phosphate solubilizing bacteria: their isolation

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 2.46 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



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



83

Figure 4.5 Neighbor joining phylogenetic tree of Ros1 strain, constructed



84

Figure 4.6 Neighbor joining phylogenetic tree of Ros2 strain, constructed



84

Figure 4.7 Neighbor joining phylogenetic tree of JA10 strain, constructed



85

Figure 4.8 Neighbor joining phylogenetic tree of R12 strain, constructed



85




86




86

Figure 4.11 Neighbor joining phylogenetic tree of SL8 strain, constructed



87

Figure 4.12 Neighbor joining phylogenetic tree of M6 strain, constructed


obtained from NCBI nucleotide data base

87

viii

Figure 4.13 Neighbor joining phylogenetic tree of L6 strain, constructed



88




88




89




89

Figure 4.17 Neighbor joining phylogenetic tree of SF strain, constructed



90

Figure 4.18 Neighbor joining phylogenetic tree of SpA strain, constructed



90

Figure 4.19 Neighbor joining phylogenetic tree of CS1 strain, constructed



91




91

Figure 4.21 Neighbor joining phylogenetic tree of S62 strain, constructed



92

Figure 4.22 Neighbor joining phylogenetic tree of W94 strain, constructed



92

ix




93




93

Figure 4.25 Neighbor joining phylogenetic tree of P1 strain, constructed from



94

Figure 4.26 Neighbor joining phylogenetic tree of U.P strain, constructed



94

Figure 4.27 Neighbor joining phylogenetic tree of C14 strain, constructed



95

Figure 4.28 Neighbor joining phylogenetic tree of C50 strain, constructed



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


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


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


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


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


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


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


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


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



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



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




tricalcium phosphate. The graph shows the mean ± standard



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



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




with ferric phosphate. The graph shows the mean ± standard



189




with tricalcium phosphate. The graph shows the mean ± standard



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).







190




with ferric phosphate. The graph shows the mean ± standard



191




with tricalcium phosphate. The graph shows the mean ± standard



191

Figure 8.13 Effect of phosphate solubilizing bacterial inoculation on protein


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





192

xv





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




tricalcium phosphate. The graph shows the mean ± standard



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



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



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



19198 on P solubilization in minimal growth medium. Plates


Error bars Mean ± standard error (n=5). Different letters on bars



215



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


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



221


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


Pseudomonas aeruginosa (SpA) with Arbuscular Mycorrhizal




223


Enterobacter aerogenes (W96) with Arbuscular Mycorrhizal




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)


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)


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)


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)


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)


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


1 Potassium iodide 2.0 g

2 Iodine 1.0 g


Table 2.9: Decolorizer

S.

No.

Components Quantity (100 mL-1)

1 Ethyl alcohol 95 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


Reagent for Catalase Test

Table 2.11: 3% hydrogen peroxide (H2O2) (Cappuccino and Sherman, 2005)


1 Hydrogen Peroxide 0.3 mL


Reagent for Cytochrome Oxidase Test

Table 2.12: 1% Oxidase reagent (Tetramethyl-para-phenylenediamine dihydrochloride)


1 Oxidase Reagent 0.1 g


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)


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)


1 95% ethanol 10 mL

2 Methyl red 0.003 g

Table 2.16: Barritt’s reagent for Voges Proskauer test

Solution A:


1 Absolute ethanol 95 mL

2 α-naphthol 5 g

Solution B:


1 Potassium hydroxide 40 g


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


100 ml-1 1 N-(1-Naphthyl)-ethylenediamine

Dihydrochloride

0.6 g

2 Acetic Acid (5 N) 100 mL

Table 2.19: Sulfanilic acid


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


Medium for Pigment Production (King et al., 1954)

Table 2.21: King’s A medium


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


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)


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)


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)


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)


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


1 Ammonium molybdate 7.5 g

2 Concentrated H2SO4 162.0 mL

Table 2.29: Chlorostanous acid


1 Stannous chloride 25.0 g

2 Concentrated H2SO4 100.0 mL

Table 2.30: Phosphate standard


1 KH2PO4 100 mg


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


1 Ethanol 50 mL


17

Table 2.33: 0.5% Phenolphthalein


1 Phenolphthalein 0.5 g

2 50% ethanol 100 mL

Table 2.34: 8.4% Ammonium hydroxide


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


1 p-nitrophenyl phosphate disodium (PNPP) 42.68 g

Table 2.36: 0.5 M sodium acetate buffer


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


1 CaCl2 55.49 g

Table 2.38: 0.5 M NaOH


1 NaOH 19.99 g

PLANT GROWTH PROMOTING ACTIVITIES

Solutions for Auxin Estimation (Brick et al., 1991)

Table 2.39: L-tryptophan stock solution


1 L-Tryptophan 1 g

18

2 Autoclaved distilled water 40 mL

Table 2.40: Solution I: 0.05M ferric chloride solution


1 FeCl3 0.08125 g

Table 2.41: Solution II: Perchloric acid


1 HClO4 50 mL

Table 2.42: Salkowski’s reagent


1 0.05M FeCl3 1 mL

2 35% HClO4 50 mL

Siderophores Production (Louden et al., 2011)

Table 2.43: Blue dye solution 1


1 Chrome Azurol S (CAS) 0.06 g

2 Double distilled water 50 mL



1 FeCl3-6H2O 0.0027 g

2 10 mM HCl 10 mL



1 Hexadecyltrimethylammonium bromide

(HDTMA)

0.073 g


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


Table 2.47: 20% Glucose stock


1 Glucose 20 g


Table 2.48: NaOH stock


1 NaOH 25 g


pH adjusted to ~12

Table 2.49: Casamino acid solution


1 Casamino acid 3 g


Extracted with 3% 8-hydroxyquinoline in chloroform to remove any trace iron and

filter sterilized

Table 2.50: CAS agar


- 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)


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


1 Peptone 4 g


Table 2.54: Nessler’s reagent (for ammonia detection)


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)


- 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)


- 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)


- 1 ACC 10.1 g


22

REAGENTS FOR PLANT MICROBE INTERACTION EXPERIMENT

Table 2.59: 0.1% HgCl2 solution for seed sterilization


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


1 Acetone 80 mL


Proline Estimation

Table 2.62: 3% Sulfosalycylic acid


1 Sulphosalycylic acid 3 g

Table 2.63: Orthophosphoric acid (6N)


1 Orthophosphoric acid 38.1 mL

2 Distilled water 61.9 mL

Table 2.64: Acid ninhydrin reagent


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)


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


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


1 C6H8O7.H2O (Citric acid) 42.0

2 NaOH (1N) 376 mL

pH was adjusted to 4.9

Table 2.70: Disodium phenyl phosphate


1 C6H5PO4Na2 (Di-sodium Phenyl

Orthophosphate)

2.54 g

Solution was cooled immediately after boiling.

Table 2.71: Phenol standard (stock)


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


1 NaOH 20.0 g

Table 2.74: 0.5N Sodium bicarbonate solution


1 NaHCO3 42.0 g

Table 2.75: 4-Amino antipyrin


1 4-amino antipyrin 6.0 g

25

Table 2.76: Potassium ferricyanide


1 Potassium ferricyanide 24.0 g

Solutions for Soluble Protein Estimation (Lowry et al., 1951)

Folin’s mixture

Table 2.77: Solution A


1 NaOH 4.0 g

2 Na2CO3 20.0 g

Table 2.78: Solution B


1 C4H4KNaO6.4H2O 4.0 g

Table 2.79: Solution C


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


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



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




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



22 W94 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

(■).




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


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


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 (▲).


84

Figure 4.5: Neighbor joining phylogenetic tree of Ros1 strain, constructed from 16S rRNA


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


Figure 4.6: Neighbor joining phylogenetic tree of Ros2 strain, constructed from 16S rRNA


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


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


Figure 4.8: Neighbor joining phylogenetic tree of R12 strain, constructed from 16S rRNA


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 (▲).


86




strain clustered together with the 16S rRNA gene sequences of Pseudomonas

plecoglossicida (▲). The 16S rRNA gene of Escherichia coli was used as out-group (■).




strain clustered together with the 16S rRNA gene sequences of Pseudomonas aeruginosa


87

Figure 4.11: Neighbor joining phylogenetic tree of SL8 strain, constructed from 16S rRNA


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


Figure 4.12: Neighbor joining phylogenetic tree of M6 strain, constructed from 16S rRNA


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


88

Figure 4.13: Neighbor joining phylogenetic tree of L6 strain, constructed from 16S rRNA


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 (▲).







89




strain clustered together with the 16S rRNA gene sequences of Pseudomonas koreensis





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


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


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


91

Figure 4.19: Neighbor joining phylogenetic tree of CS1 strain, constructed from 16S


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 calcoaceticus


92






Figure 4.22: Neighbor joining phylogenetic tree of W94 strain, constructed from 16S


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


93




strain clustered together with the 16S rRNA gene sequences of Enterobacter cloacae (▲).





strain clustered together with the 16S rRNA gene sequences of Enterobacter aerogenes


94

Figure 4.25: Neighbor joining phylogenetic tree of P1 strain, constructed from 16S rRNA


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


Figure 4.26: Neighbor joining phylogenetic tree of UP strain, constructed from 16S rRNA


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 (▲).


95

Figure 4.27: Neighbor joining phylogenetic tree of C14 strain, constructed from 16S rRNA


MEGA 7 software was used with the boot strap value based on 1000 repetitions. The C14



Figure 4.28: Neighbor joining phylogenetic tree of C50 strain, constructed from 16S rRNA


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


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,


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,


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


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.

149

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


Sh

oot

Len

gth

(cm

)

0

2

4

6

8

aabab ab ab ab

abc abcabcdabcd

abcd

abcdebcdef

bcdef cdefdefdef

ef eff

Bacterial strains


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


Bacterial Strains


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


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


156


Bacterial strain


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


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


157


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,


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


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


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


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


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


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





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





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


wheat plants in the presence of pesticide in soil amended with tricalcium phosphate. The



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 ±


(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





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


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


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


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 ±


(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





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





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


wheat plants in the presence of pesticide in soil amended with tricalcium phosphate. The



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.

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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.

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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

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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

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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).

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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.

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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


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


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


pH

0

2

4

6

8a

aa

a

a

a


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


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


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


So

lub

iliz

ed

P (

g m

L-1

)

0

5

10

15

20

25

30

35

a

a

aa

b b


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


So

lub

iliz

ed

P (

g m

L-1

)

0

20

40

60

80

100

120

140

160

a

b

c

d

e

e


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

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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

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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.

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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


220

Figure 9.12: Positive interaction between phosphate solubilizing Pseudomonas putida

(Rad2) (arrow) with Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on minimal



221

Figure 9.13: Positive interaction between phosphate solubilizing Acinetobacter baumanii

(JA10) (arrow) with Arbuscular Mycorrhizal Fungi (AMF), RiDAOM 19198 on minimal



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



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


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.

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

Munir, I., Bano, A. and Faisal, M.

Braz. J. Biol.2019, vol. 79, no. 1, pp.29-3730 30/37

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.

Impact of phosphate solubilizing bacteria on wheat


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.



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.



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.



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.



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.



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.

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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

phosphate solubilizing bacteria: their isolation

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