Effect of sugars on phosphate solubilization
To check the effect of different sugars on solubilization ability of phosphate solubilizing
isolates, Pikovskaya broth medium was prepared and pH of medium was adjusted to 7.0
before autoclaving. Sugar solutions (sucrose, glucose, maltose and galactose) were
prepared in autoclaved distilled water and filtered sterilized using 0.2 µm filters and added
to broth at the concentration of 10 gL-1. Twelve strains selected for this study were grown
in L-broth for 24 hours and were standardized by adjusting their optical density to 1.0 for
inoculating Pikovskaya broth. After inoculation, cultures were incubated on a shaker at 150
rpm at 28oC for two weeks. After completion of incubation, the cultures were checked for
change in pH. Culture were centrifuged and supernatant was used for estimating solubilized
phosphate, titrable acidity, acid and alkaline phosphatase.
Pesticide tolerance test
Bacterial ability to tolerate pesticide stress was also evaluated. Two pesticides including
Chlorpyrifos and Pyriproxyfen were selected for this study. Pesticide stock solutions were
prepared and added to L-agar and Pikovskaya agar before pouring into Perti dishes. Optical
41
density of fresh bacterial cultures was set to 1.0 at 600 nm using spectrophotometer and
agar plates were spot inoculated. Plates were incubated at 28oC and results were noted for
presence or absence of bacterial growth after 48 hours on L-agar whereas on Pikovskaya
agar plates were incubated for 7 days and presence or absence of bacterial growth and
phosphate solubilization zone around colonies was also observed. Minimal inhibitory
concentration (MIC) was also determined for each isolate.
Effect of pesticide on phosphate solubilization
To investigate the effect of pesticide stress on phosphate solubilization ability of isolates,
50 mL Pikovskaya broth was prepared in 250 mL flasks and supplemented with
recommended doses of pesticides (Dutta et al., 2010; Ahemad and Khan, 2011c) after
autoclaving. Chlorpyrifos was added to the final concentration of 0.5 µg mL-1 and for
Pyriproxyfen 1.3 µg mL-1 concentration was used. Twelve bacterial strains were included
in this study. One mL fresh bacterial culture (108 cells mL-1) was inoculated to 50 mL broth
and cultures were incubated on an orbital shaker at the speed of 150 rpm at 28oC for 7 days.
After incubation, pH of cultures was determined with the help of digital pH meter. Cultures
were centrifuged at 10000 rpm for 30 minutes. Supernatant was used to estimate
solubilized phosphate and titrable acidity.
Plant growth promoting characteristics
Biosynthesis of Indole Acetic Acid
Ability of phosphate solubilizing bacteria to produce indole acetic acid was evaluated using
colorimetric determination method as described by Patten and Glick (2002). Bacterial
cultures were grown in L-broth in the absence and presence of tryptophan (1 mg mL-1). L-
tryptophan solution was supplemented to broth medium after filter sterilization. Cultures
42
were incubated at 28oC on an orbital shaker at 150 rpm. After 48 hours of incubation,
culture supernatant was obtained by centrifugation at 10000 rpm for 5 minutes. One mL of
supernatant was mixed with two mL of Salkowski’s reagent (Gordon and Weber, 1951).
Tubes containing reaction mixture were shaken gently and placed in dark for 30 minutes.
Absorbance was measured at 535 nm with the help of spectrophotometer. Auxin
concentration of samples was calculated from standard plot by using indole acetic acid
(Sigma).
Siderophore biosynthesis
Qualitative assay was performed to check the competing abilities for iron between Chrome
Azurole S-iron complex and siderophores producing organisms. Iron removal takes place
by siderophores from Chrome Azurole S (CAS), as siderophores makes a stronger bond
with it. As a result of binding, color change appear from blue to orange (Schwyn and
Neilands, 1987).
To check siderophores production by phosphate solubilizing bacteria, 5 µL fresh bacterial
culture grown in L-broth medium was spotted onto agar surface of CAS medium. Plates
were incubated for 3 to 4 days at 28oC and results were recorded for change in color.
Hydrogen cyanide biosynthesis
To check the ability of isolated bacterial strains to produce Hydrogen Cyanide (HCN),
experiment was performed following the method of Lorck (1948). Glycine supplemented
nutrient agar plates were prepared and fresh bacterial cultures were spread inoculated.
Sterile filter papers dipped in the solution of picric acid (0.5%) and sodium carbonate (2%)
were placed asceptically onto the surface of agar. Plates were incubated at 28oC and results
were observed for orange to red color development after 4 days.
43
Biosynthesis of ammonia
Bacterial ability to produce ammonia was tested using peptone water. Fresh bacterial
cultures were inoculated in test tubes having 4% peptone water. Cultured tubes were
incubated for 3 days at 28oC. After incubation, 0.5 mL of Nessler’s reagent (Krung et al.,
1979) was added to tubes and color change of medium was recorded. Color change from
brown to yellow indicated positive result for ammonia production (Marques et al., 2010)
whereas, color intensity represented the quantity of ammonia production.
ACC deaminase biosynthesis
Utilization of ACC by phosphate solubilizing bacteria was checked by following the
method of Penrose and Glick (2003). Single bacterial colony was inoculated in L-broth and
was incubated at 28oC for 24 hours on shaker (200 rpm). Cell pellet of 2 mL culture was
obtained by centrifugation followed by washing with DF medium for two times. Pellet was
resuspended in culture tube containing ACC supplemented DF medium (2 mL) and
incubated for 24 hours at 28oC followed by centrifugation of 1.5 mL of culture at 8000 g
for 5 minutes. Supernatant (100 µL) was ten times diluted with DF medium and was used
for ninhydrin-ACC assay.
To perform the assay, sixty microliters of diluted supernatant along with 120 µL of
ninhydrin reagent was loaded to 96 well PCR plate and incubated for 30 minutes on boiling
water bath. Optical density was measured with the help of spectrophotometer at 570 nm.
Values were compared with standard plot (ACC pure) to find the concentration of ACC
deaminase produced by isolates.
44
Plant growth experiments
Inoculum preparation for seed inoculation
To prepare bacterial suspension for inoculation of seeds, bacteria were grown overnight in
conical flask having L-broth. Cultures were incubated at 28oC on an orbital shaker at the
speed of 150 rpm. Bacterial cells were harvested by the process of centrifugation and
washed twice with autoclaved distilled water to remove the growth medium. The optical
density of cultures was adjusted to 108 cells per mL at the wavelength of 600 nm.
Seed sterilization
Certified healthy seeds of Triticum aestivum var. Inqlab 97 were obtained from Punjab
Seed Corporation Lahore, Pakistan. Seeds were washed with autoclaved distilled water to
remove any possible kind of dirt. For surface sterilization, seeds were treated with the
suspension of 0.1% mercuric chloride for five minutes with continuous shaking. The seeds
were then repeatedly washed with sterile distilled water for complete removal of HgCl2
traces from seeds.
Seed inoculation
Sterilized seeds of Triticum aestivum were immersed in standardized bacterial suspension
for 25 minutes. For control, sterilized seeds were immersed in sterile water for same time
period.
Wheat root elongation assay
To evaluate the ability of phosphate solubilizing isolates to promote or elongate the growth
of wheat seeds in vitro, Penrose and Glick’s method (2003) was adapted. Twelve bacterial
strains were evaluated in this study. Bacterial treated and untreated seeds were placed in
45
sterile petri dishes lined with double layer of Whatman filter paper sheets. Plates were kept
under controlled conditions, placed in dark until start of germination and then at alternating
dark and light cycle at 25oC for seven days. At the end of experiment, results were recorded
including percentage of germination, length of root and shoot and total length of plantlet.
The rate of germination was estimated by percentage formula
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑖𝑜𝑛 =𝑁𝑜. 𝑜𝑓 𝑠𝑒𝑒𝑑𝑠 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑒𝑒𝑑𝑠 𝑝𝑒𝑟 𝑝𝑙𝑎𝑡𝑒 𝑋 100
Measurements were performed with the help of millimeter ruler. Root length was taken
from the primary root tips to hypocotyl. Total length measurement was done by
measurement from primary root tip to shoot tip. Length of shoots were determined by
deducting root lengths from total length.
Wheat root elongation assay under pesticide stress
The ability of selected strains to promote root length under pesticide stress was evaluated
by adding Chlorpyrifos solution at the concentration of 0.5 µg per mL and pryiproxyfen at
the concentration of 1.3 µg mL-1 and a combination of both instead of distilled water. Each
treatment was performed with bacterial inoculation and without inoculation of bacteria
(control). Results were recorded as described above.
Plant microbe interaction experiment with wheat
A pot experiment was conducted in experimental station of the University of the Punjab,
Lahore, Pakistan to investigate the impact of phosphate solubilizing isolates on wheat
growth. Seeds of wheat (Triticum aestivum, variety Inqalab 97) were obtained from Punjab
Seed Corporation. The experiment was started at the end of November, 2014 and plant
were harvested at maturity at the end of April, 2015. Clay pots with the diameter and height
46
of 12 and 14 inches were filled with 8.5 kilograms of unsterile natural garden soil obtained
from botanical garden of University of the Punjab, Lahore, Pakistan.
For bacterial inoculation, 12 phosphate solubilizing bacterial strains (S1, S2, Rad1, Rad2,
Ros2, JA10, R14, SL8, SpA, W95, W96 and UP) were used. For inorganic phosphate
application, three inorganic phosphate sources were used including Tricalcium phosphate
(TCP), Aluminium Phospahte (ALP) and Ferric phosphate (FP) at the concentration of 8
mg kg-1. The inorganic phosphate was mixed thoroughly in soil before filling the pots.
Pesticide effect was checked by adding the combination of pesticides including
Chlorpyrifos and Pyriproxyfen at the recommended concentrations for soil at 0.5 mg kg − 1
and 1.3 mg kg − 1, respectively (Dutta et al., 2010; Ahemad and Khan, 2011c).
The treatments used in this study are as follows:
1. PSB
2. PSB + TCP
3. PSB + ALP
4. PSB + FP
5. PSB + pesticide stress
6. PSB + TCP + pesticide stress
7. PSB + ALP + pesticide stress
8. PSB + FP + pesticide stress
The experiment was performed with three replicates for each treatment. And arranged in
completely randomized block design. Initially 10 seeds were sown in each pot and each
pot received 10 mL of respective bacterial suspension of bacteria. The bacterial cultures
were prepared by growing bacterial isolates in L-broth for 24 hours and centrifuged to get
47
cells pellets and washed with sterile water. The optical density of bacterial cultures were
adjusted to 1.0 using sterile water at the wavelength of 600 nm using spectrophotometer.
Control plants of each treatment remained un-inoculated. Plants were allowed to grow and
number of plants per pot was reduced to 5 by thinning. Plants were allowed to grow until
they got matured to record the final yield. Leaf samples were collected during growth
period and different biochemical tests were also performed including estimation of
chlorophyll, proline, peroxidase, acid phosphatase activity and soluble protein.
At maturity, plants were harvested and the data was recorded using standard methods. The
growth parameters included in this study are as follows:
a) Shoot length (cm)
b) Spike length (cm)
c) Number of tillers
d) Number of spikes per plant
e) Number of spikelets per spike
f) Weight of spikes (g)
g) Weight per hundred grains (g)
Plant biochemical tests
Chlorophyll determination
For the measurement of chlorophyll content of leaves, method described by Zhang et al.
(2009) was followed. Samples of fresh leaves were obtained and cut into smaller pieces.
One hundred milligram of sample was homogenized in 80% acetone (10 mL) using
48
Heidolph silent crusher M at 16000 rpm. Leaf homogenate was filtered using Whatman
filter paper and filterate was used to estimate chlorophyll content. Optical density of
samples was measured at the wavelengths of 470 nm, 645 nm and 663 nm against 80%
acetone as blank.
The quantities of chlorophyll a, chlorophyll b, total chlorophyll content, xanthophyll and
carotene were calculated using Arnon’s equation formulated by Arnon (1949).
𝐶ℎ𝑙𝑜𝑟𝑜𝑝ℎ𝑦𝑙𝑙 𝑎 (𝑚𝑔 𝑝𝑒𝑟 𝑔𝑟𝑎𝑚) =[(12.7 𝑋 A663) − (2.6 𝑋 A645)]
mg leaf tissue𝑋 𝑚𝐿 𝑎𝑐𝑒𝑡𝑜𝑛𝑒
𝐶ℎ𝑙𝑜𝑟𝑜𝑝ℎ𝑦𝑙𝑙 𝑏 (𝑚𝑔 𝑝𝑒𝑟 𝑔𝑟𝑎𝑚) =[(22.9 X A645) − (4.68 𝑋 A663)]
mg leaf tissue 𝑋 𝑚𝐿 𝑎𝑐𝑒𝑡𝑜𝑛𝑒
𝑇𝑜𝑡𝑎𝑙 𝑐ℎ𝑙𝑜𝑟𝑜𝑝ℎ𝑦𝑙𝑙 = 𝐶ℎ𝑙𝑜𝑟𝑜𝑝ℎ𝑦𝑙𝑙 𝑎 + 𝑐ℎ𝑙𝑜𝑟𝑜𝑝ℎ𝑦𝑙𝑙 𝑏
𝐶𝑋+𝐶 =1000 A470 − 1.90 chlorophyll a − 63.14 chlorophyll b
214
Proline content
To estimate the proline content of leaves, method described by Bates et al. (1973) was
followed. For its estimation, 500 mg leaf material was used for homogenization in 5 mL of
3% salfosalicylic acid using pastel and mortar. After homogenization, the reaction mixture
was boiled for 10 minutes in water bath at 100oC. The content in tubes was filtered using
Whatman filter paper and filterate was saved for estimation.
Reaction mixture for estimation consisted of 2 mL filtrate, 2 mL glacial acetic acid and 2
mL ninhydrin reagent. Tubes were placed in boiling water bath (100oC) for 60 minutes.
Brick red color was developed as a result of reaction after boiling. Reaction mixture was
cooled to normal temperature and 4 mL toluene was added to it. Reaction mixture was set
aside after vigorous shaking and chromophore containing layer was separated using a
49
separating funnel. Optical density of chromophore containing layer was measured at 520
nm using double beam spectrophotometer (Cecil Aquarius CE 7200). Quantity of proline
content in samples was calculated by the help of standard plot of 5-100 µg per mL proline
(Spoljarevic et al., 2011).
Peroxidases estimation
For enzymatic estimation of peroxidases, 1 gram of frozen plant leaf was grinded with the
help of pastel and mortar in 4 mL of 0.1 M phosphate buffer (pH 7.0). Sample mixture was
centrifuged for 10 minutes at the speed of 14000 rpm, obtained supernatant was used to
estimate peroxidases present in samples.
Enzyme activity was estimated following the method described by David and Murray
(1965). Experiment was performed in two sets, one set for control and other for test
reactions. Two hundred microliters of enzyme extract was added to all tubes of both sets
followed by the addition of 0.1 M phosphate buffer (2 mL). After swirling of tubes to mix
the content, 200 µL of guaiacol solution (0.1%) was added to the tubes of test reactions.
All tubes were incubated for 15-20 minutes at room temperature followed by adding 100
µL of H2O2 solution (0.3%).
To prepare blank for the experiment, 200 µL distilled water was mixed with phosphate
buffer (2.5 mL) and 100 µL of H2O2 solution. Absorbance of both sets was measured at
470 nm using double beam spectrophotometer (Cecil Aquarius CE 7200). Amount of
peroxidases was determined using following formula:
𝑃𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (𝑢𝑛𝑖𝑡 𝑝𝑒𝑟 𝑔𝑟𝑎𝑚)
=Absorbance of Test − Absorbance of Control
Absorbance of Control X 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑔)
50
Acid phosphatase activity
Enzymatic activity of acid phosphatase by plant leaf was determined by the method of
Iqbal and Rafique (1987). Frozen leaf material of plant (1 g) was finely grinded in 4 mL
Tris HCl buffer (0.1 M) with the help of pastel and mortar. Supernatant was obtained by
centrifugation at 14000 rpm for 10 minutes. Supernatant was used as enzyme for estimating
acid phosphatase activity at pH 4.9, at 37oC for 60 minutes.
To estimate the enzyme activity of control, phenyl substrate (1 mL) was mixed with citrate
buffer (1 mL) having pH 4.9. Test tubes were labelled and kept in water bath at 37oC for
60 minutes. Reaction tubes were gently mixed after the addition of 0.5 N NaOH (1 mL)
and 200 µL of enzyme extract.
For the preparation of blank, reaction mixture consisting of distilled water (1 mL), citrate
buffer (1.2 mL) and 0.5 N NaOH (1 mL) was prepared followed by gentle mixing.
For the preparation of standard, phenol standard (1 mL), citrate buffer (1.2 mL) and 0.5 N
NaOH (1 mL) were added to test tube and was mixed gently.
To perform reaction with test, substrate phenyl (1 mL) and citrate buffer (1 mL) were added
to reaction tube and mixed well. Tubes were placed at 37oC in water bath for incubation
followed by the addition of 200 µL of enzyme extract. Tubes were incubated again for 60
minutes and were added with 1 mL of 0.5 N NaOH.
Finally all reaction tubes were supplemented with 1 mL each of 0.5 N NaHCO3, 4-
aminoantipyrin and potassium ferricyanide solution. Reaction mixture was gently mixed
by shaking the tubes. Optical density was measured immediately against water with the
51
help of double beam spectrophotometer (Cecil Aquarius CE 7200) at the wavelength of
510 nm.
Enzyme activity was calculated by using the following formula:
Acid phosphatase (K. A units per 100 mL) =T − C
S − B X W
Where K.A unit represents the release of 0.001 g phenol in 60 minutes.
T= optical density of test
C= optical density of control
S= optical density of standard
B= optical density of blank
W= weight of samples in grams
Estimation of protein (soluble) content
Plant leaf samples collected after eight weeks were collected for estimation of protein,
following the method of Lowry et al., 1951. Frozen leaf material of wheat plant (1 g) was
finely grinded in 4 mL of 0.1 M phosphate buffer with the help of pastel and mortar.
Supernatant was obtained by centrifugation at 4oC and was used for estimating protein
content. Two mL of Folin’s mixture along with 400 µL of plant extract was added to test
tubes and were kept at 25oC. Folin ciocalteu’s phenol reagent was added to tubes after 15
minutes, mixed gently and placed for color development for another 45 minutes. Optical
density of reaction mixture was measured at the wavelength of 750 nm using double beam
spectrophotometer (Cecil Aquarius CE 7200). Quantity of soluble protein present in
samples was calculated using standard plot.
52
Interaction between phosphate solubilizing bacteria and arbuscular
mycorrhizal fungi
To study the interaction between phosphate solubilizing bacterial isolates and Arbuscular
Mycorrhizal Fungi (AMF), RiDAOM 19198 culture was used by grown on Agrobacterium
rhizogenes transformed roots of chicory (Cichorium intybus) maintained and grown using
minimal growth medium (Becard and Fortin, 1988). For this experiment, bi-compartment
petri dishes were used which are helpful to study interactions between bacterial cultures
and extra-radial hyphae of Ri without the interference of host roots (St-Arnaud et al., 1995).
Minimal growth medium for proximal compartment was supplemented with vitamins and
sugar whereas minimal growth medium for distal compartment was supplemented with
2500 mg per liter tricalcium phosphate as inorganic phosphate source. The pH of both
mediums was adjusted to 5.5 and solidified with Phytagel (Sigma-Aldrich) before
sterilization. Mychorrized chicory roots were grown in proximal compartment for 21 days
at 28oC.
For inoculum preparation, seven bacterial strains were selected and were grown in L-broth
for 24 hours at 28oC. Bacterial cells were harvested by centrifugation followed by washing
twice using sterile saline. Bacterial cells were resuspended in saline and number of cells
was maintained to 108 colony forming units per mL.
Roots were trimmed regularly as they were not allowed to grow in distal compartment.
Only extraradial AMF mycelium was allowed to move towards distal compartment. Plates
with extraradial mycelium in distal compartment were selected and received 50 µL of
bacterial suspension. Control plates received equal quantity of sterile saline instead of
bacterial cell suspension. The experiment was replicated ten times. Plates were placed at
53
28oC for incubation. After six weeks of bacterial inoculation, plates were analyzed for
interaction studies under stereo microscope. To check the effect of bacterial interaction
with AMF on solubilization of tricalcium phosphate, gelified medium from the distal side
was removed and transferred to falcon tubes and placed overnight at -20oC. Medium was
liquefied by thawing at room temperature. Falcon tubes containing medium were
centrifuged at 10000 g for 30 minutes. Solubilized phosphate content in supernatant was
measured by the method of King (1932) as described above and effect on pH was recorded
using digital pH meter.
Statistical analysis
Result data was analyzed by using Microsoft excel 2013 and SPSS software (Version 20.0).
T-test was applied for the comparison of two means. ANOVA was applied for comparison
of multiple means following Tukey (HSD) or post hoc Duncan with the confidence level
at 95 percent.
54
Chapter 03
Isolation and characterization of phosphate solubilizing
bacteria
Bacteria play a crucial role in soil environment. Different bacterial genera are present in
soil and are involved in biogeochemical processes and play significant role in soil.
Phosphate solubilizing bacteria are one of those bacteria that are involved in solubilization
of unavailable forms of phosphate to the forms which can be utilized by plants. In the
present study phosphate solubilizing bacteria were isolated from different soils. The soil
samples for bacterial isolation were collected from three different areas of Punjab, Pakistan
including Lahore (Figure 3.2), Kallar Kahar and Chakwal district (Figure 3.3). From
Lahore, the samples were mainly collected from rhizosphere of different plants including
Brassica compestris, Raphanus sativus, Rosa indica, Oryza sativa, Lactuca sativa,
Mangifera indica, Spinacia oleracea, Triticum aestivum and Cicer arietinum. Samples
from Kallar Kahar consisted of soil from barren areas and rhizosphere soil from Calotropis
procera. Soil sample from Chakwal were collected from rhizosphere of Sorghum bicolor.
Isolated bacterial strains were characterized morphologically, biochemically and
physiologically.
Soil analysis
Soil pH of the sample sites was recorded for all soil samples with the help of pH meter.
Variation in pH was found among different sampling sites. The samples collected from
Lahore from rhizosphere of different plants had almost neutral pH and the pH range was
7.3-7.5. Whereas pH 6.8 was recorded for the soil sample collected from Chakwal. The
55
soil collected from Kallar Kahar area was acidic in nature and recorded pH of barren soil
samples was 5.3 while soil from rhizosphere of Calotropis procera plant had pH 5.5.
Isolation of phosphate solubilizing bacteria
The soil samples from different sources were collected in sampling bags under sterile
conditions and were transferred to laboratory for the screening and isolation of phosphate
solubilizing bacteria. The soil samples were serially diluted up to 10-5 followed by spread
plating on Pikovskaya agar medium having tricalcium phosphate as a sole source for
inorganic phosphate. Phosphate solubilizing strains were screened by the formation clear
zones around the colonies as shown in figure 3.5. On the basis of solubilization ability of
inorganic phosphate and diverse morphological characteristics, twenty eight bacterial
colonies were isolated and selected for characterization studies (Table 3.1).
From rhizospheric soil sample of Brassica compestris, three phosphate solubilizing strains
(S1, S2 and S62) were isolated. Two strains from each sample of Raphanus sativus (Rad1
and Rad2) and Rosa indica (Ros1 and Ros2) were isolated. From samples of Oryza sativa,
six strains including R12, R14, R15, SF, CS1 and R2 were isolated. One strain from each
sample of Mangifera indica (M6) and Spinacia oleracea (SpA) was selected. Three strains
including W94, W95 and W96 were isolated from soil samples of Triticum aestivum. Two
strains from Cicer arietinum (P1 and UP) and two from Calotropis procera (C14 and C50)
were isolated. Four isolates were selected from soil samples of barren soil (L6, L19, L20
and L22) whereas one strains (JA10) was selected from rhizospheric soil sample of
Sorghum bicolor.
56
Characterization of phosphate solubilizing bacteria
The phosphate solubilizing isolates were characterized morphologically as well as
biochemically. All the isolates were gram negative rod. Majority of the strains were motile
while S1, S2, JA10, R12, M6, L6, L19, R2, CS1, W94, C14 and C50 were non-motile. All
the isolated bacterial strains showed catalase activity. Most of the tested bacteria exhibited
oxidase positive results except JA10, R12, L6, SF, R2, W94, W95, W96, P1 and C50 which
were found to be oxidase negative. For citrate test, positive results were observed for all
the isolates while only one isolate Rad1 showed negative result for this test. The isolates
were tested for Methyl Red and Voges Proskauer tests, only two strains W95 and W96
were positive for Methyl Red (MR) test while the rest of the isolates were negative. For
Voges Proskauer (VP) test all the isolates showed negative results except R12 strain, the
only strain which showed positive result. The nitrate reduction test was performed to check
the ability of isolates to reduce nitrate. Ros1 and Ros2 exhibited nitrate reduction activity
whereas the rest of the tested strains were not able to reduce nitrate. None of the tested
isolates showed positive result for indole production test. To check the pigment production,
isolated bacteria were gown on King’s A and King’s B agar media. Strain Rad1, Rad2,
Ros1, Ros2, R12, SL8, SF, W95 and P1 showed slight pigmentation on King’s A medium.
For King’s B medium Ros1, Ros2, SF, W96, UP and P1 showed positive result for pigment
production (Figure 3.6, Table 3.2).
57
Figure 3.1: South Asian map showing location of Pakistan.
58
Figure 3.2: Soil sampling sites (highlighted as red circle) from Lahore, Punjab, Pakistan
(31.497658, 74.296866).
Figure 3.3: Soil sampling sites (highlighted as red circle) in Chakwal and Kallar Kahar,
Punjab, Pakistan (32.781758, 72.709010 and 32.936859, 72.863817).
59
Table 3.1: Isolation of phosphate solubilizing bacteria from different sampling sites.
Sr. No Strain
code Nature of sample Source pH Locality
1 S1 Rhizospheric soil Brassica compestris 7.3 Lahore
2 S2 Rhizospheric soil Brassica compestris 7.3 Lahore
3 Rad1 Rhizospheric soil Raphanus sativus 7.5 Lahore
4 Rad2 Rhizospheric soil Raphanus sativus 7.5 Lahore
5 Ros1 Rhizospheric soil Rosa indica 7.5 Lahore
6 Ros2 Rhizospheric soil Rosa indica 7.5 Lahore
7 JA10 Rhizospheric soil Sorghum bicolor 6.5 Chakwal
8 R12 Rhizospheric soil Oryza sativa 7.5 Lahore
9 R14 Rhizospheric soil Oryza sativa 7.5 Lahore
10 R15 Rhizospheric soil Oryza sativa 7.5 Lahore
11 SL8 Rhizospheric soil Lactuca sativa 7.5 Lahore
12 M6 Rhizospheric soil Mangifera indica 7.3 Lahore
13 L6 Soil Barren soil 5.3 Kallar Kahar
14 L19 Soil Barren soil 5.3 Kallar Kahar
15 L20 Soil Barren soil 5.3 Kallar Kahar
16 L22 Soil Barren soil 5.3 Kallar Kahar
17 SF Rhizospheric soil Oryza sativa 7.3 Lahore
18 SpA Rhizospheric soil Spinacia oleracea 7.5 Lahore
19 CS1 Rhizospheric soil Oryza sativa 7.5 Lahore
20 R2 Rhizospheric soil Oryza sativa 7.5 Lahore
21 S62 Rhizospheric soil Brassica compestris 7.5 Lahore
22 W94 Rhizospheric soil Triticum aestivum 7.5 Lahore
23 W95 Rhizospheric soil Triticum aestivum 7.5 Lahore
24 W96 Rhizospheric soil Triticum aestivum 7.5 Lahore
25 P1 Rhizospheric soil Cicer arietinum 7.5 Lahore
26 UP Rhizospheric soil Cicer arietinum 7.5 Lahore
27 C14 Rhizospheric soil Calotropis procera 5.5 Kallar Kahar
28 C50 Rhizospheric soil Calotropis procera 5.5 Kallar Kahar
60
Ta
ble 3
.2: M
orp
ho
logical an
d b
ioch
emical ch
aracterization
of p
ho
sph
ate solu
bilizin
g b
acterial isolates.
Sr.
No
Stra
in
cod
e
Gra
m’s
reactio
n
Cell
sha
pe
Mo
tility
Ca
tala
se
test
Ox
ida
se
test
Citra
te
test M
R
VP
N
itrate
redu
ction
Ind
ole
test
Pig
men
t
pro
du
ction
Kin
g’s
A
Kin
g’s
B
1
S1
-v
e R
od
s -v
e +
ve
+v
e +
ve
-ve
-ve
-ve
-ve
-ve
-ve
2
S2
-v
e R
od
s -v
e +
ve
+v
e +
ve
-ve
-ve
-ve
-ve
-ve
-ve
3
Rad
1
-ve
Ro
ds
+v
e +
ve
+v
e -v
e -v
e -v
e -v
e -v
e +
ve
-ve
4
Rad
2
-ve
Ro
ds
+v
e +
ve
+v
e +
ve
-ve
-ve
-ve
-ve
+v
e -v
e
5
Ro
s1
-ve
Ro
ds
+v
e +
ve
+v
e +
ve
-ve
-ve
+v
e -v
e +
ve
+v
e
6
Ro
s2
-ve
Ro
ds
+v
e +
ve
+v
e +
ve
-ve
-ve
+v
e -v
e +
ve
+v
e
7
JA1
0
-ve
Ro
ds
-ve
+v
e -v
e +
ve
-ve
-ve
-ve
-ve
-ve
-ve
8
R1
2
-ve
Ro
ds
-ve
+v
e -v
e +
ve
-ve
+v
e -v
e -v
e +
ve
-ve
9
R1
4
-ve
Ro
ds
+v
e +
ve
+v
e +
ve
-ve
-ve
-ve
-ve
-ve
-ve
10
R
15
-v
e R
od
s +
ve
+v
e +
ve
+v
e -v
e -v
e -v
e -v
e -v
e -v
e
11
S
L8
-v
e R
od
s +
ve
+v
e +
ve
+v
e -v
e -v
e -v
e -v
e +
ve
-ve
12
M
6
-ve
Ro
ds
-ve
+v
e +
ve
+v
e -v
e -v
e -v
e -v
e -v
e -v
e
13
L
6
-ve
Ro
ds
-ve
+v
e -v
e +
ve
-ve
-ve
-ve
-ve
-ve
-ve
14
L
19
-v
e R
od
s -v
e +
ve
+v
e +
ve
-ve
-ve
-ve
-ve
-ve
-ve
15
L
20
-v
e R
od
s +
ve
+v
e +
ve
+v
e -v
e -v
e -v
e -v
e -v
e -v
e
16
L
22
-v
e R
od
s +
ve
+v
e +
ve
+v
e -v
e -v
e -v
e -v
e -v
e -v
e
17
S
F
-ve
Ro
ds
+v
e +
ve
-ve
+v
e -v
e -v
e -v
e -v
e +
ve
+v
e
18
S
pA
-v
e R
od
s +
ve
+v
e +
ve
+v
e -v
e -v
e -v
e -v
e -v
e -v
e
61
+v
e= p
ositiv
e; -ve=
neg
ative; M
R=
Meth
yl R
ed; V
P=
Vo
ges P
rosk
auer
19
R
2
-ve
Ro
ds
-ve
+v
e -v
e +
ve
-ve
-ve
-ve
-ve
-ve
-ve
20
C
S1
-ve
Ro
ds
-ve
+v
e +
ve
+v
e -v
e -v
e -v
e -v
e -v
e -v
e
21
S
62
-v
e R
od
s +
ve
+v
e +
ve
+v
e -v
e -v
e -v
e -v
e -v
e -v
e
22
W
94
-ve
Ro
ds
-ve
+v
e -v
e +
ve
-ve
-ve
-ve
-ve
-ve
-ve
23
W
95
-ve
Ro
ds
+v
e +
ve
-ve
+v
e +
ve
-ve
-ve
-ve
+v
e -v
e
24
W
96
-ve
Ro
ds
+v
e +
ve
-ve
+v
e +
ve
-ve
-ve
-ve
-ve
+v
e
25
U
P
-ve
Ro
ds
+v
e +
ve
+v
e +
ve
-ve
-ve
-ve
-ve
+v
e +
ve
26
P
1
-ve
Ro
ds
+v
e +
ve
-ve
+v
e -v
e -v
e -v
e -v
e +
ve
+v
e
27
C
14
-v
e R
od
s -v
e +
ve
+v
e +
ve
-ve
-ve
-ve
-ve
-ve
-ve
28
C
50
-v
e R
od
s -v
e +
ve
-ve
+v
e -v
e -v
e -v
e -v
e -v
e -v
e
62
Hydrolytic enzyme production by phosphate solubilizing bacteria
Some of the isolates exhibited starch hydrolyzing ability including Ros1, Ros2, JA10, R14,
R15, L19, SF, SpA, CS1 and W94. Lipid hydrolysis test was performed to check bacterial
ability to hydrolyze tributyrin and results showed that all the strains showed positive results
except one isolate (W94) which was unable to produce extracellular lipase enzyme for lipid
hydrolysis. The bacterial isolates were tested for gelatin hydrolysis and majority of them
were unable to hydrolyze it, only ten strains S2, Ros1, R12, R14, R15, L20, SpA, CS1, UP
and P1 exhibited gelatin hydrolysis by producing gelatinase enzyme. For urea hydrolysis
test, none of the tested isolates was recorded positive and all the isolates showed negative
results for this test (Figure 3.6, Table 3.3).
Bacterial growth at different pH
The pH of surrounding environment greatly influence the growth rate of microbes. To find
the optimum pH level for bacterial growth, the bacterial isolates were allowed to grow in
L-broth medium at five different pH levels (3, 5, 7, 9 and 11). The cultures were grown at
28oC for 24 hours on orbital shaker with constant agitation of 150 revolutions per minute
(rpm). For the measurement of bacterial growth, optical density of cultures was measured
at 600 nm. Majority of the isolated bacteria showed best growth at neutral pH (7.0).
However the optimum pH for Rad2, Ros2, L6, L19 and S62 was pH 5. Four strains (S2,
Ros1, R12 and SL8) showed almost similar growth at pH 5 as well as at pH7. All the strains
showed some growth at alkaline pH (9.0) with only one exception of SF strain which
showed least growth at pH 9. At extreme levels of acidity and alkalinity (pH 3 and pH 11)
least growth pattern was recorded by all the tested isolates (Figure 3.4).
63
Resistance towards antibiotics
Phosphate solubilizing bacterial isolates were tested for resistance or sensitivity against
various antibiotics (Table 3.4). Majority of the isolates showed resistance for Amoxicillin
(30 µg) whereas around 33% of isolates including R12, L6, L19, SF, R2, CS1, S62, W95,
C14 and C50 showed sensitivity against it. For Cloaxicillin, resistance was observed by all
the tested isolates. All of the strains were found susceptible to Imipenem (10 µg). In case
of Ceftazidime, strains JA10, M6, L6, L19, R2, CS1, S62, UP, P1 and C50 showed
resistance while the rest of the isolates were unable to resist it and found susceptible. Four
strains including JA10, M6, UP and P1 showed maximum resistance for tested antibiotics
other than Imipenem (Figure 3.8).
Minimum Inhibitory Concentration (MIC) for pesticides
To check the ability of phosphate solubilizing bacterial isolates to survive in the presence
of pesticides, isolates were grown with two different pesticides in in vitro conditions.
Different isolates exhibited different levels of resistance towards them. Minimum
Inhibitory concentration (MIC) was checked for pesticides applied (Table 3.5). For
Chlorpyrifos, least MIC was observed for S1, M6 and SF strains, the recorded MIC for
majority of the isolates ranged from12-60 mg mL-1. Maximum concentration of 80 mg mL-
1 was observed for only one strain (SpA). The other tested pesticide was Pyriproxyfen and
the minimum value of MIC (20 mg mL-1) was observed for S1 and SF. For most of the
isolates, the MIC ranged from 30-70 mg mL-1 of Priproxyfen. Maximum MIC value of 80
mg mL-1 was recorded for only L20 and UP strains.
64
Table 3.3: Extracellular hydrolytic enzyme production ability of isolated bacterial strains.
Sr. No Strain code Extracellular enzyme production
Starch Lipid Gelatin Urea
1 S1 -ve +ve -ve -ve
2 S2 -ve +ve +ve -ve
3 Rad 1 -ve -ve -ve -ve
4 Rad 2 -ve +ve -ve -ve
5 Ros 1 +ve +ve +ve -ve
6 Ros 2 +ve +ve -ve -ve
7 JA10 +ve +ve -ve -ve
8 R12 -ve +ve +ve -ve
9 R14 +ve +ve +ve -ve
10 R15 +ve +ve +ve -ve
11 SL8 -ve +ve -ve -ve
12 M6 -ve +ve -ve -ve
13 L6 -ve +ve -ve -ve
14 L19 +ve +ve -ve -ve
15 L20 -ve +ve +ve -ve
16 L22 -ve +ve -ve -ve
17 SF +ve +ve -ve -ve
18 SpA +ve +ve +ve -ve
19 R2 -ve +ve -ve -ve
20 CS1 +ve +ve +ve -ve
21 S62 -ve +ve -ve -ve
22 W94 +ve -ve -ve -ve
23 W95 -ve +ve -ve -ve
24 W96 -ve -ve -ve -ve
25 UP -ve +ve +ve -ve
26 P1 -ve +ve +ve -ve
27 C14 -ve +ve -ve -ve
28 C50 -ve +ve -ve -ve
+ve= positive; -ve= negative
65
Fig
ure 3
.4: E
ffect of v
ariou
s pH
levels (3
, 5, 7
, 9, 1
1) o
f med
ium
on
gro
wth
of p
ho
sph
ate solu
bilizin
g b
acterial isolates after 2
4
ho
urs o
f incu
batio
n at tem
peratu
re 28
oC. E
rror b
ars M
ean ±
stand
ard erro
r (n=
3).
0
0.2
0.4
0.6
0.8 1
1.2
1.4
OD at 600 nm
Ba
cterial iso
lates
pH
3p
H 5
pH
7p
H 9
pH
11
66
Table 3.4: Determination of antibiotic resistance profiling of phosphate solubilizing
isolates.
Sr. No Strain code
Antibiotic resistance profile
Amoxicillin
(Amc 30)
Cloxacillin
(Cx1)
Imipenem
(Ipm 10)
Ceftazidime
(Caz 30)
1 S1 R R S S
2 S2 R R S S
3 Rad 1 R R S S
4 Rad 2 R R S S
5 Ros 1 R R S S
6 Ros 2 R R S S
7 JA10 R R S R
8 R12 S R S S
9 R14 R R S S
10 R15 R R S S
11 SL8 R R S S
12 M6 R R S R
13 L6 S R S R
14 L19 S R S R
15 L20 R R S S
16 L22 R R S S
17 SF S R S S
18 SpA R R S S
19 R2 S R S R
20 CS1 S R S R
21 S62 S R S R
22 W94 R R S S
23 W95 S R S S
24 W96 R R S S
25 UP R R S R
26 P1 R R S R
27 C14 S R S S
28 C50 S R S R
R= Resistant; S= Sensitive
67
Table 3.5: Determination of Minimum Inhibitory Concentration (MIC) of pesticides.
Sr.
No Strain code
Minimum Inhibitory Concentration of
pesticide (mg mL-1)
Chlorpyrifos Pyriproxyfen
1 S1 10 20
2 S2 12 40
3 Rad1 60 70
4 Rad2 60 70
5 Ros1 60 50
6 Ros2 50 50
7 JA10 50 40
8 R12 50 60
9 R14 70 35
10 R15 12 40
11 SL8 60 50
12 M6 10 40
13 L6 20 30
14 L19 25 35
15 L20 70 80
16 L22 20 40
17 SF 10 20
18 SpA 80 70
19 CS1 12 30
20 R2 12 35
21 S62 12 35
22 W94 12 45
23 W95 50 40
24 W96 60 70
25 UP 60 80
26 P1 60 60
27 C14 70 50
28 C50 50 50
68
Figure 3.5: Crowding pattern of phosphate solubilizing bacteria after spreading of soil
samples on Pikovskaya agar plates after 7 days of incubation period at 28oC.
69
A B
C
D
E
Figure 3.6: Biochemical characterization of phosphate solubilizing bacterial isolates.
Catalase test (A), oxidase test (B), citrate utilization test (C) nitrate reduction test (D), and
indole production test (E).
70
A
B
C
Figure 3.7: Determination of extracellular hydrolytic enzymatic activities of isolated
bacteria. Starch hydrolysis (A), Gelatin hydrolysis (B), and Urea hydrolysis (C).
71
Figure 3.8: Antibiotic resistance profiling of phosphate solubilizing bacteria after 24 hours
of incubation at 28oC.
72
Discussion
Phosphate is an important macronutrient required by plants for fundamental processes. It
is a vital component of Adenosine Tri-phosphate, involved in metabolic activities of plants
(Arfarita et al., 2017). In soil, phosphate is present in very high quantities but the plant
available form of phosphate is a limiting factor. The available quantity of phosphate ranges
from 0.01 milligrams to 0.2 milligrams per kilogram of soil. The microbial activities in soil
helps to overcome the lower quantities of available phosphate. They are helpful in
conversion of un-available phosphate to available forms and also help plant roots to reach
towards available phosphate (Arfarita et al., 2017). Soil contains a huge variety of
microorganisms and in rhizospheric zone there are different microorganisms which are
involved in direct or indirect mechanisms of plant growth promotion (Ullah and Bano,
2015).
The phosphate solubilizing bacteria for this study were isolated from different soil samples
collected from rhizosphere of different plants as well as from barren soil of salt affected
area. Phosphate solubilizing bacteria are widely present in soil and they are involved in
biogeochemical cycling processes especially they transform the different forms of
phosphates present in soil (Alori et al., 2017). With this idea the phosphate solubilizing
bacteria were isolated from different soils and to identify them on the basis of phosphate
solubilizing capabilities and to characterize them to further check their role in plant growth
promotion activities.
Soil samples were collected from different sites and variation in pH of sites was observed.
Availability of phosphate also depends on many different factors among which pH levels
of soil is very crucial factor (Arfarita et al., 2017). The soil samples from rhizosphere of
73
different plants was having almost neutral pH ranging from 6.8-7.5, whereas the soil
samples collected from salt affected area were slightly acidic in nature (5.3-5.5). Twenty
eight strains (S1, S2, Rad1, Rad2, Ros1, Ros2, JA10, R12, R14, R15, SL8, M6, L6, L19,
L20, L22, SF, SpA, CS1, R2, S62, W94, W95, W96, P1, UP, C14 and C50) were isolated
from different samples on the basis of inorganic phosphate solubilization ability on
Pikovskaya agar medium. Similarly different studies have also reported the isolation of
phosphate solubilizing strains from rhizosphere of different plants including Wheat (Ogut
et al., 2010; Babana and Antoun, 2005, 2006), Rice (Rajapaksha et al., 2011), legumes,
lettuce, raddish, cowpea, pulses and other crops (Ahmad et al., 2008; Linu et al., 2009;
Iqbal et al., 2010; Baig et al., 2012) as well as from soil affected from high concentrations
of salt (Srinivasan et al., 2012).
Different characteristics of isolated bacteria were studied. All the isolates were Gram
negative rods and showed catalase positive activity. Similarly in a study conducted by
Saharan and Verma (2014) reported a plant growth promoting isolate UHI(II)7 from
rhizosphere of Ocimum sp with catalase positive activity. The majority of isolates showed
motility, oxidase activity and citrate utilization abilities. Two isolates W95 and W96 were
found positive for MR test while only R12 strain showed positive result for VP test. Ros1
and Ros2 were the only nitrate reducers among all the isolated strains. In another study
Fatima et al. (2015) reported a phosphate solubilizing Pseudomonas brassicacearum
(PKU5) as Gram negative rod with oxidase, catalase and urease activity as well as nitrate
reduction abilities. Moreover, the pigment production was also exhibited by only a few
isolates. In accordance with these results Nehra et al. (2014) has also reported a phosphate
solubilizing isolate SVC2 as gram negative bacilli with no pigment production.
74
The measurement of extracellular enzymatic activities of an organism provides an insight
of their abilities to check their performance in energy limited environments (Adnan et al.,
2016). Microorganisms use these hydrolytic enzymes for the breakdown of nutrients so
that they can be moved inside the cell for utilization by these organisms as a source of
energy (Verma et al., 2018). The isolates in this study were found to have extracellular
enzymatic abilities for starch hydrolysis, lipid and gelatin hydrolysis however urea
hydrolysis was not recorded by any of the isolates. Likewise, in a scientific report by
Kumar et al. (2016), they have reported eight bacterial strains associated with rhizosphere
of turmeric plant were good solubilizers of phosphate as well as they had the ability for
starch hydrolysis.
pH play important role in a number of processes, such as enzymatic activities, generation
of energy and expression of different genes (Casey et al., 2010; Choi and Groisman, 2016).
In general the optimum pH for majority of the bacterial isolates was neutral, at which they
exhibited maximum growth. Five isolates Rad2, Ros2, L6, L19 and S62 showed best
growth at acidic pH. While S2, Ros1, R12 and SL8 showed similar growth pattern at pH5
as well as at pH 7. All the isolates managed to grow well even at alkaline pH (9.0) except
SF strain. Least growth was observed at extreme pH levels.
In soil environments different kind of organisms are present and they continuously try to
compete with other organisms by different mechanisms. The interactions among
microorganisms and with different plants take place in soil. As a result of these interactions
bacteria develop different mechanisms to offend the competition among inter and
intraspecies. The mechanisms through which bacteria compete with others are the
production of bacteriocins, bacteriolytic enzymes and antibiotics (Sood et al., 2007;
75
Bhattacharyya et al., 2016). In response to these conditions, different microorganisms have
developed counter mechanisms to resist antibiotics. The isolated bacterial strains were
tested to check their ability for resistance or susceptibility towards antibiotics. Four
antibiotics (amoxicillin, cloaxicillin, imipenem and ceftazidime) were used in this study.
All of the isolates showed resistance to cloaxicillin and sensitivity for imipenem whereas
for amoxicillin and ceftazidime some isolates were resistant and some of them were found
susceptible. Other than imipenem, most resistant strains for other tested antibiotics were
JA10, M6, UP and P1. Similarly, de Oliveira-Longatti et al (2014) have reported a
phosphate solubilizing strain UFLA 03-84 (Bradyrhizobium sp.) which was found resistant
to twelve antibiotics including amoxicillin.
In soil another challenging condition for microbial survival is the increased applications of
large quantities of pesticides which are used to prevent plants and crops from different
infections. These pesticides are harmful for environment as well as for the microbial
communities in soil. Some microorganisms somehow manage to survive in the presence of
these harmful chemicals either by developing resistance mechanisms or by developing
mechanisms for their degradation. It is reported that in terms of sustainability the
indigenous microbes are more viable than the other induced microorganisms applied as
biofertilizers or bioremediators (Nuraini et al., 2015; Arfarita et al., 2016). The isolated
phosphate solubilizing strains were able to grow in the presence of Chlorpyrifos as well as
in the presence of Pyriproxyfen. Some of the isolates were able to resist them up to the
concentration of 80 mg mL-1. Anzuay et al (2017) has also investigated the survival and
phosphate solubilizing ability of bacterial isolates (Pantoea sp. J49 and Serratia sp. J260)
in the presence of pesticide stress.
76
Conclusion
These isolated phosphate solubilizing bacteria were Gram negative rods with catalase
activity. They also exhibited different characteristics for other biochemical tests and the
majority of them was able to produce extracellular hydrolytic enzymes. Phosphate
solubilizing bacterial isolates were able to grow remarkably between pH 5-9. They also
have abilities to resist antibiotics as well as they can survive in high concentrations of
pesticides. These characteristics of their survival in diverse environmental conditions
suggest that they can be the good candidate to be further tested for studies related to plant
growth promotions in diverse environments.
77
Chapter 04
Phylogenetic analysis of phosphate solubilizing bacteria
Bacterial isolates having good phosphate solubilization potential for inorganic phosphate
source were selected for identification studies by using molecular approach (sequences of
16S rRNA gene) and their phylogenetic studies. Besides the phenotypic studies by
biochemical and physiological characterization, the molecular characterization study
provides more appropriate insight for the phylogenetic classification of organisms into
several taxonomic levels.
For the identification of isolated phosphate solubilizing bacteria, 16S rRNA gene
sequencing was carried out. Twenty eight bacterial isolates S1, S2, Rad1, Rad2, Ros1,
Ros2, JA10, R12, R14, R15, SL8, M6, L6, L19, L20, L22, SF, SpA, CS1, R2, S62, W94,
W95, W96, P1, UP, C14 and C50 were sent to Macrogen sequencing facility in Korea for
identification. Bacterial samples were prepared by streaking the individual colonies on L-
agar plates and the plates were incubated overnight at 37oC. For the analysis of partial gene
sequence of 16S rRNA gene, a universal primer set (518F and 800R) was used and colony
PCR was performed. After sequencing, the obtained sequences were analyzed by Finch Tv
software to ensure the quality of sequencing. Classification of sequences was done by using
National Center for Biotechnology Information (NCBI) database of nucleotides. Nearest
homologues sequences were obtained and aligned with the sequence of isolated bacteria.
For the alignment of multiple sequences, MUSCLE was used and the phylogenetic tree
was computed using neighbor joining method in MEGA 7.0 (Kumar et al., 2016). To
ensure the reliability, the boot strap test was replicated 1000 times (Felsenstein, 1985). The
78
grouping of isolated phosphate solubilizing strains was compared with other sequences. In
each tree of representing phylogeny of bacterial isolates, an out-group was also used.
Accession numbers of isolated phosphate solubilizing strains were obtained by submitting
the sequences in GenBank data base of NCBI. In table 4.1, the accession numbers of
isolated bacterial strains are mentioned along with their closest homologues obtained from
Basic Local Alignment Search Tool (BLAST). Results of BLAST analysis showed that the
isolated phosphate solubilizing bacterial strains mainly belong to five different genera
including Ochrobactrum, Acinetobacter, Pseudomonas, Klebsiella and Enterobacter.
Bacterial isolates S1, S2 and S62 were isolated from rhizosphere of Brassica campestris
and these strains showed 99%, 100% and 99% similarity with Ochrobactrum
pseudogrignonense, Acinetobacter olivorans and Acinetobacter calcoaceticus,
respectively (Figure 4.1, 4.2, 4.21). Strains isolated from Raphanus sativus Rad1 and Rad2
were found to had association with Pseudomonadaceae and they showed >99% identity
with Pseudomonas putida (Figure 4.3, 4.4). The isolates of rhizospheric samples of Rosa
indica Ros1 and Ros2 were also found to be the members of the family Pseudomonadaceae
and phylogenetically they were placed them with the clad of Pseudomonas parafulva and
Pseudomonas sp. and they had 99% homology with them (Figure 4.5, 4.6).
Bacterial strain JA10 (isolate of Sorghum bicolor) was found associated to the order
Pseudomonadales and class of Gammaproteobacteria and had 100% identity with
Acinetobacter baumanii (Figure 4.7). Six strains R12, R14, R15, SF, CS1 and R2 were
isolated from Oryza sativa and they exhibited association with Klebsiella pneumoniae
(99%), Pseudomonas plecoglossicida (100%), Pseudomonas aeruginosa (99%),
Pseudomonas oryzihabitans (99%), Acinetobacter pittii (99%) and Acinetobacter
79
calcoaceticus (99%) (Figure 4.8, 4.9, 4.10, 4.17, 4.19, 4.20). Strain M6 isolated from
Mangifera indica exhibited 99% homology with Ochrobactrum sp. whereas SpA was
isolated from Spinacia oleracea had 99% relatedness with the species Pseudomonas
aeruginosa (Figure 4.12, 4.18).
Bacterial strain SL8 was the isolate of Lactuca sativa and from its phylogenetic analysis it
was revealed that it was placed next to Pseudomonas japonica (Figure 4.11) and had 99%
homology with it. Strains L6 and L19 had >99% homology with the genus of Acinetobacter
and neighbor joining tree had placed them next to Acinetobacter pittii whereas, strains L20
and L22 were found to had close relationship with the genera of Pseudomonas and from
their phylogenetic analysis it was found that L20 belonged to the species of Pseudomonas
koreensis while L22 belonged to the species of Pseudomonas frederiksbergensis (Figure
4.13, 4.14, 4.15, 4.16).
From the samples of wheat (Triticum aestivum), strain W94 exhibited more than 99%
similarity with the genus Acinetobacter (Figure 4.22) while W95 and W96 belonged to the
family Enterobacteriaceae and were found 99% similar to Enterobacter cloacae and
Enterobacter aerogenes, respectively (Figure 4.23, 4.24). Two trains P1 and UP were
isolated from Cicer arietinum were found to be the members of Pseudomonaceae and P1
exhibited 99% identity with Pseudomonas fluoresces whereas UP was 95% similar with
Pseudomonas reinekei (Figure 4.25, 4.26). Bacterial strains C14 and C50 were isolated
from the rhizospheric soil samples of Calotropis procera and showed similarity with the
genera Acinetobacter (Figure 4.27, 4.28).
80
Table 4.1: GenBank accession numbers of isolated phosphate solubilizing bacteria and
their % similarity with nearest homologues.
Sr.
No.
Strain
code
Isolation
source
Accession
number
Nearest homologues Percentage
similarity (%)
1 S1 Brassica
compestris KP241948
Ochrobactrum
pseudogrignonense 99
2 S2 Brassica
compestris KP241949
Acinetobacter
olivorans 100
3 Rad1 Raphanus
sativus KP241947 Pseudomonas putida 100
4 Rad2 Raphanus
sativus KX345931 Pseudomonas putida 99
5 Ros1 Rosa indica KX756233 Pseudomonas
parafulva 99
6 Ros2 Rosa indica KX345930 Pseudomonas sp. 100
7 JA10 Sorghum
bicolor KX345929
Acinetobacter
baumanii 100
8 R12 Oryza sativa KP241945 Klebsiella
pneumoniae 99
9 R14 Oryza sativa KP241946 Pseudomonas
plecoglossicida 100
10 R15 Oryza sativa KX756232 Pseudomonas
aeruginosa 99
11 SL8 Lactuca
sativa KY828842
Pseudomonas
japonica 99
12 M6 Mangifera
indica KX774373 Ochrobactrum sp. 99
13 L6 Barren soil KX756231 Acinetobacter pittii 100
14 L19 Barren soil KY828843 Acinetobacter pittii 99
15 L20 Barren soil KX774372 Pseudomonas
koreensis 99
81
16 L22 Barren soil KY828844 Pseudomonas
frederiksbergensis 99
17 SF Oryza sativa KX774371 Pseudomonas
oryzihabitans 99
18 SpA Spinacia
oleracea KP241950
Pseudomonas
aeruginosa 99
19 CS1 Oryza sativa KY828845 Acinetobacter pittii 99
20 R2 Oryza sativa KY828846 Acinetobacter
calcoaceticus 99
21 S62 Brassica
compestris KX774370
Acinetobacter
calcoaceticus 99
22 W94 Triticum
aestivum KP241955 Acinetobacter sp. 100
23 W95 Triticum
aestivum KP241951 Enterobacter cloacae 99
24 W96 Triticum
aestivum KX345928
Enterobacter
aerogenes 99
25 P1 Cicer
arietinum KP241944
Pseudomonas
fluorescens 99
26 UP Cicer
arietinum KY828847 Pseudomonas reinekei 95
27 C14 Calotropis
procera KY828848
Acinetobacter
calcoaceticus 99
28 C50 Calotropis
procera KY828849 Acinetobacter sp. 99
82
Figure 4.1: Neighbor joining phylogenetic tree of S1 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The S1
strain clustered together with the 16S rRNA gene sequences of Ochrobactrum
pseudogrignonense (▲). The 16S rRNA gene of Escherichia coli was used as out-group
(■).
Figure 4.2: Neighbor joining phylogenetic tree of S2 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The S2
strain clustered together with the 16S rRNA gene sequences of Acinetobacter oleivorns
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
83
Figure 4.3: Neighbor joining phylogenetic tree of Rad1 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The Rad1
strain clustered together with the 16S rRNA gene sequences of Pseudomonas putida (▲).
The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.4: Neighbor joining phylogenetic tree of Rad2 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The Rad2
strain clustered together with the 16S rRNA gene sequences of Pseudomonas putida (▲).
The 16S rRNA gene of Escherichia coli was used as out-group (■).
84
Figure 4.5: Neighbor joining phylogenetic tree of Ros1 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The Ros1
strain clustered together with the 16S rRNA gene sequences of Pseudomonas parafulva
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.6: Neighbor joining phylogenetic tree of Ros2 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The Ros2
strain clustered together with the 16S rRNA gene sequences of Pseudomonas sp. (▲). The
16S rRNA gene of Escherichia coli was used as out-group (■).
85
Figure 4.7: Neighbor joining phylogenetic tree of JA10 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The JA10
strain clustered together with the 16S rRNA gene sequences of Acinetobacter baumanii
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.8: Neighbor joining phylogenetic tree of R12 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The R12
strain clustered together with the 16S rRNA gene sequences of Klebsiella pneumoniae (▲).
The 16S rRNA gene of Escherichia coli was used as out-group (■).
86
Figure 4.9: Neighbor joining phylogenetic tree of R14 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The R14
strain clustered together with the 16S rRNA gene sequences of Pseudomonas
plecoglossicida (▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.10: Neighbor joining phylogenetic tree of R15 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The R15
strain clustered together with the 16S rRNA gene sequences of Pseudomonas aeruginosa
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
87
Figure 4.11: Neighbor joining phylogenetic tree of SL8 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The SL8
strain clustered together with the 16S rRNA gene sequences of Pseudomonas japonica
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.12: Neighbor joining phylogenetic tree of M6 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The M6
strain clustered together with the 16S rRNA gene sequences of Ochrobactrum sp (▲). The
16S rRNA gene of Escherichia coli was used as out-group (■).
88
Figure 4.13: Neighbor joining phylogenetic tree of L6 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The L6
strain clustered together with the 16S rRNA gene sequences of Acinetobacter pittii (▲).
The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.14: Neighbor joining phylogenetic tree of L19 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The L19
strain clustered together with the 16S rRNA gene sequences of Acinetobacter pittii (▲).
The 16S rRNA gene of Escherichia coli was used as out-group (■).
89
Figure 4.15: Neighbor joining phylogenetic tree of L20 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The L20
strain clustered together with the 16S rRNA gene sequences of Pseudomonas koreensis
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.16: Neighbor joining phylogenetic tree of L22 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The L22
strain clustered together with the 16S rRNA gene sequences of Pseudomonas
frederiksbergensis (▲). The 16S rRNA gene of Escherichia coli was used as out-group
(■).
90
Figure 4.17: Neighbor joining phylogenetic tree of SF strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The SF
strain clustered together with the 16S rRNA gene sequences of Pseudomonas oryzihabitans
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.18: Neighbor joining phylogenetic tree of SpA strain, constructed from 16S
rRNA gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The SpA
strain clustered together with the 16S rRNA gene sequences of Pseudomonas aeruginosa
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
91
Figure 4.19: Neighbor joining phylogenetic tree of CS1 strain, constructed from 16S
rRNA gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The CS1
strain clustered together with the 16S rRNA gene sequences of Acinetobacter pittii (▲).
The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.20: Neighbor joining phylogenetic tree of R2 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The R2
strain clustered together with the 16S rRNA gene sequences of Acinetobacter calcoaceticus
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
92
Figure 4.21: Neighbor joining phylogenetic tree of S62 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The S62
strain clustered together with the 16S rRNA gene sequences of Acinetobacter calcoaceticus
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.22: Neighbor joining phylogenetic tree of W94 strain, constructed from 16S
rRNA gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The W94
strain clustered together with the 16S rRNA gene sequences of Acinetobacter sp (▲). The
16S rRNA gene of Escherichia coli was used as out-group (■).
93
Figure 4.23: Neighbor joining phylogenetic tree of W95 strain, constructed from 16S
rRNA gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The W95
strain clustered together with the 16S rRNA gene sequences of Enterobacter cloacae (▲).
The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.24: Neighbor joining phylogenetic tree of W96 strain, constructed from 16S
rRNA gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The W96
strain clustered together with the 16S rRNA gene sequences of Enterobacter aerogenes
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
94
Figure 4.25: Neighbor joining phylogenetic tree of P1 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The P1
strain clustered together with the 16S rRNA gene sequences of Pseudomonas fluoresces
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.26: Neighbor joining phylogenetic tree of UP strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The UP
strain clustered together with the 16S rRNA gene sequences of Pseudomonas reinekei (▲).
The 16S rRNA gene of Escherichia coli was used as out-group (■).
95
Figure 4.27: Neighbor joining phylogenetic tree of C14 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The C14
strain clustered together with the 16S rRNA gene sequences of Acinetobacter calcoaceticus
(▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).
Figure 4.28: Neighbor joining phylogenetic tree of C50 strain, constructed from 16S rRNA
gene of isolate and its nearest homologues obtained from NCBI nucleotide data base.
MEGA 7 software was used with the boot strap value based on 1000 repetitions. The C50
strain clustered together with the 16S rRNA gene sequences of Acinetobacter sp (▲). The
16S rRNA gene of Escherichia coli was used as out-group (■).
96
Discussion
In microbes, the genomes inherited from their ancestors reflect their origin. The core set of
genome contains all essential genes which are required for reproducibility of the organism
and it is mostly transferred by vertical inheritance (Collins and Higgs, 2012). Due to this
vertical inheritance, these genes determine strict phylogeny of the organisms (Tamames et
al., 2016). The flexible part of genome consists of the genes which are important regarding
the environmental adaptations (including availability of specific nutrients or other
environmental factors). The balance between these adaptations has been thought to be very
important because if these adaptations were limited, each taxon would be limited only to
their own niche. While on the other hand, the limitless adaptations would have resulted in
the similarity between the other taxon rather than their ancestors (Philippot et al., 2010;
Tamames et al., 2010; Martiny et al., 2013). The sequences of rRNA, specifically 16S
rRNA has much importance with reference to the evolutionary studies of bacteria. By
targeting 16S rRNA, the phylogenetic relationships between taxon can be determined.
Furthermore, the diversity of bacteria can be explored and it also help in quantification of
relative abundances of various taxon (Vetrovsky and Baldrian, 2013).
All the bacterial isolates with the ability to dissolve inorganic phosphate were subjected to
characterization by means of their morphological and biochemical characteristics. The
isolated bacterial strains were further subjected to molecular characterization for their
identification purposes. The identification was performed by analyzing the sequence of
16S rRNA genes. The reason for using 16S rRNA gene to study phylogenetic identification
is its universal distribution, which allows the analysis of phylogenetic studies between
distant taxon. This genes is a part of core genome and it remains conserved and is less
97
likely to be affected by the transfer of genetic material horizontally. Despite having the
highly conserved region, 16S rRNA gene also contain a variable part which allows the
adequate change and is a helpful tool to classify different genera. The conserved part of
this gene is helpful for designing the primers for polymerase chain reaction as well as for
the designing of probes for several hybridization studies of several taxa at various levels of
taxonomy ranging from one strain to complete phyla (Vetrovsky and Bsldrain, 2013).
Phosphorous is present ubiquitously in environment but the concentration of soluble or
available form is very low (Maitra et al., 2015). Mostly it is present in its inorganic forms
which is not accessible to plants and hence they are not able to utilize it properly. Phosphate
solubilizing bacteria are very good converters of phosphorous to other forms. So the study
for their identification is also very important for their classification according to specific
environments. The genes present in bacterial genomes define the functionality of
organisms and it also represents their ability to live in certain environments or habitats and
to perform certain functions (Tamames et al., 2010). Some specific genes are required by
microorganisms which help their survival in different environmental conditions or stresses.
This factor play an important role in the ecology as well as in the evolution of different
bacteria and also in their distribution into different taxa. This challenge has also divided
the genome of an organism into two parts i.e. flexible and a core part (Mira et al., 2010).
According to the traditional methods of classification (morphological and biochemical), all
isolates were found Gram negative rods. All of the isolated bacteria belonged to phylum
Proteobacteria and class Gammaproteobacteria except two isolates (S1 and M6) which
belonged to the class Alphaproteobacteria. In China, the bacterial population of phosphate
solubilizing bacteria was found to be predominantly associated with phylum Proteobacteria
98
and mostly the strains were found to have association with the genera Cedecea, Raoultella,
Leclercia, Klebsiella and Burkholderia (Pei-Xiang et al., 2012).
Strain Rad1, Rad2, Ros1, Ros2, R14, R15, SL8, L20, L22, SF, SpA, P1 and UP belonged
to order Pseudomonadales and the family Pseudomonaceae. Whereas, Loper et al. (2012)
have also isolated Pseudomonas fluorescence from Triticum sp. whereas the strains
including S2, JA10, L6, L19, CS1, R2, S62, W94, C14 and C50 were associated with the
order Pseudomonadales and the family Moraxellaceae. According to a study conducted by
Azziz et al. (2012) in Uruguay the crop rotation yield the following predominant genera of
phosphate solubilizers including Burkholderia, Acinetobacter and Pseudomonas. Whereas
according to a report regarding diversity of phosphate solubilizing strains in Taiwan the
most common genera were Phyllobacterium, Gordonia, Delftia, Chyryseobacterium,
Serratia, Arthrobacter, Rhodococcus and Bacillus (Chen et al., 2006).
On the other hand, R12, W95 and W96 belonged to the order Enterobacteriales and to the
family Enterobacteriaceae. Two isolates S1 and M6 were found to be associated with the
order Rhizobiales and the family Brucellaceae. Eight strains with phosphate solubilizing
ability were isolated from the plants of palm oil and they mainly belonged to four families
including Enterococcaceae, Bacillaceae, Alcaligeneaceae and Enterobacteriaceae
(Acevedo et al., 2014).
The sequences of 16S rRNA genes of representative species were retrieved from GenBank
database. Out of twenty eight strains, thirteen isolates showed sequence homology with
genus Pseudomonas, ten strains exhibited similarity with Acinetobacter, two strains
showed identity with the genus of Enterobacter, two with the genera Ochrobacterum while
only one isolate exhibited similarity to the genus Klebsiella. In previous studies, it has been
99
documented that Rhizobium, Bacillus and Pseudomonas are the most abundant and
powerful bacteria having phosphate solubilizing abilities (Behera et al., 2014; Javadi-
Nobandegani et al., 2015).
Majority of the strains showed 99% homology to the nearest homologs while strain UP
was the only strain which showed 95% similarity to its nearest homologues. Phylogenetic
study showed that ASL12 belonged to genus Acinetobacter and had 99.4% similarity with
Acinetobacter sp when compared to 16S rRNA sequence in NCBI database (Liu et al.,
2014). 16S rRNA sequence studies of ADH302 revealed that it was 98.8% identical to
Enterobacter sp (Liu et al., 2014). In a study conducted by Singh et al. (2014), they have
reported two novel phosphate solubilizing strains PS1 and PS16 isolated from rhizosphere
of chick pea. PS1 have been reported to have 90% similarity with Pantoea cypripedii
whereas PS16 have been reported with 92% similarity with Enterobacter aerogenes.
When all isolated phosphate solubilizing strains were compared by computing their
phylogeny by neighbor joining method (Figure 4.29), two main clads appeared. From the
previous studies it seemed that the content of genome is mostly determined by phylogenetic
proximity and similar genomes are present in close species (Zaneveld et al., 2010). Strain
S1 and M6 made a separate distant clad because they belong to the class
Alphaproteobacteria while the other bigger clad represented that the rest of the isolated
bacteria are associated to the class Gammaproteobacteria.
Among the cluster of Pseudomonas species, ten strains were placed together while SF, R15
and SpA showed slightly distant grouping. The branch length of strain UP was observed to
be longer as compared to other species because it share only 95% homology to other
species while the rest of the species belonging to Pseudomonas share >98% similarity with
100
each other. Ordonez et al. (2016) have also reported that in rhizosphere of potato plant,
Pseudomonas sp. were present predominantly as compared to other genus and had plant
growth promoting abilities and were reported as good solubilizers of phosphate.
In the cluster made by Acinetobacter species, JA10 was placed above the all other
Acinetobacter species which showed that it has slightly distant origin among them.
Whereas the rest of Acinetobacter are found to be very closely related to each other. The
members of Enterobacteriaceae made a separate clad which was further divided as a cluster
of Klebsiella and Enterobacter species. Oteino et al (2015) have also conducted a research
on plant growth promoting phosphate solubilizing endophytes and reported that twelve
strains were found associated to Pseudomonas fluorescence and Pseudomonas putida and
other Pseudomonas sp. having good plant growth enhancing activities and the isolation
source of these strains were Miscanthus giganteus, Beta vulgaris, Triticum sp, Pyrus sp,
and Populus sp. The bacterial diversity of phosphate solubilizers in Oxbow lakes was
studied by Maitra et al. (2015) which reported various genus of bacteria including
Novosphingobium, Stenotrophomonas, Curtobacterium, Microbacterium, Acinetobacter,
Pseudomonas, Agrobacterium, Enterobacter, Brevibacillus and Bacillus.
Conclusion
From present study, it can be concluded that the population of phosphate solubilizing
bacteria in rhizospheric soil of different plants in Lahore, Chakwal and Kallar Kahar,
Pakistan mainly consists of five genera and the Pseudomonas and Acinetobacter species
dominate the other bacterial genera in these areas.
101
Figure 4.29: Neighbor joining phylogenetic tree of all isolated phosphate bacterial strains
constructed from 16S rRNA gene sequeces. Tree constructed by using MEGA 7 software,
boot strap values based on 1000 repetitions are mentioned at branch points. Scale bar
represents 0.02 substitutions per nucleotide position.
Pseudomonas putida Rad1 (KP241947)
Pseudomonas putida Rad2 (KX345931)
Pseudomonas parafulva Ros1 (KX756233)
Pseudomonas sp. Ros2 (KX345930)
Pseudomonas plecoglossicida R14 (KP241946)
Pseudomonas japonica SL8 (KY828842)
Pseudomonas koreensis L20 (KX774372)
Pseudomonas frederiksbergensis L22 (KY828844)
Pseudomonas fluorescens P1 (KP241944)
Pseudomonas reinekei U.P (KY828847)
Pseudomonas oryzihabitans SF (KX774371)
Pseudomonas aeruginosa R15 (KX756232)
Pseudomonas aeruginosa SpA (KP241950)
Pseudomonas sp.
Acinetobacter baumannii JA10 (KX345929)
Acinetobacter oleivorans S2 (KP241949)
Acinetobacter pittii L6 (KX756231)
Acinetobacter pittii L19 (KY828843)
Acinetobacter pittii CS1 (KY828845)
Acinetobacter calcoaceticus R2 (KY828846)
Acinetobacter calcoaceticus S62 (KX774370)
Acinetobacter sp. W94 (KP241955)
Acinetobacter calcoaceticus C14 (KY828848)
Acinetobacter sp. C50 (KY828849)
Acinetobacter sp.
Klebsiella sp. Klebsiella pneumoniae R12 (KP241945)
Enterobacter cloacae W95 (KP241951)
Enterobacter aerogenes W96 (KX345928)Enterobacter sp.
Gammaproteobacteria
Ochrobactrum sp. Ochrobactrum pseudogrignonense S1 (KP241948)
Ochrobactrum sp M6 (KX774373)Alphaproteobacteria
102
Chapter 05
Phosphate solubilization potential of bacterial isolates
Phosphorous in soil has prime importance, as it plays a very important role in the
development of plants (Arfarita et al., 2017). Most of the phosphorous remains sequestered
to cations present in soil. The cations that binds to phosphorus and make them insoluble
belongs to aluminium, ferrous, calcium and magnesium (Maitra et al., 2015). Due to the
insoluble nature, phosphorous uptake by plants becomes limited. Even if chemical
fertilizers are applied, lower quantities of phosphorous is utilized by plants. In soil, there
are several microorganisms which can solubilize the insoluble forms of phosphate into
soluble form and make them available to be easily up taken by plants (Sharma et al., 2013).
In this scenario, efficient phosphate solubilizing bacteria can increase the soluble
phosphorous quantities in rhizospheric zone to be easily up taken by plants.
Present study deal with the qualitative estimation of inorganic phosphate solubilization of
Twenty eight bacterial strains (Ochrobactrum pseudogrignonense-S1, Acinetobacter
olivorans-S2, Pseudomonas putida-Rad1, Pseudomonas putida-Rad2, Pseudomonas
parafulva-Ros1, Pseudomonas sp-Ros2, Acinetobacter baumanii-JA10, Klebsiella
pneumoniae-R12, Pseudomonas plecoglossicida-R14, Pseudomonas aeruginosa-R15,
Pseudomonas japonica-SL8, Ochrobactrum sp-M6, Acinetobacter pittii-L6, Acinetobacter
pittii-L19, Pseudomonas koreensis-L20, Pseudomonas frederiksbergensis-L22,
Pseudomonas oryzihabitans-SF, Pseudomonas aeruginosa-SpA, Acinetobacter pittii-CS1,
Acinetobacter calcoaceticus-R2, Acinetobacter calcoaceticus-S62, Acinetobacter sp.-
W94, Enterobacter cloacae-W95, Enterobacter aerogenes-W96, Pseudomonas
103
fluorescens-P1, Pseudomonas reinekei-UP, Acinetobacter calcoaceticus-C14 and
Acinetobacter sp.-C50) on agar media.
All of the isolates were able to solubilize inorganic phosphate on solid media as described
in Table 5.1. Their solubilization potential was evaluated by spot inoculating the pure
bacterial colonies on agar plates having inorganic phosphate as a sole phosphate source.
After incubation of seven days at 28 oC, the solubilization zones around bacterial colonies
were measured and solubilization index was determined (Figure 5.1, 5.2). On Pikovskaya
agar, maximum value for solubility index was 2.64 which was exhibited by strain JA10,
while SL8, L6 and C50 also showed best results and their solubilization index was recorded
as 2.53. The minimum values for solubilization index on Pikovskaya agar were recorded
for R12, UP and S62 as 2.176, 2.09 and 2.0, respectively. The order of maximum to
minimum solubilization index by phosphate solubilizing bacterial isolates on Pikovskaya
agar was: JA10 > SL8, L6, C50 > C14 > M6, L19, L22 > R2 > S2, Rad2 > Ros1 > SpA >
Ros2 > R14 > S1 > P1 > W96 > Rad1 > R15 > L20, SF > CS1 > W94, W95 > R12 > UP
> S62 (Figure 5.4). On NBRIP agar medium, maximum solubility index was recorded to
be 3.07 by R12 whereas W96, Rad2 and W95 also showed prominent results and their
solubility index were 2.93, 2.84 and 2.8, respectively. Minimum values of solubility index
was recorded by R14. The order of maximum to minimum solubilization index by isolates
on NBRIP agar was observed as R12 > W96 > Rad2 > W95 > JA10 > CS1 > L6 > M6,
C50 > S2 > SF > Rad1, SL8, R2, S62 > W94 > L19, L22 > S1, C14 > Ros1, L20 > UP >
P1 > Ros2 > R15, SpA > R14 (Figure 5.4).
Based upon solubilization and calculation of solubilization index on Pikovskaya and
NBRIP agar, the solubilization efficiency was also calculated for all the bacterial isolates
104
as well as the efficiency among both media was calculated (Figure 5.5). Among the isolated
bacterial strains, 64% isolates (R12, W96, W95, S62, CS1, Rad2, SF, Rad1, W94, M6, S2,
L6, L20, S1, C50, JA10, R2 and P1) showed increased efficiency for phosphate
solubilization on NBRIP agar as compared to Pikovskaya agar medium. The efficiency of
strain Ros1 remained same on both media. Around 28% strains showed good efficiency on
Pikovskaya agar for phosphate solubilization when compared with NBRIP medium.
Overall most of the strains showed more solubilization on NBRIP medium as compared to
Pikovskaya agar medium. In case of strain R12, solubilization efficiency was increased up
to 90% on NBRIP agar as compared to Pikovskaya agar. Similarly more than 50% increase
in solubilization efficiency was observed with strains W96, W95, S62 and CS1 on NBRIP
medium.
In order to check the production of phosphatases, bacterial isolates were subjected to spot
inoculation on Tryptic Soy Agar (TSA) having phenolphthalein indicator. The plates were
incubated for 48 hours at 28 oC. After completion of incubation, ammonium hydroxide was
added onto the lid of petri dishes and results were recorded after 15 minutes. All the tested
isolates showed pink coloration around colonies which showed that they possess
phosphatases production abilities. Strain Rad2, R12, R15, L6, L22 and SF showed strong
positive results whereas, Rad1 R14, L20, SpA, R2, W95 and P1were found to be the
moderate producers of phosphatases (Figure 5.3).
105
Table 5.1: Characterization of phosphate solubilizing bacteria based upon phosphate
solubilization in Pikovskaya agar and phosphatases production on tryptic soya agar.
Sr. No. Strain code Phosphate solubilization Phosphatase
production
1 Control - -
2 S1 + +
3 S2 + +
4 Rad1 + ++
5 Rad2 + +++
6 Ros1 + +
7 Ros2 + +
8 JA10 + +
9 R12 + +++
10 R14 + ++
11 R15 + +++
12 SL8 + +
13 M6 + +
14 L6 + +++
15 L19 + +
16 L20 + ++
17 L22 + +++
18 SF + +++
19 SpA + ++
20 CS1 + +
21 R2 + ++
22 S62 + +
23 W94 + +
24 W95 + ++
25 W96 + +
26 P1 + ++
27 UP + +
28 C14 + +
29 C50 + +
-= negative; += slight, ++= moderate; +++= strong
106
Figure 5.1: Phosphate solubilization by bacterial isolates on Pikovskaya agar after seven
days of incubation at 28oC. The clear zone around inoculation indicate the solubilization
of inorganic phosphate.
Figure 5.2: Phosphate solubilization by bacterial isolates on NBRIP agar medium after
seven days of incubation at 28 oC. The clear zone around inoculation indicate the
solubilization of inorganic phosphate.
107
Figure 5.3: Phosphatases detection on Tryptic Soy Agar (TSA) supplemented with
phenolphthalein indicator. Pink coloration shows the production of phosphatases after 48
hours of incubation at 28 oC.
108
Bacterial strains
S1 S2Ra
d1Ra
d2Ro
s1Ro
s2JA
10 R12
R14
R15
SL8
M6 L6 L19
L20
L22 SF
SpA
CS1 R2 S62
W94
W95
W96 P1 UP
C14
C50
Sol
ubili
zati
on I
ndex
(S
I)
0
1
2
3
4
Pikovskaya medium
NBRIP medium
Figure 5.4: Determination of Solubilization Index (SI) of bacterial isolates on Pikovskaya
agar and NBRIP agar after 7 days of incubation at 28 oC. Error bars Mean ± standard error
(n=3).
Bacterial strains
S1 S2R
ad1
Rad
2R
os1
Ros
2JA
10
R12
R14
R15
SL8
M6 L6 L19
L20
L22
SFSp
A
CS1 R2
S62
W94
W95
W96 P1 UP
C14
C50
So
lubil
iza
tio
n E
ffic
iency
(%
)
0
50
100
150
200
250
Pikovskaya medium
NBRIP medium
Figure 5.5: Determination of percentage Solubilization Efficiency (SE) by bacterial
isolates on Pikovskaya agar and NBRIP agar after 7 days of incubation at 28 oC. Error bars
Mean ± standard error (n=3).
109
To check the ability of isolated bacteria for inorganic phosphate solubilization in liquid
medium, three different inorganic phosphate sources were used and solubilized phosphate
was estimated quantitatively. For the estimation of solubilization of aluminium phosphate
(ALP), bacterial isolates were grown in NBRIP liquid medium having aluminium
phosphate as a sole phosphate source. The bacterial isolates were allowed to grow in liquid
medium for seven days and culture supernatant was used for estimation. It was observed
that among all the tested isolates, strain L22 had maximum potential for the solubilization
of aluminium phosphate and released 108 µg mL-1 of solubilized phosphate in the medium.
On the other hand, strains L6 and JA10 were also able to release 83 µg mL-1 and 63 µg mL-
1 of phosphate, respectively. The rest of the strains also had some ability to solubilize
aluminium phosphate but small quantities of solubilization was recorded which ranged
from 17 µg mL-1 to 51 µg mL-1 (Figure 5.6). The effect of ALP solubilization on pH and
titrable acidity of the culture medium was also observed and we found that the pH of all
strains decreased as compared to un-inoculated control. While increased titrable acidity
was recorded by different bacterial isolates when compared to control as shown in figure
5.7.
For the solubilization of ferric phosphate, NBRIP liquid media was supplemented with
ferric phosphate (FP) as a sole phosphate source and pH was adjusted to 7.0. After
incubation for 7 days at 28 oC, culture supernatant was used for the estimation of
solubilized phosphate, pH change and for the measurement of titrable acidity. From the
results, it was found that different bacterial isolates possess different dissolution potential
for ferric phosphate. The maximum solubilization for ferric phosphate was observed by
strain SF and W96 as 97.9 µg mL-1 and 92.6 µg mL-1, respectively. Whereas, the
110
solubilization potential for ferric phosphate by other isolates ranged from 55 µg mL-1 to 87
µg mL-1 (Figure 5.8). As a result of phosphate solubilization, the titrable acidity was
increased by all strains while deceased pH was recorded when compared to control as
shown in figure 5.9. The pH of un-inoculated control was decreased by 1.5 while more pH
decrease was observed for inoculated bacterial isolates. The maximum drop in pH was
observed to be -4 by three strains, R12, W95 and W96. The pH decrease of -3 was recorded
for strain Ros1, Ros2, JA10, SL8, M6, L19, R2, C14 and C50 while in rest of the isolates,
pH deceased to -2 to -2.5 (Figure 5.9).
To estimate the solubilization potential of isolates for tricalcium phosphate, isolated
bacterial strains were grown in NBRIP liquid media supplemented with tricalcium
phosphate as a sole phosphate source with pH adjusted to 7.0. All the bacterial isolates
possessed good potential for tricalcium phosphate solubilization. The dissolution ability
ranged from 618.6 µg mL-1 to 962.2 µg mL-1. The most active tricalcium phosphate
solubilizing strains were UP, SpA, Rad1, S2, JA10, W95, Ros1, R12 and L20 having the
solubilization potential of up to 962.2 µg mL-1 of solubilized phosphate (Figure 5.10). The
pH of culture medium was found to be decreased by all isolates. The maximum pH decrease
was recorded in case of Rad1, which decreased from 7.0 to 2.0. The titrable acidity was
increased in case of all the isolates and the maximum titrable acidity was observed by strain
CS1 as 28.2 (Figure 5.11).
Based on solubilization of inorganic phosphate, twelve bacterial isolates were selected for
further experiments and according to results of utilization of inorganic phosphate sources,
tricalcium phosphate was selected. The effect of four different sugars (glucose, maltose,
galactose and sucrose) as carbon sources was evaluated to check their impact on phosphate
111
solubilization. Twelve selected strains (Ochrobactrum pseudogrignonense-S1,
Acinetobacter olivorans-S2, Pseudomonas putida-Rad1, Pseudomonas putida-Rad2,
Pseudomonas sp-Ros2, Acinetobacter baumanii- JA10, Pseudomonas plecoglossicida-
R14, Pseudomonas japonica-SL8, Pseudomonas aeruginosa-SpA, Enterobacter cloacae-
W95, Enterobacter aerogenes-W96 and Pseudomonas reinekei-UP) were tested in this
study. Selected bacterial isolates were inoculated in NBRIP liquid medium supplemented
with different sugars and tricalcium phosphate as insoluble phosphate source. After
incubation of 7 days at 28 oC on rotary shaker, culture supernatant was obtained and was
used for the evaluation of solubilized phosphate content, changes in pH and titrable acidity,
and for the estimation of acid and alkaline phosphatases. From the collected result data, it
was found that among four different sugars, maximum solubilization of inorganic
phosphate was recorded in the presence of glucose. Maximum dissolution ability for
tricalcium phosphate was recorded by strain UP and W96 as 904.3 µg mL-1 and 885.455
µg mL-1, respectively. For galactose, strain S2 and Ros2 showed good results for
solubilization and dissolved 712.89 µg mL-1 and 670 µg mL-1 of phosphate, respectively.
In the presence of maltose, maximum solubility was recorded by SL8 (558.75 µg mL-1)
and R14 (555.32 µg mL-1) while for sucrose maximum solubilization activity was observed
by S1 followed by UP as 477.6 µg mL-1 and 475.89 µg mL-1, respectively (Figure 5.12).
Besides the decrease in solubilization ability, titrable acidity and pH was also affected
accordingly. Maximum titrable acidity was recorded for glucose followed by galactose,
maltose and sucrose while for pH, maximum decrease in pH was observed by glucose and
galactose whereas, in case of maltose and sucrose less decrease in pH was recorded (Figure
5.13). For glucose and galactose, pH decrease and increased titrable acidity was observed
112
for all isolates. In case of maltose, increase in pH was observed by Rad1, Rad2, Ros2 and
SpA while in case of rest of the isolates, pH decreased. Similar results were observed in
case of sucrose, whereas, increased pH was observed by control, S1, S2, Rad1, Rad2, SpA
and W96 whereas slightly decreased pH was recorded for Ros2, SL8, W95 and UP;
however no change in pH was observed in case JA10 and R14.
Variable results were observed for the production of acid and alkaline phosphatases. In
case of glucose and galactose, maximum acid phosphatase activity was recorded by Ros2
as 73.1 U mL-1 and 68.04 U mL-1. However maximum acid phosphatase activity for
maltose was observed by SpA as 68.93 U mL-1 and for sucrose similar quantity of enzyme
was produced by S1 strain (Figure 5.14). Similar variation in results were observed for
alkaline phosphatase production, maximum production of alkaline phosphatase activity
was observed by strain UP, which produced maximum quantities of acid in case of glucose,
galactose and sucrose when compared to other isolates (Figure 5.15).
The effect of pesticides on phosphate solubilization ability of isolates was also assessed.
For this purpose, NBRIP liquid media was supplemented with pesticide solutions prior to
bacterial inoculation. From the results, it was revealed that pesticide stress significantly
decrease the phosphate solubilizing ability of bacterial isolates. Bacterial strains produced
maximum quantities of solubilized phosphate without pesticide stress while in case of
stress, variable results were observed for Chlorpyrifos treatment and Pyriproxyfen as well
as for the combined effect of both pestisides. Maximum solubilization activities were
observed in the absence of any stress in all isolates whereas strain JA10 and SL8 showed
almost similar results for phosphate solubilization even in the presence of Chlorpyrifos.
When compared to the results of without stress, reduction in phosphate solubilizing activity
113
was observed. Strain S2, Rad1, Rad2, Ros2, R14, SL8 and SpA were able to produce more
solubilized phosphate in the presence of Chlorpyrifos stress. When Pyriproxyfen was
added, three strains including W95, W96, UP were able to perform well as compared to
other treatments. Least quantities of solubilized phosphate were recorded for most of the
strains when mixture of two pesticides were added only S1 was able to produce good results
for phosphate solubilization even in the presence of both pesticides (Figure 5.16).
Variable results were observed for the titrable acidity as well as for pH change as a result
of phosphate solubilization. However both in the absence and presence of pesticide stress,
titrable acidity was found to be increased by all bacterial isolates as compared to control.
For pH change as a result of phosphate solubilization, decreased pH was recorded by all
bacterial isolates as compared to control (Figure 5.17).
Bacterial isolates were evaluated for the production of acid and alkaline phosphatase
enzyme production in the presence and absence of pesticide stress. Maximum acid
phosphatase activity was observed in the absence of pesticide stress by strain S2, Rad2,
Ros2, JA10, R14, SpA and UP. Among maximum acid phosphatase producing bacteria in
the absence or presence of pesticide stress, strain SpA produced 84.46 U mL-1 while UP
produced 82.66 U mL-1 of acid phophatase production. In the presence of Chlorpyrifos,
maximum enzyme production was recorded by W95 and SpA as they produced 74.96 U
mL-1 and 73.7 U mL-1 of acid phosphatase while in the presence of Pyriproxyfen, maximum
enzyme production was recorded by strains UP and W95 which produced 79.66 U mL-1
and 72 U mL-1, respectively. When the combination of pesticides was applied, a general
reduction in activity was observed by most of the isolates except SL8 as compared to other
strains as well as other treatments (Figure 5.18). For alkaline phosphatase production, it
114
was found that maximum enzyme production was exhibited by strains UP, Ros2, R14 and
SpA in the absence of pesticides. In case of stress, maximum production of alkaline
phosphatase has been recorded by strains S1, S2, Rad1, Ros2, R14, SpA and UP. In general
the alkaline phosphatase production by isolates was affected when combination of
pesticides was applied (Figure 5.19).
115
Bacterial strain
Co
nt
S1
S2
Rad
1R
ad2
Ro
s1R
os2
JA10
R12
R14
R15
SL
8
M6
L6
L19
L20
L22 SF
Sp
A
CS
1
R2
S62
W94
W95
W96 P1
UP
C14
C50
Solu
bil
ized a
lum
iniu
m p
hosph
ate
(µ
g m
l-1)
0
20
40
60
80
100
120
140
Figure 5.6: Solubilization of aluminium phosphate by phosphate solubilizing bacteria after
7 days of incubation at 28 oC. Error bars Mean ± standard error (n=3).
Bacterial strains
Cont
S1
S2
Rad
1R
ad2
Ros1
Ros2
JA10
R12
R14
R15
SL8
M6
L6
L19
L20
L22 SF
SpA
CS1
R2
S62
W94
W95
W96 P1
UP
C14
C50
Tit
rable
aci
dit
y
-5
0
5
10
15
20
pH decrease
titrable acidity
Figure 5.7: Effect of aluminium phosphate solubilization on pH and titrable acidity of
culture supernatant after 7 days of incubation at 28 oC. Error bars Mean ± standard error
(n=3).
116
Bacterial strain
Cont
S1
S2
Rad1
Rad2
Ros1
Ros2
JA10
R12
R14
R15
SL8
M6
L6
L19
L20
L22
SF
SpA
CS
1
R2
S62
W94
W95
W96
P1
UP
C14
C50
Solu
bil
ized f
err
ic p
hosphate
(µ
g m
L-1
)
0
20
40
60
80
100
120
Figure 5.8: Solubilization of ferric phosphate by phosphate solubilizing bacteria after 7
days of incubation at 28 oC. Error bars Mean ± standard error (n=3).
Bacterial strain
Co
nt
S1
S2
Rad
1
Rad
2
Ro
s1
Ro
s2
JA10
R12
R14
R15
SL
8
M6
L6
L19
L20
L22 SF
Sp
A
CS
1
R2
S62
W94
W95
W96 P1
UP
C14
C50
Tit
rable
aci
dit
y
-10
0
10
20
30
40
pH
Titrable acidity
pH
Figure 5.9: Effect of ferric phosphate solubilization on pH and titrable acidity of culture
supernatant after 7 days of incubation at 28 oC. Error bars Mean ± standard error (n=3).
117
Bacterial strain
Cont
S1
S2
Rad1
Rad2
Ros1
Ros2
JA10
R12
R14
R15
SL8
M6
L6
L19
L20
L22
SF
SpA
CS
1
R2
S62
W94
W95
W96
P1
UP
C14
C50
Solu
bil
ized t
rica
lciu
m p
hosp
hate
(µ
g m
L-1
)
0
200
400
600
800
1000
1200
Figure 5.10: Solubilization of tricalcium phosphate by phosphate solubilizing bacteria
after 7 days of incubation at 28 oC. Error bars Mean ± standard error (n=3).
Bacterial strain
Con
t
S1
S2
Rad
1R
ad2
Ros
1R
os2
JA10
R12
R14
R15
SL
8
M6
L6
L19
L20
L22 SF
SpA CS1
R2
S62
W94
W95
W96 P1
UP
C14
C50
Tit
rable
aci
dit
y
-10
0
10
20
30
40pH
Titrabele acidity
pH
Figure 5.11: Effect of tricalcium phosphate solubilization on pH and titrable acidity of
culture supernatant after 7 days of incubation at 28 oC. Error bars Mean ± standard error
(n=3).
11
8
Ba
cte
rial stra
in
Co
nt
S1
S2
Rad
1R
ad 2
Ro
s 2JA
10
R1
4S
L8
SpA
W9
5W
96
UP
Solubilized phosphate (µg mL-1
)
0
20
0
40
0
60
0
80
0
10
00
12
00
Glu
co
se
Gala
cto
se
Malto
se
Su
cro
se
Fig
ure 5
.12
: Effect o
f differen
t carbo
n so
urces o
n p
ho
sph
ate solu
bilizatio
n ab
ility o
f isolated
ph
osp
hate so
lub
ilizing b
acteria
after 7 d
ays o
f incu
batio
n at 2
8 oC
. Erro
r ba
rs Mean
± stan
dard
error (n
=3
).
119
Glucose Galactose
Bacterial strain
Co
nt
S1
S2
Rad
1R
ad 2
Ro
s 2
JA10
R14
SL
8
Sp
A
W95
W96 UP
Tit
rable
aci
dit
y
-10
-5
0
5
10
15
20
25
30pH decrease
T it rable Acidity
pH
Bacterial strain
Co
nt
S1
S2
Rad
1R
ad 2
Ro
s 2
JA10
R14
SL
8
Sp
A
W95
W96 UP
Tit
rable
aci
dit
y
-10
-5
0
5
10
15
20
25pH decrease
T it rable Acidity
pH
Maltose Sucrose
Bacterial strain
Co
nt
S1
S2
Rad
1R
ad 2
Ro
s 2
JA10
R14
SL
8
Sp
A
W95
W96 UP
Tit
rable
aci
dit
y
-5
0
5
10
15
20pH decrease
T it rable Acidity
pH
Bacterial strain
Co
nt
S1
S2
Rad
1R
ad 2
Ro
s 2
JA10
R14
SL
8
Sp
A
W95
W96 UP
Tit
rable
aci
dit
y
-2
0
2
4
6
8
10pH decrease
T it rable Acidity
pH
Figure 5.13: Effect of phosphate solubilization on pH and titrable acidity in the presence
of different carbon sources after 7 days of incubation at 28 oC. Error bars Mean ± standard
error (n=3).
12
0
Ba
cte
rial stra
in
Co
ntS
1S
2R
ad 1R
ad 2R
os 2
JA1
0R
14
SL
8S
pAW
95
W9
6U
P
Acid phosphatase (U mL-1
)
0
20
40
60
80
Gluco
se
Galacto
se
Malto
se
Sucrose
Fig
ure 5
.14
: Acid
ph
osp
hatase p
rod
uctio
n b
y p
ho
sph
ate solu
bilizin
g b
acteria in th
e presen
ce of d
ifferent carb
on
sou
rces. Erro
r
ba
rs Mean
± stan
dard
error (n
=3
).
12
1
Ba
cte
rial stra
in
Cont
S1
S2
Rad
1R
ad 2
Ros 2
JA10
R14
SL
8S
pA
W95
W96
UP
Alkaline phosphatase (U mL-1
)
0
20
40
60
80
100
Glu
co
se
Gala
cto
se
Malto
se
Sucro
se
Fig
ure 5
.15
: Alk
aline p
ho
sph
atase pro
ductio
n b
y p
ho
sph
ate solu
bilizin
g b
acteria in th
e presen
ce of d
ifferent carb
on
sou
rces.
Erro
r ba
rs Mean
± stan
dard
error (n
=3
).
12
2
Ba
cterial stra
in
Co
ntS
1S
2R
ad 1R
ad 2R
os 2
JA1
0R
14
SL
8S
pAW
95
W9
6U
P
Solubilized phosphate (µg mL-1
)
0
20
0
40
0
60
0
80
0
10
00
12
00
Strain
con
trol
Ch
lorp
yrifo
s
Py
ripro
xyfen
Ch
lorp
yrifo
s+ P
yrip
roxy
fen
Fig
ure 5
.16
: Effect o
f pesticid
e stress on
ph
osp
hate so
lub
ilization
ability
of iso
lated p
ho
sph
ate solu
bilizin
g b
acteria after 7 d
ays
of in
cub
ation
at 28
oC. E
rror b
ars M
ean ±
stand
ard erro
r (n=
3).
123
Without stress Chlorpyrifos
Bacterial strain
Co
nt
S1
S2
Rad
1R
ad 2
Ro
s 2
JA10
R14
SL
8
Sp
A
W95
W96 UP
Tit
rable
aci
dit
y
-5
0
5
10
15
20
25pH
Titrable acidity
pH
Bacterial strain
Co
nt
S1
S2
Rad
1R
ad 2
Ro
s 2
JA10
R14
SL
8
Sp
A
W95
W96 UP
Tit
rable
aci
dit
y
-5
0
5
10
15
20
25
30
35pH
Titrable acidity
pH
Pyriproxyfen Chlorpyrifos + Pyriproxyfen
Bacterial strain
Co
nt
S1
S2
Rad
1R
ad 2
Ro
s 2
JA10
R14
SL
8
Sp
A
W95
W96 UP
Tit
rable
aci
dit
y
-10
-5
0
5
10
15
20
25
30pH
Titrable acidity
pH
Bacterial strain
Co
nt
S1
S2
Rad
1R
ad 2
Ro
s 2
JA10
R14
SL
8
Sp
A
W95
W96 UP
Tit
rable
aci
dit
y
-5
0
5
10
15
20
25
30
Col 2
Col 3
pH
Figure 5.17: Effect of phosphate solubilization on pH and titrable acidity in the presence
of different pesticides after 7 days of incubation at 28 oC. Error bars Mean ± standard error
(n=3).
12
4
Ba
cte
rial stra
in
Co
nt
S1
S2
Rad 1
Rad 2
Ro
s 2
JA
10
R1
4S
L8
SpA
W9
5W
96
UP
Acid phosphatase (U mL-1
)
0
20
40
60
80
10
0Stra
in c
on
t
Ch
lorp
yrifo
s
Py
ripro
xy
fen
Ch
lorp
yrifo
s + P
yrip
rox
yfe
n
Fig
ure 5
.18
: Acid
ph
osp
hatase p
rod
uctio
n b
y p
ho
sph
ate solu
bilizin
g b
acteria in th
e presen
ce of p
esticide stress. E
rror b
ars
Mean
± stan
dard
error (n
=3
).
12
5
Ba
cte
rial stra
in
Co
nt
S1
S2
Rad 1
Rad 2
Ro
s 2
JA
10
R1
4S
L8
SpA
W9
5W
96
UP
Alkaline phosphatase (U mL-1
)
0
20
40
60
80
10
0
Stra
in c
on
trol
Ch
lorp
yrifo
s
Py
ripro
xyfe
n
Ch
lorp
yrifo
s+
Py
ripro
xyfe
n
Fig
ure
5.1
9: A
lkalin
e ph
osp
hatase p
rod
uctio
n b
y p
ho
sph
ate solu
bilizin
g b
acteria in th
e presen
ce of p
esticide stress. E
rror b
ars
Mean
± stan
dard
error (n
=3
).
126
Discussion
In soil, phosphorous is a leading component but it remains sequestered by different
elements present in soil which are responsible for un-availability of phosphorous to plants.
This deficiency leads to reduced productivity of plants (Zhang et al., 2017). In soil,
phosphorous usually remains adsorbed by aluminium, ferrous, calcium and magnesium and
their oxides. It also lead to their gradual conversion towards more complexity. The
adsorption of phosphorous is greatly influenced by pH of soil. Calcium bound phosphorous
occur predominantly in alkaline soils while aluminium and ferric bound forms usually
occur in acidic environments (Maitra et al., 2015; Banerjea and Gosh, 1970). According to
an estimate, around 8-82 percent of total phosphorous is present in bound form. Out of
which around 50% is bound to calcium (Qian et al., 2010; Renjith et al., 2011; Rzepechi,
2010; Maitra et al., 2015). Phosphorous is an important component for growth and
development of plants and is generally used as fertilizers to enhance plant growth (Wei et
al., 2015; Wei et al., 2017). Microorganisms in soil play a crucial role in conversion or
transformation of nutrients from one form to another (Maitra et al., 2015; Gronemeyer et
al., 2011). There are several reports of isolation of phosphate solubilizing bacteria from
rhizospheric region of different plants (Singh et al., 2013; Panda et al., 2016; Tomer et al.,
2017). The occurrence of phosphate solubilizing microorganisms in soil suggests that they
can be a good option to be study and to be used as biofertilizers (Majeed et al., 2015).
Isolated bacterial strains were subjected to study their solubilization abilities for inorganic
phosphate on two media including Pikovskaya and NBRIP agar. Based on the calculation
of solubilization index, maximum index on Pikovskaya agar was exhibited by
Acinetobacter baumanii- JA10 as 2.64mm followed by Pseudomonas japonica-SL8,
127
Acinetobacter pittii-L6 and Acinetobacter sp-C50 with the solubilization index of 2.53mm.
Minimum solubility index on Pikovskaya agar was exhibited by Klebsiella pneumoniae-
R12, Pseudomonas reinekei-UP and Acinetobacter calcoaceticus-S62 as 2.176, 2.09 and
2.0mm, respectively. The solubilization index of 1.62mm has been recorded by phosphate
solubilizing Pseudomonas strain on Pikovskaya agar (Mohamed and Almoroai, 2017). On
NBRIP agar, we found better results for solubilization index, Klebsiella pneumoniae-R12
had the maximum value of 3.07mm while good results were observed by Enterobacter
aerogenes-W96, Pseudomonas putida-Rad2 and Enterobacter cloacae-W95 which
exhibited the solubilization index of 2.93, 2.84 and 2.8mm, respectively. In a current study
conducted by Tomer et al. (2017), it has been reported that isolates ST-30, N-26 and MP-
1 had solubilization index of 62mm, 8mm and 7.2mm in NBRIP agar.
The possible reason for solubilization zone formation in agar medium and phosphate
solubilization in liquid medium is due to the production of different organic acids by
bacteria. These organic acids includes malic, butyric, succinic, glyoxalic, gluconic and
citric acid (Kelel et al., 2017). When the results on both media were compared, it was found
that 64% bacterial isolates had better results for solubilization on NBRIP agar as compared
to Pikovskaya agar. Pseudomonas parafulva-Ros1 showed consistent results on both
media. Better efficiency for phosphate solubilization was observed by 28% isolates on
Pikovskaya agar as compared to NBRIP agar. For Klebsiella pneumoniae-R12 increase in
solubilization was recorded up to 89.9% on NBRIP agar as compared to Pikovskaya agar,
whereas 50% increased solubility was recorded by Enterobacter aerogenes-W96,
Enterobacter cloacae-W95, Acinetobacter calcoaceticus-S62 and Acinetobacter pittii-
128
CS1. Arfarita et al. (2017) have reported three bacterial isolates SPP1, SPP2 and SPP3 with
higher solubility index on Pikovskaya agar.
From the results of phosphatases detection on TSA plates, we found that all of the bacterial
isolates had the ability to produce phosphatases on agar plates which was indicated by pink
zone formation around point of inoculation. However, Pseudomonas putida-Rad2,
Klebsiella pneumoniae-R12, Pseudomonas aeruginosa-R15, Acinetobacter pittii-L6,
Pseudomonas frederiksbergensis-L22 and Pseudomonas oryzihabitans-SF exhibited
strong positive results for phosphatases production. Ribeiro and Cardoso (2012) have
reported that 85% of their isolates showed phosphatases production indicated by pink color
on TSA plate.
Phosphate solubilizers play an important role in the transformation of phosphorous (Wei
et al., 2016). Furthermore, bacterial isolates were tested to solubilize three different
inorganic phosphate sources including aluminium phosphate (ALP), ferric phosphate (FP)
and tricalcium phosphate (TCP) and it was observed that L22 had maximum potential to
solubilize aluminium phosphate and produced 108 µg mL-1 of solubilized phosphate. The
solubilization range for aluminium phosphate by isolated bacterial strains was 17 µg mL-1
to 51 µg mL-1. Moreover decrease in pH and increase in titrable acidity was recorded by
all isolated bacteria compared to control. According to a study related to aluminium
phosphate solubilization, Yadav et al. (2015) have reported that the isolated phosphate
solubilizing bacteria were able to solubilize 59.4 mg L-1 to 76.7 mg L-1 of phosphate.
When ferric phosphate was added to growth medium as inorganic phosphate source, it was
observed that among isolated bacterial strains SF and W96 were able to dissolve 97.9 µg
mL-1 and 92.6 µg mL-1 of ferric phosphate. Among isolates bacteria, the range of FP
129
solubilization was 55 µg mL-1 to 87 µg mL-1. On the other hand according to a study
conducted by Yadav et al. (2015), the reported range of ferric phosphate solubilization by
phosphate solubilizing bacteria was 60.1 mg L-1 to 69.2 mg L-1. From the results of our
study, dropped pH was recorded by all isolates as well as decrease in pH of control was
also observed after incubation, however increased titrable acidity was recorded in by all
isolates.
The isolated bacterial strains showed best results for dissolution of TCP. The solubilization
potential of all isolates for TCP ranged from 618.6 µg mL-1 to 962.2 µg mL-1. Best strains
for tricalcium phosphate solubilization were Pseudomonas reinekei-UP, Pseudomonas
aeruginosa-SpA, Pseudomonas putida-Rad1, Acinetobacter olivorans-S2, Acinetobacter
baumanii- JA10, Enterobacter cloacae-W95, Pseudomonas parafulva-Ros1, Klebsiella
pneumoniae-R12 and Pseudomonas koreensis-L20. Depending upon genus, different
bacterial isolates perform differently for phosphate solubilization and results vary
depending upon sources of isolation as well as inorganic sources (Zhang et al., 2017).
Likewise Tomer et al. (2017) reported the data for phosphate estimation and found that
solubilized phosphate content by isolates ranged from 314.43 - 713.11 µg mL-1. In a recent
research related to phosphate solubilizing bacteria, Zhang et al. (2017) have reported that
Ochrobactrum sp.-M11 solubilized 54.41 µg mL-1 of phosphate. Isolates belonging to
Acinetobacter sp- M01, M04, M05 solubilized 54.91 µg mL-1, 60.87 µg mL-1 and 34.88 µg
mL-1 of phosphate, respectively. Whereas, the solubilization potential of strains Klebsiella
sp-M02 and Enterobacter sp-M03 was 22.94 µg mL-1 and 25.66 µg mL-1, respectively. The
quantities of solubilized phosphate by our strains are higher than that of recently report by
Zhang et al. (2017). The pH of the culture medium as a result of phosphate solubilization
130
was decreased and most decreased pH was observed by Pseudomonas putida-Rad1 which
is 2.0 and the most volume consumption for titrable acidity was recorded by Acinetobacter
pittii-CS1 which consumed 28.2 mL to neutralize the acidity of culture supernatant. The
decreased pH of growth medium seems to be associated to solubilization of phosphate but
it is not strictly proportional (Pallavi and Gupta, 2013).
In acidic soils, phosphate gets precipitated with Al3+ and Fe3+ while in calcareous or neutral
soils, it binds to Ca2+ (de Oliveira Mendes et al., 2014). The possible reason for less
solubilization potential towards aluminium phosphate and ferric phosphate is that these
inorganic phosphate sources are abundant in acidic soils. The isolates of our study were
isolated from neutral to alkaline soils where tricalcium phosphate is present in large
quantities that is why they showed best solubilization for tricalcium phosphate. Yadav et
al. (2015) have also documented that bacterial isolates, isolated from alkaline soil were
able to solubilize tricalcium phosphate more efficiently as compared to aluminium
phosphate and ferric phosphate.
Phosphate solubilization is usually enhanced when appropriate amount of energy is present
to be utilized by organism for the production of various organic acids (Reza et al., 2017).
Carbon sources are utilized to be used as a source of energy but various sources affect the
phosphate solubilization potentials. The effect of different carbon sources (glucose,
maltose, galactose and sucrose) was checked to have any impact on phosphate
solubilization ability of isolated bacteria. The solubility of inorganic phosphate by different
strains varied in the presence of different sugars and phosphate solubilizing ability varied
significantly among strains. Maximum dissolution for insoluble phosphate was exhibited
in the presence of glucose by bacterial isolates. Maximum solubilization for glucose was
131
recorded by Pseudomonas reinekei-UP and Enterobacter aerogenes-W96 as 904.3 µg mL-
1 and 885.455 µg mL-1, respectively. For galactose, maximum dissolution was recorded by
Acinetobacter olivorans-S2 as 712.89 µg mL-1. When maltose was added maximum
solubility was recorded by strain SL8 as 558.75 µg mL-1 while in the presence of sucrose
S1 showed maximum potential for solubilization and dissolved 477.6 µg mL-1 of
phosphate. The order of maximum to minimum phosphate solubilization by isolates
depending on carbon source was found as: glucose > galactose > maltose > sucrose.
Different phosphate solubilizing bacteria show different levels of phosphate solubilization
activities for different sugars. Pallavi and Gupta (2013) have studied the effect of different
carbon sources on phosphate solubilization ability of Pseudomonas lurida and found that
most to least suitable carbon source for phosphate solubilization from tricalcium phosphate
was glucose followed by maltose, galactose, sucrose and xylose. Glucose enhanced the
production of solubilized phosphate by bacterial species from tricalcium phosphate in
NBRIP medium (Pallavi and Gupta, 2013).
The effect of phosphate solubilization on pH and titrable acidity was evaluated and it was
found that titrable acidity was increased in case of all sugars. For pH, decease was observed
by all strains in case of glucose and galactose while in case of maltose and sucrose few
strains showed increased pH as compared to control. Pallavi and Gupta (2013) have also
reported that in the presence of different sugars, bacterial isolate showed variable results
for solubilization of phosphate and it also affected the pH of culture medium. The effect of
carbon sources was evaluated to have impact on acid and alkaline phosphatases production.
Variable results were recorded for all carbon sources by isolated bacteria. The effect of
132
different sugar sources on phosphate solubilization and determination of different enzyme
production have also been reported by Qureshi et al. (2010).
Soil is a reservoir for pesticide remains and several microorganisms (Jain et al., 2015).
Besides enhancing plant growth, phosphate solubilizing bacteria have also been reported
to degrade xenobiotic compounds like pesticides. The impact of applied pesticides was
evaluated in vitro to assess the bacterial ability to solubilize inorganic phosphate in the
presence of pesticides. For this purpose, Chlorpyrifos, Pyriproxyfen, and mixture of these
pesticides was added to the culture medium of isolated bacteria. Maximum solubilization
activities were observed in the absence of any stress by all isolates, whereas JA10 and SL8
showed almost similar results for phosphate solubilization even in the presence of
Chlorpyrifos. The phosphate solubilization activity of isolates was affected in the presence
of pesticides and decrease in solubilization potential was observed by most of the isolates.
Even though the activity of phosphate solubilization by isolated bacterial stains was
decreased in the presence of pesticide stress but still they exhibited much better results for
the solubilization of inorganic phosphate when compared to control. Anzuay et al. (2017)
have studied the effect of abiotic stress and pesticide effect on solubilization activity of
phosphate solubilizing isolates and reported that Acinetobacter sp.-L176 produced 44.0 U
of acid phosphatase and 42.1 U of alkaline phosphatase. The reported acid and alkaline
phosphatase activity by Pseudomonas fluorescens was 56.1 U and 65.9 U, respectively.
Overall strain UP showed consistent results for maximum acid phosphatase activity in the
absence as well as in the presence of pesticide stress.
133
Conclusion
Phosphate solubilizing microorganisms are abundant is soil. They convert the insoluble
forms of phosphate to soluble phosphate which can be easily used by plants. The isolated
bacteria were able to convert all three tested insoluble forms of phosphate to soluble forms.
Maximum solubilization was observed for tricalcium phosphate because the isolated
bacteria of this study were isolated from normal to calcareous soils but they had ability to
solubilize acidic forms of inorganic phosphate (ALP and FP) as well. Among the evaluated
carbon sources, glucose proved to be the best source for better solubilization of phosphate.
Phosphate solubilization was slightly reduced by the application of pesticide stress but the
bacterial isolates did not lose the solubilization ability.
134
Chapter 06
Plant growth promoting attributes of phosphate
solubilizing bacteria
Rhizosphere is an important region where a large number of diversified microorganisms
exist. Some of them play a vital role in the growth and development of plants. These useful
bacteria are known as plant growth promoting bacteria. In the current scenario, due to the
increased population the demands for food are increasing which have led to the increased
use of agrochemicals which are causing deleterious effects on our environment (Namli et
al., 2017). Now the trend is shifting towards the use of plant growth promoting bacteria
because of their characteristics which are helpful in better plant growth. There are some
direct as well as some indirect mechanisms in bacteria including solubilization of some
important inorganic compounds, fixation of atmospheric nitrogen, production of
phytohormones and other compounds to prevent plants from possible pathogens (Hamuda
and Patko, 2013; Namli et al., 2017).
Isolated phosphate solubilizing bacteria including Ochrobactrum pseudogrignonense-S1,
Acinetobacter olivorans-S2, Pseudomonas putida-Rad1, Pseudomonas putida-Rad2,
Pseudomonas parafulva-Ros1, Pseudomonas sp-Ros2, Acinetobacter baumanii-JA10,
Klebsiella pneumoniae-R12, Pseudomonas plecoglossicida-R14, Pseudomonas
aeruginosa-R15, Pseudomonas japonica-SL8, Ochrobactrum sp-M6, Acinetobacter pittii-
L6, Acinetobacter pittii-L19, Pseudomonas koreensis-L20, Pseudomonas
frederiksbergensis-L22, Pseudomonas oryzihabitans-SF, Pseudomonas aeruginosa-SpA,
Acinetobacter pittii-CS1, Acinetobacter calcoaceticus-R2, Acinetobacter calcoaceticus-
135
S62, Acinetobacter sp.-W94, Enterobacter cloacae-W95, Enterobacter aerogenes-W96,
Pseudomonas fluorescens-P1, Pseudomonas reinekei-UP, Acinetobacter calcoaceticus-
C14 and Acinetobacter sp.-C50 were investigated to have plant growth promoting abilities.
To assess the ability for hydrogen cyanide (HCN) production, isolated bacterial strains
were inoculated on glycine supplemented N-agar plates and were overlaid with sterile filter
paper sheets soaked in picric acid and sodium carbonate solution. After incubation for four
days at 28oC, change in color of filter paper was observed for all tested isolates and results
were recorded. From results it was found that among all tested strains, 53% isolates were
able to produce hydrogen cyanide in in vitro conditions (Table 6.1). Strain R15 and SpA
were found strong producers of hydrogen cyanide, whereas moderate production of HCN
was observed by strain Rad2, Ros1, L6, L19, L22 and S62. Slight positive results were
recorded by strains S2, Rad1, M6, L20, CS1, R2 and UP while S1, Ros2, JA10, R12, R14,
SL8, SF, W94, W95, W96, P1, C14 and C50 were found negative for HCN production
ability (Figure 6.1).
The isolated phosphate solubilizing bacteria were evaluated for indole acetic acid (IAA)
production in the absence as well as in the presence of L-tryptophan. It was observed that
all the isolates produced greater amount of IAA in the presence of L-tryptophan (Figure
6.2). The maximum quantity of IAA was recorded by strain R12 in the presence as well as
in the absence of substrate as 74.66 µg mL-1 and 24.29 µg mL-1. Strain SL8 produced 24.44
µg mL-1 IAA in the presence of tryptophan and 68.66 µg mL-1 IAA in its absence. Whereas,
the quantities of IAA recorded by strain S1 were 8.6 µg mL-1 and 67.85 µg mL-1,
respectively. The least quantities of IAA were recorded by strain S2, SpA, S62, L6, L19,
C50, R2, C14 and CS1 as they produced less than 10 µg mL-1 of IAA (Figure 6.5).
136
Ammonia production by isolated phosphate solubilizing bacteria was checked by growing
them in peptone water. Color change indicated the production of ammonia. It was observed
that all isolates were able to produce ammonia. The quantities of ammonia production was
evaluated according to intensity of color change. High quantities of ammonia production
was observed in case of strain R12, R15, SpA and CS1 while moderate color change was
recorded by strain S2, Ros2, JA10, M6, C14 and C50 as shown in figure 6.3. The rest of
the strains produced smaller quantities of ammonia as slight color change was observed.
All the isolated bacterial strains were assessed for siderophores production. For this
purpose, bacterial isolates were spot inoculated on Chrom Azurol S (CAS) agar media and
results were observed for color change after 3-4 days of incubation at 28oC. From the
results, it was found that among 28 isolated phosphate solubilizing bacteria, only 21% of
the isolates were able to produce siderophores (Table 6.1; Figure 6.4). The majority of the
isolated bacteria were not able to produce siderophores, only strain Ros2, R14, R15, SL8,
SF and SpA were found positive for siderophores production while the rest of the isolates
were found negative for this test.
The isolated phosphate solubilizing bacteria were tested for ACC deaminase activity. The
ACC deaminase activity was quantitatively estimated and was expressed as µmol α-
ketobutyrate mg-1 protein h-1. A range of ACC deaminase activity was exhibited by the
isolates as shown in figure 6.6. The observed range of ACC deaminase activity by isolates
was 66.58 µmol- 386.35 µmol α-ketobutyrate mg-1 protein h-1. The highest activity was
detected by strain L20, S62 and UP as 386.35 µmol, 365.36 µmol and 361.76 µmol α-
ketobutyrate mg-1 protein h-1, respectively. However strain SF showed least activity which
was recorded as 66.58 µmol α-ketobutyrate mg-1 protein h-1.
137
Table 6.1: Plant growth promoting activities of isolated phosphate solubilizing bacterial
strains.
Sr.
No Strain code
HCN
production
Ammonia
production
Siderophore
production
1 Control - - -
2 S1 - + -
3 S2 + ++ -
4 Rad1 + + -
5 Rad2 ++ + -
6 Ros1 ++ + -
7 Ros2 - ++ +++
8 JA10 - ++ -
9 R12 - +++ -
10 R14 - + +++
11 R15 +++ +++ ++
12 SL8 - + +++
13 M6 + ++ -
14 L6 ++ + -
15 L19 ++ + -
16 L20 + + -
17 L22 ++ + -
18 SF - + +++
19 SpA +++ +++ +++
20 CS1 + +++ -
21 R2 + + -
22 S62 ++ + -
23 W94 - + -
24 W95 - + -
25 W96 - + -
26 P1 - + -
27 UP + + -
28 C14 - ++ -
29 C50 - ++ -
-= negative; += slight, ++= moderate; +++= strong
138
Figure 6.1: Hydrogen cyanide production by isolated phosphate solubilizing bacteria after
four days of incubation at 28oC.
Figure 6.2: Qualitative determination of Indole Acetic Acid (IAA) by isolated phosphate
solubilizing bacterial strains. T- represents IAA production in the absence of L-tryptophan,
T+ represents IAA production in the presence of L-tryptophan.
139
Figure 6.3: Ammonia production by isolated phosphate solubilizing bacterial strains after
incubation of three days at 28oC.
Figure 6.4: Siderophore production by isolated phosphate solubilizing bacterial strains on
Chrom Azurol S (CAS) agar after four days of incubation at 28oC.
14
0
Bacterial strain
s
S1
S2Rad1Rad2Ros1Ros2JA10
R12
R14
R15
SL8
M6
L6
L19
L20
L22
SFSpA
CS1
R2
S62W
94W
95W
96
P1
UP
C14C50
IAA (µg mL-1
)
0
20
40
60
80
With
out T
rypto
phan
with
Tryp
tophan
Fig
ure 6
.5: Q
uan
titative d
etermin
ation
of In
do
le Acetic A
cid (IA
A) b
y iso
lated p
ho
sph
ate solu
bilizin
g b
acterial strains in
the
absen
ce and
presen
ce of L
-tryp
top
han
. Erro
r ba
rs Mean
± stan
dard
error (n
=3
).
14
1
Bacte
rial stra
in
S1
S2Rad1Rad2Ros1Ros2JA10
R12
R14
R15
SL8
M6
L6
L19
L20
L22
SFSpA
CS1
R2
S62W
94W
95W
96
P1
UP
C14C50
ACC deaminase activity
(µmol -ketobutyrate mg-1
protein h-1
)
0
100
200
300
400
500
Fig
ure 6
.6: A
CC
deam
inase p
rod
uctio
n b
y iso
lated p
ho
sph
ate solu
bilizin
g b
acteria measu
red after 2
4 h
ou
rs of in
cub
ation in
DF
-AC
C m
ediu
m. E
rror b
ars M
ean ±
stand
ard erro
r (n=
3).
142
Discussion
Soil present around plant roots contain large number of active bacterial species. These
bacteria are also called as Plant Growth Promoting Rhizobacteria (PGPR) (Kloepper et al.,
1980; Reetha et al., 2014; Liu et al., 2018). It is estimated that above 95% of bacteria exist
in the rhizosphere of plants and are responsible to help plants in obtaining nutrients from
soil. According to current approaches, researchers are trying to isolate and study bacteria
having Plant Growth Promoting (PGP) abilities (Ullah and Bano, 2015). Plant growth
enhancement by bacteria can be due to direct mechanisms as well as it can be due to some
indirect mechanisms. Bacteria present in plant rhizosphere also help plants to survive in
stress conditions either biotic or abiotic (Park et al., 2016). The indirect mechanisms
include the production of phytohormones specifically related to stress conditions. These
stress associated phytohormones include ethylene or jasmonic acid. The other indirect
mechanisms include the induction of systemic resistance in plants and the production of
antibiotics to compete in rhizosphere. The direct mechanisms responsible for enhanced
plant growth include phosphate solubilization, fixation of atmospheric nitrogen,
siderophore production and phytohormone production including auxins, cytokinins,
gibberallins and nitric oxide (Cassan et al., 2014). These rhizospheric bacterial population
mostly include Pseudomonas, Enterobacter, Bacillus and Rhizobium. Their most common
plant growth promoting abilities include solubilization of phosphate, zinc and potassium,
auxin production, and biocontrol activities such as antibiotic production, hydrolytic
enzyme production and hydrogen cyanide production (Yadegari and Mosadeghzad, 2012;
Phua et al., 2012; Verma et al., 2012; Zhang et al., 2013; Singh et al., 2015; da-Silveria et
al., 2018).
143
HCN production by bacterial isolates have been found responsible to suppress diseases in
plants (Kumar et al., 2015). Different bacterial genera have been reported to have hydrogen
cyanide production ability. These genera mainly include Rhizobium, Enterobacter,
Pseudomonas, Bacillus, Aeromonas and Alcaligenes (Kumar et al., 2014). Isolated
bacterial strains were evaluated for their ability to produce hydrogen cyanide production
and positive results were recorded by 53% of the isolates. Best producers for hydrogen
cyanide production were R15 and SpA belonging to genus Pseudomonas. Singh et al.
(2015) have also observed HCN production by five bacterial isolates belonging to genus
Pseudomonas. Among the Pseudomonas isolates from rhizosphere, HCN production has
been found to be the most common trait. According to previous studies, around 50% of
bacterial isolates from wheat and potato rhizosphere had shown HCN production in vitro
(Kumar et al., 2015).
Previous studies have shown that IAA production by bacteria helps in better interaction
with plants as it helps in root elongation, increased root exudates and biomass production
as well as it also helps in stress tolerance (Etesami and Alikhani, 2015). During the in vitro
screening and quantification of IAA production, we found that the IAA production ability
of isolated bacteria ranged from 4.48 µg mL-1 to 74.6 µg mL-1. All isolates were able to
produce IAA whereas maximum quantity of IAA was observed in Klebsiella pneumoniae-
R12. It was observed that the isolated phosphate solubilizing bacteria were also able to
produce small quantities of IAA in the absence of tryptophan. In a recent report, Zhang et
al. (2017) have reported that 62% of studied phosphate solubilizing bacteria produced 8.06
to 62.43 mg L-1 of IAA. Klebsiella sp M-02, Enterobacter sp M-03 and Acinetobacter sp
M-04 produced 11.27 mg L-1, 13.03 mg L-1 and 10.45 mg L-1 IAA, respectively). Xu et al.
144
(2014) studied IAA production by plant growth promoting bacterial isolates and have
found that only 37% isolates were IAA producers. Reetha et al. (2014) have reported
Pseudomonas fluorescens with 15.38 µg mL-1 IAA production whereas our Pseudomonas
fluorescens-P1 produced 14 µg mL-1 and 24.32 µg mL-1 of IAA, respectively, in the
absence and presence of tryptophan. IAA level of our isolated Pseudomonas strains ranged
from 7.86 - 68.66 µg mL-1 shown to be higher than reported Pseudomonas strains which
was 10-26 µg mL-1 and IAA production range of our Enterobacter strains was 7.0- 43.6 µg
mL-1 which was lower than reported Enterobacter strain which was 126 µg mL-1
(Montanez et al., 2012; Patel et al., 2012; Ribeiro and Cardoso, 2012). IAA production by
plant growth promoting bacterial isolates including FMR15-3 and HYT-9 was 1.40 µg mL-
1 and 2.83 µg mL-1 (Xu et al., 2014). In a study conducted by Verma et al. (2013) it has
been reported that bacterial isolates from rhizosphere had the ability to produce indole
acetic acid ranged from 5.93-21.53 µg mL-1. Similarly Singh et al. (2015) also reported
Pseudomonas saponiphila with maximum indole acetic acid production (13.29 µg mL-1).
Ammonia production by plant growth promoting bacteria have been associated with
controlling of phytopathogens and high crop yield (Mota et al., 2017). Isolated phosphate
solubilizing bacteria were checked for the production of ammonia, and it was found that
all isolates had this ability. The strong production of ammonia was recorded in Klebsiella
pneumoniae-R12, Pseudomonas aeruginosa-R15, Pseudomonas aeruginosa-SpA and
Acinetobacter pittii-CS1. Pahari and Mishra (2017) have reported 4 bacterial isolates,
isolated from rice rhizosphere having ability to produce ammonia. Likewise in another
study, Nehra et al. (2014) have reported Pseudomonas fluoescens sp as a strong producer
of ammonia.
145
Siderophore production by bacterial isolates negatively influence the pathogens due to the
production of antimicrobial compounds in the surrounding of plant roots (Wahyudi et al.,
2011). Phosphate solubilizing bacterial isolates were evaluated for their ability to produce
siderophores on CAS agar media. We have observed siderophore production by 21% of
isolates including Pseudomonas sp-Ros2, Pseudomonas plecoglossicida-R14,
Pseudomonas aeruginosa-R15, Pseudomonas japonica-SL8, Pseudomonas oryzihabitans-
SF and Pseudomonas aeruginosa-SpA. According to our study, isolates belonging to genus
Pseudomonas were the only producers of siderophore while rest of the bacterial isolates
were not able to produce siderophore in in vitro conditions. Acinetobacter sp M-01 has
been reported as a strong producer of siderophore (Zhang et al., 2017). According to
previous observations, it has been proved that bacterial isolates having siderophores
production ability assist plants to uptake different metals from soil (Dimpka et al., 2009;
Gururani et al., 2012).
1-Aminocyclopropane-1-carboxylic acid (ACC) is the ethylene precursor of plants and
ACC deaminase cleaves ACC and helps lower down ethylene levels. There are a variety
of bacteria having ACC deaminase production abilities which help in growth promotion of
plant tissues, delayed flower senescence as well as they assist plants in stress conditions
(Ali et al., 2012; Barnawal et al., 2012). Phosphate solubilizing bacterial isolates were
assessed for ACC deaminase activity. ACC deaminase production by isolates ranged from
66.58 µmol to 386.35 µmol. The highest ACC deaminase activity was recorded by
Pseudomonas koreensis-L20, Acinetobacter calcoaceticus-S62 and Pseudomonas
reinekei-UP as 386.35 µmol, 365.36 µmol and 361.76 µ mol α-ketobutyrate mg-1 protein
h-1, respectively. Similarly, Xu et al. (2014) have reported bacterial isolate HYT-121, a
146
plant growth promoting endophyte as a good producer of ACC deaminase enzyme showed
112.02 nmol α-ketobutyrate mg-1 protein h-1.
In a recent study, Singh et al. (2015) have reported different phosphate solubilizing
bacterial isolates. The isolated strains were identified as Pseudomonas oryzahabitans-
BHU-10, Pseudomonas aeruginosa-BHU-3 and Enterobacter asburiae-BHU-7. These
isolates were reported to have ability to produce HCN and IAA production as 9.75 µg mL-
1, 9.87 µg mL-1 and 3.17 µg mL-1 IAA, respectively. The siderophores production has been
reported by Pseudomonas oryzahabitans- BHU-10 and Pseudomonas aeruginosa-BHU-3
as 0.28 mmh-1 and 0.32 mmh-1, respectively.
Conclusion
Soil contains a large number of different bacteria, some of them are involved in the growth
promotion of different plants. Besides phosphate solubilization, there are some other
bacterial mechanisms which are involved in growth enhancement of plants either directly
or indirectly. The isolated phosphate solubilizing bacteria were found positive for different
plant growth promoting attributes in vitro. Hydrogen cyanide, ammonia and siderophore
production was observed by majority of isolates whereas, all isolates were able to produce
indole acetic acid as well as they showed good ACC deaminase activity.
147
Chapter 07
Wheat root elongation assay in the presence and absence
of pesticide stress
Bacterial isolates involved in plant growth promotion have been isolated from different
plants (Fernandes et al., 2013; Zhao et al., 2015; Afzal et al., 2017). Phosphate solubilizing
bacteria are known to promote plant growth. The bacterial diversity in plant rhizosphere
can be related to root system as well as the nature of root exudates (Rajapaksha and
Senanayake, 2011). The presence of pesticide causes toxic effects on microbial population
in soil and they also affect their characteristics. Therefore the identification of phosphate
solubilizing bacteria having plant growth promoting abilities as well as tolerance towards
pesticides can be helpful to optimize the productivity of crops in pesticide stress conditions
(Ahemad and Khan, 2011a).
In chapter 03, bacterial strains were tested for pesticide tolerance and minimum inhibitory
concentration (MIC) was calculated. For Chlorpyrifos, MIC ranged from 10-80 mg mL-1
whereas for Pyriproxyfen, it ranged from 20-80 mg mL-1 by isolated phosphate solubilizing
strains. The effect of pesticides on plant growth promotion was checked through root
elongation assay. This chapter deals with the influence of bacterial inoculation on root
elongation both in the absence and presence of pesticides. Certified wheat seeds were
surface sterilized followed by inoculation with 12 individual bacterial strains
(Ochrobactrum pseudogrignonense-S1, Acinetobacter olivorans-S2, Pseudomonas
putida-Rad1, Pseudomonas putida-Rad2, Pseudomonas sp-Ros2, Acinetobacter baumanii-
JA10, Pseudomonas plecoglossicida-R14, Pseudomonas japonica-SL8, Pseudomonas
148
aeruginosa-SpA, Enterobacter cloacae-W95, Enterobacter aerogenes-W96 and
Pseudomonas reinekei-UP). The experiment was carried out in sterile Petri dishes lined
with double layer of Whatman filter paper. For this experiment, four treatments were
followed:
1. Bacterial inoculated
2. Bacterial inoculated + Chlorpyrifos treated
3. Bacterial inoculated + Pyriproxyfen treated
4. Bacterial inoculated + Chlorpyrifos and Pyriproxyfen treated
For all treatments, a uninoculated control experiment was also performed. Pesticide
solutions were used according to the recommended concentrations. For Chlorpyrifos and
Pyriproxyfen, final concentration of 0.5 µg mL-1 and 1.3 µg mL-1 were used, respectively.
At the end of the experiment, percentage seed germination, number of roots, length of roots
and length of shoots were measured.
Seed germination
For percentage germination, it was found that in the absence of pesticide stress, the
germination rate of wheat seeds was 90% by uninoculated control. Whereas when seeds
were inoculated with strain SpA, the germination rate was significantly increased by 11%.
Similarly, for strains Rad 2 and W96 the increased germination was 7% and for strain UP,
the 4% increased germination was recorded. Strain Ros2, SL8 and W95 had similar
germination rate as that of uninoculated control. However, strains Rad1, JA10, R14, S1
and S2 showed a decrease in the percentage germination when compared to uninoculated
control. The lowest germination rate in the case of inoculated strains was recorded with
strain S1 and S2.
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For Chlorpyrifos treatment, the percentage germination of uninoculated control was
recorded to be 100%, whereas, for the majority of the bacterial inoculated seeds, the
germination rate was found to be decreased as compared to uninoculated control. Only
strain Ros2 showed 100% germination rate in the presence of Chlorpyrifos. Stains Rad2,
SL8, S2, SpA, W96 and UP showed above 90% germination rate while W95, JA10 and S1
showed less than 90% germination. Least germination was recorded for strain S1 which is
70% in the presence of Chlorpyrifos (Figure 7.1).
In the presence of Pyriproxyfen, the germination rates of strain Rad1 and Rad2 were found
similar to uninoculated control and showed 100% germination. For the rest of the strains,
the germination rate significantly decreased when compared to uninoculated control. For
strains JA10, W96 and UP, the germination rate was found above 90% while Strain R14,
SpA and W95 showed above 80% germination. However, the least germination rate was
recorded for strain S1 and S2 as they showed 83% germination in the presence of
Pyriproxyfen.
In the presence of a combination of pesticides (Chlorpyrifos and Pyriproxyfen), the results
for germination rate of strain S1, S2 and Rad1 remained similar to uninoculated control.
The rest of the bacterial treatments showed significantly decreased germination rate as
compared to control. The germination rate was dropped by 23% in the presence of a
combination of pesticides by strain SpA.
Shoot length
The bacterial strains were evaluated for their impact on shoot length in in vitro conditions
in the absence and presence of pesticides. The shoot length was measured after seven days.
150
A general decline in shoot length was observed by most of the bacterial inoculations in the
absence of pesticide stress. However, the shoot length of strain S2 remained almost similar
to that of uninoculated control as shown in figure 7.2.
In the presence of Chlorpyrifos, the shoot length of Strain S1 and S2 were found to be
significantly increased by 10% as compared to uninoculated control. Whereas reduction in
shoot length was recorded by the rest of the bacterial strains. For Pyriproxyfen treatment,
the shoot length of the majority of the bacterial treatments was found to be decreased
significantly when compared to uninoculated control. However for strain S2, significant
increase in shoot length which is 10% was recorded as compared to control. For the
combination of pesticide treated experiment, the overall reduction in shoot length was
recorded for all bacterial inoculations as compared to uninoculated control.
Root length
Inoculation with strain W96 showed similar results for root length compared to
uninoculated control in the absence of pesticides. However, the inoculation of rest of the
bacterial isolates showed a negative impact on root length as there was a significant
decrease in root length. When Chlorpyrifos was added, it was found that none of the
bacterial isolates were able to cause an increment in root length in the presence of
Chlorpyrifos. Rather the root lengths were decreased in comparison to uninoculated
control.
In the presence of Pyriproxyfen, strain S1, S2, Rad1 and Rad2 induced a significant
increase of 20, 13, 41 and 48%, respectively, in the length of roots compared to
uninoculated control. However, the other strains had a negative impact on root length in
151
comparison to control. The inoculation of strain Rad1 and Ros2 caused a slight increment
in root length in comparison to uninoculated control in the presence of a combination of
pesticides. Whereas the majority of inoculated strains lead to the reduction in root lengths
(Figure 7.3).
Number of roots
The effect of bacterial strains was also evaluated to have an impact on the number of roots
in wheat in in vitro conditions. It was found that strain S1, S2, Rad1 and Rad2 significantly
enhanced 5, 5, 20 and 23%, the number of roots, respectively, the results are represented
in figure 7.4. Whereas a minor increase occurred in the presence of strain W96 and UP as
compared to uninoculated control. On the other hand strains Ros2, JA10, R14, SL8, SpA
and W95 showed negative impact on the number of roots when compared to uninoculated
control.
The effect of bacterial inoculation in the presence of Chlorpyrifos was also evaluated to
check their impact on the number of roots in the presence of pesticide. Increased number
of roots were observed in case of strain S1, S2, Rad1 and Rad2 to 29, 6, 38 and 33%,
respectively, compared to uninoculated control. However, strain W96 showed almost
similar results to control.
The impact of Pyriproxyfen on the number of roots of inoculated plants was estimated and
it was found that strain S1, S2, Rad1 and Rad2 caused a significant increase of 18, 10, 26
and 30%, respectively, on root number compared to uninoculated control. However, the
presence of Pyriproxyfen induced almost no impact on strain W96 whereas the rest of the
isolates showed a negative impact on root number when compared to uninoculated control.
152
When the effect of a combination of pesticides (Chlorpyrifos and Pyriproxyfen) was
evaluated it was found that majority of the strains S1, S2, Rad1, Rad2, Ros2, SL8 and UP
showed an increase in number of roots by 31, 4, 35, 37, 5, 2 and 1%, respectively, compared
to uninoculated control. Strain R14 showed similar results to control while strain JA10,
SpA, W95 and W96 showed a decrease in root number by 20, 2, 1 and 1%, respectively.
153
Bacterial strains
Cont S1 S2 Rad1 Rad2
Per
cent
age
Ger
min
atio
n
0
20
40
60
80
100
120
a
ab
abc
bcdbcd bcd
cdcd cd
d dddddddddd
Bacterial strains
Cont SpA W95 W96 UP
Per
cent
age
Ger
min
atio
n
0
20
40
60
80
100
120
a
ab abab ab ab ab ab
bbbbbb
bb
bbbb
Bacterial strains
Cont Ros2 JA10 R14 SL8
Per
cent
age
Ger
min
atio
n
0
20
40
60
80
100
120
bc
d d d
bc
d
b
aab ab
cd
aba
ab
bcc
bc
cd
abb
Figure 7.1: Effect of bacterial inoculation on wheat seeds on percentage germination in
the presence of Chlorpyrifos (0.5 µg mL-1) and Pyriproxyfen (1.3 µg mL-1). Error bars
Mean ± standard error (n=3), ANOVA followed by Duncan (p<0.05).
154
= Strain, = Chlorpyrifos, = Pyriproxyfen, = Chlorpyrifos+ Pyriproxyfen
Bacterial strains
Cont S1 S2 Rad1 Rad2
Sh
oot
Len
gth
(cm
)
0
2
4
6
8
aabab ab ab ab
abc abcabcdabcd
abcd
abcdebcdef
bcdef cdefdefdef
ef eff
Bacterial strains
Cont Ros2 JA10 R14 SL8
Sh
oot
Len
gth
(cm
)
0
2
4
6
8
aaa aaa a aa
a a a a
aa
a
b
b b
c
Bacterial strains
Cont SpA W95 W96 UP
Sh
oot
Len
gth
(cm
)
0
2
4
6
8
aa aab
abc
abcdabcdabcd
abcdbcdbcd
cdcd
cdd
de
ef
f fg
g
F Figure 7.2: Effect of bacterial inoculated wheat seeds on shoot length in the presence of
Chlorpyrifos (0.5 µg mL-1) and Pyriproxyfen (1.3 µg mL-1). Error bars Mean ± standard
error (n=3), ANOVA followed by Duncan (p<0.05).
155
= Strain, = Chlorpyrifos, = Pyriproxyfen, = Chlorpyrifos+ Pyriproxyfen
Bacterial Strains
Cont S1 S2 Rad1 Rad2
Roo
t Len
gth
(cm
)
0
2
4
6
8
a
a
aa ab
abc
abc
abc abcdabcd abcdabcd
abcdabcd
bcd
bcd
bcd
cd
cd
c
Bacterial Strains
Cont Ros2 JA10 R14 SL8
Roo
t L
engt
h (c
m)
0
2
4
6
8
10
a aab abc
abcd abcdabcd
abcdeabcde abcdef
abcdef
abcdef
abcdef
abcdef
bcdefcdef
defef
ef
f
Bacterial Strains
Cont SpA W95 W96 UP
Roo
t Len
gth
(cm
)
0
2
4
6
8
aa a
a aaa
ab
bcbc
bc
bc
cbc
bc
bc
bc
bc
bc
bc
Figure 7.3: Effect of bacterial inoculated wheat seeds on root length in the presence of
Chlorpyrifos (0.5 µg mL-1) and Pyriproxyfen (1.3 µg mL-1). Error bars Mean ± standard
error (n=3), ANOVA followed by Duncan (p<0.05).
156
= Strain, = Chlorpyrifos, = Pyriproxyfen, = Chlorpyrifos+ Pyriproxyfen
Bacterial strain
Cont S1 S2 Rad1 Rad2
No.
of
Roo
ts
0
2
4
6
8
a
a aa a
abab abcabc
abcd abcd
bcdcd
bc
dd
d d
dd
d
Bacterial strain
Cont Ros2 JA10 R14 SL8
No.
of
Roo
ts
0
1
2
3
4
5
6
ghijklab
aaa
abab
abcabc
abcd
abcd
abcd
abcd
bcdbcd
bcd bcdbcd bcd cd
d
Bacterial strain
Cont SpA W95 W96 UP
No.
of
Roo
ts
0
1
2
3
4
5
6
ghijkl
a
b bb
b b
bb
bb
bb b
bb
b bbb
b
Figure 7.4: Effect of bacterial inoculated wheat seeds on number of root in the presence
of Chlorpyrifos (0.5 µg mL-1) and Pyriproxyfen (1.3 µg mL-1). Error bars Mean ± standard
error (n=3), ANOVA followed by Duncan (p<0.05).
157
= Strain, = Chlorpyrifos, = Pyriproxyfen, = Chlorpyrifos+ Pyriproxyfen
Discussion
In different agricultural soils, phosphorus is an important limiting nutrient and its
deficiency affects plant growth. Phosphate solubilizing isolates have been reported to be
used as bio-inoculants for a number of crops. The use of microbial inoculants helps to
increase the microbial population in plant rhizosphere (Rajapaksha and Senanayake, 2011).
The development and growth of plants is a changing process that favor in adapting the
environments where the plants are restricted. Plants conform their growth according to the
external and internal stimulus by the hormonal activities. Plant growth depends on the key
phytohormones which include ethylene, auxin and abscisic acid (Vanstraelen and
Benekova, 2012; Thole at al., 2014; Khan et al., 2017). Abscisic acid responds to many
stress conditions (Culter et al., 2010; Thole et al., 2014). The application of pesticides leads
to the long term persistence of these toxic compounds in the soil which ultimately affects
the microbial communities and is also affects their functionality (Eliason et al., 2004;
Ahemad and Khan, 2012). To reduce or to overcome the harmful effects of pesticides on
plants, a good alternative is to treat the seeds with the pesticide resistant strains having
plant growth promoting abilities (Wani et al., 2005; Ahemad and Khan 2012).
Inoculated seeds with twelve strains were grown in Petri dishes supplemented with the
recommended doses of pesticide solutions. The bacterial isolates were found resistant to
the applied pesticides in in vitro condition and minimum inhibitory concentrations were
predetermined as described in chapter 03. The strains were evaluated for their abilities to
enhance the growth parameters in root elongation assay in the absence and presence of
pesticides. Pesticide resistance or tolerance in bacteria is a complicated process that can be
regulated by genetic and physiological level. The organisms having tolerance to pesticides
158
may also have the degradation capabilities to these toxic compounds (Ortiz-Hernández and
Sanchez-Salinas, 2010; Ahemad and Khan 2012).
The percentage seed germination was evaluated, and it was noticed that there was an
increase in percentage seed germination in comparison to control by 11, 7, 7 and 4% when
inoculated with Pseudomonas aeruginosa-SpA, Pseudomonas putida-Rad2, Enterobacter
aerogenes-W96 and Pseudomonas reinekei-UP, respectively, in the absence of pesticide
stress. In the presence of Chlorpyrifos, the germination rate of Pseudomonas putida-Rad1,
Pseudomonas putida-Rad2 inoculated seeds remained unaffected whereas for the rest of
the inoculations the decline in germination rate was observed as compared to uninoculated
control. Similarly, in the presence of Pyriproxyfen, the germination rate of Pseudomonas
putida-Rad1, Pseudomonas putida-Rad2 inoculated seeds remained unaffected whereas
germination decreases were observed for the rest of the isolates. Toxicity level of pesticide
varies from organism to organism, depending upon the functional group of pesticide
(Ahemad and Khan, 2011b). For the combination of pesticide (Chlorpyrifos and
Pyriproxyfen), the germination rate of seeds inoculated with Ochrobactrum
pseudogrignonense-S1, Acinetobacter olivorans-S2 and Pseudomonas putida-Rad1
remained 100%. From the results of seed germination, it was observed that with the
inoculation of Pseudomonas putida-Rad1, the results remained same both in the absence
as well as in the presence of pesticide alone and their combination.
In general, a decline in shoot length was observed by the majority of the inoculated strains
in the absence of pesticide except strain Acinetobacter olivorans-S2 treated seeds. Patel et
al. (2012) have reported the enhanced shoot and root growth by Pseudomonas and Bacillus
species in wheat plant.
159
A significantly increased shoot length was recorded by strain S1 and S2 in the presence of
Chlorpyrifos when compared to uninoculated control, while the decline in shoot length was
recorded in case of other bacterial inoculations. Ahemad and Khan (2012) have reported
the decline in plant growth promoting abilities by Mesorhizobium (MRC4) under the effect
of pesticide.
Pyriproxyfen treatment also negatively affected the shoot length of bacterial inoculations,
however, increase in shoot length was observed for Acinetobacter olivorans-S2 by 10%
when compared to uninoculated control. From many of the possible reasons of increased
or decreased percentage can be the relationship between plant and bacteria which differs
with the difference in genetic makeup. A recent study have shown that phosphate
solubilizing bacteria enhance the plant growth by direct and indirect mechanisms of plant
growth enhancement (Chauhan et al., 2013; Afzal et al., 2017).
Overall reduction was observed in root length in inoculated plants in the absence of
pesticide stress, however; Enterobacter aerogenes-W96 inoculation showed no increase or
decrease in the root length as compared to uninoculated control. Patel et al. (2012) have
reported improved root length by the bacterial isolates belonging to genus Pseudomonas.
According to a recent study, phosphate solubilizing Pseudomonas strain (B10) cause some
increment in root length when used as a bio-inoculant (Li et al., 2017). The presence of
Chlorpyrifos also negatively affected the root length of all the bacterial inoculations.
Bacterial inoculations by Ochrobactrum pseudogrignonense-S1, Acinetobacter olivorans-
S2, Pseudomonas putida-Rad1 and Pseudomonas putida-Rad2 caused a significant
increase by 20, 13, 41 and 48%, respectively, in root length in the presence of Pyriproxyfen
160
(Figure 7.5). Ahemad and Khan (2012) have also reported the production of phytohormone
in the presence and absence of pesticide by the isolated bacteria.
However, the combination of both pesticide caused reduction in root length in the majority
of the inoculations whereas Pseudomonas putida-Rad1 and Pseudomonas sp-Ros2 showed
a slight increase (1-2%) in root lengths. The functionality of the organism decrease with
the increased concentration of pesticides (Kumar et al., 2010; Ahemad and Khan, 2012b).
Auxin is involved in the regulation of cell division and elongation to control each aspect
of growth in plants including elongation of roots (Thole et al., 2014; Perrot-Rechenmann,
2010).
The effect of bacterial inoculation on number of wheat root in plate assay was also
estimated and from the results, it was noticed that Ochrobactrum pseudogrignonense-S1,
Acinetobacter olivorans-S2, Pseudomonas putida-Rad1 and Pseudomonas putida-Rad2
inoculations augmented the root number and the recorded increase was 5, 5, 20 and 23%,
respectively. A number of bacterial species belonging to genera Pseudomonas, Serratia,
Bacillus, Burkholderia, Arthrobacter, Alcaligenes, Enterobacter, Klebsiella, Azotobacter
and Azospirillum have been described to have involvement in plant growth promotion of
different plants (Ji et al., 2014; Afzal et al., 2017). A slight increase in root number was
observed by Enterobacter aerogenes-W96 and Pseudomonas reinekei-UP in comparison
to uninoculated control. Reduced root number was recorded in case of Pseudomonas sp-
Ros2, Acinetobacter baumanii- JA10, Pseudomonas plecoglossicida-R14, Pseudomonas
japonica-SL8, Pseudomonas aeruginosa-SpA, Enterobacter cloacae-W95 inoculations.
161
Figure 7.5: Effect of Pseudomonas putida-Rad2 inoculation on root length in the presence
of Pyriproxyfen (1.3 µg mL-1) on wheat compared to uninoculated control in gnotobiotic
root elongation assay.
Control Rad2
162
The increment in root number was recorded with Ochrobactrum pseudogrignonense-S1,
Acinetobacter olivorans-S2, Pseudomonas putida-Rad1 and Pseudomonas putida-Rad2
inoculations in wheat. In the presence of Chlorpyrifos, the root number increased by 29, 6,
38 and 33%, respectively. However, the increase in the presence of Pyriproxyfen was 18,
10, 26 and 30%, respectively. For the combination of pesticide a significantly enhanced
root number was recorded with Ochrobactrum pseudogrignonense-S1, Acinetobacter
olivorans-S2, Pseudomonas putida-Rad1, Pseudomonas putida-Rad2, Pseudomonas sp-
Ros2, Pseudomonas japonica-SL8 and Pseudomonas reinekei-UP as compared to
uninoculated control. The reduction in the root number was noticed in the rest of the strains
compared to uninoculated control. From the previous experiments, it was found that
Acinetobacter olivorans-S2, Pseudomonas putida-Rad1 and Pseudomonas putida-Rad2
was found positive for Hydrogen cyanide as well as for ammonia production. These
characteristics have been reported to be linked with the nitrogen accumulation and root
elongation (Marques et al., 2010).
Conclusion
In conclusion, the presence of pesticides caused a significant reduction in plant growth in
in vitro conditions in root elongation assay. The bacterial abilities for plant growth
promotion vary from strain to strain and in the presence of pesticide stress. A significant
increase in root number was found upon inoculation with phosphate solubilizing bacteria
in the presence as well as in the absence of pesticides.
163
Chapter 08
Impact of phosphate solubilizing bacteria and inorganic
phosphate on wheat (Triticum aestivum) under pesticide
stress
Increasing population, demands increased amount of food production. The urbanization
has limited the land for agricultural use (Hamuda and Patko, 2013; Namli et al., 2017). Due
to this reason, the production of damage free food with good quality is of great concern.
Chemical fertilizers are used increasingly but their increased use is raising so many
concerns. Phosphorous is the second most needed macronutrient by plants. It is involved
in different metabolic activities in plants. The phosphorous deficiency occurs due to its
fixation in soil (Khan et al., 2014; Namli et al., 2017). A good alternative to chemical
fertilizers is the use of plant growth promoting bacteria. To fulfil the needs of deficient
phosphorous in agricultural land, phosphate solubilizing bacteria can be used as an
alternate (Hamuda and Patko, 2013; Namli et al., 2017). The phosphate solubilizing
bacteria have the ability to solubilize the fixed or insoluble form of phosphorous in soil
from different bound forms (aluminium phosphate, ferric phosphate, and tricalcium
phosphate) (Sharma et al., 2013).
Phosphate solubilizing bacteria got much importance because they also have other plant
growth promoting traits as described in chapter 5, 6 and 7. Isolated bacterial strains were
tested for their tolerance towards two tested pesticides in in vitro conditions (Chapter 05).
This chapter deals with the effect of phosphate solubilizing bacteria on plant growth and
164
biochemical activities of plants in the presence of three different inorganic phosphate
sources in the absence as well as in the presence of pesticide stress.
Certified wheat seeds were surface sterilized followed by inoculation with 12 individual
bacterial strains (Ochrobactrum pseudogrignonense-S1, Acinetobacter olivorans-S2,
Pseudomonas putida-Rad1, Pseudomonas putida-Rad2, Pseudomonas sp-Ros2,
Acinetobacter baumanii- JA10, Pseudomonas plecoglossicida-R14, Pseudomonas
japonica-SL8, Pseudomonas aeruginosa-SpA, Enterobacter cloacae-W95, Enterobacter
aerogenes-W96 and Pseudomonas reinekei-UP). In this experiment, the recommended
quantities of Chlorpyrifos and Pyriproxyfen were used by mixing them with soil in each
pot. Chlorpyrifos was used at the concentration of 0.5 µg mL-1 and Pyriproxyfen was used
at the concentration of 1.3 µg mL-1, respectively. Inorganic phosphate sources (aluminium
phosphate, ferric phosphate and tricalcium phosphate) were mixed with soil at the
concentration of 8 mg kg-1. For bacterial inoculation, fresh bacterial suspension of
phosphate solubilizing bacteria was added to soil. The experiment was carried out in
earthen pots containing 8 kg of garden soil.
For this experiment, the following treatments were followed:
1. Bacteria inoculated
2. Bacteria inoculated + Pesticide stress
3. Bacteria inoculated + Aluminium phosphate (ALP)
4. Bacteria inoculated + Aluminium phosphate (ALP) + Pesticide stress
5. Bacteria inoculated + Ferric phosphate (FP)
6. Bacteria inoculated + Ferric phosphate (FP) + Pesticide stress
7. Bacteria inoculated + Tricalcium phosphate (TCP)
165
8. Bacteria inoculated + Tricalcium phosphate (TCP) + Pesticide stress
The experiment was carried out under greenhouse conditions. Initially 8 seeds were sown
in each pot. After germination, thinning was performed and number of plants was
maintained to 5 plants per pot. Plants were watered regularly according to their need. After
two months of growth, plant leaf material was collected and different biochemical tests
were performed. The wheat plants were grown till maturity and at the completion of the
experiment, plant growth promoting parameters including shoot length, spike length, spike
weight, number of spike per plant, number of spikelets, number of tillers, the weight of
shoot and grains were calculated. The impact of bacterial inoculation, inorganic phosphate
sources and pesticide stress was also evaluated for different plant biochemical parameters
including acid phosphatase production, chlorophyll content, peroxidase test, proline
content and soluble protein content estimation. The results were analyzed by using 2-way
ANOVA analysis and interaction significance (p<0.05) was determined using SPSS
software.
Plant growth parameters of wheat plant
Shoot Length
The bacterial inoculation impact on shoot length was evaluated and was compared with
uninoculated control. A significant increase in shoot length was observed by strain S1
inoculation which resulted in 13% increase in shoot length as compared to uninoculated
control. The non-significant increase of 2.5, 5.8, 2, 1.3, 2.6 and 2.7% was observed by
strain S2, Ros2, R14, SL8, SpA and W96, respectively (Table 8.1). However, strain Rad1
and Rad2 showed similar results to uninoculated control while a decrease in shoot length
166
was observed by strain JA10, W95 and UP when compared to uninoculated control. When
the soil was augmented with aluminium phosphate as inorganic phosphate source, strain
S2 produced a significant increase (12%) in shoot length compared to uninoculated control.
When plants were grown in ferric phosphate augmented soil, marked increase of 14% in
plant shoot length was recorded by strain S1 as compared to uninoculated control. The
maximum increase of 18% was observed when soil was augmented with tricalcium
phosphate as the inorganic phosphate source.
In pesticide stress conditions, among the inoculated strains, strain S1 showed a maximum
increase of 18.1% in shoot length. In case of aluminium phosphate amendment in the soil,
the highest significant increase was recorded for strain S2 and Rad2 (12 and 10%,
respectively). Among rest of the inoculated strains S1, R14, SpA, and W95 also enhanced
shoot length of plants as compared to control but the increase was non-significant. The
bacterial inoculation with strain S1 in ferric phosphate treated soil resulted in 19%
increament in shoot length in the presence of pesticide stress conditions as compared to
uninoculated control. The highest significant increase in shoot length among different
inorganic phosphate sources in stressed condition by bacterial inoculations was recorded
with strain S1 and UP by 24 and 16%, respectively, (Table 8.1).
Shoot dry weight
The effect of bacterial inoculation on shoot dry weight in the natural soil as well as in the
presence of inorganic phosphate source and pesticide stress was also evaluated. From the
experiment data, it was found that in natural soil conditions all of the bacterial inoculations
augmented in the weight of shoot (16 to 54%) as shown in table 8.1. In the presence of
aluminium phosphate, all strains caused an increment in shoot dry weight except strain
167
Ros2, which caused a decrease in weight of shoot as compared to uninoculated control
plant.
In the presence of ferric phosphate, a significant increase in shoot dry weight was observed
by strain S1, Rad1, Ros2, SpA, W96 and UP. Whereas, in the presence of tricalcium
phosphate, strain Rad1, Rad2, Ros2, R14 and W96 showed increased shoot dry weight
compared to uninoculated control plants. In pesticide-treated soil, strain S1, Rad2, Ros2,
R14, SL8 and W95 exhibited an increase in shoot dry weight, while when aluminium
phosphate was added to soil, all strains showed remarkable increase except strain S1 which
showed a non-significant increase as compared to uninoculated control plants. When ferric
phosphate and tricalcium phosphate sources were added to soil, all strains showed
remarkably increased shoot dry weight of inoculated plants compared to uninoculated
control (Table 8.1).
Spike Length
The effect of bacterial inoculation was also observed on spike length of inoculated plants
as compared to uninoculated plant in the absence and presence of inorganic phosphate
sources and pesticide stress. Without addition of phosphate to soil, it was found that strain
Rad2, JA10 and R14 showed significantly increased spike length (10, 15 and 21.6%) as
compared to uninoculated control. The addition of aluminium phosphate as inorganic
phosphate have led to increased length of spikes by 11 to 20% by strain R14, SL8, SpA,
W95, W96 and UP. When ferric phosphate was added to soil, a significant increase in spike
length was observed by strain UP only while the majority of the inoculations, a significant
decrease in spike length was observed. The significant increase in spike length was
168
observed by strain Rad2 and R14 in the presence of tricalcium phosphate as compared to
uninoculated control as shown in table 8.1.
The remarkable increase has been shown by the majority of the bacterial inoculations in
the presence of pesticide stress, the recorded substantial increase in spike length was 10-
92% with strains S1, Rad1, Rad2, Ros2, R14, SL8, SpA, W95, W96 and UP. Whereas,
when aluminium phosphate was added along with pesticide stress, strain R14, SL8, SpA,
W95, W96 and UP augmented spike length from 10 to 19%. All strains showed increment
in spike length as compared to uninoculated control when ferric phosphate was added along
with pesticide stress. While when tricalcium phosphate was added along with pesticide
stress, only three strains showed the increased length of the spike as compared to
uninoculated control (Table 8.1).
Spike weight
The effect of bacterial inoculation, addition of inorganic phosphate source and pesticides
were checked on spike weight. From the results, it was found that in the absence of
pesticide stress and inorganic phosphate source, phosphate solubilizing bacterial isolates
remarkably enhanced the spike weight in case of the majority of strains. Strain S1, S2,
Rad1, Rad2, Ros2, JA10, R14, SL8, SpA, W95, W96 and UP showed increased spike
weight (30 to 96%) as compared to uninoculated control. When aluminium phosphate was
added to soil, the bacterial inoculations significantly enhanced the spike weight. Strain R14
showed 100% increase in spike weight when compared to uninoculated control. The rest
of the strains also showed an increase in spike weight (21-77%) as shown in table 8.1. The
addition of ferric phosphate in soil also resulted in enhancement of spike weight. Strain
R14 caused marked decrease (28%) in spike weight as compared to uninoculated control.
169
However, strain JA10 and SpA had a negligible effect on spike weight. When tricalcium
phosphate was added to soil, it was found that majority of the strains significantly enhanced
the spike weight (29 to 58%) while the inoculation with strain UP resulted in decreased
spike weight (2%) as compared to uninoculated control (Table 8.1).
In the presence of pesticide stress, the inoculation of phosphate solubilizing bacteria
showed improved spike weight for strain Rad1, Ros2, JA10, R14, SL8, W95 and W96 by
26, 20, 13, 16, 31, 28 and 19%, respectively. Strain Rad2 and UP caused a negligible effect
on spike weight while strain S1, S2 and SpA caused significant decrease in spike weight
when compared to uninoculated control. The addition of aluminium phosphate as an
inorganic phosphate in stressed condition generally showed a decline in spike weight by
strain Rad1, Rad2 Ros2, JA10, R14, SL8, W95 and UP. The strains S1, S2, SpA and W96
showed a negligible increase in spike weight as compared to uninoculated control as shown
in table 8.1. Increased spike weight was noted for all the bacterial inoculations (>24%)
when ferric phosphate was added along with pesticide stress. In the presence of pesticide
stress and tricalcium phosphate, strain S1, Rad1, JA10, and R14 showed no difference in
spike weight when compared to uninoculated control. Whereas, Strain S2, Rad2, Ros2,
SL8, W95, W96 and UP showed a marked increase in spike weight. While strain SpA
caused 13% decrease in spike weight (Table 8.1).
Number of spikes per plant
The impact of bacterial inoculation and addition of inorganic phosphate source and
pesticide stress was also checked on the number of spikes per wheat plant. It was observed
that in the absence of phosphate source and stress, a negative impact on spike number per
plant was observed as compared to an uninoculated control plant (Table 8.1). The addition
170
of aluminium phosphate caused an increment in number of spikes per plant as compared
to control. Inoculation of S2, Rad1, R14, SpA W96 and UP resulted in increased number
of spikes per plant (Table 8.1). The application of ferric and tricalcium phosphate to soil
did not enhance the number of spikes per plant compared to an uninoculated control plant.
In stressed condition, spike number per plant was increased in all conditions by almost all
strains except in the presence of ferric phosphate where only strain UP showed a significant
increase in spike number per plant.
Number of spikelets per spike
The impact of phosphate solubilizing bacteria on the number of spikelets per spike was
estimated and it was found that strain R14, SL8, SpA, W95, W96 and UP caused a
significant increase in the number of spikelets per spike by 12.6, 13, 14, 14.7, 10 and
15.7%, respectively, in comparison to uninoculated control. When the soil was
supplemented with aluminium phosphate (inorganic phosphate source), significant
enhancement in the number of spikelets was recorded for strain S1, S2, Rad1, JA10, R14,
SL8 and W96 (12, 14, 18, 10, 17, 13.8 and 14%, respectively). The addition of ferric
phosphate as inorganic phosphate source to soil imposed a negative impact on the number
of spikelets in spike when inoculated with isolated bacterial strains as compared to
uninoculated control. All inoculations caused decline in number of spikelets. For tricalcium
phosphate, significant improvement in the number of spikelets was recorded with strains
S1, Rad2, Ros2 and R14 which ranged from 10 to 15%.
17
1
Ta
ble 8
.1: E
ffect of p
ho
sph
ate solu
bilizin
g b
acteria on
plan
t gro
wth
param
eters of b
acterial ino
culated
wh
eat plan
ts with
differen
t ino
rgan
ic ph
osp
hate so
urces an
d p
esticide stress. T
he d
ata sho
wn
represen
ts Mean
(n=
3) an
d ±
stand
ard d
eviatio
n. T
he
interactio
n sig
nifican
ce betw
een d
ifferent treatm
ents w
as jud
ged
by 2
-way A
NO
VA
follo
wed
by D
un
cans’s an
alysis at th
e level
of 9
5%
sign
ificance.
Trea
tmen
t
Sh
oot L
ength
(cm)
Sh
oot d
ry
weig
ht (g
)
No. o
f
tillers
Sp
ike len
gth
(cm)
Sp
ike w
eigh
t
(g)
No. o
f spik
es
per p
lan
t
No. o
f
spik
elet
Weig
ht o
f
100 g
rain
s
(g)
Non
stressed
con
trol
P0
71.9
9 ±
3.5
8
8.7
2 ±
0.3
4
1.8
0 ±
0.0
1
9.0
6 ±
0.5
8
1.2
1 ±
0.2
4
1.4
0 ±
0.2
0
27.0
6 ±
1.2
9
4.8
5 ±
0.0
0
AL
P
70.2
4 ±
4.4
7
8.3
6 ±
0.0
7
0.7
3 ±
0.0
4
8.7
4 ±
0.8
7
1.3
4 ±
0.5
0
1.4
0 ±
0.4
0
27.2
2 ±
0.4
9
4.8
5 ±
0.0
0
FP
70.4
5 ±
8.7
6
9.1
7 ±
0.0
4
1.4
6 ±
0.1
3
9.0
6 ±
0.3
4
1.5
1 ±
0.4
4
1.4
6 ±
0.5
0
32.3
0 ±
3.9
7
2.9
3 ±
0.0
2
TC
P
69.1
3 ±
2.1
3
11.0
5 ±
0.0
4
1.0
6 ±
0.0
9
9.2
0 ±
0.8
7
1.4
4 ±
0.4
6
1.6
6 ±
0.6
1
27.7
1 ±
2.8
1
3.2
3 ±
0.0
1
Pesticid
e
stressed
con
trol
P0
63.4
3 ±
3.7
8
7.2
8 ±
0.0
3
1.3
3 ±
0.0
0
8.3
6 ±
0.9
4
1.7
2 ±
0.2
2
1.0
0 ±
0.2
0
30.1
9 ±
3.7
0
3.1
6 ±
0.0
1
AL
P
70.2
4 ±
4.4
7
5.7
4 ±
0.0
5
0.7
3 ±
0.0
5
8.7
4 ±
0.8
7
2.1
1 ±
0.2
5
1.4
0 ±
0.4
0
29.5
0 ±
2.5
4
3.5
5 ±
0.0
1
FP
71.1
2 ±
7.2
8
6.4
2 ±
0.1
0
1.4
3 ±
0.0
2
7.8
2 ±
0.7
7
1.5
8 ±
0.0
6
1.4
6 ±
0.3
0
28.4
6 ±
1.0
5
2.8
5 ±
0.0
1
TC
P
64.7
6 ±
3.7
1
7.0
8 ±
0.0
1
1.3
3 ±
0.0
3
8.5
8 ±
1.0
6
1.4
7 ±
0.4
0
1.0
6 ±
0.1
1
27.1
0 ±
1.9
1
2.5
5 ±
0.0
0
Non
stressed S
1
P0
81.2
6 ±
6.4
1
11.8
6 ±
0.1
5
1.4
0 ±
0.3
1
9.4
5 ±
0.5
6
2.1
5 ±
0.5
7
1.4
0 ±
0.5
2
27.2
2 ±
0.4
4
5.0
7 ±
0.0
2
AL
P
72.2
4 ±
4.5
0
10.0
4 ±
0.0
5
0.4
0 ±
0.0
0
8.1
1 ±
1.1
4
1.7
4 ±
0.7
1
1.0
0 ±
0.0
0
30.4
2 ±
9.2
6
4.9
8 ±
0.0
2
FP
80.3
6 ±
5.4
3
10.5
4 ±
0.0
8
0.6
6 ±
0.0
0
8.5
0 ±
0.4
3
2.0
5 ±
0.2
9
1.0
6 ±
0.1
1
26.6
7 ±
1.6
4
3.1
7 ±
0.0
2
TC
P
81.5
6 ±
4.5
9
11.2
4 ±
0.0
7
0.7
3 ±
0.0
5
9.6
7 ±
0.3
8
2.0
2 ±
0.2
5
1.2
0 ±
0.2
0
30.5
6 ±
3.2
8
3.3
5 ±
0.0
2
Pesticid
e
stressed S
1
P0
74.9
0 ±
4.3
5
11.0
5 ±
0.0
4
1.2
0 ±
0.6
0
9.1
2 ±
0.2
3
1.6
5 ±
0.1
1
1.4
6 ±
0.2
3
26.4
9 ±
3.9
0
3.1
8 ±
0.0
3
AL
P
72.2
4 ±
4.5
0
5.8
6 ±
0.0
4
0.4
0 ±
0.0
0
8.1
1 ±
1.1
4
2.2
6 ±
0.2
4
1.0
0 ±
0.0
0
31.6
5 ±
3.6
2
3.1
5 ±
0.0
2
FP
84.6
1 ±
2.6
0
11.9
3 ±
0.0
6
1.1
3 ±
0.0
9
9.9
8 ±
0.6
8
2.2
7 ±
0.2
6
1.6
0 ±
0.7
2
36.5
3 ±
2.8
6
3.4
7 ±
0.0
2
TC
P
79.8
8 ±
2.9
3
10.9
4 ±
0.0
5
1.2
0 ±
0.2
0
8.0
0 ±
0.6
1
1.5
2 ±
0.1
2
1.0
6 ±
0.1
1
25.9
7 ±
1.7
1
3.0
6 ±
0.0
1
P0
83.2
6 ±
2.3
9
10.1
1 ±
0.1
0
0.7
3 ±
0.0
4
9.4
0 ±
0.1
8
2.3
7 ±
0.8
0
1.0
6 ±
0.1
1
28.3
6 ±
2.9
6
5.7
7 ±
0.0
2
17
2
Non
stressed S
2
AL
P
80.5
7 ±
1.8
3
10.8
0 ±
0.1
7
1.0
8 ±
0.0
1
9.0
9 ±
1.4
0
1.6
3 ±
0.5
0
1.5
5 ±
0.6
3
30.9
9 ±
5.1
4
4.9
9 ±
0.0
4
FP
79.5
2 ±
5.2
1
6.5
3 ±
0.0
1
0.6
0 ±
0.0
0
8.6
4 ±
1.5
4
1.7
8 ±
0.4
8
1.1
3 ±
0.2
3
25.3
7 ±
2.6
6
2.9
5 ±
0.0
8
TC
P
78.9
5 ±
7.6
1
10.0
1 ±
0.0
8
0.4
0 ±
0.0
2
9.3
4 ±
0.5
6
1.8
6 ±
0.1
2
1.2
6 ±
0.1
1
29.3
3 ±
3.1
9
3.1
7 ±
0.0
2
Pesticid
e
stressed S
2
P0
71.4
0 ±
3.8
6
7.0
4 ±
0.0
6
0.4
6 ±
0.0
6
8.9
0 ±
0.2
6
1.6
4 ±
0.2
3
1.2
0 ±
0.2
0
27.7
8 ±
3.9
1
3.3
9 ±
0.0
3
AL
P
80.5
7 ±
1.8
3
6.4
6 ±
0.0
4
1.0
8 ±
0.1
2
9.0
9 ±
1.4
0
2.2
1 ±
0.3
7
1.5
5 ±
0.6
3
31.3
3 ±
2.2
0
3.2
6 ±
0.0
4
FP
84.6
6 ±
6.0
7
10.2
9 ±
0.0
4
1.2
0 ±
0.0
2
9.2
7 ±
0.5
3
1.9
9 ±
0.4
6
1.3
3 ±
0.1
1
32.7
3 ±
7.7
5
2.9
8 ±
0.0
3
TC
P
83.0
0 ±
2.0
7
10.8
9 ±
0.1
6
1.2
0 ±
0.2
0
8.4
6 ±
0.6
7
1.7
9 ±
0.0
7
1.0
0 ±
0.0
0
25.8
7 ±
1.8
8
3.0
7 ±
0.0
2
Non
stressed
Rad
1
P0
83.1
1 ±
6.3
0
11.8
5 ±
0.0
8
1.4
0 ±
0.0
3
9.4
4 ±
0.3
1
1.3
5 ±
0.7
6
1.4
0 ±
0.4
0
24.9
1 ±
3.1
8
5.4
7 ±
0.0
2
AL
P
76.5
3 ±
4.7
2
9.7
1 ±
0 .0
3
0.6
6 ±
0.0
2
8.2
4 ±
0.9
0
1.7
7 ±
0.5
2
1.1
3 ±
0.1
1
32.1
8 ±
6.5
1
4.7
3 ±
0.1
0
FP
83.1
2 ±
3.9
2
11.5
3 ±
0.2
0
0.8
6 ±
0.1
0
9.1
5 ±
0.0
6
2.1
3 ±
0.1
5
1.0
6 ±
0.3
0
28.2
8 ±
1.1
1
3.2
5 ±
0.0
3
TC
P
82.3
6 ±
6.9
0
12.0
6 ±
0.0
6
0.2
0 ±
0.0
2
9.6
3 ±
0.9
5
2.1
9 ±
0.3
4
1.0
6 ±
0.1
1
28.6
6 ±
3.3
8
3.4
8 ±
0.0
3
Pesticid
e
stressed
Rad
1
P0
71.8
3 ±
6.2
1
7.0
5 ±
0.0
4
1.9
3 ±
0.3
0
9.1
3 ±
0.3
1
2.1
7 ±
0.3
7
1.2
6 ±
0.3
0
42.6
8 ±
5.4
0
3.0
1 ±
0.0
7
AL
P
76.5
3 ±
4.7
2
10.2
2 ±
0.1
1
0.6
6 ±
0.0
0
8.2
4 ±
0.9
0
1.8
9 ±
0.3
7
1.1
3 ±
0.1
1
30.4
8 ±
5.8
5
5.1
5 ±
0.0
0
FP
85.6
4 ±
4.0
2
10.7
4 ±
0.2
2
1.8
0 ±
0.0
1
9.4
4 ±
1.3
6
1.9
4 ±
0.1
3
1.3
3 ±
0.5
7
33.6
5 ±
2.7
9
2.9
4 ±
0.0
8
TC
P
84.2
6 ±
1.6
4
11.7
5 ±
0.2
9
1.4
0 ±
0.6
9
8.2
6 ±
1.1
8
1.6
2 ±
0.3
8
1.1
3 ±
0.2
3
26.9
9 ±
4.1
0
2.5
6 ±
0.0
1
Non
stressed
Rad
2
P0
83.2
5 ±
5.3
5
12.3
7 ±
0.1
5
1.4
6 ±
0.0
4
9.9
4 ±
0.7
2
2.2
0 ±
0.9
8
1.2
6 ±
0.1
1
28.8
8 ±
2.5
1
4.9
5 ±
0.0
9
AL
P
83.9
1 ±
2.6
7
12.0
0 ±
0.1
9
0.4
0 ±
0.0
4
8.0
6 ±
0.3
2
1.7
9 ±
0.0
7
1.2
0 ±
0.2
0
25.1
1 ±
0.5
9
4.6
7 ±
0.0
2
FP
80.7
6 ±
4.0
5
9.3
00 ±
0.0
1
0.1
3 ±
0.0
1
8.1
3 ±
0.5
7
1.7
0 ±
0.2
3
1.0
0 ±
0.0
0
24.7
2 ±
2.7
9
2.7
4 ±
0.0
8
TC
P
79.3
6 ±
1.2
8
14.3
7 ±
0.1
0
1.2
6 ±
0.0
0
10.1
2 ±
0.6
9
1.9
3 ±
0.2
4
1.7
3 ±
0.4
6
30.7
3 ±
1.6
0
3.2
9 ±
0.0
4
Pesticid
e
stressed
Rad
2
P0
72.1
3 ±
4.2
1
8.1
1 ±
0.0
9
2.3
3 ±
0.0
0
10.4
2 ±
0.2
8
1.8
8 ±
0.2
9
1.4
6 ±
0.2
3
39.9
0 ±
4.3
8
2.6
7 ±
0.0
2
AL
P
83.9
1 ±
2.6
7
7.4
5 ±
0.0
5
0.4
0 ±
0.0
3
8.0
6 ±
0.3
2
1.8
9 ±
0.0
3
1.2
0 ±
0.2
0
28.8
± 1
.83
3.0
1 ±
0.0
7
FP
83.8
6 ±
5.5
0
9.2
9 ±
0.0
4
0.6
6 ±
0.0
5
9.5
6 ±
1.4
6
2.7
3 ±
1.0
4
1.4
0 ±
0.5
2
33.2
7 ±
0.7
6
2.5
7 ±
0.0
2
TC
P
86.3
2 ±
0.5
6
9.6
2 ±
0.0
5
1.4
0 ±
0.5
2
8.5
7 ±
1.2
0
1.9
3 ±
0.2
6
1.1
3 ±
0.2
3
29.5
5 ±
2.4
4
2.5
7 ±
0.0
2
P0
88.0
7 ±
8.6
1
10.6
5 ±
0.0
8
1.2
0 ±
0.0
1
9.7
4 ±
1.7
8
2.0
4 ±
0.4
7
1.4
0 ±
0.5
2
27.3
8 ±
2.2
3
5.1
8 ±
0.0
3
17
3
Non
stressed
Ros2
AL
P
83.0
4 ±
5.7
9
8.0
4 ±
0.0
6
0.7
3 ±
0.0
4
9.4
2 ±
0.9
9
2.0
0 ±
0.3
9
1.2
6 ±
0.4
6
29.6
7 ±
2.2
1
5.0
9 ±
0.0
4
FP
81.3
1 ±
2.7
2
10.9
5 ±
0.0
7
0.3
3 ±
0.0
4
8.3
8 ±
0.6
7
1.9
1 ±
0.1
9
1.0
0 ±
0.0
0
25.9
0 ±
2.3
5
3.2
7 ±
0.0
2
TC
P
80.0
0 ±
1.3
8
13.7
7 ±
0.2
9
0.8
0 ±
0.0
9
9.8
7 ±
1.3
4
2.0
1 ±
0.2
1
1.3
3 ±
0.4
1
31.9
1 ±
3.6
4
3.2
5 ±
0.0
0
Pesticid
e
stressed
Ros2
P0
67.9
6 ±
8.1
9
10.1
1 ±
0.0
9
0.9
3 ±
0.0
1
9.6
6 ±
0.8
1
2.0
6 ±
0.1
9
1.0
6 ±
0.1
1
34.8
2 ±
4.2
2
3.1
9 ±
0.0
4
AL
P
83.0
4 ±
5.7
9
9.2
5 ±
0.0
7
0.7
3 ±
0.0
9
9.4
2 ±
0.9
9
1.8
7 ±
0.3
9
1.2
6 ±
0.4
6
41.4
5 ±
2.1
7
3.1
2 ±
0.0
8
FP
86.2
6 ±
2.0
4
10.2
1 ±
0.0
7
0.9
9 ±
0.0
0
9.0
3 ±
0.7
5
2.1
5 ±
0.3
1
1.0
0 ±
0.0
0
30.4
3 ±
3.3
3
2.9
0 ±
0.0
5
TC
P
87.7
6 ±
1.0
6
13.3
6 ±
0.1
1
1.6
6 ±
0.4
1
9.2
9 ±
0.7
1
1.8
3 ±
0.3
1
1.1
3 ±
0.2
3
30.3
3 ±
2.1
2
3.1
2 ±
0.0
7
Non
stressed
JA
10
P0
83.8
6 ±
5.7
6
12.5
0 ±
0.1
2
1.6
6 ±
0.8
5
10.4
0 ±
0.2
0
1.8
1 ±
0.5
2
1.4
6 ±
0.1
1
28.3
1 ±
0.8
8
4.9
8 ±
0.0
2
AL
P
83.0
6 ±
0.8
4
12.7
3 ±
0.2
3
1.4
6 ±
0.0
4
9.0
1 ±
0.9
1
1.6
9 ±
0.1
3
1.8
6 ±
0.6
1
29.9
7 ±
2.4
1
4.8
8 ±
0.0
3
FP
77.0
9 ±
1.9
9
9.5
9 ±
0.0
5
0.2
6 ±
0.0
3
7.8
0 ±
0.2
5
1.5
6 ±
0.1
0
1.2
0 ±
0.3
4
24.8
3 ±
2.4
1
2.9
7 ±
0.0
2
TC
P
80.2
6 ±
3.8
0
11.1
9 ±
0.0
9
0.1
3 ±
0.0
1
8.8
0 ±
0.5
5
1.7
9 ±
0.0
6
1.0
6 ±
0.1
1
26.5
2 ±
3.0
1
3.2
9 ±
0.0
3
Pesticid
e
stressed
JA
10
P0
74.3
3 ±
5.4
3
7.8
3 ±
0.0
6
0.5
3 ±
0.0
5
8.5
5 ±
0.5
7
1.9
4 ±
0.6
5
1.0
0 ±
0.0
0
35.5
8 ±
4.3
2
3.2
8 ±
0.0
2
AL
P
83.0
6 ±
0.8
4
8.1
4 ±
0.1
4
1.4
6 ±
0.4
1
9.0
1 ±
0.9
1
1.4
2 ±
0.3
4
1.8
6 ±
0.6
1
33.4
4 ±
1.3
1
2.8
1 ±
0.0
3
FP
81.4
0 ±
2.5
0
10.1
6 ±
0.0
7
0.6
0 ±
0.1
1
8.5
3 ±
0.4
0
2.1
3 ±
0.1
6
1.0
0 ±
0.0
0
32.1
2 ±
1.0
8
3.2
6 ±
0.0
2
TC
P
85.2
2 ±
3.8
4
10.3
5 ±
0.1
4
1.2
6 ±
0.1
1
8.2
2 ±
0.3
4
1.5
3 ±
0.4
6
1.0
0 ±
0.0
0
29.1
4 ±
1.9
4
3.1
8 ±
0.0
3
Non
stressed
R14
P0
85.5
4 ±
7.1
7
13.4
6 ±
0.3
7
2.3
3 ±
0.5
0
11.0
2 ±
0.7
9
1.5
5 ±
0.4
6
1.8
6 ±
0.5
0
30.4
8 ±
1.1
1
3.8
7 ±
0.0
2
AL
P
88.3
5 ±
0.5
1
12.1
6 ±
0.1
3
1.2
0 ±
0.1
0
10.3
6 ±
0.5
5
2.7
1 ±
0.2
4
1.2
0 ±
0.2
0
31.8
0 ±
1.4
5
4.9
1 ±
0.0
7
FP
73.7
8 ±
2.2
6
7.6
0 ±
0.0
7
0.2
6 ±
0.0
3
6.7
8 ±
1.0
0
1.0
8 ±
0.0
6
1.1
3 ±
0.1
1
20.3
4 ±
0.8
3
3.1
1 ±
0.0
8
TC
P
79.2
6 ±
5.3
3
12.6
2 ±
0.1
1.0
0 ±
0.5
6
10.0
6 ±
0.7
2
2.2
6 ±
0.1
6
1.5
3 ±
0.9
2
30.9
9 ±
1.4
3
3.2
7 ±
0.0
2
Pesticid
e
stressed
R14
P0
74.5
3 ±
6.2
0
9.5
1 ±
0.0
9
0.9
3 ±
0.3
0
9.1
8 ±
0.9
3
2.0
0 ±
0.3
3
1.0
0 ±
0.2
0
33.5
2 ±
3.3
0
3.1
2 ±
0.0
8
AL
P
88.3
5 ±
0.5
1
7.4
0 ±
0.1
1
1.2
0 ±
0.0
1
10.3
6 ±
0.5
5
1.5
2 ±
0.3
4
1.2
0 ±
0.2
0
24.2
6 ±
1.4
1
2.9
7 ±
0.0
2
FP
82.8
6 ±
0.9
2
8.5
4 ±
0.0
7
0.4
0 ±
0.0
3
9.3
6 ±
1.5
1
2.0
3 ±
0.3
9
1.0
6 ±
0.1
1
31.5
3 ±
3.6
0
2.9
4 ±
0.0
8
TC
P
80.7
4 ±
2.4
4
7.0
1 ±
0.0
7
2.0
0 ±
0.8
0
8.7
0 ±
0.6
8
1.4
8 ±
0.5
2
1.3
3 ±
0.4
1
26.4
5 ±
1.5
6
3.1
2 ±
0.0
8
P0
86.6
3 ±
2.8
7
13.3
5 ±
0.0
9
0.6
6 ±
0.0
2
9.3
6 ±
1.0
1
1.5
7 ±
0.6
3
1.4
0 ±
0.3
4
30.5
3 ±
6.9
9
4.5
9 ±
0.0
4
17
4
Non
stressed
SL
8
AL
P
77.5
5 ±
1.4
6
16.5
3 ±
0.0
8
1.8
6 ±
0.0
9
10.2
2 ±
0.6
2
2.2
6 ±
0.3
7
1.9
3 ±
0.5
0
31.0
0 ±
0.3
3
4.7
0 ±
0.0
4
FP
78.6
6 ±
0.7
1
7.8
6 ±
0.0
5
0.1
3 ±
0.0
1
8.2
6 ±
0.4
6
1.7
5 ±
0.7
4
1.1
3 ±
0.1
1
23.7
6 ±
0.5
0
3.0
8 ±
0.0
2
TC
P
77.5
6 ±
7.2
5
11.7
3 ±
0.2
3
0.4
6 ±
0.0
5
9.3
6 ±
1.3
9
2.0
6 ±
0.3
2
1.3
3 ±
0.4
1
30.1
7 ±
1.1
2
3.1
8 ±
0.0
2
Pesticid
e
stressed
SL
8
P0
77.9
0 ±
1.9
4
9.0
5 ±
0.1
3
1.0
0 ±
0.0
0
10.4
0 ±
1.3
8
2.2
6 ±
0.1
5
1.4
0 ±
0.4
0
32.0
0 ±
1.7
5
3.1
6 ±
0.0
1
AL
P
77.5
5 ±
1.4
6
8.5
2 ±
0.2
3
1.8
6 ±
0.0
9
10.2
2 ±
0.6
2
2.0
9 ±
0.2
3
1.9
3 ±
0.5
0
25.1
6 ±
0.8
1
3.1
7 ±
0.0
2
FP
82.5
3 ±
2.3
6
9.7
0 ±
0.0
2
0.5
3 ±
0.0
6
9.0
6 ±
0.5
5
2.1
0 ±
0.0
7
1.2
6 ±
0.3
0
31.0
5 ±
1.2
2
2.7
2 ±
0.0
6
TC
P
83.2
2 ±
2.0
0
11.9
2 ±
0.3
5
1.8
6 ±
0.5
7
9.4
9 ±
1.0
3
2.0
3 ±
0.4
3
1.0
6 ±
0.1
1
29.7
8 ±
3.2
5
2.7
8 ±
0.0
2
Non
stressed
Sp
A
P0
88.9
0 ±
2.8
7
10.4
7 ±
0.1
7
0.6
0 ±
0.0
3
9.3
9 ±
1.3
1
1.9
5 ±
0.6
4
1.0
0 ±
0.2
0
30.8
6 ±
3.5
9
4.9
2 ±
0.0
7
AL
P
84.3
6 ±
4.7
4
11.6
3 ±
0.2
1
1.4
0 ±
0.0
4
9.6
9 ±
0.8
3
1.8
5 ±
0.1
7
1.4
6 ±
0.3
0
28.5
1 ±
3.3
8
4.8
9 ±
0.0
4
FP
78.2
9 ±
3.0
6
12.3
5 ±
0.1
4
0.6
0 ±
0.0
8
8.3
0 ±
1.3
2
1.5
6 ±
0.4
3
1.1
3 ±
0.1
1
25.4
0 ±
1.4
2
3.1
2 ±
0.0
6
TC
P
78.6
6 ±
4.1
0
10.5
2 ±
0.0
9
0.4
0 ±
0.0
0
9.4
8 ±
1.2
5
1.9
4 ±
0.4
5
1.2
0 ±
0.3
4
28.2
4 ±
3.1
8
3.1
7 ±
0.0
2
Pesticid
e
stressed
Sp
A
P0
74.0
0 ±
3.9
4
7.3
0 ±
0.0
3
1.7
1 ±
0.7
0
9.5
6 ±
0.5
8
1.6
4 ±
0.1
1
1.5
3 ±
0.4
1
29.9
6 ±
1.9
5
3.1
7 ±
0.0
2
AL
P
84.3
6 ±
4.7
4
10.9
2 ±
0.0
6
1.4
0 ±
0.4
0
9.6
9 ±
0.8
3
2.2
0 ±
0.4
9
1.4
6 ±
0.3
0
29.4
2 ±
2.4
4
3.1
8 ±
0.0
3
FP
78.0
0 ±
2.2
2
12.2
3 ±
0.0
6
0.6
0 ±
0.0
2
9.8
6 ±
0.5
0
1.9
7 ±
0.0
9
1.2
6 ±
0.3
0
32.9
2 ±
1.6
8
2.1
7 ±
0.0
2
TC
P
82.1
1 ±
5.1
5
9.3
4 ±
0.1
3
1.7
3 ±
0.1
0
8.0
0 ±
1.1
3
1.2
8 ±
0.4
6
1.2
6 ±
0.3
0
24.1
4 ±
3.2
4
2.8
9 ±
0.0
3
Non
stressed
W95
P0
80.7
2 ±
2.9
7
12.5
1 ±
0.0
4
0.2
6 ±
0.4
6
9.1
5 ±
0.3
9
1.6
9 ±
0.2
2
1.0
0 ±
0.0
0
31.0
5 ±
0.0
9
5.1
7 ±
0.0
3
AL
P
88.5
4 ±
3.9
3
10.9
0 ±
0.0
8
1.6
0 ±
0.9
1
10.4
4 ±
1.3
6
2.3
8 ±
0.5
8
1.7
3 ±
0.4
1
29.7
7 ±
1.7
8
5.0
5 ±
0.1
3
FP
82.2
4 ±
3.0
1
9.7
2 ±
0.0
7
0.6
6 ±
0.0
1
8.1
9 ±
1.4
7
1.7
3 ±
0.3
9
1.0
0 ±
0.0
0
26.4
1 ±
3.1
3
3.1
3 ±
0.0
8
TC
P
78.7
8 ±
3.8
3
10.8
5 ±
0.1
5
1.0
0 ±
0.4
0
9.5
2 ±
0.9
2
2.0
0 ±
0.3
3
1.2
6 ±
0.6
4
28.4
0 ±
1.3
8
2.9
1 ±
0.0
7
Pesticid
e
stressed
W95
P0
72.8
0 ±
3.6
1
8.9
5 ±
0.0
5
0.6
6 ±
0.3
0
9.5
3 ±
0.6
1
2.2
2 ±
0.4
6
1.2
0 ±
0.2
0
31.9
4 ±
0.6
7
3.1
0 ±
0.0
5
AL
P
88.5
4 ±
3.9
3
9.5
1 ±
0.1
5
1.6
0 ±
0.9
1
10.4
4 ±
1.3
6
2.0
3 ±
0.4
7
1.7
3 ±
0.4
1
29.9
4 ±
1.9
2
2.8
9 ±
0.0
4
FP
83.7
3 ±
2.5
7
10.5
8 ±
0.0
6
0.0
6 ±
0.1
1
8.7
0 ±
0.3
6
1.9
7 ±
0.2
3
1.0
0 ±
0.0
0
31.5
2 ±
1.3
3
3.2
5 ±
0.1
3
TC
P
82.5
2 ±
2.1
2
8.5
7 ±
0.1
6
2.1
3 ±
0.1
1
9.6
6 ±
0.3
3
2.2
1 ±
0.8
6
1.2
0 ±
0.2
0
35.3
5 ±
5.2
4
3.5
5 ±
0.0
9
P0
82.8
7 ±
6.0
7
12.3
9 ±
0.1
8
0.7
5 ±
0.0
8
9.4
6 ±
2.1
1
1.8
6 ±
0.4
0
1.2
6 ±
0.4
6
29.7
7 ±
3.7
5
4.8
7 ±
0.0
1
17
5
Non
stressed
W96
AL
P
89.5
5 ±
5.2
5
12.7
4 ±
0.2
2
1.8
0 ±
0.2
1
10.3
5 ±
0.4
7
2.3
4 ±
0.2
6
1.5
3 ±
0.2
3
31.0
4 ±
1.0
0
5.4
1 ±
0.0
7
FP
84.8
6 ±
2.5
9
10.8
3 ±
0.0
7
0.3
3 ±
0.1
1
8.7
8 ±
1.3
1
1.9
3 ±
0.3
9
0.9
3 ±
0.1
1
28.6
9 ±
3.1
7
2.9
8 ±
0.1
8
TC
P
80.2
4 ±
4.1
4
12.2
0 ±
0.1
2
1.5
5 ±
0.8
9
9.0
5 ±
1.0
1
2.2
9 ±
0.4
5
1.1
3 ±
0.2
3
30.1
1 ±
2.4
1
4.1
3 ±
0.0
7
Pesticid
e
stressed
W96
P0
71.7
1 ±
3.8
7
7.8
8 ±
0.0
2
2.4
0 ±
0.0
5
9.7
1 ±
0.6
1
2.0
6 ±
0.1
1
1.3
3 ±
0.1
1
26.2
9 ±
0.7
7
3.2
9 ±
0.0
4
AL
P
88.3
9 ±
5.0
0
11.4
5 ±
0.1
7
1.8
0 ±
0.2
1
10.2
9 ±
0.5
0
2.1
6 ±
0.4
0
1.6
6 ±
0.2
3
32.1
4 ±
1.0
0
2.6
7 ±
0.1
9
FP
79.9
3 ±
4.6
7
9.3
8 ±
0.1
5
0.9
3 ±
0.6
1
10.0
1 ±
1.3
6
2.1
5 ±
0.6
4
1.4
0 ±
0.2
0
33.6
2 ±
2.4
0
2.8
7 ±
0.0
2
TC
P
70.7
3 ±
1.0
8
10.2
1 ±
0.1
1
1.8
0 ±
0.0
5
8.0
1 ±
2.9
0
2.2
6 ±
0.1
6
1.0
0 ±
0.4
0
32.1
2 ±
0.7
1
2.6
5 ±
0.1
1
Non
stressed U
P
P0
81.1
7 ±
2.7
8
11.8
5 ±
0.1
5
1.0
0 ±
0.5
2
9.7
0 ±
0.7
7
2.0
9 ±
0.3
9
1.3
3 ±
0.4
1
31.3
1 ±
0.7
5
5.2
1 ±
0.0
9
AL
P
84.2
4 ±
3.3
1
13.2
5 ±
0.0
4
1.9
3 ±
0.8
0
9.9
7 ±
0.7
6
2.0
9 ±
0.2
1
1.9
3 ±
0.2
3
28.0
4 ±
1.2
4
5.1
2 ±
0.0
8
FP
79.7
9 ±
6.1
4
12.8
0 ±
0.1
9
1.6
6 ±
0.1
3
9.9
6 ±
0.8
7
1.7
1 ±
0.3
3
1.6
6 ±
0.6
1
28.9
0 ±
2.2
7
3.0
2 ±
0.0
8
TC
P
73.9
6 ±
5.8
8
11.1
7 ±
0.1
2
0.8
6 ±
0.0
6
8.6
6 ±
1.6
8
1.4
1 ±
0.4
3
1.3
3 ±
0.2
3
27.1
3 ±
3.1
9
2.9
5 ±
0.0
8
Pesticid
e
stressed U
P
P0
74.0
3 ±
0.2
5
7.8
0 ±
0.1
2
1.1
3 ±
0.6
2
10.0
4 ±
2.3
8
1.7
9 ±
0.4
5
1.3
3 ±
0.3
0
27.6
5 ±
3.4
2
3.2
3 ±
0.0
7
AL
P
85.4
0 ±
5.2
8
8.2
3 ±
0.0
7
1.9
3 ±
0.8
0
10.0
3 ±
0.7
7
1.9
1 ±
0.2
3
1.8
0 ±
0.4
0
28.7
5 ±
1.3
2
3.0
2 ±
0.0
8
FP
72.2
3 ±
5.0
5
10.2
3 ±
0.0
9
0.8
6 ±
0.1
7
9.1
6 ±
0.5
1
2.1
5 ±
0.1
1
1.2
0 ±
0.2
0
31.8
4 ±
1.5
7
2.5
8 ±
0.0
5
TC
P
81.9
3 ±
1.6
8
10.3
7 ±
0.2
0
2.3
3 ±
0.7
0
9.3
7 ±
0.6
4
2.1
9 ±
0.3
2
1.1
3 ±
0.2
3
34.2
0 ±
9.8
7
2.7
0 ±
0.2
6
P=
0.0
5
Bacteria
<
0.0
01
<0.0
01
0.1
52
0.1
52
<0.0
01
0.1
50
0.2
81
<0.0
01
P so
urce
<0.0
01
<0.0
01
<0.0
01
<0.0
01
0.3
76
<0.0
01
0.1
00
<0.0
01
Pesticid
e 0.0
04
<0.0
01
0.0
04
0.6
47
0.1
09
0.2
72
<0.0
01
<0.0
01
B x
P so
urce
<0.0
01
<0.0
01
0.0
30
0.0
17
0.8
77
0.0
01
0.0
47
<0.0
01
B x
pesticid
e 0.4
91
<0.0
01
0.8
64
0.7
20
0.7
77
0.2
71
0.1
08
<0.0
01
P so
urce x
Pesticid
e <
0.0
01
<0.0
01
0.0
06
0.0
07
0.0
01
0.0
39
<0.0
01
<0.0
01
B x
P so
urce x
Pesticid
e 0.4
46
<0.0
01
0.9
6
0.1
64
0.0
01
0.9
04
0.0
01
<0.0
01
P0=
no
pho
sph
ate; AL
P=
Alu
min
ium
ph
osp
hate; F
P=
Ferric p
ho
sphate; T
CP
= T
ricalcium
ph
osp
hate; B
= B
acteria; P so
urce =
Pho
sph
ate sou
rce
176
The number of spikelets per spike in the presence of pesticides was remarkably increased
by 11-41.3% with strains Rad1, Rad2, Ros2, JA10 and R14. When aluminium phosphate
was added in alongwith pesticide stress, strain Ros2 and JA10 showed a notable increase
of 40.4 and 13.3 percent, respectively, as shown in table 8.1. Most remarkable increase due
to phosphate solubilizing bacteria was observed with the addition of ferric phosphate as
inorganic phosphate source, majority of the strains (S1, S2, Rad1, Rad2, JA10, R14, SL8,
SpA, W95, W96 and UP) showed marked increase in number of spikelets by 10 to 28% as
compared to uninoculated control. However, strains Ros2 and SL8 also caused minor
increase but was negligible. When tricalcium phosphate was added to soil, strain Ros2,
W95, W96 and UP showed a substantial increase of 12, 30, 18.5 and 26%, in the number
of spikelets, respectively.
Number of tillers
In natural soil, strain R14 caused a significant increase in number of tillers by 30%.
However, in the presence of aluminium phosphate, strain S2, JA10, R14, SL8, SpA, W95,
W96 and UP exhibited 47, 100, 63, 154, 90, 118, 145 and 163%, increase in the number of
tiller, respectively. In the presence of ferric phosphate, only strain UP resulted an increase
in number of tillers while in the presence of tricalcium phosphate strain Rad2 and W96
were found to have more number of tillers as compared to uninoculated control (Table 8.1).
In the presence of pesticide stress, the majority of the bacterial treated plants showed an
increased number of tillers except for strain S2, Ros2, JA10, R14 and W95. When
aluminium phosphate was added, the majority of the bacterial inoculated plants showed an
increase (47-154%) in a number of tillers as compared to control as shown in table 8.1. In
the presence of ferric phosphate, strain Rad1 showed an increase of 28% while 12-45%
177
increased numbers of tillers were observed by strain R14, SL8, W95, W96 and UP as
compared to uninoculated control (Table 8.1).
Weight of 100 grains
To check the effect of isolated phosphate solubilizing bacterial strains on the weight of
wheat grains, 100 grain were taken for each treatment for weight comparison. The weight
of grains for inoculated bacteria was compared to uninoculated control plants. It was found
that strain S2 and Rad1 caused a significant increment in grain weight by 19 and 13%,
respectively as shown in table 8.1. In the presence of aluminium phosphate as an additional
inorganic phosphate source in the soil, it was found that strain W96 augmented 12%
increase in grain weight. The strain Rad1 and Ros2 showed 12% increment in grain weight
when ferric phosphate was added to the soil. A remarkable increase of 28% was observed
in grain weight with strain W96 inoculation when tricalcium phosphate was added to the
soil.
The presence of pesticide stress caused a negative impact on the weight of grains in the
absence of additional inorganic phosphate source by bacterial inoculation (Rad1, Rad2,
R14 and W95). The weight of grains was found unaffected by strain S1, SL8 and W95.
Strain S2, Ros2, JA10, W96 and UP showed slightly increased grain weight but the
increase was non-significant as compared to uninoculated control. The addition of
aluminium phosphate in stress conditions did not positively affect the grain weight in the
majority of the bacterial inoculations. However, a noteworthy increase in grain weight was
recorded for the inoculation with strain Rad1 which enhanced the grain weight by 45% as
compared to uninoculated control plants (Table 8.1). The amendment of ferric phosphate
caused an increment of 22, 14.5 and 14% by strain S1, JA10 and W95, respectively. The
178
remarkably increased grain weight was observed for the inoculation with strain S1, S2,
Ros2, JA10, R14, SpA and W95 as 20, 20.2, 22, 24.8, 23,13.2 and 40%, respectively,
compared to uninoculated control when tricalcium phosphate was added to the soil as
shown in table 8.1. Whereas, the rest of the strains showed increased grain weight which
was non-significant.
Biochemical characteristics of wheat plants
Chlorophyll content in fresh leaves (mg g-1 fresh weight)
Phosphate solubilizing bacterial strains were inoculated to wheat plant and the impact of
inoculations on chlorophyll content of wheat plant was evaluated in different treatments.
Chlorophyll ‘A’ content was found to be remarkably increase with all bacterial inoculations
when soil was augmented with inorganic phosphates including aluminium phosphate,
ferric phosphate and tricalcium phosphate. While without phosphate addition, only 5
strains caused increased chlorophyll ‘A’ content when compared to uninoculated control.
Comparatively less increase was observed in the presence of pesticide stress when
compared to plants grown without pesticide stress as shown in table 8.2. However,
chlorophyll ‘B’ content was found to be reduced by majority of inoculations in the presence
of pesticide stress. Only a few strains caused negligible or reduction in chlorophyll ‘B’
content in natural soil conditions. However, in the inorganic supplemented soil, all bacterial
inoculations lead to a remarkable increase in chlorophyll ‘B’ content as compared to their
respective uninoculated controls. Similar results were observed when total chlorophyll
content and carotenoid content was estimated (Table 8.2).
179
Proline content in fresh leaves (µg mL-1)
In natural soil, the production of proline content was observed to be decreased significantly
as compared to uninoculated control in the absence as well as in the presence of pesticide
stress by all bacterial inoculations as compared to uninoculated control (Figure 8.1). With
the addition of aluminium phosphate in the soil, a significant decline in proline content was
found in the absence of pesticide stress while in the presence of stress only strain Rad2
inoculation showed significantly increased production of proline content (Figure 8.2). In
ferric phosphate supplemented soil, the proline content was increased in the presence of
pesticide stress in all bacterial inoculations except strain W96 when compared to non-
stressed conditions (Figure 8.3). However, in tricalcium phosphate supplemented soil,
highest enzyme production in the absence of stress was observed by strain Rad1, SpA and
Rad2 as 44, 30 and 28 µg mL-1, respectively. Whereas, in the presence of pesticide stress,
strain W96 and UP produced 45.4 and 40.1 µg mL-1 proline content, respectively, (Figure
8.4).
Peroxidase content in fresh leaves (Unit g-1)
In natural soil, the increased production of peroxidase enzyme was recorded by strain S1,
S2 and W95 as compared to uninoculated control. Whereas in the stressed conditions, the
enzyme production was observed to be increased when compared to non-stressed
conditions (Figure 8.5). In aluminium phosphate supplemented soil, the peroxidase enzyme
production was found to be increased in non-stressed treatments while in case of
inoculation with strain W96 increases enzyme was seen in stressed conditions (Figure 8.6).
Similar results were found when soil was supplemented with tricalcium phosphate (Figure
8.7). However, when the soil was supplemented with ferric phosphate, in the majority of
180
the inoculations the enzyme production was found to be increased in pesticide stressed
conditions as compared to non-stressed conditions (Figure 8.8).
Acid phosphatase content in fresh leaves (Units 100 mL-1)
The production of acid phosphatase enzyme by all bacterial inoculated wheat plants in the
absence of pesticide stress was found higher than the uninoculated control except by strain
R14 and SL8. However, less quantities of enzyme production was observed in the presence
of pesticide stress when compared to non-stressed conditions (Figure 8.9). Similar results
were recorded when aluminium phosphate was supplemented to the soil by all bacterial
inoculation except strain Rad1 which produced a higher quantity of acid phosphatase
production in the presence of stress (Figure 8.10). When ferric phosphate was added to
soil, increased quantities of acid phosphatase enzyme were produced by strains S1, S2,
Rad1, Rad2, Ros2, JA10, R14, SL8, SpA, W95, W96 and UP as compared to their
respective uninoculated control in the absence of pesticide stress. However, in the stressed
conditions, increased acid phosphatase production was recorded by strains S1, S2, Rad1,
Ros2, JA10, R14 and W95 whereas the enzyme production dropped in case of rest of the
inoculations as compared to uninoculated control (Figure 8.11). In tricalcium phosphate
supplemented soil, acid phosphatase production in bacterial inoculated wheat plants was
found to be increased without stress as compared to uninoculated control except strain W95
which showed increased enzyme production in the presence of pesticide stress (Figure
8.12).
181
Protein content (mg g-1 fresh weight)
When protein content of treated plants was estimated, it was observed that in natural soil,
the protein content was found decreased by all bacterial inoculated plants when compared
to respective uninoculated control except by strain Rad2 which showed significantly
increased protein content. While in case of pesticide treatment, strain Rad2, Ros2 and SL8
showed increased protein content as compared to their respected uninoculated control
(Figure 8.13). In the aluminium phosphate supplemented soil, all bacterial inoculated
plants showed higher protein content in the absence of pesticide stress as compared to
stressed conditions except strain SpA. Strain Rad2 and W95 produced highest quantities
of protein (152.6 and 163 mg g-1) in the absence of stress when compared to uninoculated
control (Figure 8.14). In ferric phosphate supplemented soil, the increased protein content
was observed in the majority of inoculated plants under nonstressed conditions. While in
case of tricalcium phosphate supplemented soil, the increased protein content was recorded
in pesticide stressed conditions by all of the bacterial inoculated plants except by strain UP
(Figure 8.15 and 8.16).
The properties of soil including electrical conductivity, pH, organic matter, available
phosphorous, available potassium, saturation and texture were also noted and were found
to be slightly affected as a result of phospahe solubilizing bacterial inoculation in soil
amended with different inorganic phosphate sources and pesticide stress as shown in table
in appendix III.
182
Table 8.2: Effect of phosphate solubilizing bacteria on chlorophyll content of bacterial
inoculated wheat plants in the presence of different inorganic phosphate sources alongwith
pesticide stress. The data shown represents Mean (n=3) and ± standard deviation. The
interaction significance between different treatments was judged by 2-way ANOVA
followed by Duncans’s analysis at the level of 95% significance.
Treatment
Chlorophyll content (mg g-1 FW)
Chlorophyll
‘A’
Chlorophyll
‘B’
Total
chlorophyll Carotenoid
Non stressed
control
P0 1.73 ± 0.02 0.81 ± 0.03 2.53 ± 0.06 4.51 ± 0.07
ALP 0.70 ± 0.07 0.38 ± 0.02 1.14 ± 0.10 2.29 ± 0.05
FP 0.50 ± 0.09 0.29 ± 0.01 0.77 ± 0.03 2.29 ± 0.17
TCP 0.51 ± 0.02 0.27 ± 0.01 0.77 ± 0.02 1.36 ± 0.07
Pesticide
stressed
control
P0 1.32 ± 0.01 0.66 ± 0.03 2.00 ± 0.08 3.37 ± 0.03
ALP 1.18 ± 0.01 0.47 ± 0.02 1.67 ± 0.04 4.24 ± 0.06
FP 0.90 ± 0.01 0.44 ± 0.05 1.33 ± 0.04 2.75 ± 0.06
TCP 2.07 ± 0.07 0.84 ± 0.02 2.84 ± 0.05 5.22 ± 0.09
Non stressed
S1
P0 2.35 ± 0.08 1.12 ± 0.09 3.25 ± 0.11 6.20 ± 0.18
ALP 1.72 ± 0.46 0.87 ± 0.07 2.53 ± 0.22 5.24 ± 0.23
FP 1.41 ± 0.03 0.67± 0.02 2.05 ± 0.07 4.24 ± 0.19
TCP 1.33 ± 0.07 0.55 ± 0.01 1.84 ± 0.04 3.23 ± 0.08
Pesticide
stressed S1
P0 1.66 ± 0.04 0.76 ± 0.03 2.52 ± 0.17 4.32 ± 0.16
ALP 0.79 ± 0.02 0.31 ± 0.01 1.16 ± 0.13 3.20 ± 0.01
FP 0.71 ± 0.01 0.34 ± 0.02 1.14 ± 0.11 2.62 ± 0.42
TCP 1.20 ± 0.15 0.46 ± 0.02 1.61 ± 0.10 2.91 ± 0.09
Non stressed
S2
P0 1.72 ± 0.09 0.70 ± 0.24 2.37 ± 0.08 4.52 ± 0.12
ALP 1.52 ± 0.67 0.58 ± 0.06 2.33 ± 0.08 4.56 ± 0.07
FP 1.00 ± 0.09 0.76 ± 0.03 1.56 ± 0.06 3.23 ± 0.07
TCP 0.79 ± 0.01 0.81 ± 0.07 1.17 ± 0.05 2.37 ± 0.32
Pesticide
stressed S2
P0 1.37 ± 0.06 0.62 ± 0.02 2.01 ± 0.09 3.55 ± 0.10
ALP 0.61 ± 0.01 0.26 ± 0.01 1.01 ± 0.17 3.00 ± 0.11
FP 1.53 ± 0.01 0.65 ± 0.09 1.82 ± 0.46 4.07 ± 0.16
TCP 1.67 ± 0.06 0.62 ± 0.05 2.42 ± 0.15 4.22 ± 0.03
P0 2.14 ± 0.15 1.02 ± 0.08 3.16 ± 0.09 6.44 ± 0.23
183
Non stressed
Rad1
ALP 1.24 ± 0.67 0.60 ± 0.03 1.88 ± 0.06 3.72 ± 0.08
FP 1.17 ± 0.05 0.58 ± 0.01 1.63 ± 0.04 3.36 ± 0.13
TCP 1.28 ± 0.06 0.55 ± 0.01 1.72 ± 0.05 2.69 ± 0.10
Pesticide
stressed
Rad1
P0 1.46 ± 0.01 0.70 ± 0.03 2.11 ± 0.10 3.61 ± 0.12
ALP 0.51 ± 0.06 0.25 ± 0.01 0.82 ± 0.07 2.58 ± 0.13
FP 1.00 ± 0.10 0.42 ± 0.02 1.37 ± 0.10 2.74 ± 0.22
TCP 1.72 ± 0.11 0.66 ± 0.02 2.29 ± 0.05 4.51 ± 0.21
Non stressed
Rad2
P0 2.17 ± 0.06 1.01 ± 0.09 3.11 ± 0.09 6.02 ± 0.04
ALP 1.27 ± 0.04 0.53 ± 0.07 1.88 ± 0.06 3.52 ± 0.03
FP 1.36 ± 0.07 0.62 ± 0.01 2.03 ± 0.12 3.91 ± 0.08
TCP 1.30 ± 0.07 0.52 ± 0.06 1.77 ± 0.05 2.82 ± 0.06
Pesticide
stressed
Rad2
P0 1.60 ± 0.01 0.72 ± 0.01 2.28 ± 0.17 3.99 ± 0.12
ALP 2.41 ± 0.07 1.17 ± 0.11 3.52 ± 0.08 5.82 ± 0.25
FP 0.97 ± 0.02 0.45 ± 0.02 1.52 ± 0.11 2.91 ± 0.10
TCP 1.76 ± 0.04 0.76 ± 0.04 2.48 ± 0.04 4.86 ± 0.06
Non stressed
Ros2
P0 1.75 ± 0.04 0.77 ± 0.03 2.63 ± 0.19 5.02 ± 0.08
ALP 1.51 ± 0.11 0.68 ± 0.02 2.29 ± 0.10 4.53 ± 0.10
FP 1.22 ± 0.05 0.54 ± 0.01 1.85 ± 0.12 3.38 ± 0.08
TCP 1.17 ± 0.15 0.43 ± 0.04 1.42 ± 0.07 2.54 ± 0.10
Pesticide
stressed
Ros2
P0 1.28 ± 0.05 0.59 ± 0.03 1.93 ± 0.11 3.57 ± 0.30
ALP 1.39 ± 0.05 0.66 ± 0.06 2.04 ± 0.13 3.59 ± 0.16
FP 1.55 ± 0.04 0.66 ± 0.03 2.15 ± 0.15 4.08 ± 0.11
TCP 1.68 ± 0.03 0.76 ± 0.02 2.58 ± 0.14 4.89 ± 0.11
Non stressed
JA10
P0 1.89 ± 0.30 0.80 ± 0.01 2.82 ± 0.10 5.27 ± 0.14
ALP 1.51 ± 0.12 0.73 ± 0.02 2.34 ± 0.14 4.47 ± 0.15
FP 0.91 ± 0.13 0.41 ± 0.01 1.22 ± 0.08 2.98 ± 0.05
TCP 0.93 ± 0.04 0.45 ± 0.05 1.37 ± 0.06 2.32 ± 0.05
Pesticide
stressed
JA10
P0 0.89 ± 0.04 0.46 ± 0.03 1.55 ± 0.26 2.48 ± 0.42
ALP 1.24 ± 0.05 0.60 ± 0.05 1.83 ± 0.07 3.51 ± 0.15
FP 1.20 ± 0.04 0.52 ± 0.02 1.73 ± 0.14 3.30 ± 0.19
TCP 1.18 ± 0.07 0.51 ± 0.02 1.57 ± 0.06 3.20 ± 0.15
Non stressed
R14
P0 1.75 ± 0.02 0.78 ± 0.03 2.48 ± 0.14 4.99 ± 0.19
ALP 1.04 ± 0.04 0.51 ± 0.02 1.44 ± 0.18 2.68 ± 0.28
184
FP 0.79 ± 0.06 0.38 ± 0.02 1.20 ± 0.11 2.48 ± 0.17
TCP 0.67 ± 0.03 0.35 ± 0.05 1.12 ± 0.15 1.92 ± 0.17
Pesticide
stressed R14
P0 0.68 ± 0.03 0.47 ± 0.06 1.13 ± 0.18 4.33 ± 0.09
ALP 1.57 ± 0.06 0.69 ± 0.02 2.27 ± 0.07 3.97 ± 0.06
FP 1.33 ± 0.01 0.53 ± 0.02 1.72 ± 0.18 3.58 ± 0.24
TCP 1.49 ± 0.06 0.63 ± 0.01 2.05 ± 0.06 4.17 ± 0.12
Non stressed
SL8
P0 1.38 ± 0.02 0.60 ± 0.01 2.03 ± 0.07 4.15 ± 0.16
ALP 1.20 ± 0.50 0.56 ± 0.02 1.77 ± 0.09 3.47 ± 0.15
FP 1.37 ± 0.13 0.59 ± 0.02 1.76 ± 0.15 3.62 ± 0.33
TCP 1.09 ± 0.10 0.43 ± 0.01 1.47 ± 0.04 2.55 ± 0.08
Pesticide
stressed SL8
P0 0.59 ± 0.04 0.40 ± 0.03 1.04 ± 0.16 4.52 ± 0.11
ALP 1.75 ± 0.03 0.78 ± 0.02 2.52 ± 0.03 4.67 ± 0.16
FP 1.18 ± 0.05 0.50 ± 0.02 1.59 ± 0.04 3.23 ± 0.12
TCP 1.69 ± 0.05 0.70 ± 0.01 2.21 ± 0.10 4.60 ± 0.09
Non stressed
SpA
P0 1.72 ± 0.09 0.76 ± 0.04 2.52 ± 0.08 4.98 ± 0.06
ALP 1.11 ± 0.06 0.53 ± 0.02 1.57 ± 0.02 3.23 ± 0.07
FP 1.25 ± 0.10 0.52 ± 0.01 1.45 ± 0.21 3.59 ± 0.23
TCP 1.80 ± 0.09 0.74 ± 0.03 2.68 ± 0.21 4.87 ± 0.37
Pesticide
stressed SpA
P0 0.51 ± 0.03 0.36 ± 0.03 0.76 ± 0.09 4.32 ± 0.06
ALP 1.20 ± 0.03 0.57 ± 0.06 1.73 ± 0.06 3.50 ± 0.12
FP 1.72 ± 0.02 0.70 ± 0.02 2.58 ± 0.27 4.48 ± 0.18
TCP 1.55 ± 0.08 0.67 ± 0.02 2.30 ± 0.27 4.18 ± 0.06
Non stressed
W95
P0 1.44 ± 0.07 0.66 ± 0.04 2.11 ± 0.17 4.64 ± 0.14
ALP 1.86 ± 0.03 0.85 ± 0.02 2.46 ± 0.18 5.24 ± 0.09
FP 0.99 ± 0.01 0.48 ± 0.03 1.39 ± 0.04 2.97 ± 0.15
TCP 1.20 ± 0.09 0.46 ± 0.01 1.46 ± 0.21 3.27 ± 0.17
Pesticide
stressed W95
P0 0.48 ± 0.02 0.36 ± 0.03 0.80 ± 0.01 3.69 ± 0.09
ALP 1.48 ± 0.03 0.62 ± 0.07 2.07 ± 0.04 4.19 ± 0.14
FP 0.68 ± 0.03 0.30 ± 0.02 1.08 ± 0.13 2.16 ± 0.16
TCP 2.46 ± 0.20 1.05 ± 0.13 3.07 ± 0.11 6.19 ± 0.14
Non stressed
W96
P0 1.46 ± 0.01 0.65 ± 0.03 2.20 ± 0.11 4.49 ± 0.33
ALP 1.77 ± 0.03 0.77 ± 0.15 2.68 ± 0.19 5.07 ± 0.15
FP 1.34 ± 0.22 0.54 ± 0.01 1.69 ± 0.04 3.65 ± 0.25
185
TCP 1.31 ± 0.04 0.53 ± 0.01 1.77 ± 0.12 3.25 ± 0.03
Pesticide
stressed W96
P0 0.79 ± 0.01 0.48 ± 0.01 1.38 ± 0.19 4.03 ± 0.06
ALP 2.03 ± 0.06 0.85 ± 0.01 2.68 ± 0.33 5.55 ± 0.22
FP 0.86 ± 0.02 0.38 ± 0.02 1.41 ± 0.19 2.74 ± 0.50
TCP 1.69 ± 0.04 0.77 ± 0.06 2.45 ± 0.08 4.33 ± 0.09
Non stressed
UP
P0 2.10 ± 0.10 0.93 ± 0.01 3.12 ± 0.09 6.45 ± 0.18
ALP 1.09 ± 0.01 0.52 ± 0.02 1.60 ± 0.03 2.76 ± 0.22
FP 1.73 ± 0.05 0.76 ± 0.02 2.52 ± 0.08 5.24 ± 0.30
TCP 1.87 ± 0.05 0.75 ± 0.10 2.55 ± 0.05 4.33 ± 0.17
Pesticide
stressed UP
P0 0.69 ± 0.02 0.48 ± 0.01 1.21 ± 0.08 5.75 ± 0.19
ALP 1.51 ± 0.07 0.63 ± 0.03 2.12 ± 0.08 4.26 ± 0.20
FP 1.86 ± 0.04 0.75 ± 0.04 2.72 ± 0.24 4.87 ± 0.43
TCP 1.95 ± 0.01 0.87 ± 0.04 2.82 ± 0.06 5.24 ± 0.08
p=0.05
Bacteria <0.001 <0.001 <0.001 <0.001
P source <0.001 <0.001 <0.001 <0.001
Pesticide <0.001 <0.001 <0.001 <0.001
B x P source <0.001 <0.001 <0.001 <0.001
B x pesticide <0.001 <0.001 <0.001 <0.001
P source x Pesticide <0.001 <0.001 <0.001 <0.001
B x P source x
Pesticide <0.001 <0.001 <0.001 <0.001
P0= no phosphate; ALP= Aluminium phosphate; FP= Ferric phosphate; TCP= Tricalcium
phosphate; B= Bacteria; P source = Phosphate source
186
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pro
line c
onte
nt
(µg m
l-1)
0
5
10
15
20
25
30
Without stress
Pesticide stress
Figure 8.1: Effect of phosphate solubilizing bacterial strains on proline content in wheat
plants in the presence of pesticide in natural soil. The graph shows the mean ± standard
deviation (n=3). Data judged from 2-way ANOVA followed by Duncan’s (p<0.05).
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pro
line c
onte
nt
(µg m
l-1)
0
10
20
30
40
50
60
70
Without stress
Pesticide stress
Figure 8.2: Effect of phosphate solubilizing bacterial inoculation on proline content in
wheat plants in the presence of pesticide in soil amended with aluminium phosphate. The
graph shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA
followed by Duncan’s (p<0.05).
187
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pro
line c
onte
nt
(µg m
l-1)
0
10
20
30
40
50Without stress
Pesticide stress
Figure 8.3: Effect of phosphate solubilizing bacterial inoculation on proline content in
wheat plants in the presence of pesticide in soil amended with ferric phosphate. The graph
shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA followed
by Duncan’s (p<0.05).
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pro
line c
onte
nt
(µg m
l-1)
0
10
20
30
40
50
60
Without stress
Pesticide stress
Figure 8.4: Effect of phosphate solubilizing bacterial inoculation on proline content in
wheat plants in the and presence of pesticide in soil amended with tricalcium phosphate.
The graph shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA
followed by Duncan’s (p<0.05).
188
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pero
xid
ase
conte
nt
(U g
-1)
0
20
40
60
80
100Without stress
Pesticide stress
Figure 8.5: Effect of phosphate solubilizing bacterial inoculation on peroxidase content in
wheat plants in the presence of pesticide in natural soil. The graph shows the mean ±
standard deviation (n=3). Data judged from 2-way ANOVA followed by Duncan’s
(p<0.05).
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pero
xid
ase
conte
nt
(U g
-1)
0
20
40
60
80
100
Without stress
Pesticide stress
Figure 8.6: Effect of phosphate solubilizing bacterial inoculation on peroxidase content in
wheat plants in the presence of pesticide in soil amended with aluminium phosphate. The
graph shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA
followed by Duncan’s (p<0.05).
189
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pero
xid
ase
conte
nt
(U g
-1)
0
20
40
60
80Without stress
Pesticide stress
Figure 8.7: Effect of phosphate solubilizing bacterial inoculation on peroxidase content in
wheat plants in the presence of pesticide in soil amended with ferric phosphate. The graph
shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA followed
by Duncan’s (p<0.05).
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pero
xid
ase
conte
nt
(U g
-1)
0
20
40
60
80
100Without stress
Pesticide stress
Figure 8.8: Effect of phosphate solubilizing bacterial inoculation on peroxidase content in
wheat plants in the presence of pesticide in soil amended with tricalcium phosphate. The
graph shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA
followed by Duncan’s (p<0.05).
190
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Acid
phosphata
se
(K.A
unit
s 1
00m
l-1)
0
1
2
3
4
5
6
7Without stress
Pesticide stress
Figure 8.9: Effect of phosphate solubilizing bacterial inoculation on acid phosphatase
content in wheat in the presence of pesticide in natural soil. The graph shows the mean ±
standard deviation (n=3). Data judged from 2-way ANOVA followed by Duncan’s
(p<0.05).
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Acid
phosphata
se
(K.A
unit
s 1
00m
l-1)
0
1
2
3
4
5
6
7Without stress
Pesticide stress
Figure 8.10: Effect of phosphate solubilizing bacterial inoculation on proline content in
wheat plants in the presence of pesticide in soil amended with aluminium phosphate. The
graph shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA
followed by Duncan’s (p<0.05).
191
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Acid
phosphata
se
(K.A
unit
s 1
00m
l-1)
0
1
2
3
4
5Without stress
Pesticide stress
Figure 8.11: Effect of phosphate solubilizing bacterial inoculation on acid phosphatase
content in wheat plants in the presence of pesticide in soil amended with ferric phosphate.
The graph shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA
followed by Duncan’s (p<0.05).
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Acid
phosphata
se
(K.A
unit
s 1
00m
l-1)
0
1
2
3
4
5Without stress
Pesticide stress
Figure 8.12: Effect of phosphate solubilizing bacterial inoculation on acid phosphatase
content in wheat plants in the presence of pesticide in soil amended with tricalcium
phosphate. The graph shows the mean ± standard deviation (n=3). Data judged from 2-way
ANOVA followed by Duncan’s (p<0.05).
192
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pro
tein
conte
nt
(unit
mg g
-1)
0
20
40
60
80
100
120
140Without stress
Pesticide stress
Figure 8.13: Effect of phosphate solubilizing bacterial inoculation on protein content in
wheat plants in the presence of pesticide in natural soil. The graph shows the mean ±
standard deviation (n=3). Data judged from 2-way ANOVA followed by Duncan’s
(p<0.05).
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pro
tein
conte
nt
(unit
mg g
-1)
0
20
40
60
80
100
120
140
160
180Without stress
Pesticide stress
Figure 8.14: Effect of phosphate solubilizing bacterial inoculation on protein content in
wheat plants in the presence of pesticide in soil amended with aluminium phosphate. The
graph shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA
followed by Duncan’s (p<0.05).
193
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pro
tein
conte
nt
(unit
mg g
-1)
0
20
40
60
80
100
120
140
160
180
Without stress
Pesticide stress
Figure 8.15: Effect of phosphate solubilizing bacterial inoculation on protein content in
wheat plants in the presence of pesticide in soil amended with ferric phosphate. The graph
shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA followed
by Duncan’s (p<0.05).
Bacterial strains
C S1
S2
Rad
1
Rad
2
Ros2
JA10
R14
SL8
SpA
W95
W96 UP
Pro
tein
conte
nt
(unit
mg g
-1)
0
20
40
60
80
100
120
140
160
180
200Without stress
Pesticide stress
Figure 8.16: Effect of phosphate solubilizing bacterial inoculation on protein content in
wheat plants in the presence of pesticide in soil amended with tricalcium phosphate. The
graph shows the mean ± standard deviation (n=3). Data judged from 2-way ANOVA
followed by Duncan’s (p<0.05).
194
Discussion
In soil phosphorous plays a very important role. It is present in different organic and
inorganic forms. The soluble forms are easily available to plants while the insoluble forms
are not available to plants. Among insoluble forms, the phosphorous remains sequestered
to different cations such as aluminium phosphate, calcium phosphate and ferric phosphate
(Sharma et al., 2013; Maitra et al., 2015). The phosphate solubilizing bacteria play an
important role in solubilization of these bound or inorganic phosphate forms. A number of
different plant growth promoting bacteria are present on rhizosphere (Gianfreda, 2015)
such as Pseudomonas, Enterobacter, Acinetobacter, Flavobacterium, Rhizobium and
Bacillus. Bacteria present in the soil also indicate the soil quality in different agricultural
areas (Akca and Namli, 2015; Ananyeva et al., 2016).
A number of studies has been conducted on phosphate solubilizing bacteria and it has been
found that they also showed other plant growth promoting abilities including siderophore
production, secondary metabolite and antibiotic production, ACC deaminase enzyme,
gibberellins and auxin production (Taurian et al., 2010; Namli et al., 2017). The enzymatic
activity of bacteria is greater in the rhizospheric region of soil (Gianfreda, 2015).
According to Namli et al. (2017), the inoculation of phosphate solubilizing bacteria also
statistically affected the phosphorous content in the rhizosphere (4.26 – 5.73 mg kg-1)
which was much higher than control.
Phosphate solubilizing bacterial strains belonging to different genera (Ochrobactrum
pseudogrignonense-S1, Acinetobacter olivorans-S2, Pseudomonas putida-Rad1,
Pseudomonas putida-Rad2, Pseudomonas sp-Ros2, Acinetobacter baumanii- JA10,
Pseud8omonas plecoglossicida-R14, Pseudomonas japonica-SL8, Pseudomonas
195
aeruginosa-SpA, Enterobacter cloacae-W95, Enterobacter aerogenes-W96 and
Pseudomonas reinekei-UP) have been studied in this experiment.
The agriculture sector is developing rapidly worldwide and is also addressing the toxicity
levels of pesticide compounds (Azizullah et al., 2011; Karpouzas et al., 2014). Form the
previous reports, it has been found that it could produce toxicity in soil, water, food and
ultimately cause harm to organisms (Hernández et al., 2013; Dubey et al., 2015).
Chlorpyrifos has gained great importance because of its vast use and potential toxicity in
non-target organisms (Yu et al., 2015). The production and yield of wheat crop (Triticum
aestivum L.) is affected by different pests. To protect the crops from potential pests
different pesticides are routinely used in several regions of the world including Asia,
Europe and Africa (Pansa et al., 2015). Wang and Zhang (2017) have estimated the toxicity
of Chlorpyrifos in wheat plants by estimating salicylic acid estimation. Pyriproxyfen is an
insecticide which acts as an analogue of juvenile hormone in insects and affects their
growth (Ali et al., 2016).
It was found that the inoculation with phosphate solubilizing bacterial isolates stimulated
the vegetative growth of wheat plants in different treatments. When compared to
uninoculated control plants, shoot length was significantly increased by different
inoculated strains in different treatments (Figure 8.17, 8.18, 8.19 and 8.20). In the presence
of pesticide stress, a few inoculations showed a decrease in shoot length and the increase
was less than that of non-stressed conditions. Chlorpyrifos has been reported to negatively
affect the growth of wheat plants (Wang and Zang, 2017). Increased dry plant biomass has
also been reported by Namli et al. (2017) when wheat plants were inoculated with
phosphate solubilizing bacteria.
196
Shoot dry weight was also found significantly increased for up to 54% when inoculated
with different bacterial genera in different growth conditions in the presence of inorganic
phosphate (aluminium phosphate, ferric phosphate and tricalcium phosphate)
supplemented soils and pesticide stress (Table 8.1). In a recent study, it has been reported
that a significant increase in shoot length and shoot dry weight of wheat plant by two
phosphate solubilizing strains (Pantoea cypripedii-PSB3 and Pseudomonas
plecoglossicida-PSB5) in natural soil (Kaur and Reddy, 2014). While a marked increase in
these parameters have been reported by him when inorganic phosphate (rock phosphate)
was added to soil (Gurdeep and Reddy, 2015).
Phosphorous plays an important role in plant nutrition, such as in the growth and strength
of stem, development of root, formation of seed and flower, disease resistance, improved
quality of crop and its production (Thakur et al., 2014). In general, inoculation of the wheat
plant with phosphate solubilizing bacteria lead to increase in spike length in natural and
inorganic phosphate supplemented soils. A remarkable increase of spike length up to 92%
was recorded by the majority of the inoculations including Ochrobactrum
pseudogrignonense-S1, Pseudomonas putida-Rad1, Pseudomonas putida-Rad2,
Pseudomonas sp-Ros2, Pseudomonas plecoglossicida-R14, Pseudomonas japonica-SL8,
Pseudomonas aeruginosa-SpA, Enterobacter cloacae-W95, Enterobacter aerogenes-W96
and Pseudomonas reinekei-UP. Chishti and Arshad (2013) have reported the ability of
Enterobacter spp to degrade Chlorpyrifos efficiently. According to the research reports of
Ul Hassan and Bano (2015) and Majeed et al. (2015), the phosphate and nitrogen
197
Control R14
Figure 8.17: Effect of phosphate solubilizing Pseudomonas plecoglossicida-R14
inoculation and aluminium phosphate on vegetative growth of the wheat plant after 20
weeks.
Control SpA
Figure 8.18: Effect of phosphate solubilizing Pseudomonas aeruginosa-SpA inoculation
and aluminium phosphate on vegetative growth of the wheat plant under pesticide stress
after 20 weeks.
198
Control W96
Figure 8.19: Effect of phosphate solubilizing Enterobacter aerogenes-W96 inoculation
and ferric phosphate on vegetative growth of the wheat plant after 20 weeks.
Control W95
Figure 8.20: Effect of phosphate solubilizing Enterobacter cloacae-W95 inoculation and
tricalcium phosphate on vegetative growth of wheat plant under pesticide stress after 20
weeks.
199
concentrations in aerial parts of wheat plants was higher as a result of phosphate
solubilizing bacterial inoculation.
Spike weight was increased by all bacterial inoculated wheat plants in natural soil.
However, the addition of aluminium phosphate to soil also led to significant increase in
spike weight (up to 100%). The addition of ferric phosphate and tricalcium phosphate
showed up to 77 and 58% increased spike weight in the absence of pesticide stress. Majeed
et al. (2015) have reported a study related to phosphate solubilizing Pseudomonas for
enhanced plant growth in wheat. However, with the addition of pesticide, the reduction in
increased spike weight was recorded. Chlorpyrifos is a pesticide and its higher
concentrations cause potential risk to several crop plants (Wang and Zang, 2017).
The use of phosphate solubilizing bacteria has been reported to improve the properties of
soil including fixation of nitrogen, solubilization of phosphorous, increased nutrient
uptake, physiological and agro-morphological factors (Egamberdieva, 2010; Sarker et al.,
2014; Turan et al., 2014; Zahid et al., 2015). The number of spikes per plant was increased
generally in the absence and presence of pesticide and aluminium phosphate and tricalcium
phosphate. Mineralization of organic phosphate and phosphatase enzyme production has
been reported to be increased as a result of phosphate solubilizing bacterial inoculation
(Hussain et al., 2013). However, the spike number decreased when ferric phosphate was
added as the inorganic phosphate source. Similarly, overall increase in number of spikelets
per spike and the number of tillers was recorded in all treatments. Likewise, in a research,
Sharma et al. (2011) conducted a field study related to Pseudomonas sp inoculation in
wheat and reported the significant enhancement in different enzymatic activities, uptake of
nutrients and overall yield.
200
Enhanced grain weight was recorded by different bacterial inoculations in inorganic
phosphate supplemented soil in the absence of pesticide stress. However, decrease in grain
weight was recorded by all bacterial inoculated plants when aluminium phosphate was
added along with pesticide stress. Similarly, Mukhtar et al. (2017) reported 40-86%
increase in grain yield when wheat plants were inoculated with phosphate solubilizing
bacterial isolates.
In soil, phosphate solubilizing bacteria are challenged with different ecological stresses.
As a result of these conditions, they have developed different mechanisms for their survival
(Liu et al., 2017). The potential to survive in these challenging situations helps their
survival and this idea is also useful that they can be utilized as biofertilizers in challenging
situations (de Oliveira-Longatti et al., 2014; Liu et al., 2017). Selected bacterial strains
were also subjected to different biochemical assay to measure the proline content, acid
phosphatase enzyme content and protein estimation of plant tissue with or without stress
in inorganic phosphate supplemented soil.
The chlorophyll content in fresh leaf material of bacterial inoculated plants was estimated
in different treatments. Chlorophyll ‘A’ content was found to remarkably increase by all
bacterial inoculations when soil was augmented with inorganic phosphates including
aluminium phosphate, ferric phosphate and tricalcium phosphate. Panhwar et al. (2011)
have also reported the increased chlorophyll content in plants inoculated with phosphate
solubilizing bacteria in green house conditions. A study showed that the inoculated plant
with phosphate solubilizing bacterial isolate lead to significant increase in total chlorophyll
content in leaf (Muthukumarasamy et al., 2017). Comparatively less increase was observed
in the presence of pesticide stress when compared to plants grown without pesticide stress.
201
Similarly, chlorophyll ‘B’ content was decreased when pesticide stress was applied. At
higher concentrations Chlorpyrifos causes the reduction in photosynthetic pigments,
enhanced lipid peroxidation and cause the interruption in different enzymatic activities in
wheat plant (Wang and Zang, 2017).
Foliar use of Chlorpyrifos increased the proline content and peroxidase enzyme production
in mung bean plant (Parween et al., 2012). Our study also showed that the proline content
in wheat leaves was increased when plants were treated with pesticides. However, the
concentration of peroxidase enzyme in wheat leaves was increased in non-stressed
conditions in the presence of aluminium and tricalcium phosphate whereas reverse was
observed when ferric phosphate was added to soil. The pesticide stress affect plant’s
growth and it also alter plant biochemically as well as physiologically which ultimately
affect plant yield (Parween et al., 2016). In general, variable results were found when acid
phosphatase and protein content were estimated in fresh leaves of the wheat plant.
According to a study, the application of Chlorpyrifos to Vigna radiata at the concentration
of 0.6mM and 1.5mM has been reported to lower down the levels of protein content as well
as soluble sugar (Parween et al., 2011).
Conclusion
The effect of isolated phosphate solubilizing strains inoculated wheat plant was observed
alongwith inorganic phosphate and pesticide addition in soil. It was found that different
strains showed different response towards growth parameters as well as in the enzymatic
activities. The effect of bacterial inoculation on wheat plant vary from strain to strain in
different conditions.
202
Chapter 09
Interaction between phosphate solubilizing bacteria and
arbuscular mycorrhizal fungi
Phosphorous plays an important role in the growth and development of plants. In soil, it is
present in both organic and inorganic forms. However, plants can only uptake the organic
forms of phosphorous. The availability of soluble phosphate to plants is limited due to the
formation of complexes with different elements (Sharma et al., 2013). Natural deposits of
phosphorous are depleting from soil at a very fast rate. It has been estimated that from
tropical and subtropical agricultural regions, the phosphorous reserves will be completely
depleted in next three decades (Balemi and Negisho, 2012). The limitation of phosphorous
is compensated by the application of different phosphate fertilizers (Costa et al., 2015).
The application of these chemical fertilizers are posing risks to the environment
(Pizzaghello et al., 2011).
Microorganisms present in soil play a vital role in phosphorous cycling or mobility. The
transfer of phosphorous from one form to other by microbial activity has gained a lot of
importance (Babalola and Glick, 2012). Phosphate solubilizing microorganisms convert
the organic and inorganic form of phosphorous into available forms that can be easily taken
up by plants. There are interactions between different microorganisms which directly or
indirectly influence the abilities of microorganisms. Based upon inorganic phosphate
solubilization abilities the isolated Phosphate Solubilizing Bacterial (PSB) isolates were
selected from different genera. To check the interaction with Arbuscular Mycorrhizal
Fungi (AMF), RiDAOM 19198, selected individual bacterial strains (Ochrobactrum
203
pseudogrignonense-S1, Pseudomonas putida-Rad2, Acinetobacter baumanii- JA10,
Pseudomonas aeruginosa-SpA and Enterobacter aerogenes-W96) were studied in this
experiment. The experiment was carried out in bi-compartment petri dishes. In proximal
compartment, vitamin and sugar supplemented minimal growth medium was added. In
distal compartment, tricalcium phosphate was added according to the experimental setup
(Figure 9.1 and 9.2). The pH of both mediums was adjusted to 5.5 and solidified before
sterilization. Mycorrhized chicory roots were grown in proximal compartment for 21 days
at 28oC. Roots were trimmed regularly as they were not allowed to grow in distal
compartment. Only extraradial AMF mycelium were allowed to move towards distal
compartment. Plates with extraradial mycelium in distal compartment were selected and
received 50 µL of bacterial suspension. Control plates received equal quantity of sterile
saline instead of bacterial cell suspension. The experiment was replicated five times. Plates
were placed at 28oC for incubation for six weeks.
For this experiment, four treatment were followed:
1. PSB inoculated in minimal growth medium
2. PSB inoculated in minimal growth medium supplemented with tricalcium phosphate
3. PSB and AMF inoculated in minimal growth medium
4. PSB and AMF inoculated in minimal growth medium supplemented with tricalcium
phosphate
Change in pH of minimal medium of distal compartment was measured at the completion
of incubation after six weeks of bacterial inoculation. To detect pH change, the medium of
distal compartment was removed and was added to falcon tubes followed by overnight
204
freezing at -20oC. The tubes were centrifuged and the supernatant was used to determine
the pH. The results were compared with un-inoculated control.
Impact of interaction on pH
When phosphate solubilizing bacteria were tested for pH change in minimal growth
medium, it was found that the pH for Ochrobactrum pseudogrignonense-S1, Pseudomonas
putida-Rad2 and Pseudomonas aeruginosa-SpA Pseudomonas aeruginosa-SpA was
higher than un-inoculated control whereas, decreased pH was observed for Acinetobacter
baumanii- JA10 and Enterobacter aerogenes-W96 inoculations as shown in figure 9.3.
When tricalcium phosphate was added in the absence of arbuscular mycorrhizal fungi, the
pH change of medium by phosphate solubilizing bacteria was found to be increased when
compared to un-inoculated control (Figure 9.4). In the presence of arbuscular mycorrhizal
fungi, the pH of minimal growth medium of distal compartment was found to be decreased
as compared to un-inoculated control. The maximum decrease was observed by
Pseudomonas putida-Rad2 inoculation (Figure 9.5). Similarly, in the presence of
arbuscular mycorrhizal fungi, the pH of medium supplemented with tricalcium phosphate
in distal compartment was decreased for all strains when compared to un-inoculated
control. Least pH was recorded for Pseudomonas putida-Rad2 as shown in figure 9.6.
Impact of interaction on phosphate solubilization
The interaction between phosphate solubilizing bacteria and arbuscular mycorrhizal fungi
was observed to have impact on inorganic phosphate solubilization. To check the effect of
bacterial interaction with Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on
solubilization of tricalcium phosphate, gelified medium from the distal side was removed
205
and transferred to falcon tubes and placed overnight at -20oC. Medium was liquefied by
thawing at room temperature. Falcon tubes containing medium were centrifuged at 10000
g for 30 minutes. Solubilized phosphate content in supernatant was measured by the
method of King (1932).
In minimal growth medium, the solubilization abilities of isolated bacteria were tested and
we found that highest value for solubilized phosphate was recorded for strain JA10 > W96
> S1 > SpA > Rad2 > Control. Where Acinetobacter baumanii- JA10 and Enterobacter
aerogenes-W96 produced 11 µg mL-1 phosphate and Ochrobactrum pseudogrignonense-
S1 and Pseudomonas aeruginosa-SpA produced 4.4 µg mL-1 of solubilized phosphate,
respectively. Pseudomonas putida-Rad2 produced least quantity of soluble phosphate
content (Figure 9.7).
When minimal growth medium was supplemented with tricalcium phosphate in distal
compartment in the absence of Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198,
we found the following maximum to minimum order for phosphate solubilization by strain
JA10 > Rad2 > W96 > S1 > SpA > Control. All of the tested bacterial strains showed higher
solubilization potential when compared to un-inoculated control. Acinetobacter baumanii-
JA10, Pseudomonas putida-Rad2, Enterobacter aerogenes-W96, Ochrobactrum
pseudogrignonense-S1 and Pseudomonas aeruginosa-SpA produced 90, 64, 63, 49 and 35
µg mL-1 solubilized phosphate content, respectively, (Figure 9.8).
In order to check the phosphate solubilization by phosphate solubilizing bacteria in the
presence of arbuscular mycorrhizal fungi, we found the following order of P content in
minimal growth medium: W96 > JA10 > S1 > un-inoculated control > Rad2 > SpA. The
Enterobacter aerogenes-W96, Acinetobacter baumanii- JA10 and Ochrobactrum
206
pseudogrignonense-S1 produced 31, 29 and 10 µg mL-1 of soluble phosphate content,
respectively. Whereas, Pseudomonas putida-Rad2 and Pseudomonas aeruginosa-SpA
produced less phosphate content (7 and 5 µg mL-1, respectively) when compared to un-
inoculated control (Figure 9.9).
In the presence of Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 along with
tricalcium phosphate supplementation in minimal growth medium, the order of inorganic
phosphate solubilization was Rad2 > SpA > JA10 > W96 > S1 > Un-inoculated control.
Pseudomonas putida-Rad2, Pseudomonas aeruginosa-SpA and Acinetobacter baumanii-
JA10 showed highest solubilization potential as a result of interaction with arbuscular
mycorrhizal fungi and produced 145, 135 and 103 µg mL-1, respectively. In this case, the
least producers for solubilized phosphate content were Enterobacter aerogenes-W96 and
Ochrobactrum pseudogrignonense-S1 (75 and 63 µg mL-1, respectively) (Figure 9.10).
To study the interaction between Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198
and phosphate solubilizing bacteria, plates were analyzed for interaction studies under
stereo microscope after six weeks of bacterial inoculation. The interaction of bacteria with
the hyphae of AMF was observed near the bacterial inoculation line. The movement of
bacterial cells along with the fungal hyphae was found by Pseudomonas putida-Rad2,
Acinetobacter baumanii- JA10, Pseudomonas aeruginosa-SpA and Enterobacter
aerogenes-W96 (Figure 9.12 – 9.15). Whereas, Ochrobactrum pseudogrignonense-S1 did
not show growth along with the growth of fungal hyphae (Figure 9.11).
207
Figure 9.1: Bi-compartment petri plate having mychorrized chicory roots with Arbuscular
Mycorrhizal Fungi (AMF), RiDAOM 19198, grown in proximal compartment for 21 days
at 28oC. The distal compartment containing minimal growth medium inoculated with
Acinetobacter baumanii- JA10 followed by incubation for 6 weeks at 28oC.
Figure 9.2: Bi-compartment petri plate having mychorrized chicory roots with Arbuscular
Mycorrhizal Fungi (AMF), RiDAOM 19198, grown in proximal compartment for 21 days
at 28oC. The distal compartment containing minimal growth medium supplemented with
tricalcium phosphate inoculated with Pseudomonas putida-Rad2 followed by incubation
for 6 weeks at 28oC.
208
Bacterial strains
Control S1 Rad2 JA10 SpA W96
pH
0
1
2
3
4
5
6
7
ab a ab b
c
Figure 9.3: Effect of phosphate solubilizing bacterial isolates on pH of minimal growth
medium in the absence of Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198. Plates
were incubated for six weeks after bacterial inoculation at 28oC. Error bars Mean ±
standard error (n=5). Different letters on bars indicate significant difference between
treatments using Duncan’s multiple range test (p<0.05).
209
Bacterial strains
Control S1 Rad2 JA10 SpA W96
pH
0
2
4
6
8
a
bbc bc cd
Figure 9.4: Effect of phosphate solubilizing bacterial isolates on pH of minimal growth
medium supplemented with tricalcium phosphate in the absence of Arbuscular Mycorrhizal
Fungi (AMF), RiDAOM 19198. Plates were incubated for six weeks after bacterial
inoculation at 28oC. Error bars Mean ± standard error (n=5). Different letters on bars
indicate significant difference between treatments using Duncan’s multiple range test
(p<0.05).
210
Bacterial strains
Control S1 Rad2 JA10 SpA W96
pH
0
2
4
6
8
a
bb
b b b
Figure 9.5: Effect of interaction between phosphate solubilizing bacterial isolates and
Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on pH of minimal growth
medium. Plates were incubated for six weeks after bacterial inoculation at 28oC. Error bars
Mean ± standard error (n=5). Different letters on bars indicate significant difference
between treatments using Duncan’s multiple range test (p<0.05).
211
Bacterial strains
Control S1 Rad2 JA10 SpA W96
pH
0
2
4
6
8a
aa
a
a
a
Figure 9.6: Effect of interaction between phosphate solubilizing bacterial isolates and
Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on pH of minimal growth medium
supplemented with tricalcium phosphate. Plates were incubated for six weeks after
bacterial inoculation at 28oC. Error bars Mean ± standard error (n=5). Similar letter on
bars indicate non-significant difference between treatments using Duncan’s multiple range
test (p<0.05).
212
Bacterial strains
Control S1 Rad2 JA10 SpA W96
So
lub
iliz
ed
P (
µg
mL
-1)
0
2
4
6
8
10
12
14
16
a
a
aa
b
b
Figure 9.7: Effect of phosphate solubilizing bacterial isolates on P solubilization in
minimal growth medium in the absence of Arbuscular Mycorrhizal Fungi (AMF),
RiDAOM 19198. Plates were incubated for six weeks after bacterial inoculation at 28oC.
Error bars Mean ± standard error (n=5). Different letters on bars indicate significant
difference between treatments using Duncan’s multiple range test (p<0.05).
213
Bacterial strains
Control S1 Rad2 JA10 SpA W96
So
lub
iliz
ed
P (
g m
L-1
)
0
20
40
60
80
100
a
b
c
d
d
e
Figure 9.8: Effect of phosphate solubilizing bacterial isolates on P solubilization in
minimal growth medium supplemented with tricalcium phosphate in the absence of
Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198. Plates were incubated for six
weeks after bacterial inoculation at 28oC. Error bars Mean ± standard error (n=5).
Different letters on bars indicate significant difference between treatments using Duncan’s
multiple range test (p<0.05).
214
Bacterial strains
Control S1 Rad2 JA10 SpA W96
So
lub
iliz
ed
P (
g m
L-1
)
0
5
10
15
20
25
30
35
a
a
aa
b b
Figure 9.9: Effect of interaction between phosphate solubilizing bacterial isolates and
Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on P solubilization in minimal
growth medium. Plates were incubated for six weeks after bacterial inoculation at 28oC.
Error bars Mean ± standard error (n=5). Different letters on bars indicate significant
difference between treatments using Duncan’s multiple range test (p<0.05).
215
Bacterial strains
Control S1 Rad2 JA10 SpA W96
So
lub
iliz
ed
P (
g m
L-1
)
0
20
40
60
80
100
120
140
160
a
b
c
d
e
e
Figure 9.10: Effect of interaction between phosphate solubilizing bacterial isolates and
Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on P solubilization in minimal
growth medium supplemented with tricalcium phosphate. Plates were incubated for six
weeks after bacterial inoculation at 28oC. Error bars Mean ± standard error (n=5). Similar
letter on bars indicate non-significant difference between treatments using Duncan’s
multiple range test (p<0.05).
216
Discussion
Phosphorous has been found to have importance in several metabolic activities in plants.
These activities include photosynthesis, energy transfer, respiration, biosynthesis and
signal transduction (Khan et al., 2012; Wahid et al., 2016). Soil is supplemented with
chemical fertilizers to fulfill the phosphorous requirements and the manufacture of
chemical fertilizers require enormous cost. Due to these factors, researchers are finding
cost effective and ecofriendly approaches. The symbiotic association between AMF and
plants have been reported (Zhang et al., 2016). As an obligatory biotrophs, AMF takes
carbon supplementation from the plant host and in its response, it provide different
nutrients to plants and among these nutrients, phosphate ions are the important molecules
(Karasawa et al., 2012; Zhang et al., 2017). In this regard, the microorganisms are known
as the third symbiont of AMF (Jansa et al., 2013). Several reports indicate the corporation
between them. The hyphae are the channels for photosynthetase enzyme from plants which
attract different microorganisms and also help in their growth and stimulation (Kaiser et
al., 2015). Kaise et al. (2015) reported that the presence of AMF in wheat rhizosphere
improve the cycling of mineral nutrients.
Zhang et al. (2016) have reported the positive interactions between phosphate solubilizing
bacteria and arbuscular mycorrhizal fungi. The experiment was conducted to check the
impact of hyphal growth of AMF on P solubilization and P solubilization ability of bacteria
as a result of PSB and AMF interaction. In soil, AMF have extensive network of extraradial
hyphae. These hyphae accommodate different microorganisms (Gahan and
Schmalenberger, 2015). Thus, there may exist the corporation between the associated
microorganisms and AMF. When we checked the impact of phosphate solubilizing bacteria
217
on the pH of minimal growth medium present in distal compartment. We found a slight
change in pH of medium. The evidence suggest that the bacteria do not affect the pH of
medium in the absence of AMF and inorganic phosphate source. Similarly, the pH was
found increased by all bacterial strains in the presence of inorganic phosphate
supplemented to medium in distal compartment. The Pseudomonas putida-Rad2 strain
showed lowest pH when compared to other bacterial treatments in the presence of
Arbuscular Mycorrhizal Fungi, RiDAOM 19198. In the presence of AMF and inorganic
phosphate source, decrease in pH was observed by all strains.
When phosphate content of growth medium was observed, it was found that in the absence
of inorganic phosphate supplementation, phosphate content was increased by bacteria as
compared to un-inoculated control. The fungi that can solubilize inorganic phosphorous
comprises of about 0.1-0.5% of the total fungi present in soil. According to our results,
Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 alone did not solubilized
inorganic phosphate. It has also been reported by Tisserant et al. (2013), AMF are non-
saprophytes and cannot break down the organic nutrients directly. However, microbes have
this ability and they play major role in biogeochemical cycles. Bacterial efficiency for
tricalcium phosphate solubilization was increased twice in the presence of arbuscular
mycorrhizal fungi, RiDAOM 19198 by Pseudomonas putida-Rad2. Similarly, increase was
recorded by all other strains as well.
The interactions of arbuscular mycorrhizal fungi and phosphate solubilizing was observed
under microscope (Figure 9.11-9.15). The interaction or growth of bacteria along with
arbuscular mycorrhizal fungi was not determined in the presence of tricalcium phosphate,
due to white color and opacity of the medium as a result of tricalcium phosphate
218
supplementation. Pseudomonas putida-Rad2 and Enterobacter aerogenes-W96 showed
good interaction and growth along with the mycorrhizal hyphae. Several studies showed
that the AMF associated bacteria influence AMF in different ways (Cheng et al., 2012;
Zhang et al., 2014). Acinetobacter baumanii- JA10 and Pseudomonas aeruginosa-SpA
showed slight growth along with the mycorrhizal hyphae. The analytical microscopic
studies as well as the molecular biology studies shows the colonization of different
bacterial species on hyphal surfaces of AMF and their spores (Scheublin et al., 2010;
Agnoolicci et al., 2015). Ochrobactrum pseudogrignonense-S1 did not showed interaction
or growth along with the hyphae of AMF. These evidences suggest that this selection of
microbial partner depends on AMF. Besides providing benefits to some microorganisms,
the AMF hyphae also inhibit some microbes (Nuccio et al., 2013; Bender et al., 2014).
Conclusion
The interaction between arbuscular mycorrhizal fungi and phosphate solubilizing bacteria
in the presence and absence of inorganic phosphate was observed. The studies showed that
pH was affected due to their interaction and also the phosphate solubilization by bacteria
was enhanced significantly. However, arbuscular mycorrhizal fungi alone did not
solubilize inorganic phosphate due to its non-saprophytic properties. These results suggest
that AMF provide suitable environment to bacteria for solubilization.
219
Figure 9.11: Interaction between phosphate solubilizing Ochrobactrum
pseudogrignonense (S1) with Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on
minimal growth medium. Plates were incubated for six weeks after bacterial inoculation at
28oC. The interaction was analyzed using stereo microscope.
220
Figure 9.12: Positive interaction between phosphate solubilizing Pseudomonas putida
(Rad2) (arrow) with Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on minimal
growth medium. Plates were incubated for six weeks after bacterial inoculation at 28oC.
The interaction was analyzed using stereo microscope.
221
Figure 9.13: Positive interaction between phosphate solubilizing Acinetobacter baumanii
(JA10) (arrow) with Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on minimal
growth medium. Plates were incubated for six weeks after bacterial inoculation at 28oC.
The interaction was analyzed using stereo microscope.
222
Figure 9.14: Positive interaction between phosphate solubilizing Pseudomonas
aeruginosa (SpA) (arrow) with Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198
on minimal growth medium. Plates were incubated for six weeks after bacterial inoculation
at 28oC. The interaction was analyzed using stereo microscope.
223
Figure 9.15: Positive interaction between phosphate solubilizing Enterobacter aerogenes
(W96) (arrow) with Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on minimal
growth medium. Plates were incubated for six weeks after bacterial inoculation at 28oC.
The interaction was analyzed using stereo microscope.
224
Chapter 10
Discussion
Soil present in the plant rhizosphere contains a large number of diverse microorganisms.
Some microorganisms have the ability to promote plant growth by colonizing plant roots
(Kumar et al., 2015). Diverse genera of bacteria are important for soil, performing different
biotic activities and conversion of nutrients and ultimately improve plant growth and soil
fertility (Glick, 2012; Ahemad and Kibret, 2014). Phosphorous can only be absorbed by
the plants in the form of H2PO4 and HPO2 (orthopshophates). These available forms of
phosphorous are depleting at a fast rate in the vicinity of plant rhizosphere (Richardson et
al., 2001; Kumar et al., 2015). This deficiency can be overcome by the application of
chemical phosphate fertilizers. Most of the phosphorous content of the applied phosphate
fertilizers is converted to bound forms due to the presence of some metals in soil which
affects the efficiency of applied phosphorous. Phosphate solubilizing bacteria release the
bound phosphorous by dissolving it (Ahemad and Khan, 2012b). Besides providing
available phosphorous to plants they also possess other plant growth promoting abilities.
In the present investigation, twenty eight phosphate solubilizing bacterial strains (S1, S2,
Rad1, Rad2, Ros1, Ros2, JA10, R12, R14, R15, SL8, M6, L6, L19, L20, L22, SF, SpA,
CS1, R2, S62, W94, W95, W96, P1, UP, C14 and C50) were isolated from different soil
samples on the basis of inorganic phosphate solubilization ability on Pikovskaya agar
medium. The soil samples from rhizosphere of different plants were having almost neutral
pH ranging from 6.8-7.5, whereas the soil samples collected from salt affected areas were
slightly acidic in nature (5.3-5.5). Another major purpose of the study was to determine the
225
potential of phosphate solubilizing bacteria to solubilize inorganic phosphate. All the
bacterial isolates were gram negative rods and belonged to phylum Proteobacteria and class
Gammaproteobacteria except two isolates (S1 and M6) which belonged to the class of
Alphaproteobacteria, order Rhizobiales and the family Brucellaceae. Whereas, the strains
S2, JA10, L6, L19, CS1, R2, S62, W94, C14 and C50 were associated with the order of
Pseudomonadales and the family of Moraxellaceae. On the basis of morphological,
biochemical and genetic analysis, strain Rad1, Rad2, Ros1, Ros2, R14, R15, SL8, L20,
L22, SF, SpA, P1 and UP belonged to order Pseudomonadales and the family of
Pseudomonaceae (chapter 04). However, strain R12, W95 and W96 belonged to the order
of Enterobacteriales and to the family of Enterobacteriaceae. Previous finding have also
showed that predominant bacterial genera of phosphate solubilizers include Pseudomonas,
Burkholderia, and Acinetobacter. Whereas, another study suggested that bacteria with
phosphate solubilizing abilities belong to four families including Enterococcaceae,
Bacillaceae, Alcaligeneaceae and Enterobacteriaceae (Azziz et al., 2012; Acevedo et al.,
2014; Yadav and Pandey, 2018). Different studies have reported the isolation of phosphate
solubilizing strains from the rhizosphere of different plants including wheat in the normal
soils (Ahmad et al., 2008; Linu et al., 2009; Iqbal et al., 2010; Ogut et al., 2010; Rajapaksha
et al., 2011; Baig et al., 2012; Khan et al., 2017; Liu et al., 2018) as well as from soils
affected from high concentrations of salt (Srinivasan et al., 2012; Kadmiri et al., 2018).
The majority of isolates of present study showed motility, oxidase activity and citrate
utilization abilities. Fatima et al. (2015) have reported a phosphate solubilizing
Pseudomonas brassicacearum (PKU5) as a Gram negative rod with oxidase, catalase,
urease and nitrate reduction ability. Moreover, the pigment production was also exhibited
226
by only a few isolates. In accordance with these results, Nehra et al. (2014) has also
reported a phosphate solubilizing isolate as gram negative bacilli with no pigment
production. The measurement of extracellular enzymatic activities of an organism provides
an insight of its abilities to check its performance in energy limited environments. The
isolates in this study were found to have extracellular enzymatic abilities for starch
hydrolysis, lipid and gelatin hydrolysis however urea hydrolysis was not recorded by any
of the isolates. Likewise in a scientific report by Kumar et al. (2016), they have reported
eight bacterial strains associated with rhizosphere of turmeric plant and the isolates were
good solubilizers of phosphate as well as they also had the ability for starch hydrolysis.
In natural environment, bacterial communities are exposed to several environmental
stresses including antibiotics production by other organisms and pesticide applications.
The isolated bacterial strains were tested to check their ability for resistance or
susceptibility towards antibiotics. Four antibiotics (amoxicillin, cloaxicillin, imipenem and
ceftazidime) were used in this study. All of the isolates showed resistance to cloaxicillin
and sensitivity for imipenem whereas for amoxicillin and ceftazidime, some isolates were
resistant and some of them were found susceptible. Similarly, de Oliveira-Longatti et al.
(2014) have reported a phosphate solubilizing strain UFLA 03-84 (Bradyrhizobium sp.)
which was found resistant to twelve antibiotics including amoxicillin.
In agriculture, the application of large number of pesticides is a common practice
nowadays. The application of pesticides has greatly affected the bacterial community in
the rhizosphere of different crops and plants (Holmsgaard et al., 2017). The isolated
phosphate solubilizing strains were tested for pesticide tolerance and it was found that the
isolates were able to grow in the presence of Chlorpyrifos and Pyriproxyfen. Some of the
227
isolates were able to resist them up to the concentration of 80 mg mL-1. Anzuay et al.
(2017) have also investigated the survival and phosphate solubilizing ability of bacterial
isolates (Pantoea sp. J49 and Serratia sp. J260) in the presence of pesticide stress. The
suggested reason of bacterial survival is the presence of pesticides is the pesticide
degradation potential of bacterial community (Moorman, 2018).
The bacterial isolates were subjected to 16S rRNA gene sequencing for phylogenetic
studies. When the isolated phosphate solubilizing strains were compared by computing
their phylogeny by neighbor joining method, two main clads appeared (Figure 4.29). From
the previous studies it seemed that the content of genome is mostly determined by
phylogenetic proximity and similar genomes are present in close species (Zaneveld et al.,
2010). Strain S1 and M6 made a separate distant clad because they belong to the class of
Alphaproteobacteria while the other bigger clad represented that the rest of the isolated
bacteria are associated to the class of Gammaproteobacteria. Among the cluster of
Pseudomonas species, ten strains were placed together while strains SF, R15 and SpA
showed slightly distant grouping. Likewise, Ordonez et al. (2016) have also reported that
in rhizosphere of potato plant, Pseudomonas sp. were present predominantly as compared
to other genus and had plant growth promoting abilities and were reported as good
phosphate solubilizers. Oteino et al. (2015) have also conducted a research on plant growth
promoting phosphate solubilizing bacteria and reported that twelve strains were found
associated to Pseudomonas fluorescence and Pseudomonas putida and other Pseudomonas
sp. and the strains had good plant growth promoting abilities. Pseudomonas have been
reported as a predominant genera isolated from wheat rhizosphere (Liu et al., 2018).
228
In soil, phosphorous is a preeminent component but it remains sequestered by different
elements present in soil which are responsible for un-availability of phosphorous to plants
(Zhang et al., 2017). In soil, phosphorous usually remains adsorbed by aluminium, ferrous,
calcium and magnesium and their oxides. It also lead to their gradual conversion towards
more complexity. The adsorption of phosphorous is greatly influenced by pH of soil.
Calcium bound phosphorous occur predominantly in alkaline soils while aluminium and
ferric bound forms usually occur in acidic environments (Banerjea and Gosh, 1970; Maitra
et al., 2015). Phosphorous is an important component for growth and development of plants
and is generally used as fertilizers to enhance plant growth (Wei et al., 2015; Wei et al.,
2017). Microorganisms in soil play a crucial role in conversion or transformation of
nutrients from one form to another (Gronemeyer et al., 2011; Maitra et al., 2015).
Isolated bacterial strains were studied for their solubilization abilities for inorganic
phosphate on two media including Pikovskaya agar and NBRIP agar. Based on the
calculation of solubilization index, maximum index on Pikovskaya agar was exhibited by
Acinetobacter baumanii- JA10 as 2.64. The solubilization index of 1.62 has been recorded
by phosphate solubilizing Pseudomonas strain on Pikovskaya agar (Mohamed and
Almoroai, 2017). On NBRIP agar, better results for solubilization index were found as
compared to Pikovskaya agar and it was found that Klebsiella pneumoniae-R12 exhibited
the maximum solubilization index of 3.07. In a recent study conducted by Tomer et al.
(2017), it has been reported that isolates ST-30, N-26 and MP-1 had solubilization index
of 62mm, 8mm and 7.2mm in NBRIP agar. The phosphatase enzyme activity of bacterial
strains help in the solubilization of inorganic phosphate compounds (Sharma et al., 2017;
Behera et al., 2017). From the results of phosphatases detection on TSA plates, it was
229
observed that all the bacterial isolates had the ability to produce phosphatases on agar plates
which was indicated by pink zone formation around point of inoculation. Similarly Ribeiro
and Cardoso (2012) have also reported 85% of their isolates showed phosphatases
production indicated by pink color on TSA plate.
Most of the phosphate remain sequestered to metals present in soil. The predominant metal
ions that bind with the organic phosphate are aluminium, calcium and ferrous
(Priyadharsini and Muthukumar, 2017). In the present study, bacterial isolates were tested
to solubilize three different inorganic phosphate sources including Aluminium phosphate
(ALP), Ferric phosphate (FP) and Tricalcium phosphate (TCP). The solubilization range
for aluminium phosphate by isolated bacterial strains was 17 µg mL-1 to 51 µg mL-1.
Moreover, decrease in pH and increase in titrable acidity was recorded by all isolated
bacteria compared to control. According to a study related to aluminium phosphate
solubilization, Yadav et al. (2015) have reported that the isolated phosphate solubilizing
bacteria were able to solubilize 59.4 mg L-1 to 76.7 mg L-1 of phosphate. When ferric
phosphate was added to growth medium as inorganic phosphate source, it was observed
that among isolated bacterial strains SF and W96 were able to dissolve 97.9 µg mL-1 and
92.6 µg mL-1 of ferric phosphate, respectively. The isolated bacterial strains showed best
results for dissolution of TCP. The solubilization potential of all isolates for TCP ranged
from 618.6 µg mL-1 to 962.2 µg mL-1 (Chapter 05). Depending upon genus, different
bacterial isolates perform differently for phosphate solubilization and results vary
depending upon sources of isolation as well as inorganic sources (Zhang et al., 2017). In
acidic soils, phosphate gets precipitated with Al3+ and Fe3+ while in calcareous or neutral
soils it binds to Ca2+ (de Oliveira Mendes et al., 2014). The possible reason for less
230
solubilization potential towards aluminium phosphate and ferric phosphate is their
abundance in acidic soils. The isolates of the study were isolated from neutral to alkaline
soils where tricalcium phosphate is present in large quantities that is why they showed best
solubilization for tricalcium phosphate. Yadav et al. (2015) have also documented that
bacteria, isolated from alkaline soil were able to solubilize tricalcium phosphate more
efficiently as compared to aluminium phosphate and ferric phosphate.
Phosphate solubilization is usually enhanced when appropriate amount of energy is present
to be utilized by organism for the production of various organic acids (Reza et al., 2017).
Carbon sources are utilized to be used as a source of energy but various sources affect the
phosphate solubilization potentials. The effect of different carbon sources (glucose,
maltose, galactose and sucrose) was checked to have any impact on phosphate
solubilization ability of isolated bacteria. The solubility of inorganic phosphate by different
strains varied in the presence of different sugars and phosphate solubilizing ability varied
significantly among strains. Maximum dissolution for insoluble phosphate was exhibited
in the presence of glucose by bacterial isolates. The order of maximum to minimum
phosphate solubilization by isolates depending on carbon source was found as: glucose >
galactose > maltose > sucrose. Different phosphate solubilizing bacteria showed different
levels of phosphate solubilization activities for different sugars. Pallavi and Gupta (2013)
have studied the effect of different carbon sources on phosphate solubilization ability of
Pseudomonas lurida and found that most to least suitable carbon source for phosphate
solubilization from tricalcium phosphate was glucose followed by maltose, galactose,
sucrose and xylose. Glucose enhanced the production of solubilized phosphate by bacterial
species from tricalcium phosphate in NBRIP medium (Pallavi and Gupta, 2013). The effect
231
of phosphate solubilization on pH and titrable acidity was evaluated and it was found that
titrable acidity was increased in case of all sugars. For pH, decease was observed by all
strains in case of glucose and galactose while in case of maltose and sucrose few strains
showed increased pH as compared to control. Pallavi and Gupta (2013) have also tested
that in the presence of different sugars, bacterial isolate showed variable results for
solubilization of phosphate and it also affected the pH of culture medium.
Soil is a reservoir for pesticide remains and several microorganisms (Jain et al., 2015).
Besides enhancing plant growth, phosphate solubilizing bacteria have also been reported
to degrade xenobiotic compounds like pesticides. The impact of applied pesticides was
evaluated in vitro to assess the bacterial ability to solubilize inorganic phosphate in the
presence of pesticides. For this purpose Chlorpyrifos, Pyriproxyfen, and mixture of these
pesticides was added to the culture medium of isolated bacteria. The phosphate
solubilization activity of isolates was affected in the presence of pesticides and decrease in
solubilization potential was observed by most of the isolates. Even though the activity of
phosphate solubilization by isolated bacterial stains was decreased in the presence of
pesticide stress but still they exhibited much better results for the solubilization of inorganic
phosphate when compared to control. Anzuay et al. (2017) have studied the effect of abiotic
stress and pesticide on solubilization activity of phosphate solubilizing isolates and
reported that Acinetobacter sp.-L176 produced 44.0 U of acid phosphatase and 42.1 U of
alkaline phosphatase. In the present study, the reported acid and alkaline phosphatase
activity by Pseudomonas fluorescens was 56.1 U and 65.9 U, respectively. Overall strain
UP showed consistent results for maximum acid phosphatase activity in the absence as well
as in the presence of pesticide stress.
232
Soil present around plant roots contain large number of active bacterial species. These
bacteria are also called as Plant Growth Promoting Rhizobacteria (PGPR) (Kloepper et al.,
1980; Reetha et al., 2014). It is estimated that above 95% of bacteria exist in the rhizosphere
of plants and are responsible to help plants in obtaining nutrients from soil. According to
recent approaches, researchers are trying to isolate and study bacteria having Plant Growth
Promoting (PGP) abilities (Ullah and Bano, 2015). Hydrogen cyanide (HCN) production
by bacterial isolates have been found responsible to suppress diseases in plants (Kumar et
al., 2015). Different bacterial genera have been reported to have hydrogen cyanide
production ability. Similarly, isolated bacterial strains were evaluated for their potential to
produce hydrogen cyanide. The positive results for HCN production were recorded in 53%
of the isolates (Table 6.1). According to previous studies, around 50% of bacterial isolates
from wheat and potato rhizosphere had shown HCN production in vitro (Kumar et al.,
2015).
Previous studies have shown that IAA production by bacteria helps in better interaction
with plants as it helps in root elongation, increased root exudates and biomass production
as well as it also helps in stress tolerance (Etesami and Alikhani, 2015). During the in vitro
screening and quantification of IAA production, it was observed that the IAA production
ability of isolated bacteria ranged from 4.48 µg mL-1 to 74.6 µg mL-1. In a recent report,
Zhang et al. (2017) have reported that 61.5% of studied phosphate solubilizing bacteria
produced 8.06 to 62.43 mg L-1 of IAA. Xu et al. (2014) studied IAA production by plant
growth promoting bacterial isolates and have found that only 37% isolates were IAA
producers.
233
Ammonia production by plant growth promoting bacteria is helpful for controlling of
phytopathogens as well as high crop yield (Mota et al., 2017). Isolated phosphate
solubilizing bacteria were also checked for the production of ammonia, and it was found
that all isolates had this ability. Likewise in a study, Nehra et al. (2014) have reported
Pseudomonas fluoescens sp as a strong producer of ammonia. Siderophore production by
bacterial isolates negatively influence the pathogens due to the production of antimicrobial
compounds in the surroundings of plant roots (Wahyudi et al., 2011). Phosphate
solubilizing bacterial isolates were evaluated for their ability to produce siderophores on
CAS agar media. In the present study, 21% isolates produced siderophore. According to
previous observations, bacterial isolates having siderophores production ability assist
plants to uptake different metals from soil (Dimpka et al., 2009; Gururani et al., 2013).
In different agricultural soils, phosphorus is an important limiting nutrient and its
deficiency affects plant growth. Phosphate solubilizing isolates have been reported to be
used as bio-inoculants for a number of crops. The use of microbial inoculants helps to
increase the microbial population in plant rhizosphere (Rajapaksha and Senanayake, 2011)
and the plants conform their growth according to the external and internal stimulus by the
hormonal activities. Plant growth depends on the key phytohormones which include
ethylene, auxin and abscisic acid (Vanstraelen and Benekova, 2012; Thole et al., 2014).
Inoculated seeds were grown in petri dishes supplemented with the recommended doses of
pesticide solutions and the strains were evaluated for their abilities to enhance the growth
parameters in root elongation assay both in the absence and presence of pesticides.
Increased percentage germination was recorded by majority of the strains in the absence of
pesticide stress. In general, the germination rate was found to be decreased in the presence
234
of pesticides. Toxicity level of pesticide varies from organism to organism, depending upon
the functional group of pesticide (Ahemad and Khan, 2011b). The overall decline in shoot
length was observed by the majority of the inoculated strains in the absence of pesticide
except Acinetobacter olivorans-S2 treated seeds. Patel et al. (2012) have reported the
enhanced shoot and root growth by Pseudomonas and Bacillus species. A significantly
increased shoot length was recorded with S1 and S2 in the presence of Chlorpyrifos when
compared to uninoculated control, while the decline in shoot length was recorded in case
of other bacterial inoculations. Ahemad and Khan (2012b) have reported the decline in
plant growth promoting abilities by Mesorhizobium (MRC4) under the stress of pesticide.
From many of the possible reasons of increased or decreased percentage can be the
relationship between plant and bacteria which differs with the difference in genetic makeup
(Chauhan et al., 2013; Afzal et al., 2017).
Overall reduction was observed for root length with the inoculated strains in the absence
of pesticide stress, however, Enterobacter aerogenes-W96 inoculation showed no increase
or decrease in the root length compared to uninoculated control. According to a recent
study, phosphate solubilizing Pseudomonas strain (B10) cause increment in root length
when used as a bio inoculant (Li et al., 2017). Ahemad and Khan (2012a) have also reported
the production of phytohormone in the presence and absence of pesticide by the isolated
bacteria. The combination of both pesticide caused reduction in root length by the majority
of the inoculations. The possible reason for decrease in plant growth is the decrease in
functionality of the organism in the presence of pesticides (Kumar et al., 2010; Ahemad
and Khan, 2012a). Significant increase in number of roots (23%) was observed with
Pseudomonas putida-Rad2 inoculation. A number of bacterial species belonging to
235
Pseudomonas, Serratia, Bacillus, Burkholderia, Arthrobacter, Alcaligenes, Enterobacter,
Klebsiella, Azotobacter and Azospirillum have been described to have involvement in
growth promotion of different plants (Ji et al., 2014; Afzal et al., 2017).
The agriculture sector is developing rapidly worldwide and is also addressing the toxicity
levels of pesticide compounds (Azizullah et al., 2011; Karpouzas et al., 2014). Chlorpyrifos
has gained great importance because of its vast use and potential toxicity in non-target
organisms (Yu et al., 2015). The production and yield of wheat crop (Triticum aestivum
L.) is affected by different pests. To protect the crops from potential pests, different
pesticides are routinely used in several regions of the world including Asia, Europe and
Africa (Pansa et al., 2015). Wang and Zhang (2017) have estimated the toxicity of
Chlorpyrifos in wheat plants by estimating salicylic acid estimation. Pyriproxyfen is an
insecticide which acts as an analogue of juvenile hormone in insects and affects their
growth (Ali et al., 2016). It was found that the inoculation with phosphate solubilizing
bacterial isolates stimulated the vegetative growth of wheat plants in different treatments.
When compared to uninoculated control plants, shoot length was significantly increased
by different inoculated strains in different treatments (Figure 8.17, 8.18, 8.19 and 8.20). In
the pesticide stress, a few inoculations showed a decrease in shoot length and the increase
was less than that of non-stressed conditions. Chlorpyrifos has been reported to negatively
affect the growth of wheat plants (Wang and Zang, 2017). Increased dry plant biomass has
also been reported by Namli et al. (2017) when wheat plants were inoculated with
phosphate solubilizing bacteria. Shoot dry weight was also found significantly increased
for up to 54% when inoculated with different bacterial genera in different growth
conditions in the presence of inorganic phosphate (aluminium phosphate, ferric phosphate
236
and tricalcium phosphate) supplemented soils and pesticide stress (Table 8.1). In a recent
study, it has been reported that a significant increase in shoot length and shoot dry weight
of wheat plant by two phosphate solubilizing strains (Pantoea cypripedii-PSB3 and
Pseudomonas plecoglossicida-PSB5) in natural soil.
In general, inoculation of the wheat plant with phosphate solubilizing bacteria lead to
increase in spike length in natural soil and inorganic phosphate supplemented soils. A
remarkable increase (92%) was recorded by the majority of the inoculations. Spike weight
was increased with all bacterial inoculated wheat plants in natural soil. However, the
addition of aluminium phosphate to soil also led to significant increase in spike weight (up
to 100%). Majeed et al. (2015) have reported a study related to phosphate solubilizing
Pseudomonas for enhanced plant growth in wheat. However, with the addition of pesticide,
reduction in spike weight was recorded.
The number of spikes per plant was increased generally in the absence as well as in the
presence of pesticide and aluminium phosphate and tricalcium phosphate. However, the
spike number decreased when ferric phosphate was added as the inorganic phosphate
source. Similarly, overall increase in number of spikelets per spike and the number of tillers
was recorded in all treatments. Similarly enhanced grain weight was recorded by different
bacterial inoculations in inorganic phosphate supplemented soil in the absence of pesticide
stress. However, decrease in grain weight was recorded by all bacterial inoculated plants
when aluminium phosphate was added along with pesticide stress. Similarly, Mukhtar et
al. (2017) reported 40-86% increase in grain yield when wheat plants were inoculated with
phosphate solubilizing bacterial isolates.
237
In soil, phosphate solubilizing bacteria are challenged with different ecological stresses.
As a result of these conditions, they have developed different mechanisms for their survival
(Liu et al., 2017). The potential to survive in these challenging situations helps their
survival and this idea is also useful that they can be utilized as biofertilizers in challenging
situations (de Oliveira-Longatti et al., 2014; Liu et al., 2017). Selected bacterial strains
were also subjected to different biochemical assay to measure the proline content, acid
phosphatase enzyme content and protein estimation of plant tissue with or without stress
in inorganic phosphate supplemented soil.
Chlorophyll ‘A’ content was found to remarkably increased by all bacterial inoculations
when soil was augmented with inorganic phosphates including aluminium phosphate,
ferric phosphate and tricalcium phosphate. Panhwar et al. (2011) have also reported the
increased chlorophyll content in plants inoculated with phosphate solubilizing bacteria in
green house conditions. Foliar use of Chlorpyrifos increased the proline content and
peroxidase enzyme production in mung bean plant (Parween et al., 2012). The present
study also showed that the proline content in wheat leaves was increased when plants were
treated with pesticides. However, the concentration of peroxidase enzyme in wheat leaves
was increased in non-stressed conditions in the presence of aluminium and tricalcium
phosphate whereas reverse was observed when ferric phosphate was added to soil. The
pesticide stress affect plant’s growth and it also alter plant biochemically as well as
physiologically which ultimately affect plant yield (Parween et al., 2016).
The symbiotic association between Arbuscular Mycorrhizal Fungi (AMF) and plants have
been reported (Zhang et al., 2016). As an obligatory biotrophs, AMF takes carbon
supplementation from the plant host and in its response, it provide different nutrients to
238
plants and among these nutrients, phosphate ions are the important molecules (Karasawa
et al., 2012; Zhang et al., 2015). In this regard, the microorganisms are known as the third
symbiont of AMF (Jansa et al., 2013). The hyphae are the channels for photosynthetase
enzyme from plants which attract different microorganisms and also help in their growth
and stimulation (Kaiser et al., 2015). Kaise et al. (2015) reported that the presence of AMF
in wheat rhizosphere improve the cycling of mineral nutrients. In the present study,
experiment was conducted to check the impact of hyphal growth of AMF on P
solubilization and P solubilization ability of bacteria as a result of PSB and AMF
interaction. In soil, AMF have extensive network of extraradial hyphae. These hyphae
accommodate different microorganisms (Gahan and Schmalenberger, 2015). Thus, there
may exist the corporation between the associated microorganisms and AMF. When the
impact of phosphate solubilizing bacteria on the pH of minimal growth medium of distal
compartment was checked, a slight change in pH of medium was recorded. The evidence
suggest that the bacteria do not affect the pH of medium in the absence of AMF and
inorganic phosphate source. However in the presence of AMF and inorganic phosphate
source, decrease in pH was observed by all strains.
When phosphate content of growth medium was observed, it was found that in the absence
of inorganic phosphate supplementation, phosphate content was increased by bacteria as
compared to un-inoculated control. According to the results of present study, arbuscular
mycorrhizal fungi (AMF), RiDAOM 19198 alone did not solubilized inorganic phosphate.
It has also been reported by Tisserant et al. (2013) that AMF are non-saprophytes and
cannot break down the organic nutrients directly. However, microbes have this ability and
they play major role in biogeochemical cycles.
239
The interactions of arbuscular mycorrhizal fungi and phosphate solubilizing bacteria was
also observed under microscope (Figure 9.11-9.15). Pseudomonas putida-Rad2 and
Enterobacter aerogenes-W96 showed good interaction and growth along with the
mycorrhizal hyphae. The analytical microscopic studies as well as the molecular biology
studies shows the colonization of different bacterial species on hyphal surfaces of AMF
and their spores (Scheublin et al., 2010; Agnoolicci et al., 2015).
Conclusion
In conclusion, the isolated strains are indigenous bacteria isolated from our agricultural
system and are compatible with environment and crops. These isolated phosphate
solubilizing bacterial inoculation can positively affect the growth of wheat plant. Use of
phosphate solubilizing bacteria (Ochrobactrum pseudogrignonense-S1, Acinetobacter
olivorans-S2, Pseudomonas putida-Rad2, Acinetobacter baumanii-JA10, Klebsiella
pneumoniae-R12, Enterobacter cloacae-W95, Enterobacter aerogenes-W96) is an
efficient and inexpensive way to enhance the availability of soluble phosphate to plant.
Moreover in the present study, most of the strains had good solubilization potential for
inorganic phosphate. The bacteria were able to tolerate higher concentration of
Chlorpyrifos and Pyriproxyfen and were also able to solubilizing inorganic phosphate in
their presence. The tolerance towards pesticides suggest that these isolates might be used
to study the bioremediation of pesticidal compounds. Furthermore, besides phosphate
solubilization, isolated bacteria also exhibited other plant growth promoting abilities.
Significant increase was shown by different strains for different parameters observed
during the study. In future it will be a good option to study the genetic mechanism of
phosphate solubilization by bacteria.
240
241
Chapter 11
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Appendix-I
Conference attended
Work Presented In International Conference
1. Presented this work in poster presentation in the Conference on Microbiology and
Molecular Genetics (MMG2018), organized by Department of Microbiology and
Molecular Genetics and Superior University on 7th to 9th February, 2018.
Appendix-II
Publication
Publication
From this research work following research papers has been published and accepted for
publication:
1. Iqra Murnir, Abida Bano and Muhammad Faisal. 2019. Impact of phosphate
solubilizing bacteria on wheat (Triticum aestivum) in the presence of pesticides. Brazilian
Journal of Biology. 79(1): 29-37.
2. Iqra Munir, Aqsa Zaheer and Muhammad Faisal. Diversity of Phosphate Solubilizing
Bacteria and their plant growth promoting attributes for the maintenance of sustainable
agriculture system. (in press).
Paper submitted
2. Iqra Munir, Naila Noreen and Muhammad Faisal. Isolation of phosphate solubilizing
rhizobacteria and its impact on the wheat (Triticum aestivum). Chemical Speciation and
Bioavailability.
3. Iqra Munir, Iqra Alauddin, Muhammad Farhan Nasir, Muhammad Faisal. Multifarious
beneficial attributes of phosphate solubilizing bacteria and pesticidal compounds affecting
Triticum aestivum. Punjab University Journal of Zoology.
Awards received during this research work
1. Awarded with Indigenous PhD scholarship (2012-2017) by HEC, Pakistan.
2. Awarded with IRSIP scholarship, by HEC, Pakistan for advanced research work in
Institut de Recherche en Biologie Végétales (IRBV), Département de sciences
Biologiques, Université de Montréal, Canada. 2016.
http://dx.doi.org/10.1590/1519-6984.172213Original Article
Brazilian Journal of BiologyISSN 1519-6984 (Print)ISSN 1678-4375 (Online)
Braz. J. Biol.2019, vol. 79, no. 1, pp.29-37 29/37 29
Impact of phosphate solubilizing bacteria on wheat (Triticum aestivum) in the presence of pesticides
I. Munira, A. Banoa and M. Faisala* aDepartment of Microbiology and Molecular Genetics, University of the Punjab – PU, Lahore, 54590,
Quaid-e-Azam Campus, Pakistan*e-mail: [email protected]
Received: November 19, 2016 – Accepted: May 5, 2017 – Distributed: November 30, 2018(With 3 figures)
AbstractThree phosphate solubilizing bacteria were isolated and identified by 16S rRNA sequencing as Pseudomonas putida, Pseudomonas sp and Pseudomonas fulva. The strains were subjected to plant biochemical testing and all the PGPR attributes were checked in the presence of pesticides (chlorpyrifos and pyriproxyfen). The phosphate solubilizing index of strain Ros2 was highest in NBRIP medium i.e 2.23 mm. All the strains showed acidic pH (ranges from 2.5-5) on both medium i.e PVK and NBRIP. Strain Ros2 was highly positive for ammonia production as well as siderophore production while strain Rad2 was positive for HCN production. The results obtained by the strains Rad1, Rad2 and Ros2 for auxin production were 33.1, 30.67 and 15.38 µg ml-1, respectively. Strain Rad1 showed 16% increase in percentage germination in comparison to control in the presence of pesticide stress. Most promising results for chlorophyll content estimation were obtained in the presence of carotenoids upto 6 mgg-1 without stress by both strains Rad1 and Rad2. Study suggests that especially strain Ros2 can enhance plant growth parameters in the pesticide stress.
Keywords: Pseudomonas, phosphate solubilizing bacteria, siderophore production, chlorpyrifos, pyriproxyfen.
Impacto das bactérias solubilizantes de fosfato no trigo (Triticum aestivum) na presença de pesticidas
ResumoTrês bactérias solubilizantes de fosfato foram isoladas e identificadas por seqüenciamento de rRNA 16S como Pseudomonas putida, Pseudomonas sp e Pseudomonas fulva. As estirpes foram submetidas a testes bioquímicos de plantas e todos os atributos PGPR foram verificados na presença de pesticidas (clorpirifos e piriproxifeno). O índice de solubilização de fosfato da estirpe Ros2 foi mais elevado no meio NBRIP, isto é, 2,23 mm. Todas as estirpes apresentaram um pH ácido (varia de 2,5-5) em ambos os meios, isto é PVK e NBRIP. A estirpe Ros2 foi altamente positiva para a produção de amoníaco, bem como a produção de sideróforos enquanto a estirpe Rad2 foi positiva para a produção de HCN. Os resultados obtidos pelas estirpes Rad1, Rad2 e Ros2 para a produção de auxina foram 33,1, 30,67 e 15,38 μg ml-1, respectivamente. A deformação Rad1 mostrou aumento de 16% na germinação percentual em comparação com o controlo na presença de stress de pesticida. Os resultados mais promissores para a estimativa do teor de clorofila foram obtidos na presença de carotenóides até 6 mgg-1 sem estresse por ambas as cepas Rad1 e Rad2. Estudo sugere que especialmente a estirpe Ros2 pode melhorar parâmetros de crescimento de plantas no estresse de pesticidas.
Palavras-chave: Pseudomonas, bactérias solubilizantes de fosfato, produção de sideróforos, clorpirifos, piriproxifena.
1. Introduction
Plant growth promoting rhizospheric bacteria has been used for decades in horticulture and agriculture for the improvement and productivity of crops. Biodiversity pays role in the maintenance of ecosystem, it includes variety of living organisms and genetic diversity of species (Alho, 2008). Some PGPR exert advantageous effects on the plants by the process of nitrogen fixation by delivering combined nitrogen to the plant (Jetiyanon, 2015). Microorganisms perform an important role in increasing
root growth, germination rate, leaf surface area water and mineral uptake percentages, crop yield and tolerance or resistance to stresses. Nitrifying and denitrifying microbes have great impact on ecosystem that it mediates nitrogen cycle (Medeiros et al., 2014). The most common bacterial genera involved in PGPR are Bacillus, Azospirillum and Burkholderia (Mangmang et al., 2015). Iron, nitrogen and phosphorous are important components for all life forms. Bacteria solubilize phosphorous which can be the substitute
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of synthetic phosphatic fertilizers (Ahemad and Kibret, 2014). Bacteria require iron for the chelation process and form siderophore complex. Phosphate solubilization and transport of ferric iron by siderophore released from PGPR increases the accessibility of different types of nutrients in the rhizosphere (Jetiyanon, 2015).
The area of soil near the root system is termed as rhizosphere (Ahemad and Kibret, 2014). Plants produce phytostimulators which increase the growth of plants, mostly cytokinins, indole-3-acetic acid (IAA), gibberellins, auxins and ACC deaminase (Abbamondi et al., 2016). Some microorganisms release 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme which produces α-ketobutyrate and ammonia from ACC. It decreases ethylene level in the plant which helps in the elongation of roots formation and improvement in seedlings survival. Indole-3-acetic acid (IAA) released from PGPR changes root growth and morphology as it facilitates plant’s nutrient uptake potentials (Jetiyanon, 2015). Usually the mechanisms used by the PGPR are for the enhancement of growth, but in stress conditions, strains mostly do not perform their functions efficiently because they have to compete in the harsh environment. However, many PGPR strains are able to tolerate the condition of stress and also have the ability to stimulate the growth of plants in stressful milieus (Parray et al., 2016). Microorganisms while living in pesticide stress, develop resistance to it. These microorganisms can effectively be used for bioremediation of pesticides contaminated sites along with their plant growth promoting attributes (Ahmad et al., 2015). Pesticides stress have been commonly applied to plants fruits, crops and vegetables for the protection all around the world. These pesticides due to persistence in the soil and also through direct exposure, harm the activities of bacteria which are able to solubilize phosphate. PSB Pseudomonas putida was observed for reduction in the phosphate (P) activity. Among all the pesticides, chlorpyrifos had minimum detrimental effect and can be used in the agricultural field (Kumar et al., 2015). The present study was conducted to evaluate the effect of pesticides on wheat. The pesticides chlorpyrifos and pyriproxyfen were chosen for this study as these were commonly used in Pakistan to control the pests, also it enhances crop productivity.
2. Materials and Methods
2.1. Sample collection and bacterial isolationDifferent soil samples were collected in the sterile bags
from the rhizosphere of different plants. After collection, these samples of soil were processed for the isolation of bacteria able to solubilize phosphate. After making serial dilutions the samples were spread on agar plates. The isolation of bacteria able to solubilize phosphate was done on Pikovskaya’s agar (PKV) (Pikovskaya, 1948).
Three phosphate solubilizing bacterial strains giving clear zones around the bacterial growth were selected for further analysis.
2.2. Identification by 16S rRNA gene sequencingThe 16S rRNA gene sequencing was carried out by
Macrogen, Korea. After trimming the sequences was BLAST analyzed (National Center for Biotechnology Information) in order to find the similarities between sequences. Phylogenetic trees were constructed by using MEGA 4 software by a neighbor-joining method, estimating the relationship between the halotolerant strains and reference strains.
2.3. Determination of phosphate solubilization index0.1 mL of each fresh bacterial culture was mixed
in sterile distilled water and was placed on Pikovskaya, agar plates (Pikovskaya, 1948) and incubated for 7 days. By using formula, solubilization Index was measured.
2.4. Phosphate estimationPVK medium and NBRIP liquid medium was used
for the estimation of the activity of bacterial strains for phosphate solubilization. 1 ml of 24 h bacterial suspension was added to each flask. All transfers of bacterial culture were carried out aseptically in triplicates and incubated at 28 °C on a shaker for 7 days. After centrifugation of samples, the supernatant was filtered and the filtrate was used for quantification of soluble Phosphate (Jackson, 1973)
2.5. Effect of phosphate solubilization on pH titrable acidity
pH meter was used to determine pH change in the medium after following 7 days incubation due to the growth of phosphate solubilizing bacteria. Seven days old bacterial cultures were checked for the titrable acidity and centrifugation of culture medium at 1000 rpm was carried for 10 minutes. Five millilitre of supernatant was titrated against 0.01N NaOH with a few drops of phenolphthalein indicator consumed per 5.0ml of culture filtrate.
2.6. PGPR Attributes
2.6.1. AmmonificationAmmonia production for all the isolates was checked
following manual methods (Cappuccino and Sherman, 2005). Freshly grown bacterial culture was inoculated in peptone broth and incubated at 30 ± 0.1 °C for 48 h in incubator shaking was done. 0.5 ml of Nessler’s reagent was added in the test tubes after incubation. Color change was observed from faint yellow to dark brown
2.6.2. Hydrogen cyanide productionProduction of hydrogen cyanide (HCN) from the
bacterial isolates was observed as per methodology observed by Castric and Castric (1983). Development of light brown color was observed which indicated positive results for HCN production.
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2.6.3. Siderophores productionChrome azurol sulfonate assay agar was used for
the siderophores production (Schwyn and Neilands, 1987). In order to observe qualitative assay, bacterial cultures were spotted onto the blue agar and incubated at 28 °C for 24-48 h. Color change was observed for the interpretation of results, ferric ion was transferred from strong blue complex to the siderophore. Yellow- orange zones around the growth indicated positive results for siderophore production.
2.6.4. Auxin estimationFresh bacterial cultures were inoculated in the fifty
milliliter of L-broth containing 0.1% L-tryptophan and incubated in shaker at conditions 30 ± 0.1 °C and 180 rpm for 48 h in the dark. Cultures were centrifugated at 10,000 rpm for 10 min at 4 °C. Colorimetric assay was used for the estimation of Indole-3-acetic acid (IAA) in the supernatants by adding 2 ml of Salkowski reagent in 1 ml of the supernatent. Pink color was observed and absorbance after 30 min at 535 nm in UV/Visible Spectrophotometer was read (Gordon and Weber, 1951). Regression equation was used to calculate IAA production from the standard curve and the result was expressed as µg ml-1 in comparison to control.
2.6.5. ACC deaminaseFollowing the method of (Penrose and Glick, 2003),
ACC deaminase activity for phosphate solubilizing bacteria was observed.
2.7. Effect of pesticide stress on root elongation assayThe effect of bacteria able to solubilize phosphate
on growth of wheat plant was observed under pesticide stress. For this purpose watman filter paper was fitted in the petri plates. After autoclaving plates were labelled for each strain and treatment. Inoculated seeds were evenly spread in petri plates by sterile forceps. All the plates were placed in the dark for three days. Germination was observed regularly. Plates seedlings were allowed to grow in the light after germination for 10 days and parameters were recorded.
2.8. Field experiment with wheat plantFresh fertile soil was taken from the field of New
Campus, University of the Punjab, Lahore, Pakistan. Soil was moist and brown in color. About 7-8 kg of soil was filled in each labeled pot properly. Soil was also mixed with a combination of pesticides as per recommended. Healthy seeds of Wheat (Triticum aestivum) were selected for experiment. Seeds were rinsed with tap water, discarded the floating (defective seeds) and proceeded with the healthy seeds. The seeds were dipped in 0.1% HgCl2 solution for 5 minutes and were 3-5 times washed with sterilized water for complete elimination of HgCl2 traces. The impact of phosphate solubilizing bacteria on wheat plant was observed under the stress condition i.e., pesticide stress. The three strains were observed with 3 treatment groups. Treatment group 1, 2 and 3. Treatment group 1 contained
pesticide chlorpyrifos, treatment group 2 contained pesticide pyriproxyfen while treatment group 3 contains the combination of both pesticides (chlorpyrifos and pyriproxyfen). All these 3 treatments were compared with the control in which no pesticide was used. Seeds were sown in pots labeled with respective strains. The clean conditions were maintained during sowing. Ten seeds per pot were sown with the help of forceps. The optical density of bacterial culture for inoculation was adjusted to 0.5 at 600nm and 10 ml of this bacterial suspension was added to soil. Pots were watered equally on daily basis up-to maturation of plants. Thinning of the plants was carried out after two months and only five plants which were left to grow in each pot were selected. After thinning plant material was used for biochemical analysis.
2.9. Plant biochemical assay
2.9.1. Chlorophyll contentFor the estimation of photosynthetic pigments 100 mg
of fresh leaf material was homogenized with acetone (80%). The homogenate volume was kept at 10 ml and filtered to remove the plant material. The absorbance of extracted pigments was read using UV spectrophotometer at different wavelengths (470, 645 and 663nm) following Arnon (1949) method.
2.9.2. Free Proline content determinationFresh leaves of wheat plant (Triticum aestivum) were
trimmed to smaller pieces. Proline concentration in leaves was determined by triturating frozen leaves of plants. Subsequently 500 mg of triturated leaf material was mixed in 5 ml of 3% sulfosalicylic acid. After centrifugation the supernatant was mixed in equal proportions (1:1:1) with glacial acetic acid and ninhydrin. After heating the reaction mixture at 100 °C in a boiling water bath for 60 min, the reaction mixture was cooled to room temperature and development of brick red colour was observed. Toluene was added to the above mixture and moved to a separating funnel and mixed thoroughly. Layer containing chromophore x was separated and its optical density at 520 nm was measured in comparison to blank. By taking 5-100 µg ml-1 concentration of standard proline, proline standard curve was set.
2.9.3. Peroxidase assayLeaf tissues (1 g) were crushed with 0.1 M phosphate
buffer (4 ml). The mixture of plant was centrifuged at 4 °C for 10 min at 14000 rpm. The enzyme estimation was carried out with the supernatant. Test and control reactions were conducted side by side by mixing 0.2ml enzyme extract with phosphate buffer. Guaiacol solution was added to test reaction and left at room temperature followed by addition of H2O2. Blank reaction was also performed and optical density of test and control reactions was read against blank. The enzyme units were calculated as methods were described by Racusen and Foote (1965) as units per gram.
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2.9.4. Acid phosphatase activityThe extraction of enzyme was carried out following
methods of Iqbal and Rafique (1987). Tris HCl buffer was used to crush the plant material and supernatant obtained was used for enzyme estimation. Acid phosphatase enzyme activity was determined at pH 4.9 for one hour at 37 °C. Series of reactions were carried out for qualitative estimation including control, test, blank and standard. Optical density was measured at 510nm wavelength against water and enzyme units were calculated.
2.9.5. Protein content estimationFor the estimation of soluble protein content of plant,
plant material was homogenized with phosphate buffer using pestle ad mortar and ratio of buffer to plant was 4:1. Centrifugation of samples was performed at 4 °C for 10 min at 14000 rpm. Supernatant (0.4ml) was mixed with Folin’s mixture (2ml) followed by the addition of Ciocalteu’s phenol and Folin reagent after 15 minutes at room temperature. The tubes were placed for 45 minutes at room temperature, after observing color change optical density was read at 750nm. Results were calculated with the help of standard curve.
3. Results
3.1. Isolation and 16S rRNA sequencing of BacteriaDifferent samples of soil from the rhizosphere of
vegetables were collected, bacterial strains able to solubilize phosphate were isolated. Three strains Rad1, Rad2, Ros2 gave promising results as compared to control for phosphate solubilization on PVK as well as NBRIP medium as shown in Table 1. The strains after isolation were directly sent for 16S rRNA sequencing using the service of Macrogen, Korea. The sequences retrieved after sequencing were BLAST analyzed and the homology was checked with the test organisms. Analysis showed that the strains belong to Pseudomonas genera (as shown in Table 2).
3.2. Phosphate solubilization potentialPhosphate solubilizing potential was measured for the
isolated strains as described below.
3.2.1. Determination of phosphate solubilization indexThe bacterial strains were checked for phosphate
solubilization potential by observing clear zones around the bacterial colony as compared to negative control which gave no zone around the growth of bacteria. The strain Rad1 gave zone of 2.27 mm on PVK and 2.5 mm on NBRIP medium. Strain Rad2 gave good result on NBRIP medium i.e zone of 2.84 mm while on PVK medium it gave 2.42 mm. Strain Ros2 gave good result on PVK medium i.e zone of 2.6 mm was observed, while on NBRIP medium zone of 2.23 mm was observed (as shown in Table 1).
3.2.2. Phosphate estimationThe three phosphate solubilizing strains were checked
for P estimation and were compared with the control. Strain Rad1 gave good results in NBRIP medium i.e 966 µg ml-1 while in PVK medium 126.3 µg ml-1 was observed. The most promising results were observed by strain Rad2 in NBRIP medium i.e 1163.1 µg ml-1 while in PVK medium 347.4 µg ml-1 was observed. Strain Ros2 gave values 955.6 and 648.3 µg ml-1 in NBRIP medium and PVK medium, respectively (as shown in Table 1).
3.2.3. Effect of phosphate solubilization on pH and titrable acidity
The effect of phosphate solubilization was checked on PVK medium and also in NBRIP medium and was compared with the control. The titrable acidity value for Rad1 in PVK and NBRIP medium observed was 17.2 and 21.3, respectively. Strain Rad2 and Ros2 showed decreased titrable acidity on PVK and increased titrable acidity on NBRIP medium, respectively. Strain Ros2 gave promising results in NBRIP medium (as shown in Table 1).
Table 2. Identification of phosphate solubilizing bacterial strains.Strain code Isolation source Location Identified organism Accession No.
Rad1 Raphanus sativus Lahore Pseudomonas putida KP241947Rad2 Raphanus sativus Lahore Pseudomonas sp KX345931Ros2 Rosa indica Lahore Pseudomonas fulva KX345930
Table 1. Characterization of phosphate solubilizing bacteria for solubilization potential.
Bacterial strain
Solubilization index P estimation Titrable acidity pH(µg/ml)PVK NBRIP PVK NBRIP PVK NBRIP PVK NBRIP
Control - - - - - - 7.0 7.0Rad1 2.27 2.5 126.3 966.0 17.2 21.3 4.4 5.0Rad2 2.42 2.84 347.4 1163.1 8.0 22.5 4.3 4.9Ros2 2.6 2.23 648.3 955.6 19.6 24.5 4.2 4.7
PVK: Pikovskaya agar; NBRIP: National Botanical Research Institute Phosphate.
Impact of phosphate solubilizing bacteria on wheat
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3.2.4. Effect of phosphate solubilization on pHThe negative log of hydrogen ion (pH) concentration
was checked for the phosphate solubilizing bacteria. pH was measured by using pH meter. Strains were compared with the control pH i.e 7. Strains were highly acidic on both media i.e PVK and NBRIP (as shown in Table 1).
3.3. PGPR Attributes
3.3.1. Ammonia productionPhosphate solubilizing bacterial strains were checked
for ammonia production i.e., development of the brown color was observed by the strains and was compared with the control in which no color change was observed after incubation. Strain Rad1 and Rad2 showed positive results for ammonia production whereas strain Ros2 showed highly positive results for ammonia production (as shown in Table 3).
3.3.2. HCN productionAll the three strains were also evaluated for HCN
production ability. The color change of filter paper by the strains was observed (i.e., yellow to orange). Strain Rad1 showed slight activity while strain Rad2 was highly positive for HCN production. Strain Ros2 showed negative results (as shown in Table 3).
3.3.3. Siderophore productionFor siderophore production, method of Schwyn and
Neilands (1987) was followed and bacteria were grown on blue agar. The strains were evaluated for siderophore production ability. Strains Rad1 and Rad2 were negative for siderophore production while strain Ros2 was highly positive for siderophore production (as shown in Table 3).
3.3.4. Auxin estimationThe strains were evaluated for auxin production in
the L- tryptophan presence. Production of pink color was observed after incubation, by the strain. The results obtained by the strains Rad1, Rad2 and Ros2 were 33.1, 30.67 and 15.38 µg ml-1 auxin, respectively. Maximum auxin production was observed by strain Rad1 (as shown in Table 3).
3.3.5. ACC deaminase activityThe phosphate solubilizing bacterial strains were
evaluated for ACC deaminase production. Strains Rad1, Rad2 and Ros2 showed ACC deaminase activity of 0.359, 0.301 and 0.278 mmol ml-1, respectively. The strain Ros2
showed decreased activity of ACC deaminase. Maximum activity was observed by strain Rad1 (as shown in Table 3).
3.4. Effect of pesticide stress on root elongation assayMaximum increase (16%) in the percentage
germination under pesticide stress was observed by strain Rad1 as compared to its respective noninoculated control (see Figure 1a). With the application of strains and independent or combination of fertilizers to wheat showed significant increase in its growth as compared to noninoculated control. 88% increase in root length and 33% increase in shoot length of wheat was observed in the presence of strain Ros2 and treatment group 2 in comparison to control. (see Figure 1b and 1c). There was significant 6% increase in no. of roots of wheat by strain Rad2 and treatment group 1 (see Figure 1d).
3.5. Plant biochemical tests
3.5.1. Acid phosphatase testStrain Rad2 showed good results as compared to control
with stress and without stress i.e., 0.7 and 0.5 K.A units 100ml-1, respectively. Strain Rad1 showed no prominent results as compared to control. Strain Ros2 showed decreased activity i.e., 0.15 K.A units 100 ml-1 with stress and 0.4 K.A units 100 ml-1 without stress (see Figure 2a).
3.5.2. Chlorophyll content estimationReduction in chlorophyll content was observed for
isolates Rad1, Rad2 and Ros2 under pesticide stress as compared to noninoculated control. Strains Rad1 and Rad2 were found to be the most effective isolates which gave values upto 6 mgg-1 for carotenoids over noninoculated control (see Figure 3).
3.5.3. Peroxidase testPeroxidase activity increased in stress conditions in
case of all three strains. The most promising results were obtained by strain Rad2 i.e., 72 unit gram-1 without stress and 79 unit gram-1 with stress. Strain Ros2 showed results 60 unit gram-1 without stress and 73 unit gram-1 with stress. Strain Rad1 showed 58 unit gram-1 without stress and 69 unit gram-1 with stress (see Figure 2b).
3.5.3. Proline content estimationAll the strains showed decreased proline activity as
compared to control. There was an increased activity of proline with stress by all the strains as compared to the treatment without stress (see Figure 2c).
Table 3. Plant growth promoting activities of phosphate solubilizing bacteria.Bacterial
strainAmmonia production HCN Siderophore
productionAuxin estimation ACC deaminase
(µg/ml) (mmol/ml)Rad1 + + - 33.1 0.359Rad2 + +++ - 30.67 0.301Ros2 ++ - +++ 15.38 0.278
+ slight; ++ moderate; +++ strong.
Munir, I., Bano, A. and Faisal, M.
Braz. J. Biol.2019, vol. 79, no. 1, pp.29-3734 34/37
Figure 1. Effect of phosphate solubilizing Pseudomonas inoculation and pesticide stress on germination of Triticum aestivum (a) percentage germination, (b) root length, (c) shoot length, (d) number of roots.
Figure 2. Effect of phosphate solubilizing Pseudomonas strain inoculation (a) acid phosphatase activity, (b) peroxidase activity, (c) proline concentration, (d) protein estimation of Triticum aestivum with and without pesticide stress.
Impact of phosphate solubilizing bacteria on wheat
Braz. J. Biol.2019, vol. 79, no. 1, pp.29-37 35/37 35
3.5.4. Protein estimationProtein concentration was estimated for the wheat plant
treated with pesticide stress in the presence of bacterial inoculum and the results were compared with control. Soluble protein content increased under pesticide stress i.e., 55 mg g-1 by strain Rad1 and 110 mg g-1 by strain Ros2 as compared to its control while protein concentration was decreased in the stress condition i.e 100 mg g-1 by strain Rad2 as compared to its control. (see Figure 2d).
4. Discussion
Microorganisms are present in different habitats like air, soil and water. They interact differently with host microorganisms, other microorganisms and their physiochemical environment (Reche and Fiuza, 2005). Isolation of bacteria able to solubilize phosphate from the rhizosphere of plants Rhapanus sativus and Rosa indica was done and were identified as Pseudomonas putida, Pseudomonas sp. and Pseudomonas fulva. These bacterial strains were tested in the presence of pesticide (pyriproxyfen and chlorpyrifos). Field trial was performed in the Microbiology and Molecular Genetics Department with the wheat (Triticum aestivum) plant under the stress condition. Bacterial strains P. aeruginosa, P. stutzer, P. chlororaphis and P. fluorescens are non-pathogenic biocontrol agents, also show plant growth-promoting activities (Parray et al., 2016). Pseudomonas genus give plant protection against pests, plant growth stimulation or bioremediation (Daval et al., 2011).
Phosphate solubilizing Strain Rad1 gave good results for solubilized phosphate estimation in NBRIP medium i.e., 966 µg ml-1 while in PVK medium 126.3 µg ml-1 was observed. The most promising results were observed by strain Rad2 in NBRIP medium i.e., 1163.1 µg ml-1 while in PVK medium 347.4 µg ml-1 of solubilized phosphate was recorded. All the strains showed decrease in pH after inoculation and phosphate solubilization in both medium i.e., PVK and NBRIP. Decrease in the pH after solubilization of phosphate in PVK growth medium was also reported. pH reduction indicates the production of different types
of acids like gluconic acid, citric and propionic acid etc. and acidification caused by metabolic processes play important role in phosphate solubilization (Kumar et al., 2016). According to the study conducted by Rodríguez and Fraga (1999), similar results have been reported where Rhizobia, Bacillus and Pseudomonas were able to solubilize phosphate.
According to the study done by El-Azeem et al. (2007), insoluble mineral phosphate solubilization between the range of 1.53 to 360 µg mL-1 and also the pH reduction from normal value of 7.1 to the range between 4.16 and 6.45 was reported. Strain Rad1 and Rad2 showed positive results for ammonia production while strain Ros2 showed strong positive results for ammonia production. Strain Rad1 showed slight activity while strain Rad2 was highly positive for HCN production. Strain Ros2 was highly positive for siderophore production. For IAA the results obtained by the strains Rad1, Rad2 and Ros2 were 33.1, 30.67 and 15.38 µg ml-1, respectively. Study showed low production of IAA by Pseudomonas sp. i.e., below 40.0 µg mL-1 (Anjum et al., 2011). The Solubilization Index (SI) was 2.7 for both of the strains of Pseudomonas (Ehsan et al., 2016). ACC deaminase activity observed by the strains Rad1, Rad2 and Ros2 showed results 0.359, 0.301 and 0.278 mmol ml-1, respectively. The strain Ros2 showed decreased activity of ACC deaminase. Study conducted by Ehsan et al. (2016) showed production of IAA by Pseudomonas strains in the range 54% to 88%.
In the presence of treatment group 3, strains Rad1, Rad2 and Ros2 showed percentage germination 16%, 3% and 12%. Strain Rad1 showed promising results in the presence of all the three treatment groups. When treatment group 1 was applied strain Ros2 showed 48% increase in root length while strains Rad1 and Rad2 showed no significant increase in root length. In the presence of treatment group 3, strains Rad1, Rad2 and Ros2 showed 13%, 11% and 6% increase in shoot length. The strains activity was compared with the control. Strain Rad2 showed good results for phosphatase as compared to control with stress and without stress i.e., 0.7 K.A units 100 ml-1 and 0.5 K.A units 100 ml-1, respectively. Most promising results were obtained for carotenoids production upto 6 mg g-1 without stress by strains Rad1 and Rad2. Under stress conditions all the strains (Rad1, Rad2 and Ros2) produced carotenoids for upto 3.5, 3.6 and 3.2 mg g-1 of fresh weight, respectively. Strain Ros2 showed results 60 unit gram-1 without stress and 73 unit gram-1 with stress. Strain Rad1 showed 58 unit gram-1 without stress and 69 unit gram-1 with stress for peroxidase activity. Increase activity of proline was observed with stress by all the strains as compared to without stress. Strain Rad2 gave most promising results 120 mg g-1 without stress and 100 mg g-1 with stress for protein estimation. Strain Ros2 gave results 90 mg g-1 without stress and 110 mg g-1 with stress. Use of pesticides help in causing bacteriostatic effect on microorganisms (Botelho et al., 2012). According to the study, the maximum toxicity by pesticide pyriproxyfen was observed to nodule numbers in pea, root and shoot dry
Figure 3. Effect of phosphate solubilizing Pseudomonas strain inoculation on chlorophyll content of Triticum aestivum with and without pesticide stress.
Munir, I., Bano, A. and Faisal, M.
Braz. J. Biol.2019, vol. 79, no. 1, pp.29-3736 36/37
biomass, shoot nitrogen and root phosphorus in greengram, leghaemoglobin, seed protein and chlorophyll content in chickpea. Root nitrogen, odule biomass root phosphorus, shoot phosphorus effects were also observed (Ahemad and Kibret, 2014).
Acknowledgements
This work was supported by Higher Education Commission (HEC) and Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan.
References
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Subject: Fw: Manuscript PJOES-00769-2017-02
From: [email protected]: [email protected]
Date: Friday, August 25, 2017, 11:07:02 AM GMT+5
Dr. Muhammad Faisal
Assistant ProfessorDepartment of Microbiology & Molecular Genetics
University of the Punjab Lahore, Pakistan.
Email: [email protected] Phone: +92-42-99238531
Cell: +92-346-5734332Fax: +92-42-9231879 On Tuesday, August 22, 2017 7:13 PM, Polish Journal of Environmental Studies <[email protected]> wrote:
Dear Dr Muhammad Faisal ,
I am pleased to inform you that your manuscript, entitled: Diversity of Phosphate SolubilizingBacteria and their plant growth promoting attributes for the maintenance of sustainable agriculturesystem, has been finally accepted for publication in our journal.
Thank you for submitting your work to our Journal and fruitful co-operation.
With kind regards,
Professor Hanna Radecka
Executive Editor
Professor Jerzy Radecki Editor – in – Chief
Polish Journal of Environmental Studies www.pjoes.com
Appendix-III
Soil Properties
Ta
ble: P
hysical an
d ch
emical p
rop
erties of so
il ino
culated
with
ph
osp
hate so
lub
ilizing b
acteria, ino
rgan
ic ph
osp
hate so
urce an
d p
esticide stress.
S
oil S
am
ple
Electrica
l
Co
nd
uctiv
ity
(mS
cm-1)
pH
Org
an
ic
ma
tter
(%)
Av
aila
ble
ph
osp
ho
rou
s
(mg k
g-1)
Av
aila
ble
po
tassiu
m
(mg k
g-1)
Sa
tura
tion
(%)
Tex
ture
No
n
stressed
Un
ino
culated
con
trol
1.3
8
.4
0.4
8
1.0
7
4
42
lo
amy
PS
B in
ocu
lated
1.3
8
.4
0.4
4
1.0
1
06
4
4
loam
y
Un
ino
culated
con
trol +
AL
P
1.4
8
.3
0.3
8
1.0
1
48
4
2
loam
y
PS
B in
ocu
lated +
AL
P
1.4
8
.1
0.3
0
1.0
1
74
4
2
loam
y
Un
ino
culated
con
trol +
FP
1
.4
7.9
0
.48
2
.0
13
8
40
lo
amy
PS
B in
ocu
lated +
FP
1
.3
8.2
0
.58
1
.0
14
0
42
lo
amy
Un
ino
culated
con
trol +
TC
P
1.3
8
.3
0.9
0
2.0
1
38
4
2
loam
y
PS
B in
ocu
lated +
TC
P
1.5
8
.1
0.3
4
2.0
1
68
4
0
loam
y
Pesticid
e
stressed
Un
ino
culated
con
trol
2.3
8
.2
0.5
6
2.0
1
32
4
0
loam
y
PS
B in
ocu
lated
1.3
8
.2
0.7
2
1.0
1
74
4
2
loam
y
Un
ino
culated
con
trol +
AL
P
1.3
8
.0
0.4
8
2.0
1
55
4
2
loam
y
PS
B in
ocu
lated +
AL
P
1.2
7
.9
0.8
2
2.0
1
15
4
2
loam
y
Un
ino
culated
con
trol +
FP
1
.5
7.9
0
.54
1
.0
18
8
42
lo
amy
PS
B in
ocu
lated +
FP
1
.3
7.8
0
.56
1
.0
10
4
42
lo
amy
Un
ino
culated
con
trol +
TC
P
1.2
7
.8
0.4
8
2.0
1
74
4
2
loam
y
PS
B in
ocu
lated +
TC
P
1.3
7
.8
0.3
6
2.0
9
8
42
lo
amy
PS
B=
Ph
osp
hate so
lub
ilizing b
acteria (S2
); AL
P=
Alu
min
ium
ph
osp
hate; F
P=
Ferric p
ho
sph
ate; TC
P=
Tricalciu
m p
ho
sph
ate
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