GENETIC DIVERSITY AND BENEFICIAL ROLE OF PLANT GROWTH...

GENETIC DIVERSITY AND BENEFICIAL ROLE OF PLANT

GROWTH PROMOTING RHIZOBACTERIA IN OIL SEED

PRODUCING SUNFLOWER (Helianthus annuus L.) CROP OF

AZAD JAMMU AND KASHMIR

AFSHAN MAJEED

(Regd. No. 2001-URTB-3162)

Session 2009-2012

Department of Soil and Environmental Sciences

Faculty of Agriculture Rawalakot

University of Azad Jammu & Kashmir Muzaffarabad, Azad Kashmir, Pakistan

GENETIC DIVERSITY AND BENEFICIAL ROLE OF PLANT

GROWTH PROMOTING RHIZOBACTERIA IN OIL SEED

PRODUCING SUNFLOWER (Helianthus annuus L.) CROP OF

AZAD JAMMU AND KASHMIR

by

AFSHAN MAJEED

(Regd. No. 2001-Urtb-3162)

A Thesis

Ph. D

Submitted in partial fulfilment of the requirements for the degree of

Doctor of Philosophy

in

Soil & Environmental Sciences

Session 2009-2012


Faculty of Agriculture Rawalakot

University of Azad Jammu & Kashmir Muzaffarabad, Azad Kashmir, Pakistan

CERTIFICATION

Certified that the contents and form of the thesis entitled “Genetic diversity and

beneficial role of plant growth promoting rhizobacteria in oil seed producing Sunflower

(Helianthus annuus L.) crop of Azad Jammu and Kashmir” submitted by Ms. Afshan

Majeed have been satisfactory for the requirement of the degree.

Supervisor: _______________________

Co-supervisor: _______________________

Member: _______________________

Member: _______________________

Chairman


Dean Director

Faculty of Agriculture Advanced Studies & Research

i

Dedication

I dedicate this dissertation to my mother Mrs. Zubeda Majeed for her

constant, unconditional love and support. I also dedicate it to the loving

memory of my father Sardar Abdul Majeed Khan

ii

Table of Contents

Dedication ........................................................................................................................... i

Table of Contents ............................................................................................................. ii

List of Figures ................................................................................................................. vii

List of Tables .................................................................................................................. viii

List of Abbreviations and Symbols .............................................................................. xi

Acknowledgments ......................................................................................................... xiv

Abstract........................................................................................................................... xvi

Chapter 1 ............................................................................................................................ 1

INTRODUCTION ............................................................................................................ 1

PLANT-MICROBE INTERACTIONS ............................................................... 1

1.1.1 The Rhizosphere ................................................................................................ 1

PLANT GROWTH PROMOTING RHIZOBACTERIA (PGPR)-DRIVERS OF

AGRICULTURAL SUSTAINABILITY ............................................................. 2

1.2.1 Host Specificity ................................................................................................. 4

PLANT INFLUENCE ON MICROBES.............................................................. 5

1.3.1 Plant Attributes of PGPR/the Rhizosphere Microorganisms – What are They

Doing? .............................................................................................................. 5

1.3.2 Rhizospheric Microbes; the Good .................................................................... 6

1.3.3 Rhizospheric Microbes; the Bad .................................................................... 32

COLONIZATION OF PGPR IN DIFFERENT COMPARTMENTS OF

PLANTS ............................................................................................................... 33

iii

1.4.1 Fluorescent Antibodies ................................................................................... 34

1.4.2 Specific Primers and Oligonucleotidic Probes .............................................. 35

1.4.3 Fluorescent Markers ........................................................................................ 35

1.4.4 Confocal Laser Scanning Microscope (CLSM) ............................................ 36

1.4.5 Electron Microscopy (EM) ............................................................................. 37

BIOFILM-THE CITY OF MICROBES ............................................................ 38

EFFECT OF PGPR ON PLANT GROWTH ..................................................... 40

SUNFLOWER ..................................................................................................... 43

MATERIALS AND METHODS ................................................................................. 46

2.1.1 Collection of Soil Samples ............................................................................. 46

2.1.2 Meteorological Data of Selected Sites ........................................................... 46

2.1.3 Soil Analysis .................................................................................................... 46

ISOLATION, BIOCHEMICAL AND MOLECULAR

CHARACTERIZATION OF SUNFLOWER (HELIANTHUS ANNUUS L.)

ASSOCIATED BACTERIAL POPULATION ................................................. 50

2.2.1 Isolation and Purification of Sunflower Rhizosphere/Endophytic Bacteria 50

MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERIZATION OF

BACTERIA .......................................................................................................... 51

2.3.1 Colony and Cell Morphology ......................................................................... 51

2.3.2 Gram’s Staining ............................................................................................... 51

BIOCHEMICAL CHARACTERIZATION ...................................................... 52

2.4.1 Nitrogen Fixation ............................................................................................ 52

2.4.2 Indole-3-acetic Acid Production .................................................................... 52

iv

2.4.3 Solubilization of Inorganic Phosphate ........................................................... 53

2.4.4 Production of Organic Acids .......................................................................... 54

2.4.5 Intrinsic Antibiotic Resistance ....................................................................... 55

2.4.6 Biocontrol Assay-Selection of Antagonistic Bacteria .................................. 55

2.4.7 Phenotypic Microarrays .................................................................................. 56

MOLECULAR CHARACTERIZATION OF ISOLATES .............................. 56

2.5.1 Preparation of Bacterial Colony for PCR ...................................................... 56

2.5.2 Molecular Marker ............................................................................................ 56

2.5.3 Identification of Bacterial Isolates using 16S rRNA Gene Sequence Analysis

........................................................................................................................ 57

2.5.4 Amplification of NifH Gene ........................................................................... 57

2.5.5 DNA Sequencing and Sequence Analysis ..................................................... 58

2.5.6 Phylogenetic Analysis ..................................................................................... 58

ROOT COLONIZATION STUDIES/ MICROSCOPIC STUDIES ................ 59

2.6.1 Colonization Studies ....................................................................................... 59

2.6.2 Analysis of the Bacterial Population Attached to Roots by Plate Count (CFU)

Method ........................................................................................................... 72

PLANT INOCULATION STUDIES ................................................................. 72

2.7.1 Preparation of Bacterial Inoculum ................................................................. 72

2.7.2 Experiment 1: Evaluation of the Effect of Bacterial Inoculation on Growth

of Sunflower in Growth Pouch ..................................................................... 73

2.7.3 Experiment 2: Greenhouse/Pot Experiment .................................................. 75

v

2.7.4 Experiment Details and Layout Design for N2-fixion Pot Experiment ....... 76


and Yield of Sunflower under Field Conditions .......................................... 77

2.7.6 Statistical Analysis .......................................................................................... 80

RESULTS ........................................................................................................................ 82

EXPERIMENT 3.1 BACTERIAL ISOLATION AND

CHARACTERIZATION .................................................................................... 82

3.1.1 Description and Physico-Chemical Properties of Sampling Sites ............... 82

3.1.2 Bacterial Isolation and Morphological Characterization .............................. 84

CHARACTERIZATION OF SUNFLOWER ENDO AND RHIZOSPHERE

ISOLATES FOR IN VITRO PLANT GROWTH PROMOTING TRAITS. ... 89

3.2.1 Solubilization of Inorganic Phosphate ........................................................... 95

3.2.2 Indole-3-Acetic Acid (IAA) Production ........................................................ 99

3.2.3 Nitrogen Fixation/ Nitrogenase Activity ....................................................... 99

3.2.4 Biocontrol Assay ............................................................................................. 99

3.2.5 Intrinsic Antibiotic Resistance ..................................................................... 102

3.2.6 Phenotypic Microarray Analysis .................................................................. 104

MOLECULAR CHARACTERIZATION ....................................................... 108

3.3.1 Sequence and Phylogenetic Analysis of 16S rRNA Gene.......................... 108

3.3.2 Sequence and Phylogenetic Analysis of NifH Gene ................................... 119

PLANT INOCULATION STUDIES ............................................................... 122

3.4.1 Pouch experiment 1: Evaluation of the Effect of Selected Nitrogen Fixing

Bacterial Strains on Sunflower Growth. .................................................... 122

vi

3.4.2 Pouch Experiment 2: Evaluation of the Effect of Selected Phosphate

Solubilizing Bacterial Strains on Sunflower Growth. ............................... 124

3.4.3 Pot experiment 1: Evaluation of the Effect of Selected Nitrogen Fixing

Bacterial Strains on Sunflower Growth. .................................................... 126

3.4.4 Pot experiment 2: Evaluation of the Effect of Selected Phosphate Solubilizing

Strains on Sunflower Growth. .................................................................... 130

3.4.5 Field Experiments ......................................................................................... 134

COLONIZATION STUDIES ........................................................................... 159

3.5.1 Root Colonization Studies Using Confocal Laser Scanning Microscopy . 159

3.5.2 In situ Detection of Pseudomonas sp. AF-54 Colonizing Sunflower Roots

using FISH ................................................................................................... 169

3.5.3 Colonization Study by Transmission Electron Microscopy and Immunogold

Labeling........................................................................................................ 171

3.5.4 Biofilm Formation ......................................................................................... 179

Discussion ...................................................................................................................... 185

References ...................................................................................................................... 205

vii

List of Figures

Figure 2-1 1kb DNA ladder used to measure the size of the bands. ............................. 57

Figure 2-2 Vector pBBR-MCS-4 showing restriction map. .......................................... 62

Figure 3-1 Graphical representation of the ratio of 163 bacterial isolates for different

PGP traits obtained from endo/rhizosphere of Sunflower (cv. FH-331) ...................... 89

Figure 3-2 Biocontrol, Indole-3-acetic production and phosphate solubilizing potential

of representative bacterial isolates .................................................................................. 94

Figure 3-3 Phosphate solubilization index of representative bacterial isolates on

Pikoveskya’s agar medium .............................................................................................. 95

Figure 3-4 Antibiogram of Azospirillum sp. AF-22................................................... 107

Figure 3-5 Agarose gel photograph showing amplified 16s RNA gene of

representative bacterial strains from sunflower endo/rhizosphere. ............................. 109

Figure 3-6 Phylogenetic relationship of 42 bacterial isolates obtained from

endo/rhizosphere of sunflower from 16 different sits of Dhirkot, AJK based on 16s

rRNA (1500 bp) sequences............................................................................................ 114

Figure 3-7 Phylogenetic tree based on 16S rRNA sequences (1014 bp) of sunflower

associated Azospirillum brasilense AF-22 ( ) and published sequences. .................. 115

Figure 3-8 Phylogenetic tree based on 16S rRNA sequences of sunflower associated

Pseudomonas sp. AF-54 ( ) and published sequences. ............................................... 116

Figure 3-9 Confocal microscopic image of sunflower root inoculated with YFP-

labelled Citrobacter freundii AF-56, grown in sand 21 days post inoculation. ......... 162

Figure 3-105 Confocal image of sunflower root inoculated with YFP-labelled

Enterobacter cloacae AF-31, grown in sand 21 days post inoculation. ..................... 163

viii

List of Tables

Table 2.1 Meteorological data of sites at the time of sampling .................................... 48

Table 2-2 Primers PCR conditions used for 16S rRNA pqqE and nifH genes

amplifications from selected isolates .............................................................................. 59

Table 2-3 Rabbit immunization schedule used for raising polyclonal antibodies ....... 64

Table 3-1 Physico chemical properties of soil collected from different sites .............. 83

Table 3-2 Morphological characteristics of bacterial isolates obtained from sunflower

endo/rhizosphere .............................................................................................................. 85

Table 3-3 Qualitative data of 163 bacterial isolates from sunflower endo/rhizosphere

screened for different pant growth promoting traits. ..................................................... 90

Table 3-4 Quantification of soluble P in supernatant of bacterial isolates from

sunflower rhizosphere ...................................................................................................... 96

Table 3-5 Detection of organic acids in bacteria isolated from rhizosphere and root

interior in Pikovskaya’s broth medium ........................................................................... 98

Table 3-6 Quantification of indole-3-acetic acid (IAA) produced by bacterial isolates

in LB broth medium supplemented with L-Tryptophan .............................................. 100

Table 3-7 Nitrogenase activity of bacterial isolates isolated from rhizosphere and root

interior of sunflower....................................................................................................... 101

Table 3-8 Intrinsic antibiotic resistance pattern of selected bacterial isolates from

sunflower rhizo/endosphere ........................................................................................... 103

Table 3-9 Differential metabolic profiling of selected bacterial isolates (Biolog GN2

Microplate analysis) ....................................................................................................... 104

Table 3-10 Identification of sunflower associated bacterial isolates based on 16S

rRNA gene sequence analysis ....................................................................................... 110

ix

Table 3-11 Sequence identity and closest BLASTn hits based on NifH gene of

nitrogen fixing and phosphate solubilizing PGPR strains form endo/rhizosphere of

sunflower ........................................................................................................................ 120

Table 3-12 Effect of inoculation with nitrogen fixing bacterial strains on growth of

sunflower in pouch experiment under controlled conditions ...................................... 123

Table 3-13 Effect of inoculation with phosphate solubilizing bacterial strains on

growth of sunflower in pouch experiment under controlled conditions ..................... 125


sunflower in pot experiment under controlled conditions ........................................... 127


growth of sunflower pot experiment under controlled conditions .............................. 131

Table 3-16 Mean monthly weather data for growing season of sunflower at NIBGE,

Faisalabad and Chota gala Rawalakot. ......................................................................... 135

Table 3-17 Physico-chemical analysis and bacterial population of soil from

experimental sites ........................................................................................................... 135

Table 3-18 Comparison of treatment means and location means of sunflower

inoculated with bacterial isolates under field conditions. ............................................ 137

Table 3-19 Sunflower growth parameters as affected by interaction between bacterial

treatments and locations under field conditions ........................................................... 139

Table 3-20 Effect of bacterial inoculation on sunflower agronomic and physiological

parameters under field conditions ................................................................................. 145

Table 3-21 Effect of bacterial inoculation on sunflower yield parameters under field

conditions ........................................................................................................................ 151

Table 3-22 Sunflower oil and fatty acid contents as affected by bacterial inoculation

......................................................................................................................................... 152

x

Table 3-23 Analysis of photosynthetic performance in sunflower leaves under field

conditions ........................................................................................................................ 153

xi

List of Abbreviations and Symbols

ABA Abscisic acid

ACC-deaminase 1-aminocyclopropane-1-carboxylate deaminase

A-HSLs Acyl homoserine lactones

ANOVA Analysis of variance

ARA Acetylene reduction assay

ATP Adenosine triphosphate

BLAST Basic local alignment search tool

BNF Biological nitrogen fixation

BPP ß-beta propeller

BSA Bovine serum albumin

°C Degree centigrade

cfu Colony forming unit

CLSM Confocal laser scanning microscopy

cm Centimeter

CO2 Carbon dioxide

CRD Completely randomized design

DAS Days after sowing

DGAP Diacetyl phloroglucinol

EC Electrical conductivity

ET Ethylene

FAs Fluorescent antibodies

FID Flame ionization detector

FISH Fluorescent in situ hybridization

FITC Fluorescent isothiocyanate

g Gram

GA Gluconic acid

GDH Glucose dehydrogenase

GFP Green fluorescent protein

HCN Hydrogen cyanide

HPLC High performance liquid chromatography

IAA Indole-3-acitic acid

IAM Indole-3-acetamide

xii

IAN Indole-3-acetonitrile

IgG Immunoglobulin G

IGL Immunogold labeling

IPyA Indole-3-pyruvic acid

L Litre

LASER Light amplification by stimulated emission of radiations

LB Luria Bertani

LSD Least significant difference

K Potassium

kb Kilobyte

Kg Kilogram

kpa Kilo pascal

KV Kilo volts

M Molar

mm Millimeter

mg Milligram

MPS Mineral phosphate solubilizing

N Nitrogen

N2 Nitrogen

Nr Reactive nitrogen

NCBI National center for biotechnology information

NFM Nitrogen free malate

Nif Nitrogen fixing

NSAPs Non-specific acid phosphatases

OA Organic acid

OD Optical density

OM Organic matter

P Phosphorus

PA Phytic acid

PBS Phosphate-buffered saline

PCA Principal component analysis

PCR Polymerase chain reaction

PDA Potato dextrose agar

PFA Paraformaldehyde

xiii

PGPB Plant growth promoting bacteria

PGPR Plant growth promoting rhizobacteria

pH Hydrogen ion concentration

Pi Inorganic phosphorus

Po Organic phosphorus

PPM Parts per million

PQQ Pyrroloquinoline quinone

PSB Phosphate solubilizing bacteria

RCBD Randomized complete block design

RhITC Rhodamine isothiocyanate

SDS Sodium dodecyl sulfate

SEM Scanning electron microscope

SI Solubilization index

rpm Revolution per minute

TCP Tri-calcium phosphate

TEM Transmission electron microscopy

Trp Tryptophan

VOCs Volatile organic compounds

YFP Yellow fluorescent protein

µL Micro litre

µm Micro meter

10X 10 times

xiv

Acknowledgments

All praises are to Almighty Allah who enabled me to complete this research work. I am

cordially appreciative to my parents and teachers who taught me word-by-word and enabled

me to reach at this stage. Its only my name which appears on the cover of this dissertation, but

a great many people have contributed to its production. I owe my gratitude to all those people

who have made this dissertation possible. Words cannot express my feeling of gratitude to my

father Sardar Abdul Majeed Khan(late) and my mother Mrs. Zubeda Majeed who sacrificed

their past for our future. I am especially thankful to my brothers Sardar Aitzaz Majeed Khan

and Sardar Ahtsham Majeed Khan, Sardar Shahid Imtiaz and my sisters Mrs. Mehwish

Sheraz, Parsa Fatime, Saleha Fatima and Mrs. Rehmana Aitzaz for their spiritual and moral

support during this long period of study, research work and in thesis writing.

I deem it my utmost pleasure to avail an opportunity to express my heartiest gratitude and deep

sense of obligation to my honorable supervisor, Professor Dr. Muhammad Kaleem Abbasi

for his able guidance, kind behavior, generous transfer of knowledge, moral support,

constructive criticism and enlightened supervision.

With a proud sense of gratitude, I acknowledge that this dissertation has found its way to

significant completion due to the kind supervision, and sympathetic attitude of my Co-

Supervisor, Professor Dr. Sohail Hameed, Deputy Chief Scientist, Director Academics &

Coordination (Biol.), PAEC/ National Institute for Biotechnology and Genetic Engineering

(NIBGE), Faisalabad. He has made a great contribution for planning, designing and successful

completion of this research work.

It is an immense pleasure to express my profound gratitude to Dr. Ben Raymond, Imperial

College London, United Kingdom and his team for my training in advanced molecular biology

techniques and providing research facilities to carry out a part of my research work in his lab.

Special thanks to Mr. Andrew Matthews(Lab instructor) for helping me in every aspect of science

and social life there.

I am thankful to Professor Dr. Khalid Mehmood, Dean Faculty of Agriculture, for providing

conducive environment for studies and research. Special thanks go Dr. Abdul Khaliq (Chairman

Department of Soil and Environmental Sciences,), Dr. Nasir Rahim (ex-chairman), Dr. Aqeela Shaheen, Dr. Majid

Mahmood Tahir and Dr. Mohsin Zafar and all other members of department for their

valuable support wherever I needed especially Mr. Ishfaq(Lab attendant). Thanks are also due for

Professor Dr. Dilnawaz Ahmed Gerdaizi(committee member) for support and valuable suggestions.

I am also thankful to Dr. Shahid Mansoor(Director NIBGE) and Ex-directors for kind behavior and

for providing research facilities at NIBGE. Thanks are due to Dr. Fathia Mubeen(PS) my lab

incharge who facilitated me for my academics, research and more importantly social issues.

She has been a source of moral support. I have no words to say thanks to Dr. Asma Imran(PS)

for enlightening me the first glance of research. She has taught me, both consciously and

unconsciously, how to do science, how to write it down and cheered me up at both professional

and social fronts. Undoubtedly, her presence has always been a source of inspiration and

confidence for me.

I am grateful to Dr. Sumera Yasmin(PS) for letting me perform FISH and FAs staining

techniques in her lab and thanks also go to Mr. Tariq Shah(PSA) and Mr. Tariq Mehmood (PSA)

xv

for helping me conduct these experiments. I would like to thank to all scientists in my division

for their invaluable help and cooperation whenever I needed during my study, especially Dr.

Qaisar M. Khan(Head SEBD), Dr. Sajjad Mirza(DCS), Dr. Ghulam Rasool(PS), and Mrs. Naima

Hamid(SS). I am also grateful to Mr. Asghar Ali(SA-1), and Mr. Zahid Iqbal(SSA) for their

technical help and support. Special thanks go to Mr. Ghulam Abas for helping in thesis

formatting.

The members of the Microbial Physiology Group (MPG, NIBGE) have contributed immensely to my

personal and professional time at NIBGE. The group has been a source of friendships as well

as good advice and collaboration especially Dr. Farwa Zahir, Dr. Amanat Ali, Dr. Saira Ali,

Dr. Tahir Naqqash, Dr. Kashif Hanif, Dr. Naveed Ahmed and Mr. Abdul Rauf. Special

thanks are also due for BioPower team(NIBGE), particularly Mr. M. Sarwar. I am greatly

thankful to Mr. Javed Iqbal(Electron Microscopist) for his help and valuable guidance in Transmission

Electron Microscopy. My special thanks go to Dr. Iftikhar Ali(DCS, NIFA, Peshawar) for sunflower

oil and fatty acid analysis, Dr. Yaseen Ashraf(DCS NIAB, Faisalabad) for IRGA analysis, Dr. Zahir.

A. Zahir(UAF) for root analysis.

My time at NIBGE was made enjoyable in large part due to my backpacking buddies especially

Dr. Tanveer Majeed, Dr. Hina Jabeen, Nagina Rafique, Shaista Javed, Arsh Bibi, Naila

Sarwar and many others. No acknowledgements would be complete without mentioning the

love, care and help of best friends especially Maria Gohar and Anita Kaini.

I am indebted to all my friends and family in London who opened their homes to me during my

stay at Imperial College London. Special thanks are due for Mr. Sheraz Khan(brother in law) who

was always so helpful in numerous ways and made my stay as comfortable as home. Deep sense

of thanks also goes to Dr. Farida Anjum and Mrs. Nayyar for their hospitality at Ascot,

London.

I would like to express my sincere thanks to Higher Education Commission (HEC),

Islamabad, Pakistan for awarding me funds under IRSIP fellowship to carry out a part of my

PhD research at Imperial College London, UK.

In the last, special thanks to Miss Afshan Majeed for believing in herself and not letting her

hopes die in the toughest of situations. I appreciate your patience and persistence towards the

attainment of this highest academic degree.

Afshan Majeed

xvi

Abstract

The use of plant growth promoting rhizobacteria is a promising strategy for sustainable

agriculture production. The aims of the present study were to isolate, characterize and

identify sunflower associated beneficial bacteria and to evaluate their inoculation and

colonization potential towards sunflower. Therefore, sixteen sites with varying altitudes

of Himalayan Mountain region of Dhirkot (subdivision), Azad Jammu and Kashmir

have been selected. A total of 163 isolates were obtained from rhizosphere (97) and

root interior (66 putative endophytes) of sunflower to evaluate the potential of these

beneficial root associated bacteria and their root colonization potential to improve

sunflower growth, nutrient uptake, yield and oil contents. Out of 163 screened isolates,

44 % were found positive for phosphate solubilization (9.51 to 48.80 µg mL-1), 24 %

for IAA production (1.13-24.6 µg mL-1), 20 % for nitrogen fixation (28.68-137.84

nmoles mg-1 protein h-1) and 12% for biocontrol properties against Fusarium

oxysporum detected by using standard microbiological and biochemical procedures.

Most of the phosphate solubilizing isolates were able to produce a variety of organic

acids dominated by gluconic acid (G.A) ranging between 2.17 µg mL-1 to 15.44 µg mL-

1. The isolates exhibiting multiple plant growth promoting traits in vitro were identified

as species of the genus Azospirillum, Bacillus, Enterobacter, Citrobacter,

Pseudomonas, Serratia, Stenotrophomonas and Lysinibacillus, Cellulosimicrobium,

Staphylococcus, Chryseobacterium showing 99% homology of 16S rRNA gene

sequence. Major population was dominated by Bacillus species followed by

Pseudomonas and Enterobacter. Phylogenetic analysis did not show any correlation

or distribution of specific species/genera at specific sites indicating that the distribution

of PGPR is independent of the surrounding topography. Eleven potential PGPR strains

xvii

exhibiting at least 3 of the above mentioned plant beneficial traits were further tested

for intrinsic antibiotic resistance through disc diffusion method and found to be resistant

against most of the tested antibiotics. The bacterial strains were then tested as inoculant

on sunflower (cv. FH331) in soil-free medium (growth pouches) and in sterilized soil

(pots) under controlled conditions for their N2-fixing and P-solubilizing abilities

separately, as well as in field under natural conditions at two locations i.e., Rawalakot,

AJK, and Faisalabad, Pakistan. All the eleven bacterial strains (belonging to 8 genera)

promoted the sunflower growth under controlled environmental conditions and

improved N and P uptake over non-inoculated control treatment. Out of these 11 strains,

Azospirillum brasilense AF-22, Enterobacter cloacae AF-31, Pseudomonas sp. strain

AF-54 and Citrobacter freundii AF-56 were found more effective and potent strains in

augmenting sunflower growth, yield and oil contents and NP uptake compared with 50

% (of their recommended dose) N and P fertilizers treatments. These four strains

exhibiting multiple plant growth promoting traits i.e., N2-fixation, P-solubilization,

IAA production, organic acid production and metabolic versatility, performed well in

both experimental locations at Rawalakot and Faisalabad. Principal component analysis

indicated that inoculation with these selected PGPR had better response at

Rawalakot. To confirm the efficiency of these bacterial strains for sunflower, their host

specificity and colonization potential was extensively studies in vitro and in vivo.

Bacterial population dynamics were observed at different time intervals to check the

strain persistency in sunflower rhizosphere. All the strains showed strong association

with sunflower roots up to 45 days. Their colonization potential was confirmed through

a series of high throughput microscopy techniques including yfp-labelling technique,

fluorescent antibody (FA) labelling, fluorescent in situ hybridization (FISH) techniques

coupled with confocal laser scanning microscopy and by ultrastructural and

xviii

immunogold labelling technique through transmission electron microscopy (TEM).

These biomarkers confirmed the host specificity of the applied strains in both sterilized

and natural conditions. Transmission electron microscopic studies also showed the

localization of Azospirillum brasilense AF-22 and Citrobacter freundii AF-56 both in

the rhizosphere and root interior, confirming their endophytic association with

sunflower.

Based on the results of this study, it is concluded that the potential PGPR strains

namely A. brasilense AF-22, E. cloacae AF-31, Pseudomonas sp. strain AF-54 and C.

freundii AF-56 can be used as biofertilizer for sunflower crop for enhancing yield and

to minimize the use of chemical (NP) fertilizers. It is further recommended that the

inoculum should be checked for the cross inoculation potential on other oil seed crops

in field.

Chapter 1

INTRODUCTION

PLANT-MICROBE INTERACTIONS

1.1.1 The Rhizosphere

The term ‘rhizosphere’ used first time by Hiltner in 1904, describes as ‘the compartment

influenced by the roots, where passionate interactions among microorganisms (beneficial

and pathogenic) occur all the times (Barriuso, 2008). The narrow zone of rhizosphere

serves as a home for large number of organisms (Figure 1.1), and is well known as one of

the most dynamic ecosystems on Earth (Raaijmakers et al., 2009). In Rhizosphere, plants

release root exudates that alter the physical, chemical and biological properties of

surrounding soil (Fageria and Stone 2006). Such changes support the development of huge

spectrum of microbial population, leading to the countless interactions between plants and

microorganisms, either beneficial, neutral or harmful effects (Bennett and Brever, 2007;

Richardson et al., 2009)

The well-studied rhizospheric organisms for their beneficial effects on plant health are the

mycorrhizal and/or mycoparasitic fungi, protozoa, plant growth-promoting rhizobacteria

(PGPR), and microbial biocontrol agents etc. The efficiency and structure of natural plant

community is influenced by these microorganisms directly or indirectly (Richardson et al.,

2009). Wagg et al. (2014) suggested that belowground microbial diversity acts as a

guarantee for plant productivity sustenance, as the below ground fertility serves as

predictor of above ground plant productivity (Richardson et al., 2009; Lau and Lennon,

2011).

http://femsre.oxfordjournals.org/content/37/5/634#ref-167

Chapter 1 Introduction and Review of Literature

2

PLANT GROWTH PROMOTING RHIZOBACTERIA (PGPR)-DRIVERS

OF AGRICULTURAL SUSTAINABILITY

Soil microorganisms are indispensable for the maintenance and sustainability of animal

and plant communities. They facilitate the nutrient cycling and execute many basic

biological processes.

Figure 1-1 An overview of microorganisms (with approximate values) present in the

rhizosphere zoo. Modified from Buée et al. (2009).


3

The rhizosphere and plant roots are aggressively colonized by the free-living bacteria

called PGPR, which when applied to the crop, improve its growth and yield (Kumar et al.,

2015). The activities of the rhizospheric microbial communities broadly influence plant

physiology and growth, and consequently are important for terrestrial ecosystems and

agriculture. Rhizobacteria are important catalysts involved in a range of interactions which

take place between the plant, soil and other microbes in the root zone influenced by root

exudates and compounds released by the microorganisms nourishing on them (Barriuso et

al., 2008). In general, approximately 2–5 % of the bacteria residing in rhizosphere are

PGPR (Antoun and Prévost, 2005) and are the most extensively studied group of bacteria

(PGPB). These bacteria are gifted to establish a dense population in the soil tightly adhered

to the root surface, the rhizosphere (Antoun and Prévost, 2005), directly attached to the

root cortex and epidermis, called the rhizoplane, or on leaves called phyllo-sphere bacteria

(Glick et al., 2007). They can penetrate in the root tissues and reside there, called the

endophytes. Well-reported plant beneficial rhizospheric and endophytic bacteria belong to

the genera Bacillus, Azospirillum, Pseudomonas, Enterobacter, Arthrobacter, Rhizobium,

Azotobacter, Burkholderia, Herbaspirillum, Serratia (Gray and Smith, 2005).

A bacterium meets the criteria of plant growth promoting agent when it is capable of

producing positive effect on plant growth upon inoculation, representing good quality

competitive abilities over the native microbial population present in the rhizosphere.

(Antoun and Prevost, 2005). Beneficial rhizobacteria which support the plant growth by

means of synthesis or altering the concentration of plant growth hormones like indole-3-

acetic acid (IAA) (Palaniappan et al., 2010; Marques et al., 2010; Majeed et al., 2015) and

volatile growth stimulants like ethylene (Haas and Défago, 2005), enhancing nitrogen


4

fixation (Shen et al., 2016; Naqqash et al., 2016), disease suppression by antagonism

against phytopathogens (Hassan et al., 2010; Ali et al., 2014), converting the organic and

inorganic phosphatic forms to a soluble form (Deepa et al., 2010; Hanif et al., 2015; Shen

et al., 2016), mobilization of Zn (Saravanan et al., 2007), siderophores synthesis

(particularly ferric iron) to make them plant available (Tank and Saraf, 2009; Radzki et

al., 2013), increasing photosynthetic rates (Singh et al., 2011), induced systemic resistance

(ISR) in plants (Lugtenberg and Kamilova, 2009), antibiotic synthesis, production of

enzymes and fungicidal compounds against harmful microorganisms (Lugtenberg and

Kamilova, 2009). Moreover, most of the PGPR also enhance plant tolerance against abiotic

stresses like metal toxicity, salinity and drought (Dimpka et al., 2009; Babalola, 2010). The

PGPR inoculation have the potential to increase the seedling emergence rate (Hafeez et al.,

2004), nutrient (N, P, K, Zn, Mn, Cu) uptake (Biari, et al., 2008; Shen et al., 2016), and

nutrient use efficiency (Shahid et al., 2014) and would aid to withstand environmental

health and soil productivity. Application of PGPR will result in decreased crop production

costs (Sharma et al., 2016).

1.2.1 Host Specificity

Plants are well reported to affect the native soil microbial populations and each of the plant

species is believed to select particular microbial community which add enough to their

fitness by generating an appropriate and selective environment. This selective environment

ultimately results in a narrow range of microbial diversity (de Weert et al. 2006; Berg and

Smalla, 2009). Host plant specificity of PGPB have been well reported in several reviews

and studies (Berg and Smalla, 2009; Buchan et al., 2010, Majeed et al., 2015).Moreover,

plants recruit particular defensive microbial populations to cope with harmful microbes


5

(pathogens) in stressed environments, (Berendsen et al., 2012). Zeller et al. (2007) also

reported that most of the bacteria show robust host-plant selectivity and by colonizing a

single plant species. So, the microbial diversity of environment created by the plant is very

low (Berg et al., 2006; Costa et al., 2006; Hein et al., 2008). In this perspective, culture-

dependent techniques might permit the selection of bacterial strains well-adapted to the

rhizosphere environment.

However, the performance of bacterial inoculation in filed condition are still

inconsistencies (Morrissey et al., 2002). To deal with this problem, extensive research has

been carried out to learn how to engineer the rhizospheric environment for bacterial

colonization (Ryan et al., 2009). Though, till date, the best strategy to improve the bacterial

performance in field conditions is to hunt the region-specific PGPB (Deepa et al., 2010).

PLANT INFLUENCE ON MICROBES

It is very well-known fact that rhizospheric bacteria multiply very rapidly on the roots

surfaces on which the root exudates and root cell lysates deliver plenty of sugars, growth

hormones, amino acids, organic acids, flavonoids, nucleotides, fatty acids, alkaloids,

steroids, nutrients, tannins terpenoids, and vitamins for their growth and multiplication.

Occasionally, they surpass hundred folds to those masses present in the bulk soil (Campbell

and Greaves, 1990).

1.3.1 Plant Attributes of PGPR/the Rhizosphere Microorganisms – What are They

Doing?

Based on the available literature, plant microbe interactions can be categorized into three

classes: neutral, positive or negative (Whipps, 2001). Beside these functional


6

classifications, rhizobacteria are further grouped according to the plant compartment that

they live as either intracellular (iPGPR, symbiotic) or extracellular (ePGPR, free living).

There are classification terms to describe the mechanisms by which they perform different

functions. Among the direct mechanisms, balancing phytohormones levels, plant

nutritional status enhancement and induction of systemic disease resistance are included

(Shen et al., 2016; Sharma et al., 2016). While, biocontrol associated mechanisms like

antibiotic production, Fe-chelation, production of enzymes and fungicidal compounds

against harmful microorganisms (Lucy et al., 2004) and the competition for rhizospheric

niches (Zahir et al, 2004). Next to this classification, they can also be classified with respect

to objective of their application: phytostimulators, rhizoremediators. biofertilizers, and

biopesticides (Prashar et al., 2014).

These rhizospheric microorganisms have beneficial (‘the good’) and harmful (‘the bad’),

impact on plant health. Some key functions performed by PGPR with agriculture

perspective (Figure 1.2).

1.3.2 Rhizospheric Microbes; the Good

Plant beneficial microorganisms stimulate growth and safeguard the plants from pathogen

attack by an array of mechanisms (Raaijmakers, 2009). Biofertilization, root growth

stimulation, rhizoremediation, abiotic stress control, and the control of diseases are

involved in these mechanisms and are well documented for rhizobacteria belonging to the

Proteobacteria, Deuteromycetes and Sebacinales, as well as from the Firmicutes order.

(Qiang et al., 2012).


7

Figure 1-2 Plant microbe interaction showing beneficial and harmful effects

of rhizobacteria (Pieterse et al., 2016).

1.3.2.1 Effects of Rhizospheric Microorganisms on Nutrient Acquisition by Plants

Microorganisms that possess the ability to facilitate nutrient uptake by plant roots, to make

the nutrient available and/or to stimulate the plant growth are generally referred to as

biofertilizers, and are considered as a supplement to the chemical fertilization. Well-known

attributes are nitrogen-fixing, phosphate solubilization, growth hormones production,

siderophore production


8

1.3.2.1.1 Biological Nitrogen Fixation (BNF)

Nitrogen (N) is one of the most common nutrients required for plant growth and

productivity as it is the most important pigment required for photosynthesis as well as

amino acids, the integral building blocks of nucleic acids, proteins and other vital

biomolecules, such as ATPs (Bøckman, 1997). Adequate N supply is essential for plant

metabolic processes involved in vegetative and reproductive plant growth enhancement

(Lawlor, 2002). But it is the most often limiting nutrient, as a result of excessive losses by

emission or leaching, contributing to reduced agricultural ecosystem throughout world.

Even though it is one of the most abundant (approximately 80 %) elements in the Earth’s

atmosphere, unfortunately nitrogen cannot be utilized by the plants in its molecular form.

It is essential to be converted into an available form (ammonia), to be utilized by plants

and other eukaryotes. The conversion of atmospheric dinitrogen into plant available form

can be carried out through abiotic nitrogen fixation including fixation through natural

lightening process and industrial fixation, and the biotic conversion through biological

nitrogen fixation (BNF). Greater applications of fertilizers are the typical result of

increasing demand for agricultural production, which is the leading human interference in

the nitrogen cycle. The amount of N applied in either fertilizer or manure form, could be

utilized by the plants, can be released into the atmosphere in the form of nitrogenous gases

(Flechard et al., 2007) or can be leached down into the ground water (Trindade et al., 2001),

with consequential environmental implications, soil acidification and water eutrophication.

While on the other hand, biologically fixed N is less susceptible to these losses due to its

in situ utilization. Alternative nitrogen sources are required for the development of

sustainable farming systems, rather than relying only chemical N fertilizer alone.


9

Biological nitrogen fixation (BNF) — the conversion of atmospheric elemental nitrogen

(unavailable to plant) into ammonia (available to plant) by symbiotic, associative and free-

living bacteria — is incredibly important to the global agriculture and more importantly

for the environment. Biological nitrogen fixation is considered as a vital part of the N-

cycle as it replenishes biosphere’s total N content and reimburses the denitrification losses.

N availability to the plant is dependent on the microbial activity, even if applied as chemical

fertilizer. Nitrogen is not only fixed by microbes but its availability to the plants is highly

dependent on microbial activity. Nitrogen fixation carried out by associative and free living

microorganisms, in the rhizosphere, is well-known to play a vital role in N supply for plants

growth and development (Khan, 2005).

Biological fixation can be divided into two types: symbiotic and non-symbiotic.

1.3.2.1.1.1 Symbiotic Nitrogen Fixation

The utmost important mechanism of atmospheric nitrogen fixation is symbiotic N2-

fixation. A large number of microorganisms can fix nitrogen in symbiotic association by

partnering the host plant. But it is restricted to the leguminous species and several shrubs

and trees that form actinorrhizal symbiosis with Frankia. Where symbiotic diazotrophs

include Rhizobium, Bradyrhizobium, Mesorhizobium, Ensifer (former Sinorhizobium),

Allorhizobium, Azorhizobium (Allito et al., 2015; Gourion et al., 2015)

In symbiotic relation microorganisms utilize sugars, provided by plants, for the energy

required for fixation. In return, the microbes facilitate the host plant by providing fixed

nitrogen in plant available form (Howard and Rees, 1996). Nitrogenase enzyme acts as a

catalyst in this process to split the molecular nitrogen and converts it into ammonia along

with electrons and energy supply (Postgate, 1982). The symbiosis process gets start with a


10

particular exchange of signals among macrosymbionts (plants) and microsymbiont

(rhizobia). Plants give the first signal in the form of flavonoids or isoflavonoids, as a root

exudate component (Brencic and Winans, 2005). These flavonoids and isoflavonoids when

taken up by the bacterium induce the nod genes expression (nod, noe, nol and others). The

microsymbiont’s (rhizobia) release the NOD factors (products encoded by the nod-genes)

in response to this molecular dialogue. These Nod factors are decorated with host-specific

modifications on their backbone (Oldroyd and Downie, 2008) which makes NOD factors

as first bacterial determinants of the host-specificity of the symbiosis (Op den Camp et al.,

2011; Suzaki et al. 2015). However, it is reported that nod genes are absent in certain

photosynthetic Bradyrhizobia, rely on some other plant signaling strategy, yet to be

characterized (Giraud et al., 2007). The processes and signaling responsible for symbiotic

N2 fixation (a micro scale) in legume nodules as reviewed by Garg and Geetanjali, (2007).

Many types of legumes possess this type of symbiosis, including forage legumes, grain

legumes, and some leguminous trees, contributing to build up soil N fertility (Goswami et

al., 2015).

1.3.2.1.1.1.1 Non-Symbiotic Nitrogen Fixation

Bacteria that are capable to fix inert N in a free living state with non-legumes form a non-

obligate interaction with plant. The well-studied example of diazotrophs as a nitrogen fixer

is Azospirillum sp. isolated by Beijernick in 1925 (Holguin et al. 1999). A large body of

researchers documented the importance of free living diazotrophs as growth stimulators of

pants other than legumes including sunflower (Shahid et al., 2014), potato (Naqqash et al.,

2016), rice (Mirza et al., 2006), and wheat (Majeed et al., 2015) consequently contribute

in the reduction of N-based chemical fertilizers (Bhattacharjee et al., 2008). Some


11

important examples of non-symbiotic N-fixing bacterial genera are Azospirillum,

Azoarcus, Gluconaceto bacter, Bacillus, Paenibacillus and Pseudomonas Burkholderia,

Herbaspirillum, (Bashan and de-Bashan, 2010; Naqqash et al., 2016).

These free living diazotrophs may colonize interior of plant tissues including roots, shoot

and leaves to fix nitrogen. Some examples of endophytic diazotrophs are Azotobacter

Diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. ( Xu et al., 2012).

1.3.2.1.1.1.2 Biochemistry Involved in BNF

In the complex process of N2-fixation, a number of functional and regulatory genes are

involved (Dixon and Kahn, 2004). The key player of BNF is nitrogenase enzyme which

contains 2 Fe-proteins, nitrogenase Fe-protein and nitrogenase Mo-Fe-protein (Einsle et

al., 2002, Strop et al., 2001). Nitrogen gets bound, as a substrate, to Mo-Fe-S homocitrate

part of Mo-Fe-protein (Hoffman et al., 2014) (Fig 1.3). The other protein (Fe-protein)

shuttles electrons to Mo-Fe- by consuming two Mg-ATP per electron (Halbleib and

Ludden, 2000). The complete process of BNFis described as follows:

N2 + 10H++ 8e- + 16MgATP → 2 NH4++ H2 + 16MgADP + nPi

One mol of NH4+ is produced by the consumption of 8 mol ATP, as this is an energy

demanding process. Under natural conditions, this might get higher. Acetylene reduction

assay is used to quantify the amount of atmospheric nitrogen fixed by diazotrophs

(Dilworth, 1966).


12

Figure 1-3 Protein model of molybdenum nitrogenase.

(A) One catalytic half of the Fe protein: MoFe protein complex with the Fe protein

homodimer shown in tan, the MoFe protein α subunit in green, and the β subunit in cyan.

(B) Space filling and stick models for the 4Fe−4S cluster (F), P-cluster (P), and FeMo-co

(M) (Hoffman et al., 2014).

1.3.2.1.1.1.3 The Genes Involved in BNF

Numerous gene products are involved in the complex process of N2-fixation (Figure 1-4).

The gene expression ways are diverse depending on the type of species and the dominant


13

and most important Mo-Fe protein (α2β2) is encoded by nifDK whereas nifH is involved in

Fe-protein (α2) (Halbleib and Ludden 2000). There are numerous other accessory and

regulatory genes involved in transferring electron and nif regulon synthesis. Both, nifD and

nifK are parts of same operon along with nifH (Fani et al., 2000).

Due to the highly conserved gene sequence, nifH is used as an important marker gene for

diazotrophic studies Moreover, a huge sequence data is available for comparative studies

(Wu et al., 2009).


14

Figure 1-4 Cluster of nif genes involved in atmospheric nitrogen fixation.

1.3.2.1.1.2 Agricultural Significance of Biological Nitrogen Fixation

The use of biological nitrogen fixation for crop production, in Agriculture, has been the

driving force behind N2-fixation research since long. It has been estimated that world-wide

total N-demand will increased from 1.15×108 tons (in 2015), to 1.194×108 tons in 2018 of

which 4% is required by Pakistan (FAO, 2014). The quantity of nitrogen fixed by

diazotrophs pales in importance when compared with the application of N fertilizers in a

range of 150-200 kg N ha-1 yr-1 but on the other hand excessive use of nitrogenous fertilizer

is causing serious environmental issues (Figure 1.5). So modern emphasis on sustainable

development and the known negative side effects of mineral N-fertilizer have stimulated

research in the field of BNF throughout the past century, mainly on legumes and other

symbiotic systems (Paul and Clark 1996; Naveed et al., 2015). While less attention has

been paid to free-living N2-fixers, as they mostly incorporate their fixed nitrogen into their

own biomass and it will be available to the plant indirectly through subsequent

mineralization of the biomass (Chalk et al., 2014).


15

Figure 1-5 The cascade of reactive nitrogen (Nr) forms and associated

environmental problems (Sutton et al., 2013).

Although there are many questions concerning their utility remain open. On a global scale

annual nitrogen fixed by associative diazotrophs share up to 30% of the total biologically

fixed N (Montaño et al., 2013. It can be a significant source in many terrestrial ecosystems

(van Der Heijden et al., 2008), however, in an agricultural context there is a room for more

research to explore their potential to increase crop productivity (Montaño et al., 2013;

Naveed et al., 2015).

1.3.2.1.2 Phytohormone Production

Phytohormones are the key players in plant growth and yield as these are the organic

compounds which effect physiological, biochemical and morphological plant processes in

extremely low concentrations (Fuentes-Ramírez and Caballero-Mellado, 2006). They

regulate plant growth and development as they are the signal molecules serving as chemical


16

messenger in all the processes of plant. In this way, they are also referred to plant growth

regulators. The ability of plant growth promoting bacteria to produce phytohormones or

plant growth regulators is very well discussed over the last couple of decades (Patten and

Glick, 2002; Tsavkelova et al., 2006; Spaepen et al., 2-007; Ghosh et al, 2008; Seo and

Park, 2009; Marques et al., 2010; Iqbal and Hasnain, 2013; Kang et al., 2014; Hanif et al.,

2015; Naqqash et al., 2016). Phytohormones include auxins (Iqbal and Hasnain, 2013),

gibberellins (Kang et al., 2014), cytokinins (Liu et al., 2013), or by regulating ethylene

levels in the plants and abscisic acids (Berg, 2009).

1.3.2.1.2.1 Auxins: Master Control Phytohormone

Among the various phytohormones, auxins act as a master control, regulating most of the

plant processes, directly or indirectly, thus can be considered responsible for most of the

developmental patterns in plants (Wu et al., 2011). Auxin producing bacteria fuel the

germination of seeds, accelerate the root growth process, modify its architecture and

enhance its biomass, as auxin acts as a signal molecule for plant development including

organogenesis and has a potential role in cell division, expansion, elongation and gene

regulation (Ryu and Patten 2008). Several plant growth promoting rhizobacterial genera

including Azospirillum, Enterobacter, Xanthomonas, Alcaligenes, Ochrobactrum,

Acetobacter, Rhizobium, Bradyrhizobium, Pseudomonas, and Bacillus have been reported

for the production of auxin, an established hormone which improves plant growth

ultimately (Ali et al., 2014; Imran et al., 2014; Majeed et al., 2015).


17

1.3.2.1.2.2 Mechanisms of Microbial Auxin Production.

Several pathways are involved in bacterial synthesis of IAA, three of the most common are

indole-3-pyruvic acid (IPyA), indole-3-acetonitrile (IAN) and indole-3-acetamide (IAM)

pathways (Niklas and Kutschera, 2012). ipdC is the gene involved in bacterial synthesis of

IAA, which encodes an enzyme indolepyruvate decarboxylase that acts as a catalyst in the

conversion of IPyA to indole-3-acetaldehyde (Sgroy et al., 2009). Many factors affect IAA

production like biosynthetic pathways adopted, location of the genes involved, regulatory

sequences, and the presence of enzymes to convert active free IAA into conjugated forms

and also the environmental conditions (Patten and Glick, 1996). Tryptophan (Trp), a

component of root exudates, acts as a precursor of IAA and affects IAA synthesis in the

majority of auxin producing PGPR (Merzaeva and Shirokikh, 2010). Increase in IAA

production was recorded with elevated tryptophan levels (Molina-Favero et al., 2008).

Tryptophan dependent pathways used by major IAA producing genera is shown in Figure

1.6.


18

Figure 1-6 Tryptophan dependent pathways for the production of IAA in microbes

(Adopted from Solano et al., 2008).

1.3.2.1.2.3 Cytokinins

Plant growth promoting rhizobacteria produce another class of phytohormones called

cytokinins (Cartieux et al., 2003). Naturally produced cytokinins were first introduced by

Letham in 1963 (Frankenberger and Arshad, 1995) while the history on non-natural


19

cytokinin dates back to 1955 (Miller et al., 1955). As compare o auxins, cytokinin promotes

shoot morphogenesis. The ratio between cytokinins and auxins defines the differentiation

of callus into shoot or root. Equimolar concentrations induce cell proliferation. Many

rhizobacterial strains are capable of producing cytokinins in pure culture, e.g.

Agrobacterium, Arthrobacter, Bacillus, Burkholderia, Erwinia, Pantoea agglomerans,

Pseudomonas, Serratia and Xanthomonas (García de Salome et al., 2001).

1.3.2.1.2.4 Gibberellins (GAs)

Gibberellins consist of a group of terpenoids having twenty C-atoms, and more than 130

different types of molecules (Dodd et al., 2010). The key function of GAs is internode

elongation by involving in cell division and cell elongation within the subapical meristem.

These hormones also influence the germination of seeds, growth of pollen tube and

flowering. Several bacterial genera including Acinetobacter, Agrobacterium, Arthrobacter,

Azospirillum Azotobacter, Bacillus, Clostridium, Flavobacterium, Pseudomonas,

Rhizobium and Xanthomonas are known for their GAs producing character (Tsavkelova et

al., 2006).

1.3.2.1.2.5 Abscisic Acid (ABA)

The phytohormones involved in plant response to abiotic and biotic stress is a 15-C

compound called abscisic acid. Abscisic acid involved in drought stress tolerance, as it

induces stomatal closure. This phytohormone is also involved in salt and toxic metals

tolerance present in root vicinity. Bacterial strains including A. brasilense and

Bradyrhizobium japonicum (Boiero et al., 2007) have been reported to produce ABA.


20

1.3.2.1.2.6 Ethylene (ET) and 1-Aminocyclopropane-1-Carboxylate (ACC)

Deaminase

A gaseous hormone known for its involvement in flower senescence and fruit ripening is

called ethylene. Several plant developmental processes influenced by ET are root growth

and hair formation, flowering, leaf senescence and abscission (Dugardeyn et al., 2008),

breaks seed and bud dormancy (Dodd et al., 2010). Beside direct role in plant growth, ET

serves as a virulence factor and a signaling molecule for plant protection against plant

pathogens (Weingart et al., 2001) as well as a signaling compound to induce systemic

resistance triggered by bacteria present in rhizosphere (Van Loon et al., 2007). Although

ET is essential for plant life having different plant growth promoting effects depending on

its concentration, high concentrations in root tissues is known to be harmful (Dodd et al.,

2010).

Under different types of environmental stress, along with other responses, plants respond

by synthesizing ACC, the precursor for ethylene (Glick, 2007). When ACC gets released

into the rhizosphere, plant roots absorb it and convert it into ET which leads to a detrimental

effect on plant growth due to increased ET concentration. Rhizobacteria having the ability

to produce ACC deaminase degrade this chemical to alpha-ketobutyrate and ammonium

(Mayak et al., 2004) and re-establish a vigorous root growth required to deal with the

environmental stresses. Rhizobacteria isolated from different soils belonging to the

Achromobacter, Azospirillum, Bacillus, Enterobacter, Pseudomonas, and Rhizobium are

reported for ACC deaminase production. (Ghosh et al., 2003; Mayak et al., 2004;

Govindasamy et al., 2008; Duan et al, 2009).


21

1.3.2.1.2.7 Volatiles

A wide variety of volatile compounds are produced by bacteria but their biological

functions are not completely understood. Volatile compounds (VOC) are assumed to be

involved in a number of processes responsible for plant growth promotion as they act like

microbial inhibiting agents and also involved in cell-cell and specie-specie signaling

(Ledger et al., 2016). The induction of abiotic stress tolerance by VOCs has been well

demonstrated for Bacillus subtilis (Ryu and Patten, 2008), could be a possible mechanism

shared by other growth promoting bacterial strains like Pseudomonas simiae (Vaishnav et

al., 2015). The important VOCs produced by PGPR include VOC 2- pentylfuran (Zou et

al., 2010) and 13- tetradecadien-1-ol, 2-butanone and 2-methyl-n-1-tridecene (Park et al.,

2015).

1.3.2.1.2.8 Acyl-homoserine Lactones (A-HSLs).

Another important class of intracellular lipid signal molecules produced by bacteria are N-

acyl homoserine lactones (A-HSLs), usually used by Gram negative bacteria to coordinate.

These molecules are actively involved in many processes, when their concentration reaches

a certain value, the quorum (population-density-dependent regulation of gene expression)

(von Rad et al., 2008; Vivanco, 2013), including cell motility, synthesis of

exopolysaccharide, plasmid transfer, root nodulation and N2-fixation (Appunu et al.,

2008). Many PGPR strains have been reported to produce AHls (von Rad et al., 2008;

Vivanco, 2013; Imran et al., 2014). Ali et al. (2016) characterized an endophytic

Bradyrhizobium sp. strain SR-6 from soybean nodule for the production of both short and

long chained AHLs as quorum sensing molecules, which enhanced root growth and root


22

hair development. These molecules are reported to trigger auxin-induced root formation

via hydrogen peroxide in mung bean (Bai et al., 2012).

1.3.2.1.3 Phosphate (P) Solubilization

Phosphorus is the most important key element, next to nitrogen, in plant nutrition.

Phosphorus is involved in almost all major metabolic plant processes (Pereira and Castro

2010; Bakhshandeh et al., 2014). These chief metabolic processes include cell division,

energy transfer, macromolecular biosynthesis and respiration, root development, stalk and

stem strength, photosynthesis, flower and seed formation, crop maturity in a broader

spectrum, (Khan et al., 2009). Phosphorus is also involved plant metabolic processes

responsible for disease resistance, nutrient transfer within the plant and N2-fixation in

legumes (Saber et al., 2005). Contrary to other macronutrients, P is the least mobile

element in the plants and soil.

Phosphorus has abundant natural reservoirs in primarily organic (20-80 %) and inorganic

(170 different forms of mineral) form, but unfortunately, the plant available forms (H2PO4-

and HPO4-2) are often very low (1-5 % of total soil P) as it gets fixed with iron and

aluminum ions in acidic soils while in alkaline soils, fixation occurs with Ca and Mg ions

(Rajapaksha et al., 2011. The resulted metal-ion complexes make approximately 80 % of

the total applied P unavailable for plant uptake (Bakhshandeh et al., 2014).

1.3.2.1.3.1 Phosphorus Cycle

Phosphorus cycle in the biosphere can be described as ‘open’ or ‘sedimentary’, and

unfortunately, no atmospheric phosphorus is available to be utilized by making biologically

available, like nitrogen (Ezawa et al., 2002). Phosphorus undergoes many biotic and abiotic


23

reactions, some of these reactions take only few seconds, while the others may require

years to be accomplished and redistributed into various pools. Major processes involved in

soil phosphorus cycle, were identified by Sims et al. (2005) in a review of soil phosphorus

chemistry as dissolution–precipitation (mineral equilibria), sorption– desorption

(interactions between P in solution and soil solid surfaces) and mineralization–

immobilization (biologically mediated conversions of P between inorganic and organic

forms).

When P applied as chemical fertilizer, a huge amount of it goes in to the immobile

pools/fixed through precipitation reaction with iron and aluminum ions (highly reactive) in

acidic soils, while in alkaline or normal soils it gets fixed with calcium ions. Soil

phosphorus cycle can be simply described by physicochemical (sorption-desorption) and

biological (immobilization-mineralization) processes. The biochemical and genetic

mechanisms involved in P transformations are not yet completely understood (Ohtake et

al., 1996). Soil microorganisms are the key players of soil phosphorus cycles and

consequently make the phosphate available for plant use (Richardson, 2001).

1.3.2.1.3.2 Biodiversity of P Solubilizers

A large number of microbes, in soil and plant rhizosphere, exhibit P solubilization ability

(Raghu and Macrae, 2000); including bacteria, fungi, actinomycetes and even algae (Alam

et al., 2002). Bacterial genera reported as P-solubilizers include Pseudomonas, Bacillus,

Rhodococcus, Arthrobacter, Serratia, Chryseobacterium, Gordonia, Phyllobacterium,

Delftia (Chen et al., 2006), Azotobacter (Kumar et al., 2001), Enterobacter, Pantoea,

Klebsiella (Chung et al., 2005). Pseudomonas and Bacilli among ectorhizospheric and


24

rhizobia among endophytic communities are believed to be the most effective phosphate

solubilizing bacterial strains (Zaidi et al., 2009).

The history of phosphate solubilizing bacteria (PSB) as biofertilizer dates back to 1950s

(Krasilinikov, 1957). When the efficient phosphate solubilizer applied, they increase the

availability of phosphorus from both applied and native soil phosphorus. The ability of

bacteria to dissolve the interlocked P have a significant implication (Lone et al., 2011).

They make P available to plants by mineralizing organic P in soil and by solubilizing

precipitated phosphates (Shahid et al., 2014; Majeed et al., 2015; Brígido et al., 2016).

1.3.2.1.3.3 Mechanism of P-solubilization by PSB

Primarily there are two schools of thoughts interpreting the microbial phosphate

solubilization processes (Figure 1.7)

Solubilization by production of organic acid (mainly dissolved inorganic P)

Solubilization by production of phosphatase enzymes (mainly dissolved organic P)

The main mechanisms involved in the microbial phosphate solubilization include:

1. Release of complex or mineral dissolving compounds e.g. organic acid anions,

siderophores, protons, hydroxyl ions, CO2

2. Liberation of extracellular enzymes (biochemical P mineralization)

3. The release of P during substrate degradation (biological P mineralization) (McGill

and Cole, 1981).

Microorganisms are involved in all components of soil phosphorus cycle.


25

1.3.2.1.3.4 Solubilization of Inorganic Phosphate.

The ability of bacteria to solubilize insoluble inorganic phosphate compounds, including

Al and Fe-bound phosphate, tricalcium phosphate, dicalcium phosphate, hydroxyapatite,

and rock phosphate is well reported. These microorganisms solubilize phosphate mainly

by organic acid production (Puente et al., 2009a; Hanif et al., 2015) either by: (i) decreasing

the pH, or (ii) by enhancing chelation of the cations bound to P (iii) by competing with P

for adsorption sites on the soil (iv) by forming soluble complexes with metal ions

associated with insoluble P (Ca, Al, Fe) and thus P is released (Zaidi et al., 2009). Many

heterotrophic microorganisms are reported to solubilize inorganic P by excreting organic

acids to dissolve e mineral phosphates and/or chelate cationic partners of the P ions i.e.

PO4-3 directly, releasing P into solution (He et al., 2002).

Several bacterial strains form genera Pseudomonas, Bacillus, Rhizobium and Enterobacter

could be referred as the most important strains capable of solubilizing P by organic acids

secretion (Kucey et al., 1989; Hariprasad and Niranjana, 2009; Oliveira et al., 2009).

Gluconic acid (Deubel et al., 2000), oxalic acid, citric acid (Kim et al., 1997), lactic acid,

tartaric acid (Hanif et al., 2015), formic acid, acidic, propionic, glycolic, succinic acid

(Vazquez, 2000) are the prominent acids secreted by bacteria during P-solubilization.

Inorganic acids e.g. hydrochloric acids are also involved in P-solubilization but are less

effective (Kim et al., 1997).

Glucose dehydrogenase enzyme (GDH) catalyzes the biosynthesis processes of gluconic

acid (GA) by oxidizing glucose in Gram negative bacteria. In this process pyrroloquinoline

quinone ( PQQ) acts as co-factor (Goldstein, 1994). A gene cluster of pqqA, B, C, D, E,

and F is responsible for the synthesis of PQQ (Meulenberg et al., 1992; Kim et al., 1998).


26

Among these genes, the most important gene is pqqE, being the most conserved part of the

cluster (Perez et al., 2007). Phosphate solubilizing genes were primarily cloned by

Goldstein and Liu (1987) from Erwinia herbicola, a Gram negative bacteria. Upon its

expression in E. coli HB101, it resulted in gluconic acid production and solubilization of

hydroxyapatite. Escherichia coli can synthesizing glucose dehydrogenase, but cannot

synthesize PQQ, consequently it does not produce gluconic acid (Liu et al., 1992).

1.3.2.1.3.5 Mineralization of Organic Phosphate

Organic phosphate solubilization/mineralization occurs in soil at the expense of plant and

animal remains, which are the source of large amount of organic phosphorus compounds.

Mineralization of soil organic phosphorus (Po) is another key player of phosphorus cycling.

Numerous saprophytes are involved in the decomposition of organic matter in soil, by

releasing the radical orthophosphate from the carbon structure of the molecule by the help

of the action of several phosphatases (also called phosphohydrolases). It is believed that

about half of the rhizospheric microorganisms possess phosphate mineralization potential,

regulated by phosphatases (Tarafdar et al., 2003). These phosphatases are classified as

specific or nonspecific acid phosphatases, according to their substrate specificity. Organic

phosphates are used by acid phosphatases as a substrate for mineralization (Beech et al.,

2001). The production of acid phosphatases is believed to be the principal mechanism for

organic P mineralization in soil (Hilda and Fraga, 1999). The microbes and plant roots

produce acid and/or alkaline phosphatase enzymes to hydrolyze the soil Po from organic

residues. The largest portion of extracellular soil phosphatases is derived from the

microbial population (Dodor and Tabatabai, 2003). The specific phosphohydrolases with


27

different activities include: 39- nucleotidases and 59-nucleotidases; hexose phosphatases;

and phytases (Rossolini et al., 1998).

Soil P mineralization can be carried out by three groups of enzymes: Non-specific acid

phosphatases (NSAPs), Phytases, Phosphonatases and C–P lyases.

Non-specific acid phosphatases are the group of enzymes also called (phosphohydrolases)

dephosphorylate phospho-ester or phosphoanhydride bonds of organic matter. Among the

variety of phosphatase enzyme classes (designated as class A, B and C; McGrath et al.,

1998) produced by phosphate solubilizing microorganisms, phosphomonoesterases (often

known as phosphatases) gained much attention (Nannipieri et al., 2011). Phosphatases can

be acid or alkaline depending on their pH optima (Jorquera et al., 2008). Acid phosphatases

are produced in minute quantities proposing that this is a potential niche for phosphate

solubilizers (Criquet et al., 2004; Richardson et al., 2009)

The relationship between phosphate solubilizers applied in soil, phosphatase activity and

the consequent organic phosphate mineralization is not well understood yet (Chen et al.,

2003)

About 30-50% of total soil P can be constituted by Po and the major portion of it

corresponding to phytate (Turner et al., 2002) in natural environment. In this context, there

are bacteria capable of producing phytase enzymes for the mineralization of phytates (Lim

et al., 2007). To date, there are only few studies reporting rhizobacteria capable of

mineralizing the phytate. Among the phytase producing rhizobacteria, species belonging

to Bacillus, and genera are the most common culturable bacteria.


28

Phosphate-ester linkages present in phytic acid, are rather stable be degraded naturally and

even their chemical hydrolysis is very slow (Turner et al., 2002). However, several PSM

are able to secrete phytase including Pseudomonas, Staphylococcus, Bacillus

amyloliquefaciens Serratia, Enterobacter Bacillus subtilis, Burkholderia, Sporotrichum

thermophile (Richardson, 2001; Singh and Satyanarayana, 2010; Hanif et al., 2015).

Histidine acid phosphatases (HAPs) are acidic in nature and efficiently work at pH 2.5-6.5

(Mullaney and Ullah, 2003). Histidine phosphatases share a common active site having C-

terminal motif HD and N-terminal (conserved) motif RHGXRXP, carried out hydrolysis

phosphomonoesters (a twostep process) (Mullaney and Ullah, 2003). N-terminal behaves

as nucleophile, results in the formation of phospho-histidine intermediate and C-terminal

behaves as proton donor to O2 of the scissile phosphomonoester bond (Ariza et al., 2013).

Cysteine phosphatases along with common active site motif HCXXGXXR (T/S) is capable

of utilizing phytate, was first identified by Chu, (2004) from Selenomonas ruminantium. A

particular phosphate releasing enzyme group is capable of cleaving C-P bonds from

organophosphonates (Rodriguez et al., 2006).


29

Figure 1-7 Schematic representation of the mechanisms involved in the P-

solubilization and mineralization by PSM (Adopted from Seema et al., 2013).


30

Figure 1-8 Sites of plant colonization by endophytic bacteria. Drawing adopted from

Compant et al. (2010)

1.3.2.1.4 Biocontrol of Pathogenic Micro-Organisms

Phytopathogenic microorganisms, being the cause of significant reduction of in crop yield,

are a serious threat for agriculture. Usually, chemical pesticides are used for their control

but unfortunately, this approach has led to serious environmental as well as human health

concerns. Moreover, most of the pathogenic microorganisms has developed resistance

against these chemical remedies (pesticides) over time, leading a constant development of

new pesticides (Fernando et al., 2006). When we look an alternative for chemical control

of pathogens, rhizobacteria are there with their naturally gifted ability to suppress soil

borne pathogens (Weller et al., 2010). These rhizobacteria can provide biocontrol against

a several pathogenic bacteria, fungus, nematodes, and viruses and insect pests (Zahir et al.,

2004). Both direct and indirect mechanisms are involved in bacterial biocontrol including

hydrolytic enzymes (chitinases, proteases, lipases, etc.), antagonism (by antibiotic


31

production), HCN and siderophores in direct biocontrol activities. On the other hand,

rhizobacteria act as probiotic to resist the competition of other partners, for acquiring

favorable (healthy and disease free for plant) niches in the rhizosphere. Additionally,

acquired and induced systemic resistance activation and plant tissue hormonal level

modifications also play key role in microbial biocontrol (van Loon, 2007). Rhizobacteria

belong to the genera Streptosporangium Micromonospora, Alcaligenes, Pseudomonas,

Bacillus Streptomyces, Agrobacterium and Thermobifida are known bio-control agents

(Ali et al., 2014).

1.3.2.1.4.1 Antibiotic-producing Rhizobacteria

Among the bacterial biocontrol mechanisms, the most powerful and well-studied

mechanisms is known to be the production of antibiotics (low molecular weight chemical

organic compound) (Raaijmakers et al., 2002). The most common antibiotics involved in

PGPR biocontrol are: A, phenazine-1-carboxylic acid, pyoluteorin, A, oomycin

pyrrolnitrin, butyrolactones, oligomycin viscosinamide, xanthobaccin, and 2,4-diacetyl

phloroglucinol (2,4-DAPG) (Whipps, 2001). With a wide spectrum of properties

(Rezzonico et al., 2007), 2,4-DAPG is known to be the most important antibiotic against

plant pathogens (Fernando et al., 2006). Pseudomonas is considered as largest group with

biocontrol activity (Walsh et al., 2001; Whipps, 2001; Rezzonico et al., 2007).

1.3.2.1.4.2 Hydrogen Cyanide (HCN) Producing Rhizobacteria

Hydrogen cyanide (HCN) a volatile secondary metabolite, produced by PGPR, can also be

used as biocontrol (Rezzonico et al., 2007). It is reported that HCN can also affect the plant

growth and development negatively.


32

Members of bacterial genera Pseudomonas, Bacillus, Rhizobium, Alcaligenes and

Aeromonas are known to produce HCN (Ahmad et al., 2008). Pseudomonas is reported to

be the top HCN producer among all bacterial genera with some reports showing that about

50 % rhizospheric pseudomonad are capable of producing HCN in vitro (Yasmin et al.,

2013; 14).

1.3.2.1.4.3 Siderophore-producing Rhizobacteria

Bacteria and fungi can produce iron (Fe) chelating agents called siderophores (low

molecular weight compounds) usually in response to Fe-deficiency (Sharma and Johri,

2003). Iron acquisition by siderophore production can play key role in strengthening the

ability of bacteria of root colonization and to compete for iron with other rhizospheric

microbes This iron sequestration in rhizosphere play an important role in preventing the

proliferation of pathogenic microorganisms. Bradyrhizobium, Pseudomonas, Rhizobium,

Serratia and Streptomyces are well known genera for siderophores production (Roy and

Chakrabartty, 2000; Khandelwal et al., 2002; Kuffner et al., 2008).

1.3.3 Rhizospheric Microbes; the harmful

Rhizospheric zoo contains plant beneficial and harmful microorganism side by side

(Newton et al., 2010), as the root exudates may have the equal attraction for pathogenic

population along with PGPR (Pieterse et al., 2016; Newton et al., 2010; Raaijmakers,

2015). These bacteria can harm the plant by: (a) producing type III effectors, (b)

phytotoxins e.g. coronatine, syringomycin, pectatelyases, (c) and excessive production of

auxin can lead to gall formation and (d) and nutrient competition.


33

Bacteria can be beneficial (symbionts) or harmful (pathogens), depending on prevailing

conditions (Raaijmakers, 2015) such as signals produced by the hosts, light, nutrient, water

or temperature stress, inoculum strength, set of agricultural conditions and host species.

COLONIZATION OF PGPR IN DIFFERENT COMPARTMENTS OF

PLANTS

No plant or cryptogam exists in nature without microorganisms associated with its tissues.

Plants serve as the host for microbes, are puzzles of different microhabitats, each of them

colonized by specifically adapted microbiomes. Plants constantly interact with different

types of microorganisms from soil microbial communities - the greatest reservoir of

biological diversity in the world (Berendsen et al., 2012). These interactions are believed

to have strong influence on fitness of the host. Most of the microbial mediated processes,

including plant growth promotion, plant protection, pathogenesis, competition etc. occur

in the rhizosphere - the area of soil surrounding the roots which is most exposed to the

influence of plant roots exudates (Nihorimbere et al., 2011). Effective host colonization

is the most crucial prerequisite for an agronomically important bacterium-plant

interaction. Moreover, maintenance of their population in the rhizosphere to compete the

indigenous microflora and fauna (Barea et al., 2005). A constant benefit of a plant growth

promoting rhizobacteria can only happen when bacteria enter in a close association with

plant roots. It is very important to evaluate the colonization potential of bacterial isolates

both in vitro and in vivo.

Plant-microbe interaction studies have benefitted from the development of high

throughput molecular methods, such as metagenomics and meta transcriptomics (Jansson

et al., 2012). Subsequently, the microbe-host association’s studies have become a core


34

theme in microbial ecology. But unfortunately, these methods lose the spatial information

as disadvantage, as the microbes are displaced from their original location. Due to such

reasons, the methods which allow microbial localization and visualization in microbe-host

systems are getting attention during last couple of decades. For bacterial localization

studies, several biomarkers have been established (Bais et al., 2006).

1.4.1 Fluorescent Antibodies

Immunofluorescence application is a serological technique which combines the

advantages of antibodies binding power to their target sites (Janse and Kokoskova, 2009).

Immunolocalization is based on the use of fluorescent signal molecules conjugated to the

antibodies; the emission of fluorescent light indicates the presence of a specific antigen.

Antigen-antibody interactions are detected or visualized by the use of analytical methods

including immunoassays by conjugating the antibody with signal molecule

(fluorochromes, enzymes and radioisotopes). Mono or polyclonal antibodies are used

according to the specificity required. The examples of immunolocalization used to study

root colonization include the analysis of the spatial competition between Pseudomonas

fluorescens Ag1 and Ralstonia eutropha in barley root colonization (Kragelund and

Nybroe, 1996). The use of fluorescent antibodies to localize and differentiate the target

cells from no-target cells in the rhizosphere, fluorescein isothiocyanate (FITC)-

conjugated IgG antibodies specific to the target bacterial strain is well reported

(Lemanceau et al., 1992). To study the endophytic colonization, an in situ detection with

specific labeling techniques and highly resolving microscopic methods is required.


35

1.4.2 Specific Primers and Oligonucleotidic Probes

Several primes or probes are used to monitor the inoculated bacterial cells in vitro or in

situ, as these primers/probes amplify or hybridize the strain specific sequences (Werner et

al., 1996). Usually these probes are linked covalently to a fluorochrome such as

fluorescein, rhodamine, Texas red, Cy3 and Cy5 (Amann et al., 2001).

Fluorescence in situ hybridization (FISH) and labeling with fluorescent proteins are

commonly used for the detection and localization of introduced bacterial colonization

patterns and community composition (Amann et al., 2001). Hybridized DNA-probes are

labeled with fluorochromes with the complementary target sequence in FISH technique.

As the characteristic signature sequences are present in most of rRNA genes, a specific

taxonomic range can be detected, depending on sequence specificity (Loy et al., 2007).

Assmus et al., (1995) documented the in situ localization of A. brasilense in the rhizosphere

of wheat, while the same results were reported by MacNaughton et al. (1999) with P.

syringae and R. fascians on the tomato root surface.

1.4.3 Fluorescent Markers

To monitor the introduced bacterial cells in the environment, another attractive marker

system is the use of fluorescent protein. Fluorescent proteins encoding genes are inserted

in plasmids and then cloned into competent cells (Morschhäuser et al., 1998). The

examples of fluorescent proteins used as markers are green fluorescent protein (GFP),

yellow fluorescent protein (YFP), and DsRed protein (Larrainzar et al., 2005).

All above mentioned techniques are helpful with the use of sophisticated and highly

sensitive microscopic techniques like confocal laser scanning microscope (CLSM) and


36

transmission electron microscopy (TEM) and are known for root colonization studies and

the ultrastructure of the target PGPR strains (Yasmin et al., 2012; Shahid et al.,2012).

1.4.4 Confocal Laser Scanning Microscope (CLSM)

The fluorescently labelled bacteria either by fluorescent markers, antibodies or

oligonucleotic probes, can be detected with the help of an epifluorescent microscope. But

the background fluorescence produced by root, soil particles and other contaminants is the

major drawback of direct microscopy. Confocal laser scanning microscopy has the

advantage of reducing most some of the limitations significantly (Pawley, 2006). It is a

powerful machine for visualizing fluorescently labelled microbial cells at very high

resolutions. Because three dimensional views can be generated, CLSM offers its digital

processing generating three dimensional (3D) images. The florescent signals are detected

only from the focused plane, reducing unwanted background fluorescence. Moreover,

different bacteria and/ or secondary metabolites can be detected simultaneously, with the

help of different fluorescence channels. Due to these advantages, the use of CLSM to study

the localization of introduced microbes on different compartments of plants, is growing

(Assmus et al., 1995; Hansen et al., 1997; Bloemberg et al., 2001). The cost per sample is

the major limitation of this instrument. CLSM allows the detection of Three major kinds

of fluorescently marked objects can be detected by CLSM including molecules, cells and

tissues, genetically modified organisms and autofluorescent cells, tissues and substrates.

Schloter, (1993) localized the inoculated Azospirillum brasilense SP7 on wheat roots with

the help of CLSM in plant-microbe study for the first time.


37

1.4.5 Electron Microscopy (EM)

In the field of microscopic studies, the use of electron microscope is of prime importance,

specifically, Scanning Electron Microscopy (SEM) and Transmission Electron

Microscopy (TEM). Transmission electron microscope is extensively used for

ultrastructural studies of microbial cells and plant microbe interaction studies. In TEM,

electron beam is used for specimen image formation (Bozzola and Russell, 1992) due to

their extremely short wavelength. Electron microscopes work on the princile same as that

of light miscrope but with some fundamental differenes. As they take advantage of the

much shorter wavelength of the electron (e.g., λ = 0.005 nm at an accelerating voltage of

50 kV). While, light microscope uses the wavelengths of visible light (λ = 400 nm to 700

nm) (Flagler et al., 1993). Moreover, electron microscope operates in vacuum and focuses

the electron beam coming from electron emitting gun magnifies the image with the help

of series of electromagnetic lenses (Maier, 2009). The sample preparation for TEM needs

fixation and staining by heavy metal salts (osmium tetra oxide, lead citrate and uranyl

acetate) to improve the contrast.

Rhizobacterial localization in rhizosphere, rhizoplane and in plant root cells has been

studied by using transmission electron microscope over the last many years. (Bloemberg

et al., 2000). Recently, Naqqash et al. (2016) reported strong colonization of Azospirillum

sp. TN10 to potato roots through ultrastructure studies using TEM. Transmission electron

microscopic studies revealed effective colonization of maize roots by the PGP bacterium

Bacillus amyloliquefaciens FZB42 to the roots restricted to the rhizoplane. Neither the

CLSM nor the TEM and scanning electron microscope (SEM) images indicated endophytic

presence of FZB42 suggesting that FZB42 is a true epiphyte (Fan et al., 2011).


38

On the other hand, many studies have conformed the bacterial isolations from root and

shoot interior of different crops after surface sterilization (Stoltzfus et al., 1997). However,

surface sterilization alone is not sufficient to assign the bacteria as an endophytic colonizer.

Only microscopic localization using can provide evidence of an endophytic bacterium

(James and Olivares, 1998). For example, electron microscopic observation revealed a

strong endophytic bacterial (Bradyrhizobium sp. strain SR-6) occupancy in the root nodule

cells of soybean plant (Ali et al., 2016). Similar colonization behavior studied through

TEM was described by Lee et al. (2012). Hameed et al. (2005) also observed the co-

occupancy of immunogold labeled strain of Agrobacterium and Bradyrhizobium strain in

nodule of host plant root through electron microscopy. Duijff et al. (1997) reported the

inter- and intracellular colonization by P. fluorescens WCS417r (biocontrol agent) to the

tomato root cells. Bellone et al. (1997) published sugarcane root colonization and infection

thread formation by Acetobacter diazotrophicus. The ultra-structural studies of

Samaneasaman root nodule showed the bacterial occupancy in the central region of nodule

and bacteroid tissue of nodules contained both infected and uninfected cells (Qadri et al.,

2007). Yasmin et al. (2012) visualized the localization of Bradyrhizobium and

Agrobacterium along with mycorrhizae in the root nodules of Vigna radiata through ultra-

structural studies using transmission electron microscope. They documented the presence

of both infected and uninfected cells in the root nodules tissue.

BIOFILM-THE CITY OF MICROBES

The most frequent microbial lifestyle in natural environments is the occurrence of

organized structures associated with surfaces known as biofilms (Watnick and Kolter,

2000). Plant growth promoting rhizobacteria are not the exception to this rule. Microbial


39

aggregates and floccules and also adherent populations within the pore spaces of porous

media comes under the definition of biofilm (Costerton et al., 1995). To produce beneficial

effects, PGPR have to interact with the plant surface to form those complex multicellular

assemblies/aggregates named biofilms. (Bogino et al., 2013). Biofilm formation permits

an entirely different lifestyle from the planktonic state (Bogino et al., 2013). This mode of

life is often crucial for survival of bacteria, as well as for the establishment of specific

symbiosis with legume or actinorhizal host plants or nonspecific root colonization (Bogino

et al., 2013). Bacteria in biofilms are protected from harsh environmental conditions

(Vorachit et al., 1995) and also display distinct phenotypes compared to their planktonic

counterparts including resistance to antimicrobial compounds and enhanced nutrient

uptake (Bogino et al., 2013).

The polymeric substances, mainly polysaccharides, proteins, lipids, and nucleic acids,

provide biofilm a mechanical strength, facilitate their surface adhesion, and form a

cohesive, three-dimensional polymer network that interconnects and transiently

immobilizes cells (Flemming and Wingender, 2010). Numerous microbial species,

including rhizobia, form microcolonies or biofilms when they colonize roots (Rinaudi and

Giordano, 2010). Sinorhizobium meliloti was first reported, and the rhizobia regulatory

system in this species and conditions for analyzing its ability to biofilm formation on biotic

and abiotic surfaces were investigated (Fujishige et al., 2005). The study of biofilm

development and its physiology is an emerging topic in the knowledge of plant microbe

interaction.


40

EFFECTS OF PGPR ON PLANT GROWTH

Plant growth promoting rhizobacteria are well known to improve growth and yield

attributes including plant height, fresh and dry weight, leaf area, chlorophyll content, plant

root length, number and weight of nodules, number and weight of seeds, seed oil contents

and consequently total biomass of several crop (DeKokalis-Burelle et al., 2006; Van Loon,

2007; Mia et al., 2010, Hussain et al., 2015; Imran et al., 2015; Ayyaz et al., 2016).

Bacterial inoculation plays a key role in the improvement of crop nutrition (Afzal and

Bano, 2008) enhancing growth and yield of cereal crops (Biari, et al., 2008; Baig et al.,

2014; Ali et al., 2016) and sustaining the productivity of soil and reduce costs of crop

production by reducing level of chemical fertilizers and fungicides (Yasmin et al., 2013,

Islam et al., 2016).

Anjum et al. (2007) studied the response of cotton to PGPR inoculation alone and in

combination with three levels of mineral nitrogen fertilizer. The bacterial inoculation

enhanced 21% cotton yield, 5 % plant height and the increase of microbial population was

41 % over the control. Similarly, Ashrafuzzaman et al. (2009) conducted experiments to

isolate and characterize PGPR associated to rice. They found that most of the isolates

resulted in enhanced seed germination, shoot/root length and dry weight rice seedlings.

They have concluded that the inoculation of bacterial isolates (PGB4, PGG2 and PGT3)

could be beneficial for rice due to their phosphate solubilization and indole acetic acid

production traits.

Imran et al. (2015) conducted a series of field experiments in different soils to evaluate the

differential response of different genotypes of chickpea to PGPR inoculation. They

reported pronounced soil fertility along with increased production of nodules (42 %)


41

biomass (up to 31 %), grain yield (up to 64 %), and harvest index (up to 72 %) in both

genotypes in response to PGPR inoculation over non-inoculated control treatments. Similar

effect of inoculated bacterial strains with multiple PGP traits on tomato were reported by

Sharma et al. (2015).

Majeed et al. (2015) isolated plant growth promoting bacteria from wheat root-endo and

rhizosphere to investigate their PGP traits, colonization potential and effect on wheat

growth promotion. Inoculation with multiple PGP-traits resulted insignificantly increased

root and shoot length and biomass of wheat along with shoot and root N contents up to

76% and 32% respectively.

Bacillus subtilis KPS-11 with phosphate solubilizing/phytate mineralizing and IAA

producing ability was characterized and inoculated to potato by Hanif et al. (2015).

Significantly increased root/shoot length and root/shoot fresh and dry weight of potato in

response to KPS-11 as compared to un-inoculated potato plants were recorded.

Pham et al. (2017) investigated the response of rice seedlings towards Pseudomonas

stutzeri inoculation and reported significant growth promotion over un-inoculated control

treatments. They also documented that the inoculation with P. stutzeri resulted in

significantly better performance than chemical fertilization.

The enhanced growth and yield could be result of the beneficial functions of inoculated

PGPR strains including phytohormone production, phosphate solubilization and nitrogen

fixation, disease suppression etc.

Soil erosion and soil fertility are crucial issues for sustainable agriculture in hilly areas of

Azad Jammu and Kashmir (AJK). Low inherent soil fertility, lack of sufficient external


42

inputs and high erosivity leads to non-sustainability in the region. An extensive survey in

and around Rawalakot/Dhirkot (subdivisions), Azad Jammu and Kashmir showed a severe

deficiency of most of the major plant nutrients (Malik et al., 2000). So the supplementation

of these nutrients to the plants in the form of synthetic fertilizers is a part and parcel of

existing cropping system in the region. However, these soil amendments are not only

expensive but also considered a potential source of environmental pollution because of

gaseous emissions and No3- leaching. In addition, the use of chemical fertilizer in sloppy

landscape, under high rainfall, may not be effective because of surface runoff and leaching.

Due to these pessimistic impacts of chemical fertilizers, scientists are desperate to come up

with alternative strategies that can ensure competitive yields while protecting the health of

soil and are friendlier to the environment, as well as, can maintain the long term ecological

balance of the soil ecosystem (Lucy et al., 2004).

Searching microorganisms to improve soil fertility and boost plant nutrition has continued

to magnetize the attention, as their application can cutback in the high rates of fertilizer

(Gyaneshwar et al., 2002; Shaharoona et al., 2008) and the resulting environmental

problems without compromising plant productivity. These bacteria put forth the

advantageous effects on plant and soil health (Bakker et al., 2007). It is important to search

for native or region-specific microbial strains which can be used as a potential plant growth

promoter and nutrient solubilizer/mobilizer to achieve desired production levels. Studies

are required to prove the nature of these isolates and to harness their potential as bio-

inoculants in agriculture.


43

SUNFLOWER

Sunflower (Helianthus annuus L.) is a deep rooted, 90-120 days crop and can be grown

twice a year, with an ability to extract water from below root zones of normal grain crops

(Unger 1984). It fits well as an alternative crop in crop rotations in different agricultural

regions (Amorim et al., 2008; Backes et al., 2008). Sunflower is one of the four chief

oilseed crops in the world (Škorić et al., 2008) with great potential for producing the

highest oil yield per hectare. Its economic importance is due to the nutritional quality of

its oil, beekeeping, animal feed and biofuel production. Light coloured sunflower oil is

considered a premium edible oil due to its high level of un-saturated fatty acids with a high

smoke point. An ideal combination of saturated and poly-unsaturated fatty acids is found

in this oil, which helps in reduction of blood cholesterol level (Balasubramaniyan and

Palaniappan, 2004). . Sunflower oil contains, carbohydrates, protein and vitamin E (18.6

% , 19.8 %, and 0.038 % respectively; McKevith, 2005).

Pakistan has continual deficiency in edible oil production and is the third largest edible oil

importer worldwide, resulting in a severe drain on national economy. In years 2010-11,

US$ 2.611 billion were spent on the import of edible oil (Govt. of Pakistan, 2011-12).

Therefore, concreted efforts should be made to bridge-up the gap between the edible oil

production and consumption. One of the efforts could be the production of sunflower on

large scale, as it is a high yielding non-traditional oilseed crop and can be a key player to

shrink this gap (Ehsanullah et al., 2011, Hussain et al., 2010).

There have been very few studies of the microbial diversity associated with sunflower,

and hence the bacterial diversity of sunflower in Pakistan soils remains largely unknown.

Shahid et al. (2014) isolated and characterized sunflower associated rhizobacteria from


44

different locations of Pakistan. They found 19 bacterial strains having multiple PGP-traits

and reported significant increase in sunflower growth and nutrient uptake upon the

inoculation of selected bacterial strains in field experiments conducted at Faisalabad,

Pakistan. We extended this work to explore various altitudes of Himalayan Mountains of

Dhirkot, Azad Jammu and Kashmir (AJK) to find efficient native PGPR, as the soils of this

region are completely unexplored (as per our knowledge) towards sunflower associated

bacterial population. So far, there is hardly any report discussing the use of PGPR

associated to any oil seed crops from AJK soils. Moreover, a better knowledge of these

bacteria and their implementation as biofertilizers could change traditional crop

management practices regarding plant nutrition and defense mechanisms. For a maximum

exploitation of the plant-bacterial association, effective/beneficial bacteria must be selected

that can associate with the plant roots under specific ecological conditions. To meet the

aim of the study, following working strategy was adopted

Objectives

1. Isolation, characterization and identification of plant root associated beneficial

bacteria from the soil samples collected from different sites of sub-division Dhirkot,

AJK using biochemical and molecular techniques

2. Analysis of bacterial diversity using polyphasic techniques

3. Documenting exo and endo-rhizospheric bacterial interaction in sunflower using

different microscopy techniques i.e., Transmission Electron microscopy and

Confocal Laser scanning Microscopy


45

4. Sunflower plant inoculation and evaluation of potential plant growth promoting

rhizobacteria under controlled conditions and field environment to select the

candidate bacteria for inoculum production of sunflower.

Chapter 2

MATERIALS AND METHODS

2.1.1 Collection of Soil Samples

Soil samples (0-15cm depth) were collected from 16 different sites (Table 2.1) of sub-

division Dhirkot, Azad Jammu and Kashmir, on the basis of ecological/ spatial and

temporal variation. These samples were transported to laboratory and immediately

refrigerated until utilized for soil physic-chemical properties, microbial/bacterial

population and processed for bacterial isolation.

2.1.2 Meteorological Data of Selected Sites

Meteorological data of the selected sites was recorded at the time of sampling and soil and

air temperature (°C), humidity (%), heat index (°C), barometric pressure (kpa) and altitude

were recorded with the help of weather station (Kestrel 5500, USA).

2.1.3 Soil Analysis

Soil samples were air-dried under shade and sieved through a mesh (2 mm). These samples

were analyzed for physical and chemical properties. Soil analysis were conducted in the

laboratory of Soil & Environmental Sciences, University of Poonch Rawalakot, Azad

Kashmir.

2.1.3.1 Soil Texture

To measure the particle size distribution, 50 g of soil sample was soaked (500 mL beaker)

overnight in 1% sodium hexametaphosphate solution (40 mL) with 200 mL of distilled

Chapter 2 Materials and Methods

47

water. After stirring (10 min), it was shifted to glass cylinder (1 L). The soil water mixture

was allowed to stand overnight. The suspension was dispersed in a dispersion cup for 15

min. After 40 s of shaking, preliminary reading was recorded whereas final reading was

taken after 2 h with Bouyoucous Hydrometer. Soil textural classes were determined by

ISSS triangle (Gee and Bauder, 1986).

2.1.3.2 Soil pH

To determine soil pH, a saturated soil suspension of 1:2 (soil and water) was prepared. This

suspension was allowed to stand for 1 h. The pH meter (JENCO Model-671 P) was

calibrated with the help of pH buffers (4.1 and 9.2) and used to measure pH (McLean,

1982).

2.1.3.3 Electrical Conductivity (EC)

Electrical conductivity of soil samples was determined by preparing a saturated soil

suspension 1:10 (soil and water). Electrode of EC meter (Jenway) was inserted in the

suspension and reading were recorded on EC meter (Rhoades, 1982).

2.1.3.4 Organic Matter (OM)

Soil organic carbon was determined by using a slightly modified Mebius method (Nelson

and Sommers, 1982). About 1 g soil was digested with 10 mL of 1N potassium dichromate

(K2Cr2O7) and concentrated H2SO4 (20 mL) at 150 ºC for 30 min. Digests were then titrated

with standardized FeSO4. The percent soil organic matter was calculated by multiplying

the percent organic carbon with a factor 1.724 (Brady and Weil, 2002).


48

2.1.3.5 Total Nitrogen

To determine the total N content, soil (10 g) was digested with concentrated sulfuric acid

(30 mL) and 10 g of digestion mixture contained K2SO4: FeSO4: CuSO4 = 10: 1: 0.5, in

Kjeldahl’s digestion tubes. The total volume of the digested material was then made up to

250 mL and 10 mL of it was used for ammonia distillation, with 4% boric acid solution

and indicator (boromocresol green and methyl red) in a receiver. The material was titrated

against N/10 H2SO4 by Gunning and Hibbard’s method in the receiver using micro

Kjeldahl apparatus after distillation (Bremner and Mulvaney, 1982).

Table 2.1 Meteorological data of sites at the time of sampling

Sample

No. Location Soil Temp.

0C. (20 cm)

Air

Temp 0C

Humidity

(%) Heat

Index 0C

Barometric

pressure

(kpa)

Altitude

1 U Munhasa 21 35.5 38.4 40.1 855 1130

2 Bandi 27 38.9 48.5 48.8 914.6 855

3 Hill 2 28 29.2 42.5 31.5 871 1265

4 Makhyala 26 29.7 45.5 28.7 862.5 1317

5 Chamankot 26 24.5 53.9 24.1 864.4 1496

6 Chamyati 23 26.4 51.7 25.8 839.4 1565

7 Dhirkot 25.5 27.7 37.6 24.4 843.7 1522

8 Ghaziabad 26 29.5 44.7 33.6 864.7 975

9 Keri 21 26.5 40.8 26.6 844.4 1515

10 Sanghar 23 27.7 47.8 26.5 833.2 1625

11 Hanschoki 22.5 20 52.5 16.5 804.4 1916

12 Dyar gali 24 27 39.8 30.2 819.4 1762

13 Neela but 19 19.5 61.5 20.5 803.1 1902


49

14 Kotli 21 22.8 49.8 22.6 822.8 1725

15 Narwal 22 24.7 43.6 34.7 857.1 1194

16 Arja 29 33 58.4 38.8 922 790

2.1.3.6 Extractable Potassium (K)

To measure the extractable K, 5 g soil was used for the preparation of soil solution with 1

N ammonium acetate to make up the volume up to 100 mL. After keeping this solution on

continuous shaking, it was filtered through Whatman filter paper No. 40. This filtrate was

then used to determine the extractable K Flame Photometer (Jenway, PFP7) as described

by Richard (1954).

2.1.3.7 Extractable Phosphorous (P)

Extractable P of soil samples was determined with the help of spectrophotometer

(CamSpec, M350 double beam UV-visible) following the method described by Olsen et

al. (1954).

2.1.3.8 Determination of Bacterial Population from Soil Samples

To determine the bacterial population from soil samples, viable cell counts was adopted

using serial dilution technique (Okon et al., 1977). One g soil was taken in a test tube 9 mL

of 0.89% (w/v) saline (NaCl) solution was added and followed the serial dilution

(Somasegaran and Hoben, 1994). A 100 mL of well vortexed solution from dilution 10 -3,

10-5, and 10-7 was spread on LB agar plate (Annexure 1). These plates were kept at 28 ± 2

oC for 2-4 days and colonies appeared (CFU/g) were counted.

CFU= [no. of colonies x volume spread x dilution level]/1000


50

ISOLATION, BIOCHEMICAL AND MOLECULAR

CHARACTERIZATION OF SUNFLOWER (HELIANTHUS ANNUUS L.)

ASSOCIATED BACTERIAL POPULATION

2.2.1 Isolation and Purification of Sunflower Rhizosphere/Endophytic Bacteria

Plastic pots were filled with soil (three pots/site). For the surface sterilization, sunflower

seeds (cv. FH-331) were dipped in 5% (w/v) sodium hypochlorite for 10 min. After

vigorous shaking, sodium hypochlorite was drained, followed by extensive (5-6 times)

washing of seeds with sterile water. Sterilized seeds were sown (manually) and the pots (9

cm diameter having 400 g soil) were kept in growth room (25 °C and 16/8 light and dark

periods). After 30 days of germination, plants were carefully uprooted and associated

rhizobacterial isolations were carried out. Loosely adhering soil was removed by gentle

shaking. For rhizospheric isolations, one g of soil immediately adhered to the root was

added to 9 mL 0.85 % (w/v) saline (NaCl) solution followed by serial dilution

(Somasegaran and Hoben, 1994). While, for endophytic isolations, root samples were

dipped in 70 % ethanol for surface sterilization followed by extensive washing with sterile

distilled water. Surface sterilized roots were then crushed with the help of sterile pestle and

mortar in autoclaved distilled water. The resulted root-suspension was used to make serial

dilutions. Hundred μL from each (rhizospheric and endophytic) of the dilution 10-3, 10-4

and 10-5 were spread on LB-agar plates. Plates were kept at 28±2 oC for 72 h. Bacterial

isolates from each plate were selected based on prolific growth and different colony

appearance, and purified by sub-culturing of single colonies on LB ager plates. The purified

bacterial isolates were grown at 28±2 ºC for 24 h on LB agar plates and preserved in 20 %

glycerol at stored at -80 ºC.


51

MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERIZATION OF

BACTERIA

2.3.1 Colony and Cell Morphology

Pure bacterial colonies were characterized for their morphology on the basis of colour,

shape, size and margins through stereo-microscopy (BAUSCH & LOMB, ASZ30E, USA).

The cell size, shape and motility of bacterial isolates were studied under light microscope

(Nikon LABOPHOTO-2, Japan), by placing a drop of an overnight grown bacterial culture

on glass slide or by suspending the bacterial colony in a drop of sterilized de-ionized water

on glass slide. Microscopic observations were made at 100X magnification.

2.3.2 Gram’s Staining

The Gram’s reaction was carried out in accordance with the method described by (Vincent,

1970). A loopful of pure bacterial culture was spread on glass slide in a drop of saline in

the form of thin smear. This smear was then dried in air, fixed on heat and stained with few

drops of crystal violet (Annexure 2) for one min. Distilled water was then poured over the

slides to wash it. The smear was then flooded with few drops of Gram’s iodine (Annexure

2) for another min. After that ninetyfive % (v/v) ethanol was dropped over the slide to

decolorize for 30 s and washed with distilled water immediately. It was finally treated with

safranin solution (Annexure 2), washed with water, air-dried and observed under light

microscope.


52

BIOCHEMICAL CHARACTERIZATION

2.4.1 Nitrogen Fixation

Atmospheric N2-fixing ability of the bacterial isolates was assessed using acetylene

reduction assay (ARA), following the method proposed by Hardy et al. (1968). Fully

grown bacterial isolates were inoculated in 12 mL vials containing 6mL semisolid nitrogen

free (NFM) malate media (Annexure 3; Okon et al., 1977) and incubated at 28 ± 2oC for

74 h. Sterile rubber septa were then used to replace the vial caps and 10% (v/v) acetylene

gas was used to replace the same amount of air from the vials with sterile syringe. The vials

were incubated for 24 h at the same temperature. The amount of acetylene (C2H2) reduced

to ethylene (C2H4) was measured by gas chromatograph (Thermoquest, Trace GC, Model

K, Rodon Milan, Italy) equipped with Porapak N column and flame ionization detector

(FID), following the standard protocol described by Park et al. (1976). The nitrogenase

activity was expressed as nmoles of ethylene formed per hour per mg of the protein. Protein

concentration was calculated by the method described by Bradford (1976).

2.4.2 Indole-3-acetic Acid Production

The ability of bacteria to produce indole-3-acetic Acid (IAA) was qualitatively determined

by colorimetric method (spot test). Sterilized Eppendorf tubes were filled with LB (200

μL) supplemented with tryptophan (100 mgL-1) which served as IAA-precursor. These

tubes were inoculated with pure bacterial cultures (100 μL) and incubated at 28±2 ºC

(without shaking). After 48 h, bacterial culture (100 μL) was spotted on white Perpex sheet

and 100 μL of Salkowski’s reagents (Annexure 4) was added and mixed with bacterial

culture. For positive control, hundred microliter of IAA (1000 ppm) standard (Sigma) was

mixed with same volume of Salkowski’s reagent at the same time. The colour development


53

was noted after 20 min and pink/purple colour was considered as positive result for IAA

production (Sachdev et al., 2009). For the quantification of IAA produced, bacterial

cultures were grown (in triplicate) in LB-broth (50 mL) supplemented with tryptophan (100

mgL-1) for 7 days. The grown culture was then centrifuged (10,000 rpm) at 4 °C and

supernatant was collected in fresh falcon tubes. Hydrochloric acid was used to acidify the

supernatant up to pH 2.8 and equal volume of ethyl acetate was used for the extraction

(twice) (Tien et al., 1979). The resulted extract was evaporated and collected in ethanol (1

mL) in an Eppendorf and filtered with 0.2 μm nylon filters (Millipore, USA). Finally,

filtrate was then run on high-performance liquid chromatography (HPLC, λ = 260 nm),

equipped with Turbochrom software (Perkin Elmer, USA) and C-18 column. The flow rate

kept was 0.5 mL min-1 and the mobile phase used was 30:70 (v/v) methanol: water as

described by Shahid et al. (2015).

2.4.3 Solubilization of Inorganic Phosphate

To evaluate the inorganic phosphate solubilization potential, the bacterial isolates were

checked as described by Pikovskaya, (1948) on Pikovskaya’s agar (Sigma, USA)

containing tricalcium phosphate as insoluble P source (Annexure 5). For qualitative

analysis, overnight grown bacterial culture (10 µL) was spot inoculated onto Pikovskaya’s

agar plates in triplicate and the plates were kept in incubator (28±2 ºC) for 19-12 days. The

plates were daily observed for clear zone formation around spots. The clear zones formed

were considered as positive for phosphate solubilization activity.

The solubilization index (SI) was determined by measuring the halo (clear zone) diameter

and the colony diameter, and the formula (Edi-Premono et al., 1996) used for SI calculation

is


54

SI= Colony diameter+Holozone diameter

Colony diameter

For quantification of phosphate solubilized buy bacterial isolates, Pikovskaya’s broth (50

mL in 250 mL Erlenmeyer flask) was inoculated in triplicate with overnight grown pure

culture. The flasks were kept on an orbital shaker (150 rpm at 28±2 oC) for 8-10 days. The

cultures were centrifuged at 12000 rpm for 8 min and cell free supernatant was collected

and filtered through 0.22 µm filter (Millipore, USA) and pH was recorded with a pH meter.

Two mL of standard reagent B (Annexure 5) was mixed with equal volume of distilled

water to make the blank. This blank was used to adjust spectrophotometer readings at zero.

Solubilized P was determined by recording optical density on spectrophotometer (Camspec

M350-Double Beam UV-Visible Spectrophotometer, UK) at 882 nm and comparing it to

standard curve of 2, 4, 6, 8, 10, 12 ppm solutions of KH2PO4, following phosphomolybdate

blue colour method (Murphy and Riley, 1962).

2.4.4 Production of Organic Acids

Pikovskaya’s broth (100 mL in 500 mL Erlenmeyer flask) was inoculated (triplicate) with

overnight grown single colony of each bacterial isolate. The flasks were kept on an orbital

shaker (150 rpm at 28±2 oC) for 8-10 days. The cultures were centrifuged at 12000 rpm for

8 min and cell free supernatant was collected followed by pH measurement with the help

of pH meter (pH/ion analyzer 350, CORNING). The supernatant was then filtered through

0.22 µm filter (Millipore, USA). Twenty mL of this filtrate was injected to HPLC equipped

with Turbochrom Software (Perkin Elmer, USA) and C-18 column. The flow rate kept was

0.6 mL min-1 and the mobile phase used was 30:70 (v/v) methanol: water. Signals were

detected at 210 nm. The peaks of unknown samples were compared to the standard organic


55

acids including gluconic acid, lactic acid, malic acid, tartaric acid, acetic acid and citric

acid (Sigma chemicals Ltd) as described by Park et al. (2010)

2.4.5 Intrinsic Antibiotic Resistance

Fully grown bacterial cultures were spread on solid ASS agar (Antibiotic sensitivity

sulphonamide agar; Merck, Germany) plates (40 g/L and the pH was kept 7.4). Ready-to-

use antibiotic discs (Bioanalyse®, Turkey) were used to determine the intrinsic antibiotic

resistance pattern by the bacterial isolates, following disc diffusion method (Valverde et

al., 2005). Antibiotic discs used were: streptomycin S (10 µg), erytomycin E (15 µg),

gentamycin CN (10 µg), cephradine (CE) (30 µg), carbenicillin PY (100 µg), ceftriaxone

CRO (30 µg), nalidixic acid NA (30 µg), doxicycline DO (30 µg), paratam CES (105 µg,

rifampicin RA (5 µg), cefixime CFM (5 µg), amikacin AK (30 µg), neomycin N (30 µg),

ofloxacin OFX (5 µg), trimethoprim (1.25 µg)/sulfamethoxazole (23.76 µg) SXT (25 µg),

ciprofloxacin CIP (5 µg), tetracycline TE (30 µg), aztreonam ATM (30 µg), erythromycin

E (15 µg), kanamycin (30 µg) ampicillin AM (10 µg) and chloramphenicol C (30 µg),).

Antibiogram (clear zone formation around the antibiotic disc) was observed after 1-2 days

of incubation at 28±2ºC.

2.4.6 Biocontrol Assay-Selection of Antagonistic Bacteria

Purified bacterial isolates were tested for in vitro antagonistic activity against the selected

Fusarium oxysporum (fungal phytopathogens) by using dual-culture assay (Sakthivel and

Gnanamanickam, 1987). About 5 mm2 fungal disc, taken from growing fungal mat

cultures, was placed near one edge on potato dextrose agar (PDA) plates. Pure bacterial

colony was streaked 3 cm away from the fungal plug towards the outer edge. Control plates


56

were just with the fungal plug and no bacteria and all the plates were in triplicates. The

plates were incubated at 28 ±2 ºC for 3-5 days and observed for antifungal activity.

2.4.7 Phenotypic Microarrays

Metabolic potential of bacterial isolates was checked by using BIOLOG GN2 micro-plates.

Fully grown cultures (on LB-agar paltes) were inoculated to Eppendorf tubes containing 1

mL DEPC H2O. Tubes were kept at room temperature for 3 h to starve the cells and drain

most of the stored energy. The starved cultures were added with inoculation fluid (IF-0a)

and redox indicators as instructed by the manufacturer. This mixture was then added (100

mL) to each well of micro-plate (Biolog, Hayward, CA) and incubated at 28±2°C for 24 h.

MOLECULAR CHARACTERIZATION OF ISOLATES

2.5.1 Preparation of Bacterial Colony for PCR

The freshly grown colonies were inoculated into LB broth in a 96 well plate and incubated

at 30°C with modest shaking overnight. Fifty μL of this culture was diluted in 50 μL of

10mM Tris (pH 8) again in 96 well plate. The plates were covered with lid and were boiled

for 10 min at 100 °C and centrifuged at 13,000 rpm for 1 min. Supernatant was used as

template for PCR, 2 μL/reaction.

2.5.2 Molecular Marker

1 kb ladder (Fermentas, Germany) was used as marker to compare and analyze the DNA

products (Figure 2-1).


57

Figure 02-1 1kb DNA ladder used to measure the size of the bands.

2.5.3 Identification of Bacterial Isolates using 16S rRNA Gene Sequence Analysis

The primer set fD1 and rD1 (Table 2.2) was used in polymerase chain reaction (PCR) to

amplify the 16S rRNA gene of the bacterial isolates as described earlier (Weisburg et al.,

1991) with slight modifications. A reaction mixture (50 µL) was used for amplification and

the conditions used in PCR are given in Table 2.2. The amplified product was separated on

1% (w/v) agarose Tris-acetate-EDTA (TAE) gel electrophoresis. The gel was

supplemented with 5 % ethidium bromide (stain) and visualized under ultra violet light and

photographed using gel documentation system (Vilbour Lourmat, France).

2.5.4 Amplification of NifH Gene

Amplification of nifH gene was carried out through a nested PCR (Silva et al., 2011). A

reaction mixture (25 µL) including primer set FGPH19 (Simonet et al., 1991) and PolR


58

(Poly and Bally, 2001) was prepared and incubated in a thermal cycler (PeQLab, advanced

Primus 96) following the conditions mentioned in table 2.2. A second PCR was conducted

using the product yielded by the 1st PCR as a template along with the primer set of PolF

and AQER (Poly and Bally, 2001).

2.5.5 DNA Sequencing and Sequence Analysis

The PCR products yielded by all the PCRs conducted for 16S rRNA and nifH genes

amplifications were purified using Wizard® SV Gel and PCR Clean-up System (Promega)

as instructed by the manufacturer. The purified products were then sent for sequencing to

LGC Genomics, Berlin, Germany. The resulted sequences were analyzed using Sequence

scanner software package and phylogeny was determined by BLASTn technique. The

sequences were submitted to GenBank EMBL.

2.5.6 Phylogenetic Analysis

The software package MEGA6 was used for phylogenetic analysis. The sequences of the

isolates were compared and analyzed using alignment tool CLUSTAL W, by downloading

the closely related sequences from NCBI data base (Tamura et al., 2013). Phylogenetic

analysis were carried out using maximum likelihood (ML), however the bootstrap values

of 70 % or greater were maintained to represent well supported nodes (Hillis and Bull,

1993).


59

Table 2-2 Primers PCR conditions used for 16S rRNA pqqE and nifH genes

amplifications from selected isolates

Primers 16S rRNA (5’-3’) PCR mixture Thermal conditions

fD1

(AGAGTTTGATCCTGGCT

CAG)

rD1

(AAGGAGGTGATCCAGC

C)

1X buffer (Fermentas),

3.5 µL MgCl2 (50 mM),

0.4 µL dNTP’s (25 mM),

0.5 µL formamide, 0.05 µL T4,

0.5μM each primer,

0.1U Taq polymerase

(Fermentas).

95 °C, 5 min

94 °C 1 min, 57 °C 1

min, 72 °C 2 min

35 cycles

final extension 72 ºC

10 min

Primers nifH (5’-3’) PCR mixture Thermal conditions

FGPH19

(TACGGCAARGGTGGNA

THG)

PolR

(ATSGCCATCATYTCRCC

GGA)

PolF

(TGCGAYCCSAARGCBG

ACTC)

AQER

(GCCATCCATCTGTATGT

CCA)

0.20 mM dNTPs, 1x buffer

(Roche),

0.01 mg BSA (20 mg/mL),

0.5 μM each primer,

0.5U Taq polymerase (Roche)

0.25 mM dNTPs, 1x buffer

(Roche),

0.01 mg BSA (20mg/mL),

0.5 μM each primer,

0.8 U Taq polymerase (Roche)

94 °C 2 min,

95 °C 60 s, 55 °C 2

min, 72 °C 2 min

30 cycles


30 min

94 °C 5 min,

94 °C 60 s, 48 °C 1

min, 72 °C 2 min

30 cycles


30 min

ROOT COLONIZATION STUDIES/ MICROSCOPIC STUDIES

2.6.1 Colonization Studies

The root colonization ability of selected PGPR strains (based on their performance in

different plant growth promoting biochemical tests in laboratory and infectivity tests in

controlled and field experiment) was investigated in both sterile and natural conditions

and was monitored by both the cultivation dependent (viable cell count for quantification

of colonizing bacteria) and cultivation independent method (YFP-Labeling,

immunofluorescence marker as well as fluorescence in situ hybridization used along with


60

confocal laser scanning microscopy and immunogold labeling by using transmission

electron microscope).

2.6.1.1 Labeling of Bacteria with Yellow Fluorescent Protein (YFP)

Four bacterial strains (Azospirillum brasilense AF-22, Enterobacter cloacae AF-31,

Pseudomonas sp. AF-54 and Citrobacter freundii AF-56) found best for their growth

promoting attributes in vitro and in vivo, were used in this study.

2.6.1.1.1 Preparation of Electro-Competent Cells

The electro-competent cells of Azospirillum brasilense AF-22, Enterobacter cloacae AF-

31, Pseudomonas sp. AF-54 and Citrobacter freundii AF-56 were prepared and

transformed through electroporation. Forty mL of fully grown bacterial cultures (1 × 108

CFU mL-1) were taken in ice cold falcon tubes (45 mL). These tubes were chilled on ice

for half an hour and harvested at 6000 × g at 4 °C for 15 min and cell free supernatant was

carefully drained. The harvested cells were very gently re-suspended in 20 mL cold 10 %

(v/v) glycerol and harvested again following the same temperature and speed of the

centrifuge machine. The re-suspension and centrifugation steps were repeated; the amount

of 10 % glycerol was reduced with every step of resuspension and centrifugation (15 mL,

10 mL, 5 mL in 2nd, 3rd and 4th wash, respectively). In the last step, 250 µL of 10 % glycerol

was used to re-suspend the cells. Aliquots (30 µL) of all the processed samples were flash

frozen in liquid nitrogen and stored at -80 oC.

2.6.1.1.2 YFP-Plasmid Isolation

Plasmid DNA was isolated from E. coli strain DH5α, containing 4.95 kb vector (Fig. 2.3)

pBBRIMCS-4 (well reported as a broad host range ampicillin resistant vector) along with


61

YFP cassette (Kovach et al., 1995), using QIAGEN QIA Miniprep kit. The plasmid DNA

was quantified by ultraspec™ 3100 (OD260, 260/280).

2.6.1.1.3 Transformation through Electroporation

The aliquots of electro-competent cells (section 2.6.1.1.1) and yfp-plasmid DNA (5 µL;

section 2.6.1.1.2) were gently mixed and electroporated on a Gene Probe set at 200 Ω

resistor, 25 µF capacitor and 12.5 KV cm-1 field strength (Wu, Matand et al., 2010) at pulse

length 5-6 msec. One mL of LB broth was added in each transformation voile immediately

after this short pulse and gently mixed and placed on a shaker incubator (28 ±2 oC) for 45

min. Finally, 50, 100 and 200 µL from each vial was spread on LB agar plates (amended

with ampicillin @ 50 µg/ mL) and incubated at 28 ±2 oC for 2 days. Transformed colonies

were selected in UV light at 4X using fluorescent microscope (Leica DMLS) and grow in

LB broth (amended with ampicillin @ 50 µg/ mL). The transformation was then conformed

by the observations under confocal laser scanning Microscope (CLSM, Olympus Fluoview

Ver.1.3). The transformed cells were preserved in 20 % (v/v) glycerol at -80 oC.

2.6.1.1.4 Inoculation of Sunflower with YFP-Transformed Bacterial Strains and

Root Colonization Studies

yfp-transformed bacterial strains were grown well (≈ 108 CFU mL-1) in Erlenmeyer flask

and transferred in 45 mL falcon tube. These tubes were centrifuged (8000 x g; 4 °C) and

the cell free supernatant was drained carefully. The pellets were suspended in the same

amount of 0.85 % saline (NaCl). Surface sterilized (as described in section 2.2.1) sunflower

(cv. FH-331) seeds were dipped in the freshly prepared inoculum for 25-30 and germinated

on 1.5 % (w/v) water agar plates. Five days old seedlings were then transplanted aseptically

to sterile sand pots and kept in growth chamber (day/night temperature 25/20 oC, light/dark


62

periods 16/8). Roots of 21 days old plants were taken out after a little washing with sterile

water roots were cut (1/2 cm) and placed on glass slides with a drop of sterilized water

covered with the cover slip. The slides were visualized under CLSM (Olympus fluoview

Ver. 1.3) to observe colonization of yfp-labelled strains in the form of fluorescence.

Figure 2-2 Vector pBBR-MCS-4 showing restriction map.

2.6.1.2 Colonization Studies of FITC-labeled Bacteria

Fluorescent antibodies were raised against two plant growth promoting rhizobacterial

strains i.e. Enterobacter cloacae AF-31 and Citrobacter freundii AF-56 for colonization

studies following the method as described by Somasegaran and Hoben (1994b).


63

2.6.1.2.1 Preparation of Antigen

Both strains (Enterobacter cloacae AF-31 and Citrobacter freundii AF-54) were grown in

LB broth and the cells were harvested by centrifugation (Vincent and Humphrey, (1970)

and supernatant was carefully drained and resulted pellets were washed with sterile saline.

This process was repeated three times O.D. was adjusted to 0.45 at λ600 on a

spectrophotometer to adjust 1x109 cell/mL. These cultures were then steamed (without

pressure) for one h at 100 ºC to inactivate the flagellar antigens. 1 % Merthiolate (1 mL/100

mL cell suspension in saline) was added to preserve the antigens and kept in refrigerator

until use.

2.6.1.2.2 Raising of Antibodies

Female albino rabbits, at the age of six months, were immunized with the antigens in

triplicate according to the schedule given in Table 2.3 After seven days of last injection, a

test bleed was conducted from marginal ear-vein for titer determination through

agglutination process. In case of satisfactory titter (>1200), antiserum was collected from

the heart, while 1.5 mL of antigen was given subcutaneous as a booster dose to raise the

titer if it was less than 1200. The final bleed was conducted after one week of booster dose.

2.6.1.2.3 Collection and Processing of Antiserum

From each rabbit 30-50 mL blood was taken by cardiac puncture and incubated at room

temperature for approximately 2 h and refrigerated over-night. When the red blood cells

were settled in the bottom of tube, the serum was carefully separated and centrifuged at

5000 x g for 15 min (4 ºC) for the removal of red blood cells. One percent Merthiolate was

added for serum preservation and stored at -20 ºC for further use.


64

Antiserum was serially diluted in 10 test tubes by taking 9.6 mL of 0.85 % saline in tube 1

and 2.5 mL in rest of the tubes. Serum (0.4 mL) was added to tube 1, mixed well by gentle

pipetting and 2.5 mL was transferred from first tube to the second. The dilution was

repeated down the series by transferring 2.5 mL of the diluted serum successively to the

next tube up to tube ten as 1/25, 1/50, 1/100 and so on.

Table 2-3 Rabbit immunization schedule used for raising polyclonal antibodies

Days Injection routes Amount of antigen

(mL)

One Intravenous 0.5

Subcutaneous 1.5

Intramuscular 1.0

Two Intravenous 1.0

Three Intravenous 1.5

Four-Six Rest 0

Seven Intravenous 1.5

Eight Intravenous 2.0

Nine Intravenous 2.0

For agglutination confirmation, 80 μL from each of the test tubes was then transferred in

the wells of 96-well microtiter plates A1-A10 respectively. Each well was then added with

80 μL of antigen and mixed well with a sterile wooden stick (e.g., tooth picks). The controls

for the reaction were: serum-saline control (80 μL serum + 80 μL saline), and antigen-


65

saline control (80 μL antigen + 80 μL saline). Parafilm was used to seal the wells and plate

was incubated at 37 ºC overnight. Before interpreting the results, the plate was refrigerated

for 3-4 h. White precipitates at the bottom of each well were considered as positive results

for the agglutination.

2.6.1.2.4 Purification of Immunoglobulin G (IgGs)

Antiserum stored at -20 ºC was gently thawed and processed for purification. An equal

volume of chilled 3.9 M (NH4)2SO4 was added drop wise into the serum with constant

stirring. The resulting white cloudy mixture was stirred overnight at 4 ºC to precipitate

completely. The precipitated globulins were centrifuged for 30 min (12000 rpm at 4 °C),

clear supernatant was carefully drained the resulted pellet was suspended in chilled saline

to get the initial volume. This precipitation and resuspension of ammonium sulphate was

repeated three time. In the last step the resulted globulin precipitates were snow white and

the globulins were free of hemoglobin.

Finally, the pellets were dissolved in distilled water (minimum volume) and dialyzed

against 0.85 % NaCl in a cold cabinet at 4ºC. Saline was frequently (3-4 times) changed

during dialysis, until ammonium sulphate was no longer detectable in the dialysate. A

saturated solution of barium chloride was mixed with the equal volume of dialysate to

determine the presence of ammonium sulfate in it. A colorless mixture was the indicator

of no more sulphate, while cloudy mixture indicated its presence. Glycerol (100 μL) was

added in purified IgGs (30 % v/v) and stored at 4 °C in 500 μL screw cap tubes until used.


66

2.6.1.2.4.1 Protein Estimation

Biurete method was followed to estimate the globulin protein concentration (Somasegaran

and Hoben, 1994). The color development intensity of the solutions in each tube was

determined by spectrophotometer at 540 nm. Standard solution of bovine serum albumin

(BSA) was prepared by adding BSA in distilled water (200 mg/ mL). Protein (antibodies)

concentration from each sample was estimated by determining their OD and compared

them with the standard curve.

2.6.1.2.5 Conjugation of the Globulins with Fluorescein Isothiocynate (FITC)

A rate of 0.03 mg FITC/ mg protein was used for protein conjugation with FITC (Sigma).

In a beaker (50 mL) 10 mL of immunoglobulin solution (10 mg/mL) was taken and 4 mL

of filter sterile phosphate buffer (0.15 M; pH 9) was added in it. In another beaker (50 mL)

4 mL of filter sterile phosphate buffer (0.1 M; pH 8) was taken and 3 mg of FITC powder

was carefully added in it and dissolved by stirring on a very slow speed. Then

immunoglobulin solution and dissolved FITC solutions were pooled together in a beaker

and kept on slow stirring for overnight at room temperature after adjusting pH 7.1 by 1M

NAOH (filter sterile). Merthiolate was added as preservative at a concentration of l:10000

(Somasegaran and Hoben, 1994b)

2.6.1.2.5.1 Column Chromatography

Column chromatography was then carried out to collect the conjugated fluorescent

antibodies (FA) separated from non-conjugated FITC. Sephadex G25 (15 g) was poured

gently in the column after soaking (30-60 min) in 100 mL of phosphate buffered saline

(PBS) in 500 mL beaker. The PBS level was maintained by adjusting the lower valve of


67

column to control the flow rate at 40 drops/ min. the mixture of FITC: FA was carefully

added in to the column with the help of 1.5 mL pipette. The continuous addition of PBS

was managed to maintain its level in the column. When the first dark yellow band fraction

(FA) was appeared, it was collected into 3 parts (initial band, second and the last band) in

three tubes separately. Finally collected fluorescent antibodies were aliquoted into cryo

vials and refrigerated till further use. Specific bacterial cultures were stained to determine

the quality and specificity of the FA. Working dilution was also optimized by making

twofold dilution series of FA with PBS in microtiter trays (Somasegaran and Hoben,

1994b).

2.6.1.2.5.2 Processing of Pure Culture for FA Staining

Freshly grown cultures of selected PGPR were mounted on sterile glass sides in a smear

form and the slides were air dries and heat fixed. Then gelatin-rhodamine isothiocyanate

(RhlTC) conjugate was dropped on the fixed cells for the suppression of non-specific

adsorption of stain (Bohlool and Schmidt, 1968). The working solution of FA was dropped

on the smear after getting dried. The slide was kept under A moist chamber was made in a

Petri dish with wet filter paper and the slides were placed in this chamber for 20 min at 37

ºC. The slides were taken out and the excessive FA solution was carefully drained by tilting

the slides on a piece of tissue. A 15minute washing was given with PBS and a second wash

with distilled water and slides were then air dried. A drop of mounting solution containing

1 part of PBS and 9 parts of glycerol was placed on the smear and covered with cover slip.

Slide observations were made on confocal laser scanning microscope (Olympus FV 1000,

Japan). The appearance of green fluorescence is the indicator of positive results; on the

other hand, the absence of green color indicates the negative results.


68

The specificity of FA was checked by double staining of mix cultures (antigen) with FA

and ethidium bromide. The FA specific cells produce a green color, while red color

indicated nonspecific population (result of ethidium staining) To confirm the specific

discrimination of the target bacterial strains, purified labeled sera were cross-incubated

with the cell extracts of the non-target bacterial strain e.g. (i) serum against Citrobacter

freundii AF-56 with Enterobacter cloacae AF-31, (ii) serum against Citrobacter freundii

AF-56 with a mixture of Citrobacter freundii AF-56, Enterobacter cloacae AF-31 and

Pseudomonas sp. AF-54. In later case, the smear of liquid culture of bacteria was stained

with ethidium bromide and incubated for 5 min (in dark). Slides were washed with distilled

water and then covered with EA. Further processing was performed in the similar way as

mentioned in the single staining method.

2.6.1.2.5.2.1 Processing of Root Samples for FA Staining.

Small pieces of specific stain inoculated roots (14days old) were placed on a clean glass

slide and a few drops of RhITC conjugate was added to cover the root pieces. The slides

were placed in oven at 60 0C for drying. Further processing was same as used for pure

cultures.

The appearance of fluorescence around the cells were detected by a confocal laser scanning

microscope (Olympus FV 1000, Japan). An Argon-Ion laser facilitated the wavelength of

488 nm and 525 run for absorption and emission of FITC, respectively. Ethidium bromide

was detected at 510 nm and 595 nm absorption and emission wavelengths, respectively

using Argon-Ion laser (Roger et al., 2006).


69

2.6.1.2.6 Colonization Studies of Inoculated Bacterial using Fluorescence in Situ

Hybridization (FISH) Analysis

The root colonization potential of a potential PGP rhizobacterial strain Pseudomonas sp.

AF-54 was determined by using fluorescence in situ hybridization (FISH). Fluorescently

labelled oligonucleotide probes were used for FISH analysis. These oligonucleotide

synthesized probes contain Cy3 at 5’ end (Interactiva Biotechnologie GmbH, Ulm,

Germany). FISH was performed according to already published method (Amman et al.,

1995) with some modifications.

2.6.1.2.6.1 Bacterial Cell Fixation

Freshly grown bacterial culture (1.5 mL, 1 x 108 CFU/ml) was centrifuged (7000 rpm, 5

min) to get cell pellet. Supernatant was discarded and pellet was re-suspended in 800 µl

1X PBS and centrifuged for five minutes at 11000 rpm. Re-suspension of pallet was done

in 200 µl 1X PBS and 600 µl of 4 % para formaldehyde (PFA). The samples were

incubated at 4 °C for 1 to 2 hours. After incubation, centrifugation of samples was done at

11000 rpm for 5 min. Supernatant was discarded and re-suspended the pallet in 1:1 PBS-

Ethanol. Samples were stored at -20 °C for further use (Amman et al., 1995).

2.6.1.2.6.2 In situ Hybridization

Hybridizations were carried out on six welled slides to keep the cells isolated from each

other. Two μL fixed cells were mounted on the wells air dried or incubated at 46 °C until

dry. Slide with fixed cells was washed subsequently with 50 %, 80 % and 100 % ethanol

separately for 3 minutes each. After getting the wells air dried, 18 µl hybridization buffer

Annexure 6) and 2 µl (5 ng) oligonucleotide probe was added in each well. The slide was


70

washed with distilled water, air dried and embedded with mounting solution (1 ml glycerol,

9 ml 1X PBS). Prepared slide was observed using CLSM (Amman et al., 1995).

2.6.1.2.6.3 Preparation of Root Sample for FISH Analysis

Fourteen days old inoculated plants were carefully uprooted from pots containing both

sterilized and natural soils separately. The roots were washed with sterilized PBS cut in to

15-30 long pieces and refrigerated for 1 h in fixation buffer (4 % paraformaldehyde in

PBS). The samples were washed subsequently with 50 %, 80 % and 100 % ethanol

separately for 3 minutes each, at room temperature. For hybridization, the root samples

were fixed on a glass slide and treated with hybridization buffer (30 °C) and 15pmol of

each FLUOS-labeled EUB 338 specific for bacteria and Cy3-labelled probe GAM42a

specific for gamma Proteobacteria were for Pseudomonas sp. AF-54. Hybridization was

carried out at 46 °C for 3 h or overnight. These samples were then incubated in washing

buffer (Appendix 6) for 20 min, and after that washed with sterilized water. Finally, the air

dried hybridized root samples were shifted on a microscopic slide in citifluor (mounting

buffer). Observations were made on CLSM (Olympus FV 1000, Japan). An Argon-Ion

laser facilitated the wavelength of 488 nm and 525 run for absorption and emission of

FITC, respectively.

2.6.1.2.6.4 Tissue Processing for Immunogold Labelling

Ultrathin sections of fixed roots were cut and mounted on plastic (pyroxylin) coated nickel

grids (foamwax grids) in triplicate. A piece of wet filter paper was placed in a petri dish to

make an incubation chamber and glass slides were coated with wax for the treatment drops.

Grids were floated on 15-20 μL drops of IGL buffer (Annexure 7) and left for 1h. The grids

were picked and after draining carefully placed on the drops of drops (15-20 μL) of


71

antiserum diluted in IGL buffer and incubated in antiserum overnight at room temperature.

The grids were then removed and after draining the IGL buffer, were washed in fresh IGL

buffer for ten min (twice) in microtiter plate wells (430 μL). Grids were then transferred

on 15-20 μL drops of gold probe (diluted in IGL buffer) and incubated on room temperature

for 4–6 h.

Washing of the grids was done sequentially on IGL buffer and distilled water for 10 min

each on microtiter plate wells, and then dried. Grids were double stained with uranyl

acetate (30 min) and lead citrate (10 min). Some of the grids left unstained and used to

assess the background labelling. Finally, the grids were washed with deionized water, and

air-dried. The observations were made under transmission electron microscope (Jeol, JEM-

1010, Japan).

2.6.1.2.7 Biofilm Formation Assay

A time course study was conducted to access the in vitro biofilm formation ability of

selected bacterial strains as described by Fatima et al. (2015). LB broth was inoculated

with freshly grown selected strains (AF-22, AF-31, AF-54 and AF-56) in 45 mL falcon

tubes in triplicate. Sterile cover slips (22-mm) were immersed in inoculated falcon tubes

and the tubes were then incubated for 96 h at 28±2 ºC in slanting position. The formation

of biofilm on thin glass cover slips was studied for development of film by the selected

strains at 4 different time intervals (24th, 48th, 72th and 96th h) of growth. The cover slips

were recovered from the culture tubes, washed thoroughly with PBS (1X) thrice and

stained with 0.005 % acridine orange (w/v) for 5 min in dark and washed twice with

distilled water. Biofilm load was estimated by using confocal laser scanning microscopic

(Fluo view, FV 1000, Olympus) under 100× oil immersion objective lens (Dasgupta et al.,


72

2013). The imageJ software was used to estimate the intensity of bacterial population

attached to the slide.

2.6.2 Analysis of the Bacterial Population Attached to Roots by Plate Count (CFU)

Method

Root colonization assay was performed as described previously by Majeed et al. (2015).

Root samples were collected from inoculated sunflower plants at every 15 days. For

rhizospheric strains, one g tightly adhered soil was taken for serial dilution. On the other

hand, for putative endophytic strains, root surface was washed with running tap water to

remove weakly bound soil and roots were blotted dry. One g of root along with tightly

adhered soil was homogenized with 9 mL autoclaved saline (0.85 %) using sterile mortar

and pestle. Serial dilutions (10−3, 10−5 and 10−7) were then spread on LB agar plates and

incubated at 30 °C for 24–48 h. Colony forming unit (CFU) per g soil/root was determined

as described previously by Somasegaran and Hoben, 1994). Each treatment was replicated

thrice and mean was calculated.

PLANT INOCULATION STUDIES

Plant growth promoting potential of bacterial strains was studied by inoculating respective

crop i.e. sunflower (cv. FH-331) under controlled and field conditions through a series of

experiments carried out in pouch, pot and field.

2.7.1 Preparation of Bacterial Inoculum

Cells of well grown bacteria strains (~109 viable cells /mL) were harvested at 6000 rpm in

centrifuge (Beckman Coulter, USA) at 4 °C for 10 min. the supernatant was carefully

drained and the resulted pellets were washed twice with 0.85% sterilized saline. The


73

washed pellets were then re-suspended in equal volume of saline/sterilized distilled water.

This inoculum was used for seed pelleting or for direct application at the base of

germinating seedling @ 1 mL per seedling rate.

2.7.1.1 Seed Sterilization

For surface sterilization, sunflower seeds (cv. FH-331) were dipped in 5 % (w/v) sodium

hypochlorite for 10 min. After vigorous shaking, sodium hypochlorite was drained and

seeds were washed extensively with sterile water (5-6 times), following the method

described by Shahid et al, (2014).

2.7.1.2 Seed Bacterization

Sunflower seeds were dipped in bacterial inoculum and fairly grinded filter mud (sterilized)

was gradually added with gentle mixing. This process was done until the seeds were coated

with a thin layer of bacterial suspension and filter mud.

2.7.1.3 Reference Strains

Two previously characterized sunflower associated strains Fs-9 (Pseudomonas trivialis)

and Fs-11 (Enterobacter sp.) obtained from the BIRCERN culture collection NIBGE,

Faisalabad, Pakistan were used as positive control, as efficient nitrogen fixer and phosphate

solubilizer respectively.

2.7.2 Experiment 1: Evaluation of the Effect of Bacterial Inoculation on Growth of

Sunflower (Growth Pouches)

11 beneficial bacteria with the most efficient growth promotion characteristics, found best

in biochemical tests on the basis of in vitro studies, were inoculated on sunflower plant in

growth pouches under controlled environmental conditions. Two experiments were setup


74

separately to evaluate the effect of bacterial isolates on sunflower crop as phosphate

solubilizers (AF-6, AF-9, AF-21, AF-22, AF-31, AF-54, AF-56, AF-95, AF-146, and AF-

163) and nitrogen fixers (AF-6, AF-9, AF-21, AF-22, AF-31, AF-48, AF-54, AF-56, AF-

95, AF-146).

2.7.2.1 Experimental Set-up

Surface sterilized seeds (as described in section 2.2.1) of sunflower (cv. FH-331 were

germinated on water agar plates. The seedlings were then aseptically transferred to the

autoclaved growth pouches (Waver and Frederick, 1982). Commercial growth pouches are

16×17 cm autoclaveable plastic bags provided with a water absorbent paper. A plastic

straw was also placed in pouches to facilitate the application to nutrient solution/water.

Twenty mL of Hoagland’s nutrient solution (Annexure 8) was added to the growth pouches

and its level was maintained throughout the experiment. In the experiment conducted to

evaluate P-solubilization potential of bacterial strains, Hoagland’s nutrient solution was

used without phosphate source (Arnon and Hoagland 1940) i.e. KH2PO4, whereas, Tri-

calcium phosphate (TCP, Sigma) @ 1.239mL (g)/pouch was used as an insoluble form of

P. Enterobacter sp. FS-11 was used as inoculated positive control (Section 2.7.1.3) In

second growth pouch experiment Hoagland’s nutrient solution was used without nitrogen

sources i.e. Ca(NO3), KNO3. A positive inoculated control (FS-9, described in section

reference strains) was used. Three replication for each isolate were kept with a negative

and positive controls in both experiments.

The pouches were kept in growth chamber (day/night temperature 25/20 oC, light/dark

periods 16/8). One mL inoculum of each isolate was applied to the seedling roots

immediately after shifting to the pouches. Growth parameters like shoot length (cm), root


75

length (cm), shoot fresh and dry weight (g), root fresh and dry weight (g) and viable cell

count (as described in section 2.1.3.8) were recorded after 30 days of

germination/transplantation.

2.7.3 Experiment 2: Greenhouse/Pot Experiment

Two separate experiments were conducted to investigate the phosphate solubilizing and

nitrogen fixing potential of selected bacterial isolates on growth of sunflower and P and N

uptake by the plants (from respective experiment).

2.7.3.1 Growth Conditions, Sowing and Parameters Studied

Surface sterilized (as described in the section 2.7.1.1) sunflower seeds were bacterized (as

described in the section 2.7.1.2) with well grown inoculum (as described in section 2.7.1)

and sown in sterile soil in pots each of 9 cm diameter. Each treatment was replicated thrice

for each data collection. Completely randomized design (CRD) design was used for both

experiments. Pots were placed in growth room (adjusting the temperature at 25 °C and 16/8

light and dark periods). Pots were daily watered with sterile water. After 45 days of

transplanting, data was recorded on growth parameters including root and shoot length,

fresh and dry weight and P/N contents (in respective experiments).

Growth parameters like shoot length (cm), root length (cm), shoot fresh and dry weight (g),

root fresh and dry weight (g) and bacterial population from sunflower rhizosphere (as

described in section 2.1.3.8) were recorded after 15, 30 and 45 days of germination for both

experiments (pot experiments). Phosphorus uptake by root and shoot was measured in first

experiment and root and shoot N uptake was accessed from second experiment.


76

2.7.3.2 Experiment Details

Ten P-solubilizing potential strains used in the pouch experiment were used in a pot

experiment. Tri-calcium phosphate (TCP, Sigma) was mixed in soil (200 mg/kg soil) as

insoluble inorganic P source. A positive control bacterial treatment (Fs-11 as described in

section 2.7.1.3 was also used. Treatment detail is described below:

T1= Pseudomonas thivervalensis strain AF-6

T2= Bacillus safensis AF-9

T3= Citrobacter braakii AF-21

T4= Azospirillum brasilense strain AF-22

T5= Enterobacter cloacaeAF-31

T6= Arthrobacter sp. AF-163

T7= Pseudomonas sp. strain AF-54

T8= Citrobacter freundii AF-56

T9= Pseudomonas brassicacearum AF-95

T10= Lysinibacillus sp. strain AF-146

T11= Fs-11 (positive control)

T12= Half dose of TSP (Recommended)

T13= Full dose of TSP (Recommended)

T14= Control (P0)

2.7.4 Experiment Details and Layout Design for N2-fixion Pot Experiment

In this experiment ten nitrogen fixing bacterial strains were used along with a previously

known nitrogen fixing bacterial strain Fs-9 (as described in section 2.7.1.3) and three un-

inoculated control treatments (0%, 50% and 100%) of recommended chemical fertilizers.

Treatment details are described below:

T1= Pseudomonas brassicacearum strain AF-6

T2= Bacillus subtilis strain AF-9


77

T3= Citrobacter freundii strain AF-21

T4= Azospirillum brasilense strain AF-22

T5= Enterobacter cloacae strain AF-31

T6= Bacillus cereus AF-48

T7= Pseudomonas sp. strain AF-54

T8= Citrobacter freundii strain AF-56

T9= Bacillus safensis strain AF-95

T10= Lysinibacillus sp. strain AF-146

T11= Fs-9 (positive control)

T12= ½ Urea (Recommended)

T13= Full Urea (Recommended)

T14= Control (N0)


and Yield of Sunflower under Field Conditions

Two field experiments were conducted to investigate in detail the effect of best selected

strains on growth of sunflower at Faisalabad, Pakistan and Rawalakot, AJ&K, Pakistan.

2.7.5.1 Experiment Details

The experiments were conducted in randomized complete block design (RCBD) with three

replications. There were 16 treatments; 13 inoculated and 3 un-inoculated control as

described below:

T1= Half dose of recommended TSP and Urea + Pseudomonas thivervalensis strain AF-6

T2= Half dose of recommended TSP and Urea + Bacillus safensis AF-9

T3= Half dose of recommended TSP and Urea + Citrobacter braakii AF-21

T4= Half dose of recommended TSP and Urea + Azospirillum brasilense AF-22

T5= Half dose of recommended TSP and Urea + Enterobacter cloacae AF-31

T6= Half dose of recommended TSP and Urea + Bacillus cereus AF-48


78

T7= Half dose of recommended TSP and Urea + Pseudomonas sp. strain AF-54

T8= Half dose of recommended TSP and Urea + Citrobacter freundii AF-56

T9= Half dose of recommended TSP and Urea + Pseudomonas brassicacearum AF-95

T10= Half dose of recommended TSP and Urea + Lysinibacillus sp. strain AF-146

T11= Half dose of recommended TSP and Urea + Arthrobacter sp. AF-163

T12 = Half dose of recommended TSP and Urea + Fs-9 (inoculated control treatment)

T13 = Half dose of recommended TSP and Urea + Fs-11(inoculated control treatment)

T14= Half dose of recommended TSP and Urea (un-inoculated control treatment)

T15= Full dose of recommended TSP and Urea (un-inoculated control treatment)

T16 = No fertilizer (un-inoculated control treatment)

Locations:

L1 = Rawalakot, AJK

L2 = NIBGE, Faisalabad

2.7.5.2 Crop Husbandry and Parameters Studied

The soil was cultivated (2-3 times) with tractor mounted cultivator followed by planking

before the seedbed preparation 3 plot per treatment were prepared (3×2 m2). Sunflower

(cv. FH-331) seeds (8 kg ha-1) were sown manually by dibbling method with 75 cm row to

row (R×R) and 25 cm plant to plant (P×P) distance and thinning was done where required.

Nitrogen fertilizer in the form of urea was applied in two splits. Whole of phosphorus in

the form of triple super phosphate (TSP), along with half of nitrogen was incorporated in

the soil at the time of sowing by means of single row drill. The other part of N was applied

at first irrigation. To keep the crop safe from weeds, insects, pests and diseases, the

respective measures were followed throughout the crop period. After harvesting, the crop


79

was kept exposed to sun and air for drying and then threshing was carried by physical

beating.

Parameters studied were plant height (cm), head diameter (cm), number of achene per head,

1000 achene weight (g), achene yield (kg ha-1), biological yield (kg ha-1), harvest index

(%) Achenes were analyzed for their N and P content (g kg-1), protein (g kg-1), oil content

(%), palmitic acid C16:0 (%) stearic acid C18:0 (%), linoleic acid C18:2 (%), oleic acid

C18:1.

2.7.5.2.1.1 Estimation of Total N in Plant Samples

Oven dried (60 °C for 72 h) and finally ground (in stainless steel grinding mill) plant (0.5

g) samples (root and shoot) were used for the estimation of total N-uptake by the plants

following Kjeldahl method (Sparks et al., 1996). The plant samples were mixed with

digesting mixture (2.5 g) containing K2SO4, CuSO4 and Se (100:10:1) and concentrated

H2SO4 (5-7 mL) was added and the mixture was then digested (Digestion system 40,

Digester 1016, Tecator). The digestion was stoped after attaining green color. The digested

material was steam distilled in the presence of concentrated NaOH solution and NH3

evolved was collected into 2% boric acid solution. The H2SO4 solution was used for

ammonium-boric acid titration to calculate the total nitrogen content, using following

equation;

N (mg g-1) = acid used (blank sample) × 14 × normality of acid used for titration

Weight of sample


80

2.7.5.2.1.2 Determination of P Content in Plant Material

Vanadium phospho-molybdate yellow color method (Yoshida et al., 1971) was adopted to

measure the root and shoot p content. Dried ground plant material (0.5 g) was taken in

digestion tube and 3 mL of concentrated H2SO4 was added. The mixture was incubated

over night at room temperature; 3 mL of H2O2 was then added and tubes were covered.

Samples digestion was carried out at 350 oC until no more fumes were produced. An

additional heat for 30 min was also given. One mL of H2O2 was added to the tubes (after

getting cooled) digestion was continued until the digested mixture become colorless. The

volume was made up to 20 mL by adding dH2O. The extract was then filtered and 2 mL of

it was dissolved in the same volume of Vanadomolybdate reagent containing 200 g

ammonium molybdate (NH4)6MO7O2.4H2O) (Ashraf et al., 1992). These samples were

incubated at room temperature (30 min) and OD was recorded at 420 nm on M 350 double

beam UV-visible spectrophotometer (Camspec). The standard curve was prepared and the

actual values were computed from the equation derived from standard curve.

2.7.5.2.2 Achene Oil Content Analysis

Oil content and fatty acid analysis of sunflower achenes was carried out commercially by

Oil Seed Brassica Lab, Plant Breeding & Genetics Division, Nuclear Institute for Food and

Agriculture (NIFA), Peshawar, Pakistan through Gas chromatography as described

(Erickson et al., 1980).

2.7.6 Statistical Analysis

Analysis of variance (ANOVA) technique (Steel et al., 1997) was used to analyze data

regarding pot, green house and field experiments using Statistix (version 8.1) software. For


81

pot and green house experiment one-way ANOVA was used whereas in field experiments

to find the interaction between different treatments and locations 2-way-ANOVA was

used. Likewise, PCA analysis to study the effect of different treatments according to

experimental sites were used with the help of SPSS17 software. To compare the difference

between treatments means ‘least significant difference’ (Fisher’s LSD) test was used at 5

% probability.

Chapter 3

RESULTS

BACTERIAL ISOLATION AND CHARACTERIZATION

3.1.1 Description and Physico-Chemical Properties of Sampling Sites

Meteorological and physico-chemical analysis of the soils collected from 16 sites of sub-

division Dhirkot, Azad Jammu and Kashmir, located between (altitude 790 to 1916)

30o34’699” to 32o03’509”N and 72o16’080” to 74o07’400” E geographical coordinates

represents that soil and air temperatures ranged between 19 to 29 °C and 19.5 to 38.5 °C

respectively. Sites showed a wide range of heat index (16.5-48.4 °C) and humidity 61%-

37.6 % (Table 2.1). Maximum pH (7.51) was recorded in Keeri, while minimum pH (6.34)

was found in Bandi. Soil EC ranged (0.64- 1.34 dS m-1) with minimum at Chamyati and

maximum at Arja. Soils across the sampling sites showed a wide variation in soil organic

matter ranged between a minimum of 1.36 % at Upper Munhasa to a maximum of 2.68 %

at Dyar Gali (Table 3.1). Available P was maximum (14.72 mg kg-1) at Chamman Kot and

available K was highest (113.68 mg kg-1) at Neela but, while minimum available K (43.0

mg kg-1) was found in Ghaziabad. Total N ranged between 0.09 to 0.272%, the later was

found in Keri. Total bacterial population analysis showed a wide range () with maximum

((2×107) in Keeri and minimum (0.2×107) in Makhyala (Table 3.1). The textural classes

also showed a wide range of textural classes (loam, sandy loam, clay loam, silt loam etc.).

Chapter 3 Results

83

Table 3-1 Physico chemical properties of soil collected from different sites

Location Soil pH ECe (dsm

-1)

O.M (%)

Total N (%)

Available P (mg/kg)

Available K (mg/kg)

Textural class *CFU

U. Munhasa 7.26 0.86 1.36 0.104 8.41 74.48 Sandy loam 4×106

Bandi 6.34 0.73 1.47 0.156 7.36 57.39 Clay loam 6×106

Hill 2 6.67 0.99 2.34 0.211 10.93 94.62 Clay loam 1.4×107

Makhyala 6.48 1.02 2.09 0.091 9.72 69.78 Sandy loam 2×106

Chamankot 6.96 0.87 2.08 0.098 14.72 53.14 Loam 1×107

Chamyati 6.59 0.64 2.36 0.181 13.48 72.19 Silt loam 9×106

Dhirkot 6.66 0.86 2.14 0.259 7.48 58.73 Silt loam clay 6×106

Ghaziabad 7.12 0.81 1.56 0.27 8.86 43.25 Sandy loam clay 7×106

Keeri 7.42 0.97 2.17 0.272 13.97 68.96 Loam 2.2×107

Sanghar 6.66 1.13 2.43 0.217 14.42 71.834 Clay loam 1.4×107

Hanschoki 7.16 0.92 2.19 0.202 11.34 87.16 Clay loam 7×106

Dyar gali 6.84 0.640 2.68 0.224 9.46 96.53 Loam 7×106

Neela but 6.64 0.85 2.63 0.213 7.66 113.68 Sandy loam clay 9×106

Kotli 6.86 0.98 2.59 0.213 12.28 78.82 Silt loam 7×106

Narwal 7.33 1.12 1.54 0.102 6.93 76.94 Loam 1×107

Arja 7.51 1.34 1.43 0.092 6.72 57.78 Clay loam 4×106

*CFU= Colony forming unit

Chapter 3 Results

84

Bacterial Isolation and Morphological Characterization

Initially, a total of 163 bacterial isolates were isolated and purified from sixteen sampling

sites. Ninety seven bacteria were ectorhizospheric while, 66 were obtained from root

interior (putative endophytic). These isolates were designated site wise as AF-1 to AF-16

from Arja, AF-17 to AF-27 from Bandi, AF-28 to AF-38 form Chamman Kot, AF-39 to

AF-47 form Dhirkot, AF-48 to AF-62 from Diyar Gali, AF-63 to AF-73 from Ghazi abad,

AF-74 to AF-85 from Hanschoki, AF-86 to AF-93 from Hill 2, AF-94 to AF-98 from Keri,

AF-99 to AF-111 from Kotli, AF-112 to AF-119 from Makhyala, AF-120 to AF-127 form

Munhasa, AF-128 to AF-136 from Narwal, AF-137 to AF-146 from Neela but, AF-147 to

AF-155 from Sanghar, and AF-156 to AF-163 from Upper Chamyati (Table 3.3)

All of the isolates were studies for their morphological traits (colony and cell morphology,

cell motility and Gram’s reaction etc.). The bacteria showed a range of colony colors from

white to milky-white, gray, yellow, red, brown and translucent with variable sizes and

margins on LB agar plates. Most of the bacterial cells were short rods followed by medium

and long rods, a few were cocci in shape and were dominated by motile with a few non-

motile bacteria. Except six, all the isolates showed negative G- reaction (Table 3.2).

Chapter 3 Results

85

Table 3-2 Morphological characteristics of bacterial isolates obtained from sunflower endo/rhizosphere Isolate

codes

Colony

Color

Colony

Size

Colony

Shape

Colony

Margins

Cell Shape Isolate

codes

Colony

Color

Colony

Size

Colony

Shape

Colony

Margins

Cell Shape

AF-1 Milky white Small Round Smooth Short rod AF-83 Translucent Medium Round Wavy Long rods

AF-2 Milky white Large Round Rough Long rod AF-84 Orang Tiny Round Smooth Short rods

AF-3 Cream Medium Irregular Rough Short rod AF-85 Yellow Tiny Round Smooth Short rod

AF-4 Cream Medium Round Rough Short rod AF-86 Translucent Tiny Round Loopy Medium rods

AF-5 Translucent Large Round Rough Short rot AF-87 Off-white Large Irregular Wavy Long rods

AF-6 White Large Irregular Smooth Short rot AF-88* Yellow Medium Flower Smooth Short rods

AF-7 Light Gray Small Round Rough Oval AF-89 Dark brown Medium Round Smooth Long rods

AF-8 Translucent Medium Round Smooth Short rod AF-90 Dark brown Medium Round Smooth Coccus

AF-9* Cream Small Irregular Rough Long rods AF-91 Dark brown Tiny Round Smooth Medium rods

AF-10 Milky white Medium Wavy Rough Short rods AF-92 Off-white Large Round Wavy Long rod

AF-11 Light brown Small Round Smooth Long rod AF-93 Off-white Medium Round Smooth Medium rods

AF-12 Dark brown Large Round Rough Short rods AF-94 Dark brown Medium Round Smooth Short rod

AF-13 Pink Large Irregular Loopy Short rod AF-95 Red Medium Round Smooth Short rod

AF-14* Yellow Small Round Smooth Coccus AF-96 Dark brown Small Round Smooth Coccus

AF-15 Gray Large Round Smooth Long rods AF-97 Brown Medium Round Smooth Short rod

AF-16 Mily-white Tiny Round Smooth Short rods AF-98 Brown Large Round Wavy Long rods

AF-117 White Small Flower Rough Short rods AF-99 Transparent Small Round Smooth Short rods

AF-18** Dark brown Small Round Smooth Short rods AF-100 Dark brown Medium Round Smooth Short rods

AF-19 Cream Medium Wavy Rough Coccus AF-101 Brown Tiny Round smooth Short rods

AF-20* Cream Large Wavy Smooth Long rod AF-102 Translucent Large Round Wavy Medium rods

AF-21 Milky white Large Round Smooth Short rods AF-103 White Small Flower Rough Coccus

AF-22 Light yellow Small Round Smooth Small rods AF-104 Dark brown Medium Round Smooth Long rod

Continued…

Chapter 3 Results

86

Isolate

codes

Colony

Color

Colony

Size

Colony

Shape

Colony

Margins

Cell Shape Isolate

codes

Colony

Color

Colony

Size

Colony

Shape

Colony

Margins

Cell Shape

AF-23 Light brown Medium Round Wavy Short rod AF-105 Milky white Small Round Smooth Short rods

AF-24 Translucent Medium Irregular Smooth Coccus AF-106 Brown Medium Flower Rough Short rod

AF-25 Translucent Medium Round Smooth Long rod AF-107 Cream Tiny Round Smooth Short rod

AF-26** Cream Medium Round Smooth Short rod AF-108 Translucent Tiny Round Smooth Short rods

AF-27 Off-white Large Irregular Smooth Short rod AF-109 Off-white Small Round Wavy Short rod

AF-28** Yolk Small Round Smooth Coccus AF-110 Milky white Medium Round Smooth Short rod

AF-29 Dark brown Medium Round Smooth Medium rods AF-111 Gray Large Irregular Smooth Coccus

AF-30 Cream Medium Round Smooth Short rod AF-112* Brown Medium Round Smooth Long rod

AF-31 Cream Small Round Smooth Short rods AF-113 Off-white Medium Flower Rough Medium rods

AF-32 Dark brown Small Round Smooth Small rods AF-114 Dark brown Large Round Smooth Short rod

AF-33 Off-white Large Leafy Irregular Medium rods AF-115 Milky white Large Round Rough Medium rods

AF-34 Brown Medium Round Wavy Short rods AF-116 Translucent Medium Round Smooth Short rods

AF-35 Translucent Medium Round Irregular Short rods AF-117 Transparent Small Round Smooth Medium rods

AF-36 Dark brown Medium Round Smooth Medium rods AF-118 White Small Flower Wavy Long rods

AF-37* Cream Medium Round Smooth Small rods AF-119 Brown Small Round Smooth Small rods

AF-38 Cream Large Irregular Rough Thin rods AF-120 Cream Medium Round Smooth Medium rods

AF-39 Yellow Small Round Smooth Small rods AF-121 Off-white Large Flower Rough Oval

AF-40 Off-white Small Wavy Rough Medium rods AF-122 Translucent Large Irregular Wavy Medium rods

AF-41 Off-white Tiny Round Smooth Long rod AF-123 Brown Small Round Smooth Medium rods

AF-42* Light brown Small Round Smooth Short rod AF-124 Cream Medium Round Smooth Small rods

AF-43** Translucent Medium Round Smooth Coccus AF-125 Off-white Tiny Round Smooth Medium rods

AF-44* Milky white Small Irregular Rough Long rod AF-126 Brown Large Round Smooth Long rods

AF-45 Orange Small Round Smooth Short rods AF-127 White Large Irregular Rough Long rods

AF-46 Brown Medium Round Smooth Coccus AF-128 Yellow Small Round Smooth Medium rods

Continued…

Chapter 3 Results

87

Isolate

codes

Colony

Color

Colony

Size

Colony

Shape

Colony

Margins

Cell Shape Isolate

codes

Colony

Color

Colony

Size

Colony

Shape

Colony

Margins

Cell Shape

AF-47 Translucent Medium Round Smooth Short rod AF-129 Milky white Small Round Smooth Medium rods

AF-48* White Small Round Smooth Short rods AF-130 Off white Medium Round Smooth Medium rods

AF-49 Cream Medium Round Smooth Long rods AF-131 Cream Medium Round Smooth Medium rods

AF-50 Milky white Medium Irregular Rough Short rods AF-132 Brown Small Round Rough Long rods

AF-51 Cream Medium Irregular Rough Long rods AF-133 Milky white Small Round Rough Medium rods

AF-52 Cream Medium Round Rough Short rod AF-134 Yellow Small Round Smooth Medium rods

AF-53 Off-white Large Irregular Wavy Coccus AF-135 White Medium Round Rough Thin rods

AF-54 Dark brown Small Round Smooth Long rod AF-136 Off white Medium Round Smooth Short rods

AF-55 Brown Medium Flower Wavy Short rod AF-137 Brown Medium Round Smooth Medium rods

AF-56 Brown Medium Round Smooth Short rods AF-138 Milky white Small Round Smooth Tiny rods

AF-57 Brown Small Flower Wavy Coccus AF-139 Light yellow Medium Irregular Rough Short rods

AF-58 Cream Tiny Round Smooth Short rods AF-40 Cream Small Round Smooth Short rods

AF-59 Cream Medium Irregular Rough Long rod AF-141 Red Small Irregular Rough Medium rods

AF-60 Cream Medium Round Rough Short rods AF-142 Dark brown Medium Round Smooth Medium rods

AF-61* Cream Medium Irregular Rough Long rods AF-143 Yellow Medium Round Smooth Small rods

AF-62 Cream Medium Round Smooth Short rods AF-144 Yellow Small Round Rough Tiny rods

AF-63 Gray Small Round Smooth Long rod AF-145 Brown Medium Flower Rough Short rods

AF-64 Brown Medium Round Smooth Long rod AF-146* Off-white Small Round Smooth Medium rods

AF-65 Cream Medium Irregular Rough Short rods AF-147 Off-white Small Round Smooth Medium rods

AF-66 Orange Medium Round Smooth Short rod AF-148 Brown Small Round Smooth Medium rods

AF-67 Off-white Medium Flower Wavy Short rods AF-149 White Medium Round Smooth Short rods

AF-68 Translucent Small Round Smooth Short rods AF-150 Brown Large Round Smooth Tiny rods

AF-69 Brown Large Round Rough Coccus AF-151 Cream Medium Irregular Rough Medium rods

Continued…

Chapter 3 Results

88

Isolate

codes

Colony

Color

Colony

Size

Colony

Shape

Colony

Margins

Cell Shape Isolate

codes

Colony

Color

Colony

Size

Colony

Shape

Colony

Margins

Cell Shape

AF-70 Yolk Medium Round Smooth Short rod AF-152 Off-white Small Round Rough Medium rods

AF-71* Orange Small Round Smooth Short rods AF-153 Brown Large Irregular Rough Medium rods

AF-72 Off-white Medium Irregular Wavy Long rod AF-154 Brown Medium Round Smooth Medium rods

AF-73 Brown Small Round Smooth Short rod AF-155 Off-white Large Wavy Rough Short rods

AF-74 Brown Medium Irregular Wavy Short rods AF-156 Cream Small Round Smooth Medium rods

AF-75* Brown Medium Round Smooth Oval AF-157 Cream Large Flower Rough Medium rods

AF-76 Milky-white Medium Irregular Wavy Short rods AF-158 White Small Round Smooth Tiny rods

AF-77 White Small Flower Wavy Long rod AF-159 Translucent Medium Round Smooth Short rods

AF-78 Translucent Small Round Smooth Short rod AF-160* Milky white Medium Flower Wavy Short rods

AF-79 White Tiny Round Smooth Short rod AF-161 Off white Medium Round Smooth Medium rods

AF-80 Dark brown Medium Irregular Wavy Short rods AF-162 Cream Small Round Smooth Medium rods

AF-81 Off-white Medium Irregular Wavy Short rods AF-163 Milky white Small Round Smooth Short rods

AF-82 Cream Medium Round Smooth Long rods

*Strains showed Gram negative reaction while rest were Gram positive.

** Isolates were non-motile

Chapter 3 Results

89

CHARACTERIZATION OF SUNFLOWER ENDO AND RHIZOSPHERE

ISOLATES FOR IN VITRO PLANT GROWTH PROMOTING TRAITS

After morphological characterization, the rhzio/endophytic bacterial isolates were screened

(in vitro) for their qualitative biochemical traits (P-solubilization, IAA, N2 fixation, and

biocontrol activities). Out of 163 screened isolates 40 % were positive for phosphate

solubilization, 24 % for IAA production, 20% for N2 fixation and 12% were biocontrol

agents against Fusarium oxysporum (Figure 3.1). Isolates showing positive response for

more than one trait were further screened for qualitative analysis (Table 3.3).

Figure 3-1 Graphical representation of the ratio of 163 bacterial isolates for different

PGP traits obtained from endo/rhizosphere of Sunflower (cv. FH-331)

Chapter 3 Results

90

Table 3-3 Qualitative data of 163 bacterial isolates from sunflower endo/rhizosphere screened for different pant growth

promoting traits.

Isolate

codes

Sites *Endo/

Rhizo

P-

sol

IAA Biocontr

ol Assay

Isolate

codes

Sites Endo/

Rhizo

P-sol IAA Biocontro

l Assay

AF-1 Arja E + - - AF-83 Hanschoki R +++ - +

AF-2 Arja R - + - AF-84 Hanschoki E - + -

AF-3 Arja E - - W AF-85 Hanschoki R + +

AF-4 Arja R + - - AF-86 Hill 2 R - W -

AF-5 Arja R - - - AF-87 Hill 2 R - - W

AF-6 Arja R ++

+

+ ++ AF-88 Hill 2 E - W -

AF-7 Arja E - W - AF-89 Hill 2 E W - -

AF-8 Arja R - + - AF-90 Hill 2 R + - -

AF-9 Arja E ++

+

+++ W AF-91 Hill-2 E - - W

AF-10 Arja R - - - AF-92 Hill-2 R +++ - -

AF-11 Arja E + - + AF-93 Hill-2 E + W -

AF-12 Arja E + - - AF-94 Keri R - - -

AF-13 Arja R - W - AF-95 Keri R +++ ++ +

AF-14 Arja E + - - AF-96 Keri E + - +

AF-15 Arja R + - - AF-97 Keri E + - -

AF-16 Arja E - + - AF-98 Keri R + - -

AF-117 Bandi E - ++ - AF-99 Kotli E - - W

AF-18 Bandi R + + - AF-100 Kotli R + - -

AF-19 Bandi E - - + AF-101 Kotli E - + -

AF-20 Bandi R + + - AF-102 Kotli R ++ - -

Continued…

Chapter 3 Results

91

Isolate

codes

Sites Endo/

Rhizo

P-

sol

IAA Biocontr

ol Assay

Isolate

codes

Sites Endo/

Rhizo

P-sol IAA Biocontro

l Assay

AF-21 Bandi R W ++ ++ AF-103 Kotli R - W -

AF-22 Bandi R ++ ++ +++ AF-104 Kotli R + - -

AF-23 Bandi R + - - AF-105 Kotli E W + -

AF-24 Bandi E - + - AF-106 Kotli R + + -

AF-25 Bandi R + - - AF-107 Kotli R ++ - +

AF-26 Bandi E W - - AF-108 Kotli R - - -

AF-27 Bandi R W - - AF-109 Kotli R + ++ +

AF-28 Chamankot E - - W AF-110 Kotli E - - -

AF-29 Chamankot E - W - AF-111 Kotli E - + -

AF-30 Chamankot R - - - AF-112 Makhyala R - - W

AF-31 Chamankot R +++ ++ + AF-113 Makhyala E + + -

AF-32 Chamankot R + - - AF-114 Makhyala E - W -

AF-33 Chamankot R - - W AF-115 Makhyala R W - -

AF-34 Chamankot E + - - AF-116 Makhyala R - W -

AF-35 Chamankot R ++ + - AF-117 Makhyala E W - -

AF-36 Chamankot R - + - AF-118 Makhyala E + - -

AF-37 Chamankot E - W - AF-119 Makhyala R + - -

AF-38 Chamankot R - - W AF-120 Munhasa R + ++ -

AF-39 Dhirkot E - +++ - AF-121 Munhasa R + - -

AF-40 Dhirkot R - + - AF-122 Munhasa E + - -

AF-41 Dhirkot E - + - AF-123 Munhasa R - + -

AF-42 Dhirkot R + - - AF-124 Munhasa R + + -

Continued…

Chapter 3 Results

92

Isolate

codes

Sites Endo/

Rhizo

P-

sol

IAA Biocontr

ol Assay

Isolate

codes

Sites Endo/

Rhizo

P-sol IAA Biocontro

l Assay

AF-43 Dhirkot E - + - AF-125 Munhasa R - - -

AF-44 Dhirkot R +++ W - AF-126 Munhasa E + - -

AF-45 Dhirkot R ++ - - AF-127 Munhasa R + + -

AF-46 Dhirkot R + - - AF-128 Narwal E - - W

AF-47 Dhirkot R - + + AF-129 Narwal E - W -

AF-48 Diyar Gai E ++ - +++ AF-130 Narwal R +++ - W

AF-49 Diyar Gali R + - - AF-131 Narwal R - + -

AF-50 Diyar Gali R W - - AF-132 Narwal R + - -

AF-51 Diyar Gali E - W - AF-133 Narwal E - - +

AF-52 Diyar Gali R + - - AF-134 Narwal E + - -

AF-53 Diyar Gali E - - - AF-135 Narwal R ++ - -

AF-54 Diyar gali R +++ ++ ++ AF-136 Narwal R ++ - -

AF-55 Diyar gali R ++ - - AF-137 Neela but E + - +

AF-56 Diyar Gali E ++ +++ + AF-138 Neela but R - ++ -

AF-57 Diyar Gali R + - + AF-139 Neela but E - - W

AF-58 Diyar Gali R + - - AF-141 Neela but R + + -

AF-59 Diyar Gali R - + - AF-142 Neela but E - W -

AF-60 Diyar Gali E W - - AF-143 Neela but E + + -

AF-61 Diyar Gali R - + - AF-144 Neela but R +++ - +

AF-62 Diyar Gali R + - - AF-145 Neela but R W - -

AF-63 Ghazi abad E + - - AF-146 Neela but R - + -

AF-64 Ghazi abad R + - - AF-147 Sanghar E ++ + +

Continued…

Chapter 3 Results

93

Isolate

codes

Sites Endo/

Rhizo

P-

sol

IAA Biocontr

ol Assay

Isolate

codes

Sites Endo/

Rhizo

P-sol IAA Biocontro

l Assay

AF-65 Ghazi abad R + - - AF-148 Sanghar R - ++ -

AF-66 Ghazi abad E - - W AF-149 Sanghar R + - -

AF-68 Ghazi abad R +++ W - AF-151 Sanghar E + + -

AF-69 Ghazi abad R - - - AF-152 Sanghar R + - W

AF-70 Ghazi abad R - W - AF-153 Sanghar E - W -

AF-71 Ghazi abad E - W W AF-154 Sanghar E + - -

AF-71 Ghazi abad R +++ - - AF-155 Sanghar R + - -

AF-73 Ghazi abad R + - - AF-156 Upper Chamyati R - - W

AF-74 Hanschoki E - + - AF-157 Upper Chamyati E + - +

AF-75 Hanschoki E W - - AF-158 Upper Chamyati R + - -

AF-76 Hanschoki R + - - AF-159 Upper Chamyati E + - -

AF-77 Hanschoki R + - + AF-160 Upper Chamyati E +++ + -

AF-78 Hanschoki E - - W AF-161 Upper Chamyati R - W W

AF-79 Hanschoki R - + - AF-162 Upper Chamyati R - + -

AF-80 Hanschoki E + - - AF-163 Upper Chamyati ++ +++ + ++

AF-81 Hanschoki E - W -

AF-82 Hanschoki R +++ - -

E: Endophytic, R: Rhizospheric, P-sol: Phosphate solubilization, +: Halo zone >1mm, ++: Halo zone >2mm, and +++: Halo zone

>3mm

W: Weak positive, -: Negative, Endo: Endosphere, Rhizo: Rhizosphere

Chapter 3 Results

94

`

Figure 3-2 Biocontrol, Indole-3-acetic production and phosphate solubilizing

potential of representative bacterial isolates

Panel A and B indicating biocontrol activity of representative bacterial isolate AF-83 and AF-22

through dual plate assay against fungus (Fusarium oxysporum) respectively, as compared to control

(panel b), after 5 days of inoculation.

The IAA production of representative bacterial strains (panel d) after 25 minutes of reaction, LB

broth was supplemented with L-tryptophan as a precursor of IAA, compared with standard.

Halo zones formation by bacteria isolates on Pikovskaya’s agar medium indicating P- solubilizing

ability of AF-54 (panel E), AF-48 (panel F) and AF-56 (panel G). Observation were made after 7

days of inoculation.

Chapter 3 Results

95

3.2.1 Solubilization of Inorganic Phosphate

Out of 163 sunflower associated endo and rhizospheric bacterial isolates, 84 developed

halo-zone on Pikovskaya’s agar plates with varied range (Figure 3.2) after 7 days of

incubation representing their phosphate solubilizing ability (Table 3.3). Among the

positive isolates, 23 were endophytes while the rest were isolated form rhizosphere.

Isolates found positive for plates assay were then quantified spectrophotometrically by

phospho-molybdate blue color method. Inorganic phosphate was solubilized (Table 3.4) in

the range of 9.51 to 48.80 µg mL-1 by bacterial isolates. Maximum inorganic phosphate in

culture medium was solubilized by rhizospheric isolate AF-54 (59.37 µg mL-1) from Diyar

Gali followed by rhizospheric isolate from Chamankot AF-31 (47.86 µg mL-1), and another

rhizospheric isolate from Diyar Gali AF-48 (44.58 µg mL-1).

Solubilization index

Potential phosphate solubilizers were checked for their solubilization index (Figure 3.3).

Bacterial isolate AF-54 (Diyar Gali) showed highest solubilization index (3.03) followed

by AF-48 (2.95) while AF-9 a putative endophyte from Arja showed lowest index (0.98).

Figure 3-3 Phosphate solubilization index of representative bacterial isolates on

Pikovskaya’s agar medium

Values are average of three replicates, Bars show the standard deviation of data

2.73

0.98

2.32 2.26

2.02 2.07

3.032.81

2.41

2.95

2.19

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

AF-6 AF-9 AF-56 AF-163 AF-21 AF-22 AF-54 AF-31 AF-142 AF-48 AF-95

Pho

sph

ate

solu

bili

zati

on

ind

ex

Chapter 3 Results

96

Table 3-4 Quantification of soluble P in supernatant of bacterial isolates from

sunflower rhizosphere

Isolate

Cods Sites Soluble P

(µg/mL) Isolate

codes Sites Soluble P

(µg/mL)

AF-6 Arja 32.58±1.7 AF-72 Ghazi abad 27.37±2.6

AF-7 Arja 33.37±2.0 AF-75 Hanschoki 28.82±2.7



AF-16 Arja ND AF-86 Hill 2 23.96±1.8

AF-19 Bandi 33.11±1.9 AF-88 Hill 2 20.18±1.7

AF-20 Bandi 38.31±6.2 AF-90 Hill 2 24.40±2.2

AF-21 Bandi 38.94±2.5 AF-95 Keri 40.45±3.7

AF-22 Bandi 40.11±2.3 AF-98 Keri 18.33±2.2

AF-24 Bandi ND AF-105 Kotli 27.19±2.0

AF31 Chamman

Kot

47.86±3.2 AF-106 Kotli 21.59±1.9

AF-33 Chamman

Kot

23.56±2.5 AF-108 Kotli 31.14±1.3

AF-36 Chamman

Kot

29.68±2.3 AF-112 Makhyala 26.80±2.5

AF-41 Dhirkot 27.93±1.5 AF-115 Makhyala 25.54±1.5

AF-42 Dhirkot 26.91±2.1 AF-122 Munhasa 17.89±1.1

AF-43 Dhirkot 11.59±1.3 AF-133 Narwal 38.35±2.3

AF-48 Diyar Gali 44.58±4.1 AF-139 Narwal 28.11±2.1

Continued…

Chapter 3 Results

97

Isolate

Cods

Sites Soluble P

(µg/mL)

Isolate

codes

Sites Soluble P

(µg/mL)

AF-52 Diyar Gali 11.77±1.4 AF-144 Neela but 26.24±2.5



AF-58 Diyar Gali 13.12±0.8 AF-155 Sanghar 22.24±2.4

AF-62 Diyar Gali 32.30±1.9 AF-160 Upper Chamyati 28.30±2.2

AF-66 Ghazi abad 12.59±1.1 AF-163 Upper Chamyati 40.50±1.6

AF-69 Ghazi abad 30.61±3.0

The phosphate solubilization activity was determined by Spectrophotometer. Data was

taken after 7 days of inoculation. Values are average of three replicates ± indicates the

standard deviation of data. ND: Not detected

*Values <10 (µg/mL) are not presented here.

3.2.1.1 Detection of Organic Acid Production Involved in P-solubilization Process.

Organic acid production by the top phosphate solubilizing isolates was detected on HPLC.

Chromatographic results showed that most of the tested bacterial isolates were able to

produce several organic acids in different ranges. Malic acid was produced by 8 bacterial

isolates in a range of 1.96 µg mL-1 (AF-62, endophytic) to 12.11 µg mL-1 (AF-31,

rhizospheric isolate from Chamankot). Maximum number of bacteria produced gluconic

acid (G.A) ranged between 2.17 µg mL-1 to 15.44 µg mL-1 produced by AF-21

(rhizospheric isolate from Bandi) and AF-54 (rhizospheric isolate form Diyar Gali)

respectively. Only AF-95 (endophytic isolate from Keri; 1.09 µg mL-1), AF-54

(rhizospheric; 5.2 µg mL-1) and AF-31(rhizospheric; 3.01 µg mL-1) were able to produce

tartaric acid (T.A). Seven bacterial isolates showed ascorbic acid (A.A) production in a

range of 1.2 to 15.02 µg mL-1 with highest value detected in case of rhizospheric isolate

AF-31. AF-54 again produced maximum amount of oxalic acid (O.A) 11.76 µg mL-1

among 9 oxalic acid producing bacterial isolates (Table 3.5).

Chapter 3 Results

98

Table 3-5 Detection of organic acids in bacteria isolated from rhizosphere and root

interior in Pikovskaya’s broth medium

Isolates Malik

Acid

Gluconic

Acid

Tartaric

Acid

Ascorbic

Acid

Lactic

Acid

Oxalic

Acid

AF-6 ND 12.61±1.02 ND 1.20±0.05 NA 8.72±132

AF-21 10.78±0.82 3.31±0.03 ND ND 17.90±1.33 1.25±0.06

AF-95 ND 5.01±0.41 1.09±0.04 ND ND ND

AF-146 ND 14.61±1.73 ND ND 22.09±1.51 2.12±0.04

AF-163 10.60±1.96 ND ND 1.69±0.02 ND 3.25±0.21

AF-9 ND 10.79±0.77 ND ND 7.38±1.23 ND

AF-48 11.50±1.23 12.04±1.7 ND 1.33±0.07 3.99±0.09 ND

AF-22 5.25±0.12 3.47±0.05 ND ND ND 1.97±0.21

AF-54 ND 15.44±0.91 5.20±0.33 ND 12.18±1.89 11.76±0.88

AF-56 9.24±0.73 ND ND 9.14±0.76 31.93±2.17 0.17±0.04

AF-31 12.11±0.82 5.00±0.21 3.01±0.52 15.01±1.12 13.21±1.44 2.95±0.07

AF-19 ND 2.46±0.04 ND ND ND ND

AF-108 6.01±0.21 ND ND 5.01±0.08 ND ND

AF-49 ND ND ND ND 27.31±1.94 1.29±0.08

AF-7 ND 2.17±0.02 ND ND ND ND

AF-62 1.96±0.07 ND ND 10.01±1.3 ND ND

ND: Not detected. ±: Standard deviation

Values are the means of three replicates.

Chapter 3 Results

99

3.2.2 Indole-3-Acetic Acid (IAA) Production

The in vitro qualitative analysis of indole acetic acid synthesis showed 46 bacterial isolates

produced pink color hence found to be positive for IAA production (Figure 3.2).

Quantification of IAA production using HPLC revealed that 19 bacterial isolates produced

IAA in the range of 1.13-24.6 µg mL-1. Highest amount of IAA (24.6 µg mL-1) was

produced by a putative endophytic isolate from Bandi AF-22 followed by rhizospheric

isolate from Diyar Gali AF-54 (23.9 µg mL-1). Minimum quantity of IAA was detected in

another putative endophytic isolate from Bandi AF-24 (1.13 µg mL-1) Table 3.6).

3.2.3 Nitrogen Fixation/ Nitrogenase Activity

Gas chromatographic analysis of the isolates showed 14 isolates exhibited nitrogenase

activity in acetylene reduction assay (ARA) in the range of 28.68-137.84 nmoles mg-1

protein h-1. Out of these 14 isolates, 9 showed fairly substantial amount of nitrogenase

activity. The maximum nitrogenase activity (137.84 nmoles mg-1 protein h-1) was found

in a putative endophytic isolate AF-22 from Bandi, which was even greater than the

nitrogenase activity shown by Azospirillum brasilense Er-20 used as positive control

(Table 3.7) followed by another putative endophyte AF-146, from Sanghar, by producing

129.46 nmoles mg-1 protein h-1. 10 more isolates showed nitrogenase activity less than 25

nmoles mg-1 protein-1 and were considered as weak- nitrogen fixers.

3.2.4 Biocontrol Assay

The purified bacterial strains were tested for in vitro antagonistic activity against Fusarium

sp. (fungal phytopathogens) using dual-culture plate assay. Results revealed that out of 163

bacterial isolates, 23 isolates inhibited fungal growth on PDA plate showing biocontrol

activity. (Table 3.3). Maximum inhibition zone was produced by AF-54 (rhizospheric

isolate from Diyar Gali) followed by a putative endophyte AF-146 from Sanghar (Figure

3.2)

Chapter 3 Results

100

Table 3-6 Quantification of indole-3-acetic acid (IAA) produced by bacterial isolates

in LB broth medium supplemented with L-Tryptophan

Isolate

Code

Site IAA

Production

(µl mL-1

)

Isolate Code Site IAA

Production

(µl mL-1

)

AF-6 Arja 11.53±1.21 AF-54 Diyar Gali 23.90±1.76

AF-9 Arja 16.30±2.27 AF-56 Diyar Gali 22.87±1.10

AF-20 Bandi 1.17±0.25 AF-69 Ghazi abad 4.30±0.78

AF-21 Bandi 14.67±0.87 AF-75 Hanschoki 2.17±0.25

AF-22 Bandi 24.67±1.91 AF-95 Keri 6.67±0.25

AF-24 Bandi 1.13±0.29 AF-105 Kotli 3.33±0.60

AF-31 Chamman

Kot

20.43±1.57 AF-115 Makhyala 3.57±0.21

AF-43 Dhirkot 12.47±1.22 AF-146 Sanghar 19.93±1.37

AF-48 Diyar Gali 16.23±0.45 AF-163 Upper

Chamyati

12.77±1.11

AF-49 Diyar Gali 2.70±0.61

Values are average of three replicates

± indicates the standard deviation of data

*Values <1 are not presented here.

Chapter 3 Results

101

Table 3-7 Nitrogenase activity of bacterial isolates isolated from rhizosphere and

root interior of sunflower

The nitrogenase activity was detected by acetylene reduction assay. Bacterial isolates were

inoculated to NFM medium. Values are the mean of 3 replicates. ± shows the standard

deviation of the replicates.

* Nitrogenase activity<30 nmoles is not presented here

Selection of Potential PGPR for Further Studies

On the basis of in vitro biochemical screening including phosphate solubilization,

phytohormones synthesis, N2 fixation and biocontrol activity, 11 potential endo and rhizo-

Isolate Code Sites Nitrogenase activity (nmoles mg

-1

protein h-1

)

ER-20 ---------- 133.31±9.85

AF-22 Bandi 137.84±14.77

AF-21 Bandi 117.16±13.79

AF-95 Keri 126.00±13.88

AF-146 Sanghar 129.46±13.45

AF-163 Upper Chamyati 107.24±12.38

AF-9 Arja 93.09±8.77


AF-6 Arja 76.33±8.34

AF-54 Diyar Gali 44.28±12.44


AF-31 Chamman Kot 56.07±5.78

AF-72 Ghazi abad 62.50±4.82

AF-77 Hanschoki 42.78±3.68

AF-111 Kotli 91.33±6.27

Chapter 3 Results

102

bacterial isolates (AF-6, AF-21, AF-22, AF-95, AF-148, AF-54, AF-163, AF-9, AF-56,

AF-48 and AF-31) were selected for further detailed in vitro and in vivo studies.

3.2.5 Intrinsic Antibiotic Resistance

Disc diffusion method was used to determine the intrinsic antibiotic resistance pattern by

the selected isolates (Figure 3.4) and antibiosis (clear zone formation around the antibiotic

disc) showed that AF-6, AF-54, AF-95, AF-31, AF-56, AF-9, AF-163 and AF-48 were

resistant to carbenicillin (100µg). AF-6, AF-54, AF-95, AF-31, AF-9, AF-56, AF-163, AF-

146 and AF-48 were found resistant to aztreonam (30µg). AF-6, AF-56, AF-163 and AF-

22 were resistant to nalidixic acid (30 µg). AF-54, AF-95, AF-9, AF-163, AF-21 and AF-

31 were resistant to gentamicin (10µg). AF-6, AF-95, AF-31, AF-56, AF-163, AF-148,

AF-22 were resistant to rifampicin (5µg). Only AF-22 was found resistant to

chloramphenicol (30 µg) and ciprofloxacin (5µg). AF-54, AF-31, AF-9, AF-163, AF-148,

AF-21 were resistant to streptomycin (10µg). AF-95, AF-31, AF-163 and AF-22 were

resistant to cephradine (30µg). AF-95, AF-31, AF-9, AF-163, AF-22, AF-48 were resistant

to erythromycin (15 µg). AF-31, AF-9, AF-22 and AF-31 were resistant to tetracyclin (30

µg). Only AF-9 and AF-22 found resistant to amikacin (30µg) and AF-48 and AF-22 were

resistant to kanamycin (30µg). While bacterial isolates were susceptible to the rest of the

antibiotics tested (Table 3.8).

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103

Table 3-8 Intrinsic antibiotic resistance pattern of selected bacterial isolates from sunflower rhizo/endosphere

Isolates AK PY CN CIP CE ATM TE NA K RA S E C CFM

AF-6 S R S S S S R R R S S S S S

AF-22 S S R R R R R R R S R R R S

AF-54 S R S S S S R R R S R S R S

AF-95 S R R S R R S S S R S R S R

AF-31 S S R R R R R R R S R R S S

AF-56 S S R R S S R R R S S S S S

AF-9 S S R R R R R R R S R R R S

AF-63 R R R R S R R S R S R R R R

AF-146 S S S S S S R R R S R S S S

AF-21 S S R R R S R R R S S S S S

AF-48 S R R S S R R S R S S R S R

S= susceptible, R=resistant

AK: Amikacin (10 µg), PY: Carbenicillin (100 µg), CN: Gentamicin (10 µg) CIP: Ciprofloxacin (5 µg), CE: Cephradine (30 µg), ATM: Aztreonam

(30 µg), CFM: Cefixime (5 µg), TE: Tetracycline (30 µg), NA: Nalidixic acid (30 µg), K: Kanamycin (30 µg), RA: Rifampicin (5 µg), S:

Streptomycin (10 µg), E: Erthromycin (15 µg), C: Chloramphenicol (30 µg)

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104

3.2.6 Phenotypic Microarray Analysis

Phenotypic microarray represents the metabolic potential of various isolates to utilize

different carbon sources. BIOLOG GN-2 system was employed to conduct phenotypic

microarray of four finally selected isolates. Isolate AF-54 metabolized maximum number

of carbon sources (77) out of 96 which were evident from the conversion of colorless

tetrazolium redox dye to purple color and minimum carbon sources were metabolized by

AF-31. All the isolates were positive for Tween 80, Acetic Acid, Hydroxybutyric Acid, γ -

Hydroxybutyric Acid, p-Hydroxy Phenylacetic Acid, D,L-Lactic Acid, Malonic Acid,

Propionic Acid, Succinic Acid, D-Alanine, L-Asparagine, L-Aspartic Acid, L-Glutamic

Acid, L-Leucine, L-Phenylalanine, L-Proline, L-Pyroglutamic Acid, while negative for D-

Galactonic Acid Lactone, D-Saccharic Acid, Sebacic Acid, Urocanic Acid, Putrescine, 2-

Aminoethanol, 2,3-Butanediol (Table 3.9)

Table 3-9 Differential metabolic profiling of selected bacterial isolates (Biolog GN2

Microplate analysis)

Sr. No. Substrate AF-22 AF-31 AF-56 AF-54

4 Glycogen - - + +

5 Tween 40 - + - +

9 Adonitol - - + +

10 L-Arabinose - - + -

12 D-Cellobiose - - + +

13 i-Erythritol + - - -

16 D-Galactose + - - +

17 Gentiobiose + - - -

18 α-D-Glucose + - - +

Continued…

Chapter 3 Results

105


19 m-Inositol - - - -

20 α-D-Lactose - - + +

24 D-Mannose - - + +

28 D-Raffinose + - + -

30 D-Sorbitol + - - +

31 Sucrose + - - +

35 Pyruvic Acid Methyl Ester - + + +

36 Succinic Acid Mono-

Methyl-Ester

+ + - +

38 Cis-Aconitic Acid - + + +

39 Citric Acid - + + +

40 Formic Acid - - + +

42 D-Galacturonic Acid + - - -

44 D-Glucosaminic Acid - - + +

46 α-Hydroxybutyric Acid - - - +

51 α-Keto Butyric Acid - - + +

53 α-Keto Valeric Acid - + + -

61 Bromosuccinic Acid - - + +

62 Succinamic Acid + + - -v

64 L-Alaninamide + + + +

66 L-Alanine + + - +

71 Glycyl-Laspartic Acid + - - +

Continued…

Chapter 3 Results

106


73 L-Histidine - + - -

74 Hydroxy-LProline + - + -

87 Uridine - - + +

88 Thymidine - - + +

89 Phenyethyl Amine + + - -

+ = Substrate metabolized; ‒ = substrate not metabolized

*Water used as control.

The reactions for 2,3-Butanediol, 2-Aminoethanol, Putrescine, Urocanic Acid, Sebacic

Acid, D-Saccharic Acid, D-Galactonic Acid Lactone was negative in all isolates

Tween 80, Acetic Acid, Hydroxybutyric Acid, γ-Hydroxybutyric Acid, p-Hydroxy

Phenylacetic Acid, D,L-Lactic Acid, Malonic Acid, Propionic Acid, Succinic Acid, D-

Alanine, L-Asparagine, L-Aspartic Acid, L-Glutamic Acid, L-Leucine, LPhenylalanine, L-

Proline, L-Pyroglutamic Acid, showed reaction + for all bacterial isolates.

All strains were positive for reactions including α-Cyclodextrin, Dextrin, N-Acetyl-

DGalactosamine, N-Acetyl-DGlucosamine, D-Arabitol, D-Fructose, L-Fucose, Lactulose,

Maltose, D-Mannitol, D-Melibiose, β-Methyl-D-Glucoside, D-Psicose, L-Rhamnose, D-

Trehalose, Turanose, Xylitol, D-Gluconic Acid, D-GlucuronicAcid, Itaconic Acid, α-Keto

Glutaric Acid, Quinic Acid, Glucuronamide, L-Alanylglycine, Glycyl-Lglutamic Acid, L-

Ornithine, D-Serine, L-Serine, L-Threonine, D,L-Carnitine, γ-Amino Butyric Acid,

Inosine, Glycerol, D,L-α-Glycerol Phosphate, α-D-Glucose-1-Phosphate, D-Glucose-6-

Phosphate except AF-31.

Chapter 3 Results

107

Figure 3-4 Antibiogram of representative bacterial isolate AF-22

AK (Amikacin)

PY (Carbenicillin)

CN (Gentamicin)

CIP (Ciprofloxacin)

CE (Cephradine)

ATM (Aztreonam)

CFM (Cefixime)

TE (Tetracycline)

NA (Nalidixic acid)

K (Kanamycin)

RA (Rifampicin)

S (Streptomycin)

E (Erthromycin)

C (Chloramphenicol)

Chapter 3 Results

108

MOLECULAR CHARACTERIZATION

3.3.1 Sequence and Phylogenetic Analysis of 16S rRNA Gene

The 16S rRNA gene sequence analysis (Figure 3.5) of selected plant growth promoting

rhizobacterial isolates revealed that isolates belong to the already reported bacterial genera

i.e. Azospirillum, Bacillus, Enterobacter, Citrobacter, Pseudomonas, Serratia,

Stenotrophomonas and Lysinibacillus Cellulosimicrobium, Staphylococcus,

Chryseobacterium etc. with up to 98-100 % sequence similarity. (Table 3.10). Bacillus

found to be the prominent among all genera followed by Pseudomonas then comes

Enterobacter. AF-9, AF-14, AF-20, AF-42, AF-44, AF-48, AF-72, AF-75, AF-88, AF-

112, AF-144, and AF-160 belong to different Bacillus species including B. safensis, B.

thuringiensis, B. subtilus, B. cereus, B. licheniformis, B. pumilus, B. megaterium. These

strains clustered together in a combined phylogenetic tree (Figure 3.6). AF-6, AF-16, AF-

54, AF-62, AF-66, AF-81, AF-86, AF-95, AF-108, AF-122 and AF-161 belong to different

Pseudomonas species including P. thivervalensis, P. chlororaphis, P. brassicacearum, P.

fluorescens, P. putida, P. azotoformans with 98-99 % similarity (Table 3.10) and formed

second major cluster of combined phylogenetic tree (Figure 3.6). AF-31, AF-32, AF-115

and AF-145 were identified as Enterobacter sp. (98 %) Enterobacter cloacae (99 %)

Enterobacter asburiae (98 %) and Enterobacter sp. (98 %) respectively, clustered together

and Serratia marcescens AF-43 also clustered with Enterobacter species (Table 3.10;

Figure 3.6). Members of genus Citrobacter (98-99 %) AF-21, AF-56 and AF-129 clustered

together. The combined tree did not show any biased distribution of specific genera to the

sites.

Phylogenetic relationship of selected sequenced bacterial strains were developed for each

genus separately, by taking relevant sequences from NCBI Genbank. Among the selected

4 isolates, Isolate AF-22 revealed up to 98 % similarity with Azospirillum brasilense strain

Gr22. This isolate also clustered with A. brasilense when phylogenetically analyzed with

other species of genera Azospirillum where A. halopraeferens (NR_044859) was taken as

root (Figure 3.7). Isolates AF-54 showed 99 % similarity with Pseudomonas sp. TY1205

and in phylogenetic tree AF-54 was clustered with Pseudomonas fluorescens

(EU854429.1) (Figure 3.8). Isolate AF-31 was identified as Enterobacter cloacae with 99

Chapter 3 Results

109

% sequence identity with already reported Enterobacter cloacae strain CMM1 (Table 3.10)

and in phylogenetic three Enterobacter cloacae clustered with Enterobacter cloacae

(KJ607594) (Figure 3.9). Isolates AF-56 showed 99 % similarity with Citrobacter freundii

strain SL151 and in phylogenetic tree it was clustered with Citrobacter freundii

(CP018810.1) (Figure 3.10).

Figure 03-5 Agarose gel photograph showing amplified 16s RNA gene of

representative bacterial strains from sunflower endo/rhizosphere

1 = AF-6, 2 = AF-9, 3 = 1 kb ladder,

4= AF-14, 5 = AF-16, 6 = AF-19,

7 = AF-20, 8 = AF-21, 9- AF-21,

10 = Blank

Chapter 3 Results

110

Table 3-10 Identification of sunflower associated bacterial isolates based on 16S

rRNA gene sequence analysis

Strain

code

Strain origin 16S rRNA based

identification

Closest GenBank match (%

identity)

AF-6 Arja P. thivervalensis P. thivervalensis strain s59 98%

AF-9 Arja Bacillus safensis Bacillus safensis strain SL-40 (98)

AF-14 Arja Bacillus sp. Bacillus sp. Ba10b (99)

AF-16 Arja P. thivervalensis P. thivervalensis strain MRC33

(99)

AF-20 Bandi B. thuringiensis B. thuringiensis strain L15 (99)

AF-21 Bandi Citrobacter braakii Citrobacter braakii strain A8 (99)

AF-22 Bandi A. brasilense A. brasilense strain Gr22 (98)

AF-24 Bandi Stenotrophomonas sp. Stenotrophomonas sp. strain

UYSB33 (98)

AF-31 Chamman

Kot

Enterobacter cloacae Enterobacter cloacae strain CMM1

(99)

AF-32 Chamman

Kot

Enterobacter asburiae Enterobacter asburiae strain

IAC/BECa-123(99)

AF-35 Chamman

Kot

Hafnia alvei Hafnia alvei strain C10 (99)

Continued…

Chapter 3 Results

111

Strain

code


identification


identity)

AF-41 Dhirkot Staphylococcus sp. Staphylococcus sp. strain JSM

101067 (98)

AF-42 Dhirkot Bacillus thuringiensis Bacillus thuringiensis strain

BPR162 (100)

AF-43 Dhirkot Serratia marcescens S. marcescens strain RM142 (99)

AF-44 Dhirkot Bacillus subtilus Bacillus subtilus strain THJ-E28

AF-48 Diyar Gali Bacillus cereus Bacillus cereus strain S72 (98)

AF-54 Diyar Gali Pseudomonas sp. Pseudomonas sp. TY1205 (99)

AF-56 Diyar Gali Citrobacter freundii C. freundii strain SL151 (99)

AF-60 Diyar Gali Delftia sp. Delftia sp. strain AER321 (98)

AF-61 Diyar Gali Bacillus licheniformis B. licheniformis strain BAB-1833

(99)

AF-62 Dyar Gali Pseudomonas

chlororaphis

Pseudomonas chlororaphis strain

IARI-DHD-9 (99)

AF-66 Ghaziabad Pseudomonas

brassicacearum

Pseudomonas brassicacearum

strain BNHKY-2 (99)

AF-72 Ghaziabad Bacillus pumilus B. pumilus strain X-A12 (98)

AF-75 Hanschoki Bacillus safensis B. safensis strain F-82 (98)

AF-81 Hanschoki Pseudomonas

brassicacearum

Pseudomonas brassicacearum

strain: AF129 (99)

Continued…

Chapter 3 Results

112

Strain

code


identification


identity)

AF-86 Hill-2 P. fluorescens P. fluorescens strain R15 (99)

AF-88 Hill-2 Bacillus subtilis Bacillus subtilis strain SA4 (98)

AF-95 Keri P. brassicacearum P. brassicacearum strain 36D4 (98)

AF-98 Keri Pseudomonas putida P. putida strain IARI-KP6 (99)

AF-108 Kotli P. brassicacearum P. brassicacearum strain EPR3 (98)

AF-112 Makhyala Bacillus sp. Bacillus sp. S22913 (98)

AF-115 Makhyala Enterobacter sp. Enterobacter sp. SJZ-5 (98)

AF-122 Munhasa P. azotoformans P. azotoformans EXXP-1

AF-129 Narwal Citrobacter sp. Citrobacter sp. XT-10 (99)

AF-139 Neela but Chryseobacterium sp. Chryseobacterium sp. CH16 (99)

AF-144 Neela but B. megaterium B. megaterium strain SBA (99)

AF-145 Neela but Enterobacter cloacae Enterobacter cloacae strain ST15

(99)

AF-146 Neela but Lysinibacillus sp. Lysinibacillus sp. strain P22 (99)

AF-151 Sanghar Pseudomonas sp. Pseudomonas sp. BMO1_1D7 (99)

AF-155 Sanghar Cellulosimicrobium

sp.

Cellulosimicrobium sp. TV30bNov

(98)

Continued…

Chapter 3 Results

113

Strain

code


identification


identity)

AF-160 Upper

Chamyati

Bacillus licheniformis Bacillus licheniformis strain GR36

(98)

AF-161 Upper

Chamyati

Pseudomonas

fluorescens

P. fluorescens strain J13 (98)

AF-163 Upper

Chamyati

Arthrobacter sp. Arthrobacter sp. strain M18-2 (99)

*Isolates with less than 98 % similarity to already reported bacterial strains are not

presented here.

Chapter 3 Results

114

Figure 3-6 Phylogenetic relationship of 42 bacterial isolates obtained from

endo/rhizosphere of sunflower from 16 different sits of Dhirkot, AJK based on 16s

rRNA (1500 bp) sequences

*Different colors indicate the sites of isolation.

Chapter 3 Results

115

Figure 03-7 Phylogenetic tree based on 16S rRNA sequences (1014 bp) of sunflower

associated Azospirillum brasilense AF-22 ( ) and published sequences.

*Neighbor joining method was adopted.

*Bootstrap values greater than 50 were given and were based on 1000 replicates.

*Azospirillum halopraeferens (NR_044859) was used as outgroup.

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116


Pseudomonas sp. AF-54 ( ) and published sequences.


*Bootstrap values greater than 50 were given and were based on 1000 replicates

Chapter 3 Results

117


Enterobacter cloacae AF-31 ( ) and published sequences


*Bootstrap values greater than 50 were given and were based on 1000 replicates

Chapter 3 Results

118

Figure 03-10 Phylogenetic tree based on 16S rRNA sequences of sunflower

associated Citrobacter freundii AF-56 ( ) and published sequences


*Bootstrap values greater than 50 were given and were based on 1000 replicates.

*Escherichia coli Nov. (U019111) was used as out group

Chapter 3 Results

119

3.3.2 Sequence and Phylogenetic Analysis of NifH Gene

Four out of 14 top nitrogenase activity showing isolates gave positive amplification for

nifH (360 bp) gene (Figure 3.11). But only three isolates gave significant percent homology

after sequencing. The nifH sequences of Azospirillum brasilense strain AF-22 revealed 99

% sequence similarity with already reported nifH gene of Azospirillum brasilense strain

GR59. While the other two PGPR strains Enterobacter cloacae AF-31 and Citrobacter

freundii AF-56 showed 99 % and 98 % similarity to the uncultured bacterial clones CO43

and SIS-5 respectively (Table 3.11)

Phylogenetic analysis of nifH gene sequences showed that Azospirillum brasilense AF-22

clustered with A. brasilense (X51500) and Enterobacter cloacae AF-31 and Citrobacter

freundii AF-56 branched with the nifH gene sequence of Brevundimonas sp. strain TN37,

whereas nifH sequence of Azospirillum doebereinerae (FJ799358) was used as outgroup

in the phylogenetic tree (Figure 3-12).

Chapter 3 Results

120

Figure 3-11 Agarose gel photograph showing amplified nifH gene from sunflower

associated endo/rhizospheric bacterial strains (representative)

M = 1 kb ladder, 2 = Bacillus safensis AF-9, 3= Azospirillum brasilense AF-22, 4 =

Enterobacter cloacae AF-31, 5 = Citrobacter freundii AF-56, 6 = negative control (blank).

Table 3-11 Sequence identity and closest BLASTn hits based on NifH gene of

nitrogen fixing and phosphate solubilizing PGPR strains form endo/rhizosphere of

sunflower

Strain Closest GenBank match based on nifH

sequence

(%

identity)

Gene: NifH

Azospirillum brasilense

AF-22

Azospirillum brasilense strain

GR59

99

Enterobacter cloacae AF-

31

Uncultured bacterium clone

CO43

99

Citrobacter freundii

AF-56

Uncultured bacterium clone

SIS4-5

98

Chapter 3 Results

121

Figure 3-12 Phylogenetic tree based on nifH sequences of sunflower associated

bacterial strains (●) compared with related published sequences

* Neighbor joining method was adopted.

* Bootstrap values greater than 50 are given and were based on 1000 replicates.

Chapter 3 Results

122

PLANT INOCULATION STUDIES

3.4.1 Pouch Experiment 1: Evaluation of the Effect of Selected Nitrogen Fixing

Bacterial Strains on Sunflower Growth

In growth pouch experiment, comparisons were made among 10 bacterial strains,

Pseudomonas trivialis Fs-9 (inoculated as positive control) and two non-inoculated control

treatments (with a full strength Hoagland and negative control). Generally inoculated

bacterial isolates exerted a significant influence on sunflower growth characteristics with

varied efficacy (Table 3.12).

Among the bacterial strains applied, Azospirillum brasilense AF-22 (T4) was the most

efficient PGPR strain by producing maximum root dry weight, shoot length, shoot fresh

and dry weight (0.263 g, 19.1 cm, 2.27 g and 1.83 g respectively). Root dry weight, shoot

length and shoot dry weight were statistically similar to the un-inoculated positive control

(full strength Hoagland, T13). Maximum root length (11.9 g) and root fresh weight (0.687

g) were produced by Pseudomonas sp. AF-54 (T7) that makes about 121 % and 93.94 %

increase over control treatment (T13). The minimum root dry weight was recorded in case

of Citrobacter braakii AF-21 (T3, 0.14 g) inoculation. Overall, the effect of bacterial

inoculation was more pronounced on shoot than root.

Chapter 3 Results

123


sunflower in pouch experiment under controlled conditions

Treatment

Root

length

(cm)

Shoot length

(cm)

Root fresh

weight (g)

Root dry

weight (g)

Shoot

fresh

weight (g)

Shoot dry

weight (g)

AF-6 7.80 ef 16.47 def 0.3233 fgh 0.1633 bcd 1.179 cd 1.6867 c

AF-9 10.33 c 17.57 bcde 0.4433 d 0.1900 bcd 2.227 b 0.5467 de

AF-21 7.13 f 14.93 fg 0.3033 ghi 0.1433 cde 1.163 cd 1.7100 c

AF-22 10.83 bc 19.13 ab 0.5567 c 0.2633 ab 2.277 b 1.8267 ab

AF-31 9.77 cd 18.47 abc 0.6633 b 0.2067 bcd 2.245 b 1.7200 c

AF-48 8.67 de 15.87 efg 0.2700 hi 0.1267 de 1.137 cd 0.4300 fg

AF-54 11.90 ab 17.20 cde 0.6867 b 0.2533 ab 2.263 b 1.8232 ab

AF-56 10.80 bc 18.11 bcd 0.5833 c 0.2233 bc 2.255 b 1.7633 bc

AF-95 6.80 fg 14.27 g 0.3800 def 0.1633 bcd 1.188 c 0.6267 d

AF-146 8.40 e 17.13 cde 0.3700 efg 0.1367 cde 1.181 cd 0.5967 de

FS-9 9.87 cd 16.63 def 0.4067 de 0.1667 bcd 1.197 c 0.5100 ef

Full Hoag. 12.53 a 20.17 a 0.9233 a 0.3433 a 3.017 a 1.8900 a

Control 5.83 g 12.33 h 0.2433 i 0.0533 e 1.109 d 0.3767 g

LSD 5% 1.2073 1.792 0.0677 0.0900 0.074 0.1143

SEC 0.5873 0.872 0.0329 0.0438 0.036 0.0556

LSD=Least Significant Difference, SEC=Standard Error for Comparison.

Values (mean of 3 replicates) in same column sharing same letter do not differ significantly

(P≤ 0.05) according to Fisher’s LSD

AF-6= Pseudomonas thivervalensis, AF-9= Bacillus safensis, AF-21= Citrobacter braakii,

AF-22= Azospirillum brasilense, AF-31= Enterobacter cloacae, AF-48= Bacillus cereus,

AF-54= Pseudomonas sp., AF-56= Citrobacter freundii, AF-95= Pseudomonas

brassicacearum, AF-146= Lysinibacillus sp., FS-9 (positive control). Full Hoag= Full

strength Hoagland

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124

3.4.2 Pouch Experiment 2: Evaluation of the Effect of Selected Phosphate

Solubilizing Bacterial Strains on Sunflower Growth

Statistical analysis of pouch experiment regarding phosphate solubilization showed that

inoculated P-solubilizers exerted a significant influence on all growth parameters of

sunflower plant in general with varied efficiency over negative control (Table 3.13). The

maximum inoculation response was recorded in the plants inoculated with Pseudomonas

sp. AF-54 (T7) that produced maximum root length (17.4 cm), root fresh weight (0.851 g)

and root dry weight (0.297 g) followed by and Enterobacter cloacae AF-31. While, the

minimum increase in root length was exhibited by the inoculation of Arthrobacter sp. AF-

163 (T6, 9.3 cm).

In case of shoot length, Azospirillum brasilense AF-22 (T4) displayed the highest shoots

length (22.2 cm) followed by AF-31 (21. 63 cm) after soluble P supplemented treatment

(T12, 23.47 cm), while Lysinibacillus sp. AF-146 (T10) produced statistically greater shoot

fresh (2.18 g) and maximum shoot dry weights (1.5 g) was produced by Enterobacter

cloacae AF-31 (T5).

Chapter 3 Results

125


growth of sunflower in pouch experiment under controlled conditions

Treatments Root length

(cm)

Shoot length

(cm)

Root fresh

weight (g)

Root dry

weight (g)

Shoot fresh

weight (g)

Shoot dry

weight (g)

AF-6 12.73 ef 19.17 def 0.720 de 0.23 abcd 1.225 fg 0.503 ef

AF-9 11.57 fg 17.57 fg 0.527 hi 0.143 cd 1.187 fg 0.373 g

AF-21 10.13 gh 21.40 abcd 0.583 gh 0.263 abc 1.354 de 0.890 c

AF-22 15.97 bc 22.20 ab 0.783 bcd 0.287 ab 1.266 ef 0.737 d

AF-31 16.83 abc 21.63 abc 0.807 bc 0.287 ab 2.049 c 1.493 b

AF-163 9.300 h 18.73 ef 0.470 i 0.130 cd 1.297 ef 0.493 ef

AF-54 17.40 ab 20.60 bcde 0.851 ab 0.297 ab 2.079 bc 1.503 b

AF-56 10.90 gh 19.90 cde 0.793 bcd 0.277 abc 1.174 fg 0.737 d

AF-95 13.87 de 18.83 ef 0.690 ef 0.21 abcd 1.473 d 0.603 e

AF-146 16.87 abc 20.33 bcde 0.637 fg 0.200 bcd 2.186 ab 1.443 b

FS-11 14.00 de 16.20 g 0.770 cd 0.133 cd 1.197 fg 0.447 fg

Full Hoag. 18.77 a 23.47 a 0.913 a 0.337 a 2.291 a 1.703 a

Control 6.567 i 10.03 h 0.230 j 0.047 e 1.120 g 0.213 h

LSD 5% 1.7296 2.2978 0.0791 0.1366 0.1228 0.1138

SEC 0.8414 1.1178 0.0385 0.0664 0.0597 0.0554

LSD=Least Significant Difference, SEC=Standard Error for Comparison.


(P≤ 0.05) according to Fisher’s LSD

AF-6= Pseudomonas thivervalensis, AF-9= Bacillus safensis, AF-21= Citrobacter braakii,

AF-22= Azospirillum brasilense, AF-31= Enterobacter cloacae, AF-163= Arthrobacter

sp., AF-54= Pseudomonas sp., AF-56= Citrobacter freundii, AF-95= Pseudomonas

brassicacearum, AF-146= Lysinibacillus sp., FS-11 (positive control). Full Hoag= Full

strength Hoagland

Chapter 3 Results

126

3.4.3 Pot experiment 1: Evaluation of the Effect of Selected Nitrogen Fixing

Bacterial Strains on Sunflower Growth

All inoculated strains increased root length as compared to negative control. Among the

inoculated treatments, highest root length (16.9 and 16.33 cm) was recorded in plants

inoculated with Azospirillum brasilense AF-22 and Pseudomonas sp. strain AF-54

respectively. Significantly (P ≤ 0.05) higher shoot length (26.3, 26.0 cm) was produced in

plants inoculated with AF-22 (Azospirillum brasilense) and AF-31 (Enterobacter cloacae).

Inoculation with Enterobacter cloacae AF-31 resulted in a maximum increase in root fresh

weight (3.2 g) among the inoculated treatments, while the maximum root dry weight (0.503

g) and shoot fresh weight (11.9 g) were in strain Citrobacter freundii AF-56 (T8), followed

by Pseudomonas sp. (0.41 g) AF-54 and Azospirillum brasilense AF-22 (0.406 g).

Maximum shoot dry weight (3.23 g) was exhibited by Azospirillum brasilense AF-22 (T4),

while shoot dry weight was recorded minimum responsive to inoculation of Pseudomonas

brassicacearum AF-95 (Table 3.14). The highest N-uptake by plants (0.91 %) was

recorded in plants treated with Azospirillum brasilense AF-22 followed by Enterobacter

cloacae AF-31 and Citrobacter freundii AF-56 among the inoculated treatments (Figure

3.13).

Chapter 3 Results

127


sunflower in pot experiment under controlled conditions

Treatment

Root length

(cm)

Shoot length

(cm)

Root fresh

weight (g)

Root dry

weight (g)

Shoot fresh

weight (g)

Shoot dry

weight (g)

AF-6 13.27 def 21.83 def 1.933 ef 0.347 cde 11.90 ab 2.267 ef

AF-9 12.87 efg 20.96 ef 2.183 de 0.273 fg 8.983 de 2.000 g

AF-21 12.26 fg 20.87 ef 1.667 fg 0.330 def 8.363 ef 2.133 fg

AF-22 16.96 ab 26.36 ab 2.646 c 0.406 c 10.23 cd 3.233 ab

AF-31 15.57 bc 26.03 ab 3.223 b 0.383 cd 11.70 abc 3.133 b

AF-48 12.26 fg 22.97 cde 2.38 cd 0.346 cde 10.82 bc 2.733 cd

AF-54 14.60 cde 23.10 cde 2.383 cd 0.410 c 10.24 cd 2.633 d

AF-56 16.33 abc 24.76 bc 2.633 c 0.503 b 12.10 ab 2.867 c

AF-95 11.77 fg 20.06 f 1.547 g 0.223 gh 7.030 f 1.600 h

AF-146 10.96 g 19.33 f 1.483 g 0.310 ef 8.240 ef 1.467 h

FS-9 15.13 bcd 24.36 bcd 2.213 de 0.350 cde 11.54 abc 2.400 e

½ Urea 13.06 ef 23.76 bcd 2.540 c 0.323 def 11.34 bc 2.733 cd

Full urea 17.60 a 27.43 a 3.720 a 0.570 a 13.03 a 3.366 a

Control 7.166 h 13.50 g 0.790 h 0.173 h 6.160 g 0.933 i

LSD 5% 2.0055 2.608 0.315 0.0646 1.5334 0.1879

SEC 0.9791 1.2735 0.1541 0.0315 0.7486 0.0917

LSD= Least Significant Difference, SEC= Standard Error for Comparison. Values (mean of 3 replicates) in same column sharing same letter do not differ significantly (P≤

0.05) according to Fisher’s LSD AF-6= P. thivervalensis, AF-9= B. safensis, AF-21= Citrobacter braakii, AF-22= Azospirillum brasilense, AF-31= Enterobacter cloacae, AF-48= B. cereus, AF-54 = Pseudomonas sp., AF-56=

Citrobacter freundii, AF-95= P. brassicacearum, AF-146= Lysinibacillus sp., FS-9= positive control

Chapter 3 Results

128

Figure 3-13 Effect of inoculation with different bacterial strains on plant N-uptake

Values are the mean of three replicates

Bars represent standard deviation

Chapter 3 Results

129

Figure 3-14 Population dynamics of inoculated PGPR strains in rhizosphere of

sunflower at different time intervals after sowing.

Value are average of 3 replicates. Each. Bar represents standard deviation of the replicates.

Chapter 3 Results

130

3.4.4 Pot experiment 2: Evaluation of the Effect of Selected Phosphate Solubilizing

Strains on Sunflower Growth

Ten potential P solubilizing bacterial strains were tested to evaluate their effect on

sunflower growth. Control treatments were kept same as in pot experiment 1 and P replaced

N in these controls. The comparison of treatment means (Table 3.15) showed that

inoculation of Pseudomonas sp. AF-54 resulted in a maximum root length (20.7 cm), shoot

length (33.6 cm) and shoot fresh weight (21.7 g). Enterobacter cloacae strain AF-31

produced 32.9 cm shoot length which is statically at par with shoot length recorded in

Pseudomonas sp. AF-54. The maximum root fresh weight (3.08 g) was produced in

response to Citrobacter freundii AF-56 inoculation. All the strains significantly (P ≤ 0.05)

enhanced root dry weights over negative control and maximum root dry weight (0.75 g)

was produced by the inoculation of both E. cloacae strain AF-31 and Arthrobacter sp. AF-

163 (T6). A. brasilense AF-22 was found to be responsible for maximum shoot dry weight

(4.61 g) production followed by C. freundii AF-56 (Table 3.15). The plants inoculated with

Pseudomonas sp. AF-54 resulted in maximum P-uptake by sunflower plants followed by

C. freundii AF-56 (Figure 3.15).

Chapter 3 Results

131


growth of sunflower pot experiment under controlled conditions

Treatment

Root

length(cm)

Shoot length

(cm)

Root fresh

weight (g)

Root dry

weight (g)

Shoot fresh

weight (g)

Shoot dry

weight (g)

AF-6 16.967 cd 27.833 cdef 2.4117 cd 0.60 def 19.967 bcd 3.5133 cde

AF-9 15.800 de 26.233 ef 1.7767 e 0.506 fg 17.300 d 3.3300 de

AF-21 15.600 de 26.900 def 2.2733 d 0.576 efg 21.167 abc 3.1467 ef

AF-22 20.067 b 31.233 abcd 2.4277 cd 0.740 abc 19.700 bcd 4.6133 b

AF-31 19.033 bc 32.933 ab 2.3637 d 0.753 ab 21.367 ab 3.4233 cde

AF-163 18.700 bc 32.000 abc 2.7867 bc 0.750 ab 19.767 bcd 3.6333 cd

AF-54 20.700 ab 33.600 ab 2.8433 b 0.713 bc 21.767 ab 3.7433 c

AF-56 19.167 bc 31.767 abc 3.080 b 0.700 bcd 19.900 bcd 4.3233 b

AF-95 12.567 f 31.033 abcd 1.6233 e 0.490 g 17.133 d 2.1900 h

AF-146 13.867 ef 24.100 f 1.4063 e 0.673 bcde 18.033 d 2.6167 g

FS-11 17.067 cd 29.333 bcde 2.3233 d 0.726 bc 18.867 bcd 3.3200 de

½ SSP 17.200 cd 30.833 abcd 2.3133 d 0.637 cde 18.300 cd 2.8000 fg

Full SSP 23.200 a 35.033 a 3.5973 a 0.833 a 23.633 a 5.3633 a

Control 8.0767 g 15.033 g 0.8567 f 0.280 h 8.100 e 1.1200 i

LSD 5% 2.6205 4.4102 0.3942 0.1034 3.1037 0.4047

SEC 1.2793 2.1530 0.1924 0.0505 1.5152 0.1976

LSD=Least Significant Difference, SEC=Standard Error for Comparison. Values (mean of 3

replicates) in same column sharing same letter do not differ significantly (P≤ 0.05) according to

Fisher’s LSD, SSP= Single super phosphate

AF-6= P. thivervalensis, AF-9= B. safensis, AF-21= Citrobacter braakii, AF-22= Azospirillum

brasilense, AF-31= Enterobacter cloacae, AF-163= Arthrobacter sp., AF-54 = Pseudomonas sp.,

AF-56= Citrobacter freundii, AF-95= P. brassicacearum, AF-146= Lysinibacillus sp. FS-11=

positive control

Chapter 3 Results

132

Figure 3-15 Effect of inoculation with different bacterial strains on plant P-uptake


Bars represent standard deviation

Chapter 3 Results

133

Figure 03-16 Population dynamics of inoculated bacterial strains in rhizosphere of

Sunflower at different time intervals after sowing.

Value are average of 3 replicates. Bar represents standard deviation of the replicates.

Chapter 3 Results

134

Bacterial Population in Sunflower Rhizosphere

In both pot experiment all the potential PGPR established successful colonization with

sunflower rhizosphere up to 45 days of inoculation. Bacterial strains showed differential

colonization potential with sunflower roots. Overall a trend to decline with time interval

was recorded. Azospirillum brasilense AF22, Enterobacter cloacae AF-31, Citrobacter

freundii AF-56 and Pseudomonas sp. AF-54 showed maximum stable association in terms

of CFU g-1. While Citrobacter braakii AF-21, Bacillus safensis AF-9 and Arthrobacter sp.

AF-163 showed a gradual decrease in population over time (Figure 3.14; 3.16).

3.4.5 Field Experiments

All the isolates used in pouch and pot experiments were further evaluated for their PGP

potential in field conditions. Monthly weather data (mean) for sunflower growing season

at both sites showed greater rainfall (mm) and humidity (%) levels at Rawalakot, while

greater temperature at Faisalabad (Table 3.16). Soil of experimental site Chota gala,

Rawalakot was found to be loam with high organic matter percentage (1.72 %), while the

soil of NIBGE, Faisalabad was sandy loam with organic matter 0.67 %. Pre-sowing soil

analysis for physico-chemical properties and bacterial population of both sites is presented

in Table 3.17.

3.4.5.1 Response of Inoculation on Plant Height (cm)

Plant height showed significant response to inoculation to all inoculated treatments except

the treatment with P. brassicacearum AF-95 inoculation. (T9) (Table 3.18). Among the

inoculated treatments, the highest plant height (96.15 cm) was recorded in plants treated

with Citrobacter freundii AF-56 (T8), followed by Azospirillum brasilense AF-22 (T4,

90.30 cm), B. safensis AF-2, AF-5, and B. cereus AF-6 produced statistically same plant

height. With regard to the experimental locations, plant height was significantly higher at

Rawalakot (L1) than that recorded at Faisalabad (L2). Interaction between experimental

locations and treatments revealed that the application of full dose of recommended NP

fertilizes produced maximum plant height (117 cm) at Chota gala, Rawalakot (L1×T15)

(Table 3.19).

Chapter 3 Results

135

Table 3-16 Mean monthly weather data for growing season of sunflower at NIBGE,

Faisalabad and Chota gala Rawalakot.

Month Maximum

Temperature

(oC)

Minimum

Temperature

(oC)

Average

Temperature

(oC)

Rainfall

(mm)

Relative

Humidity

(%)

Chota gala, Rawalakot

June 34.3 18 26.1 59.1 69.5

July 31.5 16.1 23.8 526.1 78.5

August 27.4 17.2 22.3 169.9 87

September 26.2 13.9 20.05 25.9 77.5

October 23.5 11.7 17.25 78.9 67.5

NIBGE, Faisalabad

February 22.1 9.9 16 18.2 64.1

March 27.5 14 20.8 14 53.5

April 33.5 19.1 26.3 22.9 41.7

May 40.1 24.8 32.4 9.1 31.4

June 40.7 27 33.8 9.6 33.6

Table 3-17 Physico-chemical analysis and bacterial population of soil from

experimental sites

Parameter Rawalakot NIBGE

Soil bulk density (mg m-3) 1.22 1.39

Particle density (mg m-3) 2.55 2.66

Porosity (%) 51.2 59.7

Soil pH 7.1 7.9

Soil texture Loam Sandy loam

Organic matter (%) 1.72 0.67

Total N (g kg-1) 0.74 0.58

Available P (mg kg-1) 6.6 4.3

Bacterial population (g-1 soil) 7× 106 6 × 106

Chapter 3 Results

136

Among the inoculated treatments, Citrobacter freundii AF-56 (T8) produced maximum

plant height (111.83 cm) which is statically at par with the full dose of NP fertilizer at

location 1 (Chota gala), followed by Azospirillum brasilense AF-22 (98.17 cm), and

Enterobacter cloacae AF-31 (T5), B. cereus (T6), Pseudomonas sp. AF-54 (T7) plus half

dose of NP fertilizer were statistically at par with each other at location 1 and produced

plant height 94.33, 94.17 and 94.67 cm, respectively (Table 3.19).

3.4.5.2 Response of Inoculation on Root Length (cm)

Root length was generally increased in response to all inoculated treatments over un-

inoculated without fertilizer treatments except inoculation with P. brassicacearum AF-95

(T9). (Table 3.18). The application of full dose of recommended NP fertilizers (T15)

resulted in statistically (P ≤ 0.05) longest roots (37.683 cm), while among the inoculated

treatments, maximum root length was recorded in Pseudomonas sp. AF-54 (T7) and

Citrobacter freundii AF-56 (T8) inoculated treatments with half dose of NP fertilizers as

33.4 cm and 32.9 cm respectively. In case of experimental locations, statically maximum

root length (31.32 cm) was recorded in L2 (NIBGE, Faisalabad) (Table 3.18). Interaction

studies between experimental locations and treatments (Table 3.19) showed that the plants

treated with full dose of recommended NP fertilizes produced maximum root length (40.87

cm) at NIBGE, Faisalabad (L2×T15). Among the inoculated treatments, Pseudomonas sp.

AF-54 (L2×T7) resulted in maximum plant height (36.467 cm) at L2 followed by

Azospirillum brasilense AF-22 (T4, 34.4 cm), at the same site, which is statically at par

with the application of full dose of NP fertilizers at Chota gala, Rawalakot (L1×T15) (Table

3.19).

3.4.5.3 Response of Inoculation on Plant Fresh Weight (g)

Bacterial inoculation with half dose of NP fertilizers significantly (P≤0.05) increased plant

fresh weigh (Table 3.18).as compared to un-inoculated plants with half dose of

recommended NP fertilizer (T14) and un-inoculated without fertilizer (T16), except P.

thivervalensis AF-6, Citrobacter braakii AF-21 which produced plant fresh weight

statically similar to the half dose of NP fertilizer application (T14). Among the inoculated

treatments, statistically highest plant fresh weight was recorded in plants inoculated with

Azospirillum brasilense AF-22 and Citrobacter freundii AF-56 with half dose of

recommended fertilizer (391.34 and 386.19 g respectively) statically similar, followed by

Chapter 3 Results

137

Enterobacter cloacae AF-31 (32.842 g) (Table 3.18). Minimum response to inoculation

and fertilizer application was recorded in the plants treated with P. brassicacearum AF-95

(T9). When experimental locations were compared, significantly greater plant fresh weight

(319.93 g) was recorded in experiment conducted at location 2 (NIBGE, Faisalabad) (Table

3.18). When the interaction of inoculated treatments with locations was studied, maximum

fresh weight (395.28 g) was recorded in Citrobacter braakii AF-56 inoculation with half

dose of recommended fertilizer in the experiment conducted at Rawalakot (L1×T8)

followed by AF-22 (389.73 g) at location 1 (L1×T4) which is statically at par with the same

Table 3-18 Comparison of treatment means and location means of sunflower

inoculated with bacterial isolates under field conditions.

Continued…

Treatments Plant

height (cm)

Root length

(cm)

Plant fresh

weight (g)

Plant dry

weight (g)

Root

fresh

weight

(g)

Root dry

weight (g)

AF-6 78.95 defg 26.525 ghi 272.52 fg 35.893 hij 34.818 f 11.285 h

AF-9 84.86 cd 30.617 bcde 337.79 cd 41.585 def 41.133 cd 15.157 de

AF-21 77.32 efg 26.267 ghi 264.64 fg 35.473 ij 40.080 de 9.957 i

AF-22 90.30 bc 32.842 bc 391.34 ab 47.180 bc 44.227 bc 17.742 bc

AF-31 85.87 cd 31.525 bcd 357.03 bc 43.615 cde 47.067 b 16.622 c

AF-48 86.12 cd 30.792 bcde 343.04 cd 43.320 de 41.238 cd 15.368 d

AF-54 84.51 cde 33.400 b 364.72 bc 50.408 ab 46.471 b 18.083 b

AF-56 96.15 ab 32.967 b 386.19 ab 45.260 cd 46.971 b 18.215 b

AF-95 71.52 gh 24.333 ij 219.11 hi 28.490 k 29.027 gh 9.028 ij

AF-146 83.20 cde 27.700 efgh 291.26 ef 38.830 fghi 37.253 ef 12.933 fg

AF-163 79.91 def 27.092 fghi 285.69 ef 37.362 ghij 36.795 ef 12.250 gh

Chapter 3 Results

138

L1= Chota gala, Rawalakot, L2= NIBGE, Faisalabad, LSD=Least Significant Difference.


(P≤ 0.05) according to Fisher’s LSD.

AF-6= P. thivervalensis, AF-9= B. safensis, AF-21= Citrobacter braakii, AF-22=

Azospirillum brasilense, AF-31= Enterobacter cloacae, AF-48= B. cereus, AF-54 =

Pseudomonas sp., AF-56= Citrobacter freundii, AF-95= P. brassicacearum, AF-146=

Lysinibacillus sp. AF-163= Arthrobacter sp., FS-9=positive control, FS-11= positive

control * Half dose of recommended NP fertilizer was added in all inoculated treatments.

Treatments Plant height

(cm)

Root length

(cm)

Plant fresh

weight (g)

Plant dry

weight (g)

Root fresh

weight (g)

Root dry

weight (g)

FS-9 82.87 cde 29.242 defg 298.79 ef 39.445 fgh 37.063 ef 13.775 f

FS-11 83.63 cde 29.750 cdef 317.65 de 40.313 efg 39.530 de 14.067 ef

½ NP

fertilizer

74.48 fg 25.633 hi 248.84 gh 33.705 j 30.998 g 9.455 i

Full NP

fertilizer

102.51 a 37.683 a 413.62 a 53.722 a 51.493 a 19.657 a

Negative

control

66.06 h 21.983 j 194.26 i 23.285 l 26.283 h 7.850 j

LSD 5% 7.4652 3.1967 34.919 3.7387 3.3664 1.2264

Effect of locations

L1 90.448 a 27.225 b 303.38 b 31.058 b 43.105 a 15.217 a

L2 75.581 b 31.319 a 319.93 a 48.678 a 35.700 b 12.463 b

LSD 5% 2.6393 1.1302 12.346 1.3218 1.1902 0.4336

Chapter 3 Results

139

Table 3-19 Sunflower growth parameters as affected by interaction between

bacterial treatments and locations under field conditions

Treatments

Plant height

(cm)

Root length

(cm)

Plant fresh

weight (g)

Plant dry

weight (g)

Root fresh

weight (g)

Root dry

weight (g)

L1×T1 84.67 cdefghi 24.000 klmn 256.87 mnop 44.637 efgh 31.733 lmn 11.647 lmno

L1×T2 93.00 bcd 29.333 efghij 328.32 defghij 49.860 cde 37.023 hijk 16.403 efg

L1×T3 83.17 defghij 23.833 klmn 248.92 nopq 44.097 fgh 45.213 cdef 11.283 mno

L1×T4 98.17 b 31.33 cdefgh 389.73 abc 57.477 b 34.810 ijklm 19.907 bc

L1×T5 94.33 bc 30.23 cdefgh 345.53 cdefgh 52.147 cd 45.627 cde 18.277 cd

L1×T6 94.17 bc 29.333 efghij 333.10 defghi 52.123 cd 37.353 hijk 16.627 defg

L1×T7 94.67 bc 30.33 cdefgh 371.00 abcde 54.420 bc 41.015 efgh 19.413 c

L1×T8 111.83 a 31.833 cdefg 395.28 ab 63.067 a 38.998 ghi 21.330 ab

L1×T9 73.33 jklmn 21.833 mn 202.95 qr 34.647 klmn 26.673 op 10.157 op

L1×T10 86.83 cdefgh 25.700 ijklm 277.33 klmno 47.277 defg 33.820 jklm 13.743 ijk

L1×T11 86.00 cdefgh 24.833 jklm 270.12 lmno 45.790 efgh 32.643 klmn 13.107 jkl

L1×T12 90.83 bcdef 27.333 ghijkl 287.33 ijklmn 48.007 defg 30.630 mno 15.107 fghi

L1×T13 91.83 bcde 27.833 fghijk 309.50 fghijkl 48.893 def 35.683 ijkl 15.540 efgh

L1×T14 78.67 ghijklm 23.167 lmn 240.50 nopq 42.743 ghi 28.495 nop 10.573 no

L1×T15 117.00 a 34.500 bc 417.05 a 65.560 a 47.063 cd 22.240 a

L1× T16 68.67 mn 20.167 n 180.52 r 28.103 opq 24.425 p 8.123 q

L2× T1 73.23 jklmn 29.050 efghij 288.17 ijklmn 27.150 pqr 37.903 hij 10.923 mno

L2× T2 76.72 hijklm 31.900 cdef 347.27

bcdefgh

33.310 lmno 45.243 cdef 13.910 hij

Continued…

Chapter 3 Results

140

Treatments

Plant height

(cm)

Root length

(cm)

Plant fresh

weight (g)

Plant dry

weight (g)

Root fresh

weight (g)

Root dry

weight (g)

L2× T3 71.47 klmn 28.700 efghij 280.36 jklmno 26.850 pqr 34.947 ijklm 8.630 pq

L2× T4 82.43 efghij 34.350 bc 392.95 abc 36.883 jkl 53.643 a 15.577 efgh

L2× T5 77.40 hijklm 32.817 bcde 368.54 abcde 35.083 klmn 48.507 bc 14.967 ghi

L2× T6 78.07 ghijklm 32.250 bcdef 352.99 bcdefg 34.517 klmn 45.123 cdef 14.110 hij

L2× T7 74.35 ijklm 36.467 ab 358.43 bcdef 36.100 klm 51.927 ab 16.753 def

L2× T8 80.47 fghijk 34.100 bcd 377.09 abcd 37.750 ijk 54.943 a 15.100 fghi

L2× T9 69.70 lmn 26.833 hijkl 235.28 opq 22.333 rs 31.380 lmno 7.900 q

L2× T10 79.57 ghijkl 29.700 defghi 305.20 ghijklm 30.383 nop 40.687 fgh 12.123 klmn

L2× T11 73.82 jklmn 29.350 efghij 301.26 hijklm 28.933 opq 40.947 efgh 11.393 lmno

L2× T12 74.90 ijklm 31.15 cdefgh 310.24 fghijkl 30.88 nop 43.497 defg 12.443 jklm

L2× T13 75.42 ijklm 31.667 cdefg 325.79 efghijk 31.73 lmnop 43.377 defg 12.593 jklm

L2× T14 88.02 bcdefg 28.100 fghijk 257.18 mnop 24.667 qr 33.500 jklm 8.337 q

L2× T15 70.30 klmn 40.867 a 410.18 a 41.883 hij 55.923 a 17.073 de

L2× T16 63.45 n 23.800 klmn 207.99 pqr 18.467 s 28.140 nop 7.577 q

LSD 5% 10.557 4.5208 49.383 5.2873 4.7608 1.7344

L1= Chota gala, Rawalakot, L2= NIBGE, Faisalabad, LSD=Least Significant Difference.



T1= P. thivervalensis AF-6, T2= B. safensis AF-9, T3= Citrobacter braakii AF-21, T4=

Azospirillum brasilense AF-22, T5= Enterobacter cloacae AF-31, T6= B. cereus AF-48,

T7= Pseudomonas sp. AF-54, T8= Citrobacter freundii AF-56, T9= P. brassicacearum

AF-95, T10= Lysinibacillus sp. AF-146, T11= Arthrobacter sp. AF-163, T12= FS-9

(positive control), T13= Fs-11 (positive control), T14= Half dose of recommended

fertilizer, T15=Full dose of recommended fertilizer, T16= Negative control.

* Half dose of recommended NP fertilizer was added in all the inoculated treatments.

Chapter 3 Results

141

bacterial inoculation with half dose of recommended fertilizer at the other location

(L2×T4). While minimum fresh weight (180.52 g) was recorded in an interaction of un-

inoculated without fertilizer treatment at Chota gala, Rawalakot (L1×T16, Table 3.19).

3.4.5.4 Response of Inoculation on Plant Dry Weight (g)

Data regarding plant dry weight shows a significant inoculation response over the control

treatments (Table 3.18) with varied efficiency. The most responsive inoculation treatment

was Pseudomonas sp. AF-54 (50.4 g) followed by Azospirillum brasilense AF-22 (47.2 g).

P. brassicacearum AF-95 showed non-significant response (28.5 g). As for as locations

are concerned, inoculation on plant dry weight was most effective in the experiment

conducted at NIBGE, Faisalabad (Table 3.18). Relationship between experimental sites

and treatments calculated by two-way ANOVA (Table 3.19) showed that plants inoculated

with Citrobacter freundii AF-56 with half NP at Rawalakot (L1×T8) produced

significantly maximum plant dry weight (63.067 g) followed by the inoculation with

Azospirillum brasilense AF-22 (57.47 g) at same location (L1×T2). Least responsive

inoculated treatment was again P. brassicacearum AF-95 (22.3 g) at NIBGE (L2×T9)

which is even lesser than the negative control at the other location (L1×T15). Minimum

plant dry weight was produced by un-inoculated with no fertilizer treatment at location 2

(L2×T16).

3.4.5.5 Response of Inoculation on root Fresh Weight (g)

Inoculation with Enterobacter cloacae AF-31, Citrobacter freundii AF-56, and

Pseudomonas sp. AF-54 along with half NP fertilizer found to be most responsive by

producing maximum root fresh weight (47.1, 46.97, 46.4 g respectively) which were

statically at par with each other (Table 3.18) in contrast with un-inoculated treatments

having zero, half dose of NP fertilizer. Inoculations with P. brassicacearum AF-95 along

with half dose of NP fertilizer again did not show significant response. Experiment

conducted at Chota gala, Rawalakot (L1) was significantly responsive (43.105 g) with

regard to root fresh weight when compared with NIBGE, Faisalabad (L2) (Table 3.18).

Two-way analysis of variance on interaction between experimental sites and treatments

(Table 3.19) revealed that, plants treated with Citrobacter freundii AF-56 and Azospirillum

brasilense AF-22 displayed significantly (P ≤ 0.05) higher root fresh weight (54.9, 53.6 g

Chapter 3 Results

142

respectively) at NIBGE, Faisalabad (L2×T8, L2×T4), statically at par with full fertilizer

treatment at the same site (L2×T15). Minimum root dry weight was recorded in case of

negative control at Chota gala, Rawalakot (L1×T16) (Table 3.19).

3.4.5.6 Response of Inoculation on Root Dry Weight (g)

The most inoculation-responsive treatments were Citrobacter cloacae AF-56 (18.2 g) and

Pseudomonas sp. AF-54 (18 g) in case of root dry weight (Table 3.18) resulted in about 92

% increased weight as compared to un-inoculated control with half NP fertilizer treatment.

When effect of location was compared, experiment conducted at Rawalakot (L1) was

significantly greater responsive to the bacterial inoculation (15.2 g) (Table 3.18).

Relationship between experimental sites and treatments (two-way ANOVA) showed that

application of full dose of NP fertilizer produced significantly (P ≤ 0.05) maximum root

dry weight (22.2 g) at Rawalakot (L1×T15). Among inoculated treatments, Citrobacter

cloacae AF-56 resulted in maximum root dry weight (21.3 g) followed by Azospirillum

brasilense AF-22 (19.9 g) and Pseudomonas sp. AF-54 (19.14 g) at the same site and all

these bacterial strains produced statically greater root dry weigh than the plants

supplemented with full dose of recommended NP fertilizer at Faisalabad (L2×T15).

Overall L1 was the most effectively responsive to inoculation regarding root dry weight

(Table 3.19).

Chapter 3 Results

143

Figure 3-17 Principal component analysis showing the relationship between

different experimental locations and bacterial treatments

L1= Chota gala, Rawalakot, L2= NIBGE, Faisalabad.

T1= Pseudomonas thivervalensis AF-6, T2= Bacillus safensis AF-9, T3= Citrobacter

braakii AF-21, T4= Azospirillum brasilense AF-22, T5= Enterobacter cloacae AF-31,

T6= Bacillus cereus AF-48, T7= Pseudomonas sp. AF-54, T8= Citrobacter freundii AF-

56, T9= Pseudomonas brassicacearum AF-95, T10= Lysinibacillus sp. AF-146, T11=

Arthrobacter sp. AF-163, T12= FS-9 (positive control), T13= Fs-11 (positive control),

T14= Half dose of recommended fertilizer, T5= Full dose of recommended fertilizer, T16=

Negative control.

* Half dose of recommended NP fertilizer was added in all the inoculated treatments.

Chapter 3 Results

144

3.4.5.7 Effect of Bacterial Inoculation on Sunflower Agronomic and Physiological

Parameters under Field Conditions

3.4.5.7.1 Plant Height at Maturity (cm)

The plant height at maturity was maximum 191.3 cm in treatment supplemented with full

NP fertilizer. However, seven bacterial strains i.e., AF-9, AF-22, AF-31, AF-54, AF-56,

AF-95 and AF-163 were able to produce plant height (179.3 cm to 188.1 cm) statically at

par to that recorded from full NP fertilizer treatment. The most efficient inoculation

response (188.1 cm) was observed in case of Azospirillum brasilense AF-22 inoculation

and Enterobacter cloacae AF-31 (186.8 cm) was second to that. The plant height recorded

in these inoculated treatments was significantly (P≤0.05) higher than that recorded for half

NP fertilizer treatment (Table 3.20).

3.4.5.7.2 Plant Fresh Weight at Maturity (kg)

Plant fresh weight showed variable response towards different inoculated bacterial strains.

Among the inoculated treatments, statically improved fresh weight was recorded in case of

Arthrobacter sp. AF-163 (1.84 kg). In this case, the relative increase on half dose of

recommended NP fertilizer is about 24 % followed by the application of Citrobacter

freundii AF-56 with 18 % greater fresh weight as compared to half dose of recommended

NP fertilizer. Citrobacter braakii AF-21 and Lysinibacillus sp. AF-146 with half dose of

NP did not show significant effect on respectively pant fresh weigh and produced (1.37,

1.4 kg) statically lesser than the un-inoculated control treatment with half dose of NP

fertilizer (data provided in Table 3.20).

3.4.5.7.3 Plant Dry Weight at Maturity (kg)

Inoculation effect on plant dry weight was more or less similar to that on plant fresh weight

(Table 3.20). Inoculation of Arthrobacter sp. AF-163 with half dose of recommended NP

resulted in the maximum plant dry weight (193 g) which makes about 29 % increase on

un-inoculated half NP control, followed by Enterobacter cloacae AF-31 plus half NP

which produced 25 % increase over half fertilizer alone. Inoculation with Citrobacter

braakii AF-21 with half dose of NP showed inoculation response similar to plat fresh

Chapter 3 Results

145

weight by producing statically lesser dry weight (130.12 g) than the un-inoculated control

treatment with half dose of NP fertilizer (149.9 g) (Table 3.20).

3.4.5.7.4 Head Diameter (cm), Fresh and Dry Weight (g).

Statistically maximum head fresh weight (613.7 g) was recorded in the plants treated with

Pseudomonas sp. AF-54 plus half dose of fertilizer after maximum weight gained by full

dose of NP fertilizer (645.6 g). While in case of head dry weight Citrobacter freundii AF-

56 was the most efficient bacterial strain (92.8 g) produced 44.8 % greater head dry weight

as compared to un-inoculated treatment with half dose of NP (64.07 g). Application of

Citrobacter braakii AF-21 and B. cereus AF-48 did not show significant effect on head

dry weight (Table 3.20). Among the inoculated strains, B. safensis AF-9 along with half

NP produced statically maximum head diameter (24.1 cm) followed by Citrobacter

freundii AF-56 (23.8 cm). Inoculation with Citrobacter braakii AF-21, B. cereus AF-48,

Lysinibacillus sp. AF-146, and Arthrobacter sp. AF-163 did not show significant response

to inoculation (Table 3.20).

Table 3-20 Effect of bacterial inoculation on sunflower agronomic and physiological

parameters under field conditions

Treatments

Plant

height (cm)

Plant fresh

weight (kg)

Plant Dry

weight (g)

Head fresh

weight (g)

Head dry

weight (g)

Head

diameter

(cm)

AF-6 175.78 bcde 1.5133 gh 168.88

cdef

539.45 cdef 81.167 def 22.113 bcdef

AF-9 179.3 abcde 1.5567 fg 152.52 fghi 490.11 fghi 77.267 ef 24.110 ab

AF-21 173.67 de 1.3767 i 130.12 jk 437.78 jk 56.467 i 20.333 fg

AF-22 188.11 ab 1.7567 c 175.87 bcde

542.22 cde 84.233 cde 22.667 abcde

AF-31 186.89 abc 1.6667 de 188.38 ab 574.89 bc 88.833 abc 22.777 abcde

AF-48 177.78 bcde 1.6333 e 136.74 ij 475.00 ghij 53.867 i 20.443 fg

AF-54 182.7 abcde 1.7133 cd 181.92 bcd 613.78 ab 86.533 bcd 23.523 abcd

AF-56 185.11 abcd 1.7667 c 186.44 bc 551.67 cd 92.800 ab 23.890 abc

AF-95 183.4 abcde 1.4667 h 163.80 defg

515.67 defg 79.600 def 21.553 defg

AF-146 171.89 e 1.4000 i 144.32 hij 455.56 ijk 69.200 gh 21.333 efg

Continued…

Chapter 3 Results

146

Treatments

Plant

height (cm) Plant fresh

weight (kg) Plant Dry

weight (g) Head fresh

weight (g) Head dry

weight (g) Head

diameter

(cm)

AF-163 181.4 abcde 1.8400 b 193.57 ab 512.2 defgh 78.833 ef 20.667 efg

FS-9 174.78 cde 1.4933 gh 160.92 efgh

500.56 efghi 75.133 fg 21.667 def

FS-11 180.8 abcde 1.6133 ef 157.70 efgh

463.00 hij 65.933 h 21.110 efg

½ NP

fertilizer

176.67 bcde 1.4833 h 149.93 ghi 470.00 ghij 64.067 h 21.887 cdef

Full NP

fertilizer

191.33 a 2.0100 a 206.28 a 645.56 a 95.067 a 24.643 a

Negative

control

152.00 f 1.2833 j 114.40 k 406.11 k 43.333 j 19.447 g

LSD 5% 13.074 0.0651 18.281 50.157 7.5553 2.1484

SEC 6.4019 0.0319 8.9513 SEC 6.4019 0.0319


Azospirillum brasilense, AF-31= Enterobacter cloacae, AF-48= B. cereus, AF-54=


Lysinibacillus sp. AF-163= Arthrobacter sp., FS-9= positive control, FS-11= positive

control


LSD=Least Significant Difference and SEC= standard error for comparison.


(P≤ 0.05) according to Fisher’s LSD. * Half dose of recommended fertilizer was added in all inoculated treatments.

* Representative Data from L2 (Faisalabad)

3.4.5.7.5 Number of Achenes per Head

Bacterial inoculation exerted positive effect on number of achenes per head as compared

with un-inoculated control treatments with no fertilizer and half dose of NP fertilizer (Table

3.21). Inoculation with Azospirillum brasilense AF-22 along with 50 % of recommended

NP fertilizer was found to be the most effective bacterial strain to produce maximum

number of achenes (2237) per head that is about 41 % increase over application of half

dose of fertilizer alone. However, non-significant inoculation response was recorded in

case of Citrobacter braakii AF-21 and B. cereus with 50 % of recommended fertilizer

application to sunflower plant (Table 3.21).

Chapter 3 Results

147

3.4.5.7.6 1000 Achene Weight (g)

Statistical analysis of field data conformed the positive effective of bacterial inoculation

on sunflower achene weight (Table 3.21) as compared to the control treatment with half

dose of recommended fertilizer except inoculation with Citrobacter freundii AF-2 and

Lysinibacillus sp. AF-146. The most efficient bacterial strain was Citrobacter freundii AF-

56 along with half dose of recommended NP fertilizer produced statically maximum weight

(56.6 g) followed by A. brasilense AF-22 plus half NP.

3.4.5.7.7 Achene Yield (kg ha-1)

Most of the inoculated plant growth promoting rhizobacterial strains in combination with

half dose of recommended NP fertilizers, found efficient in improving sunflower achene

yield in comparisons with un-inoculated control treatments (half NP and no fertilizer). The

highest achene yield (1717.5 kg ha-1) was recorded in case of full dose of recommended

NP fertilizer application alone. The most effective bacterial strain was Azospirillum

brasilense strain AF-22 plus half NP (1477.1 kg ha-1), followed by Citrobacter freundii

AF-56 (1406.4 kg ha-1) in combination with half dose of NP. Among the inoculated

treatments, least achene yield (772.2 kg ha-1) was produced by the inoculation of

Citrobacter braakii AF-21 with half dose of fertilizer (Figure 3.19).

3.4.5.7.8 Harvest Index (HI)

Statistically improved harvest index was recorded in most of the inoculated plants as

compared to the un-inoculated treatments (Figure 3.21) Maximum harvest index was

recorded in the plants supplemented with full fertilizer dose (19574) while among the

inoculated plates, maximum inoculation effect was recorded in case of Azospirillum

brasilense AF-22 (18298) followed by Enterobacter cloacae AF-31 inoculated plants

(18108). Minimum HI was recorded in P. brassicacearum (11487).

Chapter 3 Results

148

3.4.5.8 Achene Analysis

3.4.5.8.1 Achene Protein Contents (%)

Bacterial inoculations resulted in achene protein content statistically greater than or equal

to full dose of recommended NP fertilizer (Table 3.22). Azospirillum brasilense AF-22

inoculated seed analysis showed the highest protein content (23.5 %) which is makes about

11% increase over the plants supplemented with full dose of recommended fertilizer (21

%). Among the inoculated treatments, B. cereus AF-48 produced least protein content (19.7

%) which was statically greater than half dose of recommended fertilizer (18.9 %).

3.4.5.8.2 Achene Nitrogen and Phosphorus Contents (g kg-1)

Results showed positive effect of bacterial inoculation on achene nitrogen and phosphorus

contents. Data presented in Figure 3.18 (A) shows that the highest achene N contents (55.5

g kg-1) was recorded in the plants supplemented with full NP fertilizer. Among the

inoculated treatments, maximum achene N contents (41.3 g kg-1) were found in

Azospirillum brasilense AF-22 inoculation in combination with half NP fertilizer, that

makes about 120 % increase over the plants supplemented with half dose of fertilizer alone.

It was followed by Citrobacter freundii AF-56 that resulted in an increase in achene N

content about 74 % over T14 while, T3, T9 and T11 did not show significant response to

inoculations regarding achene N contents.

Parallel data fashion was recorded in case of achene phosphorus (P) content (Figure 3.18,

B). The highest P contents were recorded in achenes of the plants treated with hundred

percent recommended NP fertilizers (7.32 g kg-1) followed by Pseudomonas sp. AF-54

with half dose of recommended fertilizer (6.12 g kg-1) produced 37.84% increase over the

treatment where half dose of NP was applied alone (4.44 g kg-1). As in case of achene N

content, Arthrobacter sp. AF-163 again did not show positive inoculation regarding achene

P content (3.07 g kg-1 7).

3.4.5.8.3 Achene Oil Contents (%)

Achene oil contents were significantly affected by different bacterial treatments (Table

3.22). Most of the inoculated treatments produced sunflower achene oil contents at par with

Chapter 3 Results

149

full fertilizer treatment while, Enterobacter cloacae AF-31 along with half NP fertilizer

produced 12 % increase in oil content (38.1 %) over full fertilizer treatment (34 %)

followed by Bacillus cereus AF-48 (36.5 %). Application of P. brassicacearum AF-95, on

the other hand, did not work well and produced statistically (P≤0.05) minimum achene oil

contents (31 %) among the inoculated treatments. While, over all minimum oil contents

were observed in negative control treatment un-inoculated unfertilized treatment.

3.4.5.8.4 Palmitic Acid C16:0 (%)

Analysis of variance revealed that different bacterial inoculations affected palmitic acid

content of sunflower achene with variable efficiency (data presented in table 3.22) as

compared to negative control. Statistically (P≤0.05) maximum palmitic acid (5.7 %) was

recorded in P. thivervalensis AF-6 inoculation along with half dose of recommended NP

fertilizer that is even greater than the un-inoculated plants supplemented with full dose of

recommended fertilizer. It was then followed by Enterobacter cloacae AF-31 (5.2 %) with

half dose of NP fertilizer which is statistically at par with plants grown with 100 %

recommended fertilizer. In this case, least effective bacterial strain was B. safensis AF-9

(3.1 %) by producing palmitic acid lesser than the plants treated with half dose of fertilizer

alone (Table 3.22).

3.4.5.8.5 Oleic Acid C18:1 (%)

A data trend parallel to palmitic acid was observed regarding oleic acid (Table 3.22). The

highest oleic acid was produced by in inoculation of Pseudomonas sp. AF-54 (T7, 15.8 %)

which is statically similar to AF-63, FS-11 and the plants treated with full dose of

recommended fertilizer. Among the inoculated treatments, T9 was the least effective on

oleic acid contents of sunflower achene (13.9 %) even lesser than the treatments

supplemented with half dose of fertilizer alone (14.0 %.).

3.4.5.8.6 Linoleic Acid C18:2 (%)

A pattern similar to oleic acid was recorded in case of linoleic acid (Table 3.22).

Inoculation with Citrobacter freundii AF-56 resulted in maximum linoleic acid (73.4 %)

which is statically at par with B. safensis AF-9, Citrobacter braakii AF-21, Azospirillum

Chapter 3 Results

150

brasilense AF-22, Lysinibacillus sp. AF-146, Arthrobacter sp. AF-163 along with half dose

of recommended NP fertilizer and un-inoculated treatment with full dose of fertilizer

treatment.

3.4.5.8.7 Stearic Acid C18:0 (%)

Azospirillum brasilense AF-22 inoculation resulted in highest achene stearic acid (6.3 %)

along with half dose of recommended NP fertilizer, followed by the plants supplemented

with full fertilizer without inoculation, the treatments inoculated with P. thivervalensis AF-

6, Enterobacter cloacae AF-31, Pseudomonas sp. AF-54 and Citrobacter freundii. All of

these treatments are statically at par (Table 3.22).

Effect of bacterial inoculation on photosynthetic performance of leaf under field

condition

The analysis of photosynthetic performance of leaf under field condition showed that leaf

internal temperature and photosynthetically active radiations were not influenced by

bacterial inoculation or fertilizer application as compared to un-inoculated control

treatments (Table 3.23). While net photosynthesis rate was recorded in the plants

inoculated with Bacillus cereus AF-48 (15.72 µmol-2s-1) followed by Azospirillum

brasilense AF-22, which produced statically similar to that of AF-54. Inoculation with

Pseudomonas sp. AF-54 resulted in maximum transpiration rate (4.05 µmol-2s-1) which was

greater than the plants treated with hundred percent recommended fertilizer. Statically

highest stomatal conductance was recorded in the plants treated with Pseudomonas sp. AF-

54 (4.98 µmol-2s1).

Chapter 3 Results

151

Table 3-21 Effect of bacterial inoculation on sunflower yield parameters under field

conditions

Treatments No. of

Achenes/head

Achene

weight/head

(g)

1000

Achene

weight (g)

Achene

weight/16

heads (kg)

Plant weight

at harvest

(kg)

AF-6 1891.9 de 80.44 de 49.6 defg 1.1433 ef 6.6700 hij

AF-9 1710.0 fg 85.33 cd 51.0 cdef 0.8500 hi 6.9533 gh

AF-21 1412.8 i 77.56 def 48.0 fg 0.7300 j 6.3767 jk

AF-22 2237.9 b 100.44 a 56.0 abc 1.3967 b 8.0767 bc

AF-31 1948.3 d 97.22 ab 53.6 abcde 1.1933 de 8.2833 b

AF-48 1913.7 de 91.89 bc 52.6 abcdef 1.2267 d 7.8267 cd

AF-54 2035.6 cd 102.00 a 55.0 abcd 1.2700 cd 7.5800 de

AF-56 2094.8 c 98.00 ab 56.6 ab 1.3267 bc 7.9300 cd

AF-95 1469.4 hi 81.89 de 50.6 cdefg 0.7467 j 6.8633 ghi

AF-146 1674.1 fg 70.55 f 45.3 gh 0.9133 gh 5.8700 l

AF-163 1781.6 ef 91.11 bc 52.0 bcdef 1.1267 ef 7.4100 ef

FS-9 1740.2 f 76.34 ef 47.6 fgh 0.9933 g 6.2233 k

FS-11 1567.2 gh 90.44 bc 51.6 bcdef 1.0833 f 7.1433 fg

½ NP

fertilizer

1582.8 gh 79.45 de 49.3 efg 0.7900 ij 6.5600 ijk

Full NP

fertilizer

2492.2 a 105.44 a 58.0 a 1.6233 a 8.7700 a

Control 1214.8 j 54.78 g 1214.8 j 0.6367 k 5.7067 l

LSD

5%

144.21 8.3893 5.3872 0.0805 0.3522

SEC 70.612 4.1078 2.6379 0.0394 0.1725


Azospirillum brasilense, AF-31= Enterobacter cloacae, AF-48= B. cereus, AF-54=


Lysinibacillus sp. AF-163= Arthrobacter sp., FS-9= positive control, FS-11= positive

control.

LSD= Least Significant Difference and SEC= standard error for comparison.


(P≤ 0.05) according to Fisher’s LSD. * Half dose of recommended fertilizer was added in all inoculated treatments. *Representative Data from L2 (Faisalabad)

Chapter 3 Results

152

Table 3-22 Sunflower oil and fatty acid contents as affected by bacterial inoculation

AF-6= P. thivervalensis, AF-9= B. safensis, AF-21= Citrobacter braakii, AF-22= Azospirillum brasilense, AF-31= Enterobacter cloacae, AF-48= B. cereus, AF-54 = Pseudomonas sp., AF-56=

Citrobacter freundii, AF-95= P. brassicacearum, AF-146= Lysinibacillus sp. AF-163= Arthrobacter sp., FS-9=positive control, FS-11= positive control. LSD=Least Significant Difference and SEC= standard error for comparison. Values (mean of 3 replicates) in same column sharing same letter do not differ significantly (P≤ 0.05) according to Fisher’s LSD.

*Representative Data from L2 (Faisalabad) *Half dose of recommended fertilizer was added in all inoculated treatments.

Treatment

Achene oil

contents

(%)

Protein

content (%)

Palmitic

acid C16:0

(%)

Stearic

acid

C18:0

(%)

Oleic acid

C18:1(%)

Linoleic

acid C18:2

(%)

AF-6 33.4 abc 21.0 abc 5.7 a 5.7 ab 15.4 ab 69.0 ab

AF-9 35.5 abc 22.2 ab 3.1 de 5.3 abc 14.5 abc 73.0 a

AF-21 32.7 bc 19.9 bc 3.9 cd 4.5 bc 14.1 bc 72.5 a

AF-22 35.3 abc 23.5 a 4.6 abc 6.3 a 15. abc 71.9 ab

AF-31 38.1 a 20.6 abc 5.2 ab 5.6 abc 14.8 abc 72.7 a

AF-48 36.5 ab 21.6 abc 4.3 bc 5.5 abc 15.2 abc 71.5 ab

AF-54 36.0 abc 19.7 bc 4.8 abc 5.3 abc 15.8 a 71.7 ab

AF-56 34.0 abc 22.2 ab 4.6 abc 5.0 abc 15.3 abc 73.4 a

AF-95 31.0 cd 20.4 bc 3.9 cd 4.4 bc 13.8 c 71.7 ab

AF-146 33.3 abc 22.0 ab 4.0 cd 4.6 bc 15.3 abc 73.0 a

AF-163 35.9 abc 20.3 bc 4.0 cd 4.8 bc 15.7 a 72.8 a

FS-11 32.9 bc 21.9 ab 4.5 bc 4.9 abc 15.8 a 68.5 ab

½ NP

fertilizer

31.1 cd 18.9 c 4.5 bc 4.2 c 14.0 bc 70.8 ab

Full NP

fertilizer

34.0 abc 21.0 abc 4.8 abc 5.2 abc 16.0 a 73.3 a

Control 26.6 d 13.9 d 2.7 e 2.5 d 9.9 d 62.7 c

LSD 5% 5.0875 2.9898 1.1740 1.4528 1.5547 4.3951

SEC 2.4911 1.4640 0.5749 0.7114 0.7612 2.1521

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153

Table 3-23 Analysis of photosynthetic performance in sunflower leaves under field

conditions

Treatments

LIT °C PAR

µmol-2

s-1

NPR

µmol-2

s-1

TR

µmolm-2

s-

1

SC

µmol-2

s-1

AF-6 43.80 a 1510.6 a 13.01 abc 2.54 cde 3.69 abc

AF-9 43.60 a 1642.2 a 14.52 ab 3.02 abcde 3.99 abc

AF-21 43.20 a 1436.8 a 11.79 abc 3.79 abc 4.78 ab

AF-22 44.50 a 1741.0 a 15.25 a 2.62 bcde 3.42 bc

AF-31 42.23 a 1652.9 a 15.72 a 2.88 abcde 4.02 abc

AF-48 43.80 a 1605.7 a 13.49 abc 3.19 abcde 4.02 abc

AF-54 43.40 a 1662.2 a 13.02 abc 4.05 a 4.98 a

AF-56 44.10 a 1203.0 a 12.50 abc 3.17 abcde 4.12 abc

AF-95 44.40 a 1724.1 a 11.97 abc 2.34 de 3.76 abc

AF-146 44.20 a 1701.5 a 14.52 ab 2.98 abcde 4.11 abc

AF-163 43.67 a 1760.9 a 12.87 abc 3.61 abcd 4.34 abc

FS-11 44.40 a 1207.7 a 12.26 abc 3.17 abcde 4.19 abc

½ NP fertilizer 43.63 a 1159.1 a 9.65 bc 2.92 abcde 3.71 abc

Full NP fertilizer 43.30 a 1100.4 a 14.74 ab 3.99 ab 4.81 ab

Negative control 45.33 a 1300.1 a 13.57 abc 2.99 abcde 4.23 abc

FS-9 44.63 a 1106.3 a 8.99 c 2.18 e 3.14 c

LSD 5% NS NS 5.1355 1.4172 1.3990

SEC 1.6640 348.27 2.5146 0.6939 0.6850


Azospirillum brasilense, AF-31= Enterobacter cloacae, AF-48= B. cereus, AF-54 =


Lysinibacillus sp. AF-163= Arthrobacter sp., FS-9= Positive control, FS-11= Positive

control.

LT: Leaf internal temperature, PAR: Photosynthetically active radiations, NPR: Net

Photosynthesis rate, TR: Transpiration rate, SC: Stomatal conductance

LSD=Least Significant Difference and SEC= standard error for comparison.



*NS= Non significant

*Representative Data from L2 (Faisalabad)

* Half dose of recommended fertilizer was added in all inoculated treatments.

Chapter 3 Results

154

Figure 03-18 Effect of inoculation with different PGPR on sunflower achene

nitrogen (A) and phosphorus (B) contents

*Values are the mean of three replicates.

*Bars represent standard deviation.


Chapter 3 Results

155

Figure 03-19 Sunflower achene yield (kg ha-1) as affected different bacterial

inoculations.





(positive control), T13= Fs-11(positive control), T14= Half dose of recommended


*Half dose of recommended fertilizer was added in all the inoculated treatments.


Chapter 3 Results

156

Figure 03-20 Sunflower biomass yield (kg ha-1) as affected by different bacterial

inoculations.







*Half dose of recommended NP fertilizer was added in all the inoculated treatments.


Chapter 3 Results

157

Figure 3-21 Sunflower harvest index as affected by different bacterial inoculations.







*Half dose of recommended fertilizer was added in all the inoculated treatments.


Chapter 3 Results

158

Principal component analysis (PCA) was also carried out to summarize the relationship

among agronomic parameters and experimental locations. A similar data fashion regarding

different treatment was recorded at both sides. Maximum readings in every parameter was

recorded in the plants supplemented with full dose of recommended NP fertilizers (T15),

followed by Citrobacter freundii AF-56 (T8), Azospirillum brasilense AF-22 (T4),

Pseudomonas sp. AF-54 (T7) and Enterobacter cloacae AF-31 (T5) at both locations but

with varied efficiency. Principal component analysis showed that these potential strains

performed well at Rawalakot site as compared to the experiment conducted at Faisalabad.

All the treatments except T16 (un-inoculated control with zero NP fertilizer) at Rawalakot

site, were loaded positively to the PC1 showed positive correlation within the parameters

(Figure 3.17).

Chapter 3 Results

159

COLONIZATION STUDIES

3.5.1 Root Colonization Studies Using Confocal Laser Scanning Microscopy

Fluorescence of YFP-tagged bacterial cells were observed on roots inoculated with

different strains, confirmed their presence in close vicinity to root cells, while no

fluorescence was observed in un-inoculated plants.

Azospirillum brasilense AF-22 showed heavy colonization with root hair (Figure 3.22 a)

as well as on the whole surface of root cells (Figure 3.22 b, c). Extensive bacterial masses

were attached to the root cells forming macro-colonies (Figure 3.22 d) that showed the

close association of applied bacterial strain with host plant and is the clear evidence of

biofilm formation by inoculated strain on the root cell.

Pseudomonas sp. AF-54, yfp-labelled cells were found in significantly higher number

scattered all over the root surface (Figure 3.23 a, b, c). Confocal laser scanning microscopic

analysis revealed that bacteria are attached to the population attached to the root hair

forming macro-colonies and also in close vicinity (rhizosphere) (Figure 3.23 a). AF-54

formed aggregates onto the epidermal cells of root which were detected in the form of

strings and macro-colonies (Figure 3.23 b). This aggregation of sessile bacterial

community existence (shown in Figure 3.23 panel c and d), is the confirmation of

successful colonization of Pseudomonas sp. AF-54 and its biofilm forming ability as well.

In case of Citrobacter freundii AF-56, specific colonization could not be detected as

bacterial were observed in the form of strings and aggregates on root hair on root hair

(Figure 3.24 a) and all over the root surface (Figure 3.24 b, c d). Evidences of biofilm

formation are also clear in panel b and d (Figure 3.24).

Chapter 3 Results

160


labelled Azospirillum brasilense AF-22, grown in sand culture 21 days post

inoculation.

Panel a-d showing lesser to very high population of plant root associated bacteria with

characteristic N2-fixation, P-solubilization, IAA production and biocontrol activities.

B = Bacteria,

RH = Root hair,

MC = Macro colonies

Magnification=100X

Chapter 3 Results

161


labelled Pseudomonas sp. AF-54, grown in sand 21 days post inoculation.



B = Bacteria,

RH = Root hair,

MC = Macro-colonies

Magnification=100X

Chapter 3 Results

162


labelled Citrobacter freundii AF-56, grown in sand 21 days post inoculation.



B = Bacteria,

RH = Root hair,

MC = Macro-colonies

Magnification=100X

Chapter 3 Results

163

Figure 3-10 Confocal image of sunflower root inoculated with YFP-labelled

Enterobacter cloacae AF-31, grown in sand 21 days post inoculation.



B = Bacteria,

RH = Root hair

Magnification=100X

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164

Among four strains inoculated to sunflower roots Enterobacter cloacae AF-31 found to be

the most aggressive root colonizer. Massive aggregates of bacteria were spotted in the form

of bacterial strings and also micro and macro-colonies (Figure 3.25 a, b, c, d), that shows

the establishment of biofilm formation and strong bipartite relationship between

Enterobacter cloacae AF-31 and sunflower host.

Fluorescent Antibody Staining

Results of immunofluorescent marker employed in combination with confocal laser

scanning microscopy (CLSM) showed the specificity of Enterobacter cloacae AF-31 and

Citrobacter freundii AF-56 strain (pure culture) and their attachment on the roots of host

plant.

In case of pure cultures of Citrobacter sp. strains AF-56 cells stained with their respective

FA (Figure 3.26 a), when observed under CLSM, green fluorescence of bacterial cells

showed the specificity of the cells to the specific antibodies used. Fluorescent antibodies

of AF-56 (Citrobacter freundii) when applied to non-target cells (Enterobacter cloacae

AF-31) to check their cross reactivity, no fluorescence was detected (Figure 3.26 panel b)

which shows strain specificity of the FAs used. To further validate the results, a consortium

of different mixture of different strains (Citrobacter freundii AF-56, Pseudomonas sp.

AF-54 and an Enterobacter cloacae AF-31) was double stained with FA and ethidium

bromide stain. When this consortium was observed under CLSM, some of the stained cells

produced yellow colored fluorescence (positive result) of FAs which is due to the overlay

of red and green fluorescence while the non-target cells showed only the red fluorescence

(no signals of FAs, figure 3.26 panel c). Red color of nucleic acid staining became visible

Chapter 3 Results

165

with ethidium bromide and apple green fluorescence of FA appeared around the cell

showed the specific binding of the antigen determined.

FAs were used for root colonization of plants grown in both in sterile and natural

conditions. The primary antiserum raised against Citrobacter freundii AF-56 and

Enterobacter cloacae AF-31 were found effective as the cells were detected on the roots

inoculated with both strains both in sterile and natural soils. A dense bacterial population

is assembled in a strings tightly associated to the epidermal root cells along with bacterial

population aggregated in macro-colonies in sterilized soil of both AF-56 and AF-31 (Figure

3.27 panel b; Figure 3.28 respectively). While in case of roots grown in non-sterile soil,

red, green and yellow fluorescence was found, which indicated the presence of target

(Citrobacter freundii AF-56) and non-target cells (indigenous) in rhizosphere (Figure 3.27

panel c). Roots of control plants (non-inoculated control) showed no green fluorescence

(Figure 3.27 panel a), that ruled out the chances of false positive results. Data regarding

FA staining of AF-31 is not presented here.

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166

Figure 3-26 Confocal laser scanning micrographs of fluorescent antibody stained cells of Citrobacter sp. AF-56

FAs of AF-56+Citrobacter freundii strain AF-56 (positive control to check strain specificity) (a), FAs of AF-56+ Enterobacter cloacae

strain AF-31 (negative control to check cross-reactivity) (b), FAs of AF-56+ Citrobacter freundii strain AF-56+ Enterobacter cloacae

strain AF-31+Pseudomonas sp. AF-54+Ethidium bromide (c)

Key: FITC-labeled strain specific antibody = gives green fluorescence

Nucleic acid stain with ethidium bromide = gives red fluorescence

Overlay of red & green = gives yellow fluorescence

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167

Figure 3-27 Confocal scanning image of sunflower roots inoculated with Citrobacter freundii AF-56 stained with fluorescent

antibody staining coupled with CLSM.

Root of control plant (non-inoculated) treated with FAs of AF-56+C. freundii strain (a), C. freundii strain inoculated roots of sunflower

grown in sterile conditions treated with respective FAs (b), C. freundii strain inoculated roots of sunflower grown in natural soil treated

with FAs of AF-56+ C. freundii strain + ethidium bromide staining(c).

*Observations were made 21 days after inoculation.


Nucleic acid stain with ethidium bromide = gives red fluorescence

Overlay of red & green = gives yellow fluorescence

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168

Figure 3-28 Confocal scanning image of sunflower roots inoculated with

Enterobacter cloacae AF-31 stained with fluorescent antibody staining coupled with

CLSM.

Enterobacter cloacae strain inoculated roots of sunflower grown in sterile conditions

treated with respective FAs. Bacteria found in the form of strings and macro-colonies are

the evidence of successful detection of fluorescently labelled bacteria and also the

indication of biofilm formation.

*Observations were made 21 days after inoculation


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169

3.5.2 In situ Detection of Pseudomonas sp. AF-54 Colonizing Sunflower Roots using

FISH

Pseudomonas sp. AF-54 were documented in the rhizoplane of sunflower through

fluorescence in situ hybridization carried out using the oligonucleotide bacteria-specific

FLUOS-labeled EUB 338 and gamma Proteobacteria specific Cy3-labelled GAM42a

probes. When pure culture was observed under CLSM, results showed green fluorescence

in the cells, revealed the successful labeling of Pseudomonas sp. AF-54 (Figure 3.29 panel

a). AF-54 inoculated roots were when hybridized and observed under CLSM, results

exposed the successful colonization of inoculated strain with the root of sunflower grown

in both, sterile (Figure 3.29 panel b) and natural soils (Figure 3.29 panel c)

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170

Figure 3-29 Fluorescent in situ hybridization of Pseudomonas sp. AF-54

FLOUS-labeled EUB 338 pure culture of Pseudomonas sp. AF-54 (panel a), FLOUS-labeled EUB 338 root inoculated by Pseudomonas sp. AF-54

in sterile soil (panel b), FLOUS-labeled EUB 338 root inoculated by Pseudomonas sp. AF-54 in natural soil (panel c). White arrow heads in panel

D, E and F are also showing successful colonization of Pseudomonas sp. AF-54 on different parts of roots.

Bars represent 100 µm. Arrows show bacterial attachment to the roots in the form of strings and microcolonies

Chapter 3 Results

171

3.5.3 Colonization Study by Transmission Electron Microscopy and Immunogold

Labeling Technique

Polyclonal antisera were produced specific for the Azospirillum brasilense AF-22,

Enterobacter cloacae AF-31 and Citrobacter freundii AF-56. Inoculated plants were

grown for 3 weeks under greenhouse conditions.

Plastic embedded thin (transverse) sections of 3 weeks old inoculated sunflower root

tissues (stained with 1% toluidine blue) were examined by light microscopy to specify the

regions of bacterial localization (data not shown) These light microscopic observations

enabled the study of the organization of tissues in young sunflower roots.

Transmission electron micrographs (Figure 3.30 panel a, b, c) show that sunflower roots at

3 weeks after inoculation with AF-22, colonized intercellular spaces, but none have

penetrated the intra cellular spaces (panel b, c). Higher magnification shows dense bacterial

colonization in intercellular (panel d).

Figure 3.31 panel c shows rhizospheric bacterial colonization to sunflower roots inoculated

by Azospirillum brasilense AF-22. Dense bacterial population localized along with root

hair as well as with root cell wall. While panel a and b show their presence inside the root

cell, conforming the endophytic localization of Azospirillum brasilense AF-22. It is evident

from set of panels in figure 3.31 that these bacteria multiplied vigorously in endophytic

state (a, b) as compared to their counter parts (c).

The bacteria were immunogold labeled using a primary antibody raised against

Azospirillum brasilense AF-22, and a secondary goat anti-rabbit antibody conjugated to

15-nm gold particles (transmission electron micrographs of serial sections to panels a and

b, figure 3.28). Panel (a) shows endophytic mode of bacterial root colonization, while panel

b is the extension of the same area of thin section at higher magnification. Bacteria showing

internal immunogold labeling (the deep black dots), indicating the specificity of the target

bacteria (Figure 3.32).

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172

Figure 3-30 Transmission electron micrograph of a root cross-section from a

sunflower plant inoculated with Azospirillum brasilense AF-22.

Ultrastructural localization of Azospirillum brasilense AF-22 within the sunflower root

cortical zone providing its endophytic nature. Panel a indicating hardly and bacteria within

the cortical cells. Panel b, c and d (lower to higher magnifications) indicate that the bacteria

occupied mostly the inter cellular space only.

CW= cell wall, B= bacteria, RC= root cell, ICS= inter cellular spaces

*Roots were taken 21 days post inoculation

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173

Figure 3-31 Transmission electron micrograph of a root cross-section from a

sunflower plant inoculated with Azospirillum brasilense AF-22.

Ultrastructural localization of Azospirillum brasilense AF-22 both inter cellular (panel a

and b) as well as in the rhizospheric portion, close to the root hair tip (c)

CW= cell wall, B= bacteria, RC= root cell, ICS= inter cellular spaces. RS= rhizosphere

*Roots were taken 21 days post inoculation

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174

Figure 3-32 Transmission electron micrographs of immunogold labeled cells of

Azospirillum brasilense AF-22

Electron micrographs at higher magnifications show endophytic localization of

Azospirillum brasilense AF-22 (panel a) and antigen antisera specificity (panel a, b)

CW= cell wall, B= bacteria, IGL=immunogold labeled. Bold arrows show the labeling of

bacteria with IGL

*Roots were taken 21 days post inoculation.

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175

Figure 3-33 Transmission electron micrographs of a root cross-section from a sunflower plant inoculated with Citrobacter

freundii AF-56 and its endophytic colonization.

Panel a, b, c showing the characteristic inter cellular endophytic nature of AF-56 while panel d, e and f showing immunogold labelled

specific Citrobacter freundii as endophytes

CW= cell wall, B= bacteria, RC= root cell, ICS= inter cellular spaces. RS= rhizosphere, IGL= immunogold labeled *.Roots were taken 21 days post inoculation.

X 1K

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176

Figure 3-34 Transmission electron micrographs of a root from a sunflower plant

inoculated with Citrobacter freundii AF-56

Micrographs showing rhizospheric and endophytic colonization (panel A, B) and antigen

antisera specificity (panel c)

CW= cell wall, B= bacteria, RC= root cell, ICS= inter cellular spaces. RS= rhizosphere,

IGL= immunogold labeled.

*Roots were taken 21 days post inoculation.

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177

Figure 3-35 Transmission electron micrographs of a root cross-section from a

sunflower plant inoculated with Enterobacter cloacae AF-31.

Micrographs showing rhizospheric colonization (panel a, b, c) and antigen antisera

specificity (panel d)

CW= cell wall, B= bacteria, RC= root cell, RS= rhizosphere, IGL= immunogold labeled.

*Roots were taken 21 days after inoculation.

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178

Transmission electron micrographs (Figure 3.30 panel a, b, c) of sunflower roots inoculated

with Citrobacter freundii AF-56 showing successful colonization of target bacterial cell to

the host plant root. In figure 3.33, panel a divulges the over view of sunflower root showing

level of bacterial occupancy in the rhizosphere. Profuse population of Citrobacter cells

localized in intercellular spaces are shown in panel b, while panel c is the extension of

panel b at higher magnification. These results confirmed the endophytic nature of

Citrobacter freundii AF-56.

An immunogold labeling, followed by an electron microscopic detection of the colonized

root samples revealed the antigen antisera specificity of AF-56 (Figure 3.33, panel d, e)

and allowed the documentation of several intercellular spaces are almost completely filled

with bacteria (Figure 3.33 panel b and panel c). Single bacterial cells at higher

magnification (Figure 3.33 panel f) showing copious immunogold labeling (white arrow

heads), clearly revealing the specificity of localized bacterial strain to the antisera produced

against that bacteria. Figure 3.34 showing endophytic localization of AF-56 at different

magnifications (panel a, b) and immunogold labeling (panel c).

Electron microscopic studies of sunflower roots inoculated with Enterobacter cloacae AF-

31 revealed the successful colonization potential of the target bacteria to its host plant. The

micrographs in figure 3.35 panel a, b and showing the presence of bacteria in rhizosphere

and rhizoplane at different magnifications. While panel d reveals the antigen antisera

specificity of Enterobacter cloacae AF-31. These results clearly revealing the rhizospheric

nature of inoculated Enterobacter strain.

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179

3.5.4 Biofilm Formation

A time course study was conducted to investigate biofilm formation ability of 4 potential

bacterial strains i.e. Azospirillum brasilense AF-22, Enterobacter cloacae AF-31,

Pseudomonas sp. AF-54, and Citrobacter freundii AF-56 at 24, 48, 72 and 96 h. Confocal

laser scanning microscopic observations showed the initial event of bacterial (AF-22)

attachment in the first 48h of growth (Figure 3.36, panel b), and bacterial cells

subsequently assembled on the solid substratum. At about 72 h of growth, cells were

cemented in the form of clusters (Figure 3.36, panel c). After 96 h of incubation (Figure

3.36d), cell clusters were fully grown and more aggregated to form a well-defined biofilm.

Same pattern of biofilm formation found in all bacterial strains but Enterobacter cloacae

AF-31, when observed under confocal laser scanning microscope and quantified using

imageJ (Figure 3.40), found to be the most powerful biofilm forming bacterial strain at

72 and 96 h (Figure 3.39 panel c and d respectively) followed by Pseudomonas sp. AF-

54 showing extensive biofilm formation (Figure 3.37) On the other hand Azospirillum

brasilense AF-22 and Citrobacter freundii AF-56 produced comparatively lesser biofilm

on glass walls up to 96 h. (Figure 3.36 and 3.38).

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180

Figure 3-36 Time course study to detect biofilm formation activity Azospirillum sp.

AF-22

Biofilm formation on thin cover slip (22 mm2) by Azospirillum brasilense AF-22 grown in

LB broth. Observations were made after 24h of incubation (a), at 48h (b), at 72h (c) and at

96h (d) under confocal laser scanning microscope.

B= Bacteria

BF= Biofilm

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181

Figure 3-37 Time course study to detect biofilm formation activity Pseudomonas sp.

AF-54

Biofilm formation on thin cover slip (22 mm2) by Pseudomonas sp. AF-54 grown in LB

broth. Observations were made after 24h of incubation (a), at 48h (b), at 72h (c) and at 96h

(d) under confocal laser scanning microscope.

B= Bacteria

BF= Biofilm

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182

Figure 3-38 Time course study to detect biofilm formation activity Citrobacter

freundii AF-56

Biofilm formation on thin cover slip (22 mm2) by Citrobacter freundii AF-56 grown in LB



B= Bacteria

BF= Biofilm

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183

Figure 3-39 Time course study to detect biofilm formation activity Enterobacter

cloacae AF-31

Biofilm formation on thin cover slip (22 mm2 by Enterobacter cloacae AF-31grown in LB



B= Bacteria

BF= Biofilm

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184

Figure 3-40 Quantification of biofilm formed by selected PGPR strains

Biofilm formed by Azospirillum brasilense AF-22, Pseudomonas sp. AF-54, Enterobacter

cloacae AF-32 and Citrobacter freundii AF-56 at different time intervals was measure

through ImageJ software.

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Chapter 4

Discussion

World population growth in general and in Asia in particular is surpassing food supply.

The horizontal expansion of food growing lands is seized because of limited and non-

renewable sources. The prevailing agriculture expansion has led to co-saturation of many

ecosystems with extensive use of chemical fertilizers and pesticides which possess serious

threats to soil and environmental degradation (Tilman et al., 2001). This has led to a strong

interest in alternative strategies to improve crop yields and pathogen control without

compromising the environment. The plant growth promoting rhizobacteria (PGPR) fulfill

this criteria being potential tools for plant growth promotion, soil health, and non-toxic -

towards the ecosystem. This environment friendly process or strategy for farming system

has been suggested after quoting successful world class laboratory and field experiments

by various researchers around the world. The PGPR have proved their worth in plant

growth promotion and environment protection through conservation of natural resources

with decreased reliance on synthetic chemicals. Mechanisms behind PGPR action are

mainly fixation of atmospheric nitrogen, solubilization of inorganic and organic phosphate,

production of various plant growth - hormones, control of diseases by producing various

secondary metabolites, amelioration of abiotic stress, induction of systematic resistance

and many still unknown phenomenon (Morrone et al., 2009; Mehta et al., 2010).

Sunflower being high yielding crop has great potential for improving local edible oil

production (Hussain et al., 2010; Ehsanullah et al., 2011). Its oil is rich in vitamin E, low

saturated fat, contains monounsaturated (80 %) and polyunsaturated fats which make it one

of the healthiest oils for consumption that helps in lowering cardiovascular diseases, chance

Chapter 4 Discussion

186

of heart attack, an antioxidant which helps in neutralizing cancer-causing free radicals.

Unfortunately, there is a large gap between local oil production and annual oil import.

Pakistan meets only 34 % of edible oil requirement with local oil production of only 0.696

million tons out of more than 2.045 million tons required annually (Govt. of Pakistan,

2011-12).

Subdivision Dhirkot is completely unexplored area of Azad Jammu and Kashmir, Pakistan,

with reference to soil microfauna, particularly plant growth promoting rhizobacteria

(PGPR). This area is usually cultivated with fruit orchids especially apple, walnut, pear etc.

and partially maize and wheat, on small scale along with some leafy vegetables. Sunflower

had never been grown in this region and even cultivation history of other oil seed crops

hardly exist. However, the climate of Dhirkot area is quite suitable for growth of sunflower.

We targeted 16 argo-ecologically different sites located between (Altitude 790 to 1916),

high variation in soil temperature (19 to 29 °C), air temperatures (19.5 to 38.5°C), heat

index (16.5-48.4°C), humidity (37.6 to 61%), soil pH (6.34 to 7.51) and electric

conductivity (0.64- 1.34 dS m-1), organic matter (1.36 to 2.68 % ), available phosphorus

(6.72 to 14.72 mg kg-1), available potassium (43 to 113.68 mg kg-1), total nitrogen (0.09 to

0.272%) and total soil bacterial population (2×106 to 22×106) and with a wide range of soil

textural classes (loam, sandy loam, clay loam, silt loam etc.). Samples were collected from

undisturbed grasslands dominated by rough meadow grass (Poatrivialis L.)/ Bermuda

grass (Cynodondactylon L.) and orchard grass (Dactylis glomerata L.) at all sampling sites.

To obtain maximum sunflower-associated (rhizospheric and edospheric) bacterial diversity

and to hunt for ‘the best’ PGPR strains, sunflower plants were grown in the soils collected

from all above mentioned sites. Although it is very well known that plants affect the native


187

soil microbial populations and each of the plant species select specific microbial

populations contributing enough to their fitness, generating a very selective environment

and limited diversity (Berg and Smalla, 2009). But keeping in mind the variation in the soil

nutrients, atmospheric temperatures and altitude of the sampling sites, a high variation in

structure and function of the bacterial population in these areas was anticipated. It was also

hypothesized that sunflower-associate bacterial population in the hilly areas may be

different from already known sunflower-associate bacterial population growing in plains.

Rigorous host plant specificity of plant growth promoting bacteria have been well reported

in several studies (Berg and Smalla, 2009; Buchan et al., 2010). Out of 163 bacterial

isolates, 97 were obtained from root surface/ecto-rhizosphere while 66 were obtained from

root interior (putative endophytic). Morphologically they displayed a range of colony

colors, sizes, margins and cell shapes. Dominant population was motile and Gram negative.

Although, non-motile bacteria are reported to colonize roots of host plant (de Weert et al.,

2002) but bacterial motility has been documented as an obligatory feature of root

colonization (Sakai et al., 1996). Ambrosini et al., (2012) also documented the prevalence

of Gram negative bacteria among rhizospheric bacterial population. Diversity and

abundance of Gram negative bacteria might be due to the availability of specific amino

acids or organic acids along with particular signaling molecules in the rhizosphere.

Functional diversity of bacteria from sunflower rhizosphere

Out of 163 sunflower associated putative endo and rhizospheric bacterial isolates, 84

showed the ability to solubilize inorganic phosphate on Pikovskaya’s agar plates showing

a high percentage (51 %) of P-solubilizers in our study. A high percentage of P-solubilizers

have also been reported by others in-soil and plant rhizosphere (Raghu and Macrae, 2000,


188

Hanif et al., 2015). The potential of P-solubilization in different bacteria was variable i.e.

9.51 to 48.80 µg mL-1. This difference/variation may be attributed to the variation in the

organic acid (tartaric, malic, lactic, gluconic, and oxalic acids) production by these bacteria.

These organic acids are known to have variable influence on P-solubilization mechanism

(Patel et al., 2008). Among the detected organic acids, the most prevalent one was gluconic

acid which has been reported to play the prime role in solubilization of inorganic P (de

Werra et al., 2009). These low molecular weight organic acids result in the acidification of

the soil or media (Puente et al., 2004; Khan et al., 2014) and chelate cationic partners of

the P ions i.e., PO43- with their hydroxyl and carboxyl groups directly releasing P into

solution. A large number of scientists reported that solubilization of inorganic forms of P

is the combined effect of decrease in pH and production of organic acids (He et al., 2002;

Sahin et al., 2004; Richardson et al., 2009; Hanif et al., 2015). When the efficient phosphate

solubilizers are applied, they increase the availability of phosphorus from both applied and

native soil phosphorus. The ability of bacteria to dissolve the interlocked P have a

significant implication (Pradhan and Sukla, 2005; Chen et al., 2006; Lone et al., 2011;

Oteino et al., 2015).

The variation in P-solubilization by different bacterial strains under conditions was also

reported earlier (Hameeda et al., 2006; Gupta et al, 2007; Oliveira et al., 2009; Qian et al.,

2010; Islam et al., 2010; Bakhshandeh et al., 2014; Shahid et al., 2014; Majeed et al., 2015,

Hanif et al., 2015),

Nitrogen supply to the plant is mainly dependent on the microbial activity, even if it is

applied in the form of chemical fertilizers. The N transformation and cycling in soil and its

subsequent fate i.e., plant availability is highly dependent on microbial activity (Khan,


189

2005). In this study, from a total of 163 bacterial isolates, 14 isolates exhibited nitrogenase

activity through acetylene reduction assay (ARA) in the range of 28.68-137.84 nmoles mg-

1 protein h-1. The maximum nitrogenase activity (137.84 nmoles mg-1 protein h-1) was

exhibited by the isolate AF-22. While, the remaining isolates could be the efficient

scavengers of traces of reduced nitrogen or they may be dependent on nitrogen fixed and

released by other diazotrophs (Tripathi et al., 2002). Nitrogenase activity have also been

documented by a vast range of N2-fixing bacterial isolates in many studies (Islam et al.,

2016; Shen et al., 2016).

Phytohormone production by PGPR is considered to be one of the most important

mechanisms of plant growth promotion by rhizobacteria (Spaepen et al., 2007). These

hormones are the organic compounds which effect various physiological, biochemical and

morphological processes of plants in extremely low concentrations. They serve as chemical

messengers and are considered as the key players in plant growth and yield (Fuentes-

Ramírez and Caballero-Mellado, 2006). Several bacterial species have been well reported

to produce phytohormones (Tsavkelova et al., 2006; Majeed et al., 2015). In the present

study, 46 bacterial isolates were found to produce indole-3-acetic acid (IAA) of which 19

showed IAA production in the range of 1.13-24.6 µg mL-1. Indole acetic acid production

is a common phenomenon among PGPR (Islam et al., 2016), and is synthesized through

several pathways, three of which are commonly observed i.e., IAM (indole-3-acetamide),

IAN (indole-3-acetonitrile) and IPyA (indole-3-pyruvic acid) pathways (Fu and Wang,

2011; Niklas and Kutschera, 2012). Tryptophan (Trp), present in the root exudates, acts as

a precursor of IAA and affects IAA synthesis in the majority of auxin producing PGPR

(Merzaeva and Shirokikh, 2010). In the present study, Azospirillum brasilense AF-22


190

produced the highest IAA and among PGPR species, Azospirillum is one of the best studied

IAA producers (Saharan and Nehra, 2011, Naqqash et al., 2016). Bacteria belonging to

Azotobacter (Ahmad et al., 2008), Bacillus (Swain et al., 2007), Burkholderia (Hal da-

Alija, 2003), Enterobacter (Shoebitz et al., 2009), Pseudomonas (Hariprasad and

Niranjana, 2009) and Rhizobium (Ghosh et al., 2008) have been reported to produce a

considerable amount of IAA in vitro and in vivo (Chenet al., 2006, Nahas, 1996, Venieraki

et al., 2011).

Bacterial strains were tested for in vitro antagonistic activity against fungal phytopathogens

(Fusarium sp.) using dual-culture plate assay. Out of 163 bacterial isolates, 23 isolates

inhibited fungal growth on PDA plate showing biocontrol activity. Plant growth promoting

rhizobacteria control the phytopathogens by producing antifungal metabolites, or through

induction of systemic resistance (Haas and Defago, 2005; Medeiros et al., 2011) and by

modification of hormonal levels (van Loon, 2007), in the plant tissues. Hence, they can be

the key players in the sustainable agriculture (Bhardwaj et al., 2014) and are considered an

alternative to chemical pesticides (Zahir et al., 2004). Ali et al. (2014) also reported the

broad spectrum antifungal activity of Bacillus sp. RMB7 along with its plant growth

promoting potential.

Taxonomic diversity of bacteria from sunflower rhizosphere

The most authentic taxonomic marker for bacterial identification at genus and species level

is 16S rRNA gene. Therefore, the potential plant growth promoting bacterial isolates were

identified through 16S rRNA gene sequence analysis. The sequence analysis showed that

these PGPR belong to genera Azospirillum, Bacillus, Enterobacter, Citrobacter,

Pseudomonas, Serratia, Stenotrophomonas and Lysinibacillus Cellulosimicrobium,


191

Staphylococcus, Chryseobacterium which are commonly found in the rhizosphere of

various crops (Yasmin et al., 2013; Ali et al., 2014; Hanif et al., 2015; Naqqash et al.,

2016; Sharma et al., 2016; Henagamage et al., 2016). Similarly, many strains belonging

to these genera have been isolated from sunflower roots and studied for their plant growth

promoting characteristics (Ambrosini et al., 2012; Roberts et al., 2011, Shahid et al., 2015)

from plain areas. Many Bacillus spp. have also been isolated as endophytes in sunflower

(Forchetti et al., 2007).

Putative diazotrophs (showing ARA activity) were confirmed by nifH gene amplification.

Nitrogenase enzyme plays key role in nitrogen fixation whose various subunits are encoded

by nif gene i.e. nifD, nifH and nifK (Halbleib and Ludden, 2000). Due to the highly

conserved regions of gene sequence, nifH is used as an important marker gene for

diazotrophic studies. Moreover, a huge sequence data is available for comparative studies

(Wu et al., 2009). Out of nine strains, nifH gene was successfully amplified in three strains.

NifH gene of Azospirillum brasilense AF-22 showed similarity with Azospirillum

brasilense. This result is very much similar with recent finding of Naqqash et al. (2016),

who reported that phylogenetic analysis of the nifH gene of A. brasilense TN10, conformed

its clustering with the nifH gene from respective strain A. brasilense FR669137.

Metabolic diversity of bacteria from sunflower rhizosphere

Bacteria develop metabolic adaptations to inhabit special niches as individual plants

produce specific carbon sources (Berg and Smalla, 2009). A great diversity in carbon

source utilization pattern exists (metabolic potential) in a variety of bacteria from different

habitats (soil, rhizospheric and aquatic). Metabolically versatile bacterial strains are the

most successful competitor in plant microbe interaction (Wielbo et al., 2007). Biolog GN2


192

and GP2 microplate technique has been introduced to determine the metabolic potential of

microbial communities (Stefanowicz, 2006). To determine the ability of PGPR strains to

utilize different carbon sources, bacterial strains from Enterobacter, Pseudomonas,

Citrobacter and Azospirillum genera were tested on BIOLOG GN2 system (Line et al.,

2011). These bacterial strains metabolized variable number of substrates from microplates.

Diversity in utilizing the various carbon sources and chemicals indicates the competency

and adoptability of the introduced bacterial strains in diverse environments (Garland and

Mills, 1991). Pseudomonas sp. AF-54 utilized maximum (77 of 96) carbon sources which

supports the competency and adoptability of this strain in the rhizosphere of the host plants

over other microbes. Recently, Naqqash et al, (2016) and Hanif et al, (2015) reported

Azospirillum sp. TN10 and Bacillus subtilis strain KPS-11showing a very diverse

metabolic potential and resisting against 20 chemicals using same BIOLOG system.

Oksinska et al. (2011) also suggested that more substrates utilizing bacteria can lead to the

successful colonization. The strains were also resistant to many of the tested antibiotics.

Antibiogram reflects that the bacterial isolates showed complete resistance against many

antibiotics. The resistance to different antibiotics also reflects the ecological fitness of these

strains in a range of environments. Our results are in line with the previous studies

conducted by Imran et al, (2014). Kuykendall et al. (1988) reported that B. elkanii showed

a higher intrinsic resistance to several antibiotics and Chen et al. (2001) documented

different bacterial strains resistant to several antibiotics.

Colonization potential of bacteria in sunflower rhizosphere

Effective host colonization is the most crucial prerequisite for an agronomically important

bacterium-plant interaction and maintenance of their population in the rhizosphere to


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compete the indigenous microflora and fauna (Barea et al., 2005). A progressive and

consistent advantage of any plant beneficial bacterium is only materialized when it enters

in a close association with plant roots. Evaluation of roots colonization studies both under

in vitro and in vivo conditions is important. Subsequently, the microbe-host association

studies have become a core theme in microbial ecology, as their role for the macroscopic

hosts was increasingly recognized. In present study, four potential PGPR strains

(Azospirillum brasilense AF-22, Enterobacter cloacae AF-31, Pseudomonas sp. AF-54

and Citrobacter freundii AF-56) were studied for their colonization potential by using

various biomarkers coupled with different imaging techniques. Various biomarkers have

been established to study the localization and identification of the introduced bacteria in

the tissues of host plant (Bais et al., 2006) along with sophisticated and highly sensitive

microscopic techniques like TEM and CLSM (Yasmeen et al., 2012; Shahid et al., 2012;

Naqqash et al., 2016; Ali et al., 2016).

An attractive marker system for monitoring bacterial cells in the environment is the

fluorescent protein such as Yellow fluorescent protein (yfp) (Leveau and Lindow, 2002) in

non-destructive manner. In present study 4 yfp-labelled strains Azospirillum brasilense AF-

22, Enterobacter cloacae AF-31, Pseudomonas sp. AF-54 and Citrobacter freundii AF-56

were inoculated to sunflower. Strong fluorescent signals under CLCM observations

conformed resilient root colonization ability of all bacterial strains to their host plant and

authenticating their suitability as a bio-inoculant for the host plant. These results are

strongly supported by the previously reported results of yfp labeled Enterobacter sp. FS-

11 to sunflower roots (Shahid et al., 2012). To further document the colonization, the

inoculated roots were stained with a fluorescent stain, acridine orange. The staining clearly


194

showed the association of the inoculated bacteria with the roots. Root hair zones were

found to be highly populated with inoculated strains. Root hairs are reported to be sites

involved in eliciting chemotaxis and specific attachment (Fournier et al., 2008). Bacterial

cells were distributed all over the root zone in the form of micro and macro colonies

showing their biofilm formation potential, yet another factor considered to be requisite for

successful bio-inoculant (Bogino et al., 2013).

The immunofluorescence is a serological technique which combines the advantages of

antibodies binding power to their target sites (Janse and Kokoskova, 2009).

Immunolocalization is based on the use of fluorescent signal molecules conjugated to the

antibodies; the emission of fluorescent light indicates the presence of a specific antigen.

The use of fluorescent antibodies for bacterial colonization based on the use of fluorescein

isothiocyanate (FITC)-conjugated IgG antibodies specific to the introduced bacterial strain

to distinguish the target and non-target colonies is well reported (Leeman et al., 1991;

Lemanceau et al., 1992). The examples of immunolocalization used to study root

colonization include the analysis of the spatial competition between Pseudomonas

fluorescens Ag1 and Ralstonia eutropha in barley root colonization (Kragelund and

Nybroe, 1996).

Using immunofluorescence marker employed in combination with CLSM, specificity of

best selected bacterial strain i.e., Enterobacter cloacae AF-31 and Citrobacter freundii AF-

56 strain (pure culture) and attachment of these cells on roots (root colonization) was

examined. Green fluorescence of bacterial cells under CLSM revealed the specificity of

the target cells towards the respective antibodies applied. While no fluorescence was

observed when FAs were cross reacted to the non-target cells further authenticating the


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analyses. Similar observations were made when FAs were applied to sunflower roots

inoculated with the respective strains in sterile soil. A dense bacterial population was

assembled in strings tightly associated to the epidermal root cells. Immunofluorescence

staining although results in a weak auto-fluorescence from the root epidermal cells, but did

not seriously affect detection of the target bacteria. Auto-fluorescence was removed by

background staining or increasing contrast which made possible to visualize the root cells

together with bacteria, as reported previously (Hansen et al., 1997). When roots were

grown in non-sterile soil, the total bacterial population (target and non-target) was red

(stained with ethidium bromide), target population was green (FA’s signal) and some were

yellow (fluorescence overlay of red and green where target population was also stained

with ethidium). These results are the strong evidence of the specificity of our strains and

their root colonizing potential to the host plant. These results are in line with the previous

studies conducted by Leeman et al. (1991) on immunofluorescence colony staining for

monitoring pseudomonads introduced into soil and Mahaffee et al. (1997) in comparative

analysis of antibiotic resistance, immunofluorescent colony staining, and a transgenic

marker for monitoring the environmental fate of rhizobacterium. Our studies are also

supporting the results of Raaijmakers and Punte (1994) about siderophore receptor PupA

as a marker to monitor wild-type Pseudomonas putida WCS358 in natural environments.

To visualize Pseudomonas sp. AF-54 in the rhizoplane of sunflower, fluorescence in situ

hybridization (FISH) was carried out using the oligonucleotide probe EUB 338 and Cy3-

1abeled probe GAM42a that detects several Pseudomonas sp. This technique is based on

the hybridization of DNA-probes labeled with fluorochromes with the complementary

target sequence. It is most frequently used technique for the detection and localization of


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introduced bacterial colonization patterns and community composition (Amann et al.,

2001). Results showed green fluorescence in the cells, revealed the successful labeling with

EUB 338 in case of pure culture hybridization. Pseudomonas sp. AF-54 inoculated roots

were then hybridized and CLSM results revealed the successful colonization of inoculated

strain with the root of sunflower grown in both, sterile and natural soils, further confirming

the specificity of our strains and authenticating its colonizing ability both in sterile and

natural soils. Our results are supporting various reports about the root colonization by

PGPR using FISH successfully (Buddrus-Schiemann et al., 2010; Yasmin et al., 2013).

Oliveira et al., (2009) reported the wheat root colonization potential of two Azospirillum

brasilense strains with specific monoclonal antibody by using combination of FISH and

epifluorescence. Wheat root colonization by Azospirillum brasilense using FISH was also

documented by Assmus et al. (1995). Localization of the introduced Pseudomonas

fluorescence RF13H on tomato root surface using FISH with the help of universal

oligonucleotide probe EUB338 with Cy3 fluorochrome was reported by Shcherbakov et

al. (2013).

Surface sterilization alone is not sufficient to assign the bacteria as an endophytic colonizer.

Only ultrastructural localization can provide direct evidence of an endophytic bacterium

(James, and Olivares. 1998). Rhizobacterial localization in rhizosphere, rhizoplane and in

different compartments of plant especially within the root and nodule cells is studied by

using transmission electron microscopy (Schloter et al., 1997, Hameed et al., 2005; Imran

et al., 2014, Ali et al., 2016; Naqqash et al., 2016). For unambiguous studies of endophytic/

rhizospheric colonization, immunological labeling techniques using monospecific and

polyclonal antibodies against two putative endo-rhizobacterial strains (Azospirillum


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brasilense AF-22 and Citrobacter freundii AF-56 and one ecto-rhizobacterial strain

(Enterobacter cloacae AF-31) was done as well as to confirm their host specificity. Similar

experiments were conducted earlier by Rothballe et al. (2008) to demonstrate the

endophytic localization of H. frisingense strains and reported that polyclonal antibodies

enabled specific in situ identification and very detailed localization of these isolates within

roots of Miscanthus giganteus. When the ultra-thin sections were observed under TEM,

bacteria within sunflower roots as well as in rhizoplane were detected and showed specific

reactions with strain-specific gold-labelled antibodies (IGgs). Shahid et al. (2014) reported

endophytic localization of sunflower associated bacterial strains on the surface of root and

in the root interior. It has been reported that bacterial cells upon inoculation into the soil

first colonize the rhizosphere and then enter into the root cells (Garnalero et al., 2009).

Immunogold labeling was very evident on the bacterial walls. Indeed, within the root cells,

bacteria were observed between the host cells aligned in rows or aggregated into large

colonies in the intercellular spaces. Similar observations were reported by Caiola et al.

(2004) for the localization of endophytic bacteria. Electron microscopic observation

revealed a strong endophytic bacterial (Bradyrhizobium sp. strain SR-6) occupancy in the

root nodule cells of soybean plant (Ali et al., 2016). A number of earlier studies carried out

with TEM and immunogold labeling techniques revealed the successful localization of

PGPR strains in the rhizosphere, root cortical cells and nodules of various crops (Hameed

et al., 2005; Yasmeen et al., 2012). The wide application of TEM was reported by Duijff

et al. (1997) in the study of inter- and intracellular root colonization by P. fluorescens

WCS417r with tomato and Bellone et al. (1997) in studying colonization potential of

Acetobacter diazotrophicus with sugar cane roots. The current results confirmed that both


198

the rhizobacterial strains Azospirillum brasilense AF-22 and Citrobacter freundii AF-56

are the true endophytes and they also colonized the sunflower rhizosphere. On the other

hand, bacterial population could only be localized in rhizoplane/ rhizosphere in case of

Enterobacter cloacae AF-31. Sunflower roots inoculated with Enterobacter cloacae AF-

31 showed strong adhesion of bacterial cell with host plant roots. Multiple cell aggregates

were observed in extracellular matrix on the root surface. The presence of fairly high

number of bacterial cells in the rhizosphere may be related to the root area being rich in

nutrients (Jeunet al., 2008; Guerrero-M et al., 2012. Similar to the results, rhizospheric

bacterial localization with potato roots through TEM has been recently reported by

Naqqash et al. (2016). These ultra-structural studies further validated that strains AF-22,

AF-31 and AF-31 are the true sunflower root-colonizing PGPR, which is of particular

significance as PGPR strains can stimulate plant growth when they have an optimum

colonization concentration.

Both TEM and CLSM observations for colonization studies provided the evidence that the

tested strains with multiple plant growth promoting activities are greatly interacting with

sunflower roots, very well rhizospheric competent, able to survive and colonize in the

rhizosphere, which suggested them suitable candidate as bio-inoculant, particularly for

sunflower plants.

Biofilm formation

The most frequent microbial lifestyle in natural environments is the occurrence of

organized structures associated with surfaces known as biofilms (Watnick and Kolter,

2000) and PGPR are not the exception to this rule. To produce beneficial effects, PGPR

have to interact with the plant surface to form those complex multicellular assemblies/


199

aggregates named biofilms (Danhorn and Fuqua, 2007) which allows a lifestyle that is

entirely different from the planktonic state (Bogino et al., 2013). A time course study was

conducted to investigate biofilm forming ability of 4 potential bacterial strains i.e.

Azospirillum brasilense AF-22, Enterobacter cloacae AF-31, Pseudomonas sp. AF-54,

and Citrobacter freundii AF-56 at 24, 48, 72 and 96 h. CLSM observations showed the

initial event of bacterial attachment in the first 48h of growth and bacterial cells

subsequently assembled on the solid substratum. These results are supported by very recent

similar results documented by Reyes-Pérez et al. (2016) on biofilm formation processes of

R. etli CFN42 were analyzed at an early (24-h incubation) and mature stage (72 h), through

CLSM. After 96h of incubation, cell clusters were fully grown and more aggregated to

form a well-defined biofilm. Same pattern of biofilm formation was found in all bacterial

strains but Enterobacter cloacae AF-31, when observed under confocal laser scanning

microscope, found to be the most powerful biofilm forming bacterial strain at 72 and 96 h.

Similarly, yet in another study, an optimal attachment was observed when cells were

harvested at the late log or the early stationary phase (Dardanelli et al., 2003). The biofilm

formation ability of bacterial strains used in present study again conforms their bio-

inoculant potential, being a mode of life, often crucial for survival of the bacteria, as well

as for the establishment of specific symbiosis with legume or actinorhizal host plants or

nonspecific root colonization (Danhorn and Fuqua, 2007). As bacteria in biofilms are

protected from harsh environmental conditions (Vorachit et al., 1995) and also display

distinct phenotypes compared to their planktonic counterparts including resistance to

antimicrobial compounds and enhanced nutrient uptake (Danhorn and Fuqua, 2007).

Biofilm formation through various bacterial strains is well reported by number of scientists


200

(Ramey et al., 2004; Fujishige et al., 2005; Flemming and Wingender, 2010; Rinaudi and

Giordano, 2010). The study of biofilm development and its physiology is an emerging topic

in the knowledge of plant microbe interaction.

Sunflower growth and yield promoting potential of selected PGPR

In in vivo (pouch and pot) experiments regarding P-solubilization, tricalcium phosphate

(TCP) was used as sole P source (unavailable for plant uptake) to evaluate plant growth

promotion potential of P-solubilizing rhizobacterial strains in the presence of insoluble P

source. It was observed that most of the strains produced significant (P ≤ 0.05) positive

effects on plant growth parameters like root/shoot length, root/shoot fresh weights,

root/shoot dry weights and nutrient uptake with varied efficiency. Differential specificity

of a particular bacterial strain might be articulated by several growth promoting traits like

plant growth hormone production, nitrogen fixation, phosphate solubilization, disease

suppression and biocontrol activity etc.

Field experiments were planned at two different locations (Rawalakot and Faisalabad) to

evaluate the effect of 11 potential PGPR strains on sunflower growth and development in

different agro-ecological zones with combinations of half dose of recommended NP

fertilizers. The performance pattern of all the inoculated strains was almost same at both

sites, but principal component analysis showed that at Rawalakot site these isolates

performed better than Faisalabad. Although the difference between both sites regarding

different growth parameters is not statically much different, showing their competency to

promote sunflower growth at different environmental and soil conditions. Sunflower

requires a temperature range between 25-35 °C for its optimal growth and development;

however, it can tolerate lower temperatures as well. At Rawalakot, the average temperature


201

from June to October was more suitable for sunflower growth and development. Although

during this period, rainfall was high but as the hilly topography of Rawalakot having a

good drainage; it did not affect the crop or crop yield. The relative humidity at Rawalakot

was also favorable to support healthy crop growth. In case of Faisalabad, suitable

temperature for sunflower growth and development was from February to June. In June the

temperature at this location was little high but sunflower is very well reported to tolerate

slight fluctuations in temperature. However, during the crop field testing rainfall and

relative humidity did not remain optimum due to a dry spell throughout the crop growth

period. This might be one of the contributing reasons for an imbalanced performance of

PGPR strains at Rawalakot as compared with that of Faisalabad.

Comparisons among the inoculated PGPR strains in all inoculation experiments ( in vivo

studies) showed that two of the rhizospheric bacterial strains Pseudomonas sp. AF-54 and

Enterobacter cloacae AF-31 and two of the endophytic bacterial strains Azospirillum

brasilense AF-22 and Citrobacter freundii AF-56 were superior in enhancing the growth

(plant height, head diameter and dry matter production) and yield (number of achenes head-

1, 1000 achene weight, biomass yield and achene yield). In addition, these strains resulted

in better mobilization of N, P to sunflower stover and achenes, achene oil content and

quality as well, providing the evidence of competitiveness and ability of these strains to

promote growth and yield of sunflower under field conditions. Joe and Sivakumaar (2009),

and Ambrosini et al. (2012) also reported the overall growth promotion of sunflower along

with improved nutrient uptake by plants under field conditions. Most of the strains

performed better in controlled conditions (pouch and pot experiments), but upon

inoculation in field, some of them did not show efficient growth promotion including


202

Citrobacter braakii AF-21, B. cereus AF-48 and P. brassicacearum AF-95. Rhizobacteria

equipped with plant growth promoting attributes in vitro and capable to trigger plant

growth under controlled environments, sometimes failed to perform in field conditions

(Datta et al., 2011). One of the main reasons could be the extremely un-predictable and

heterogeneous nature of soil, whereas, it is impossible to obtain results similar to controlled

conditions (Lucy et al., 2004). Under controlled conditions hydroponic system was used in

pouch experiments while in pot experiments sterilized soil was used, indicating that the

inoculated bacteria might not have faced competition of indigenous micro-flora under

sterile and controlled conditions. In case of natural soils, indigenous microflora could have

out-competed the inoculated bacterial strains (Lucy et al., 2004).

The growth promotion of sunflower by bacterial inoculation can be credited to the multiple

PGP traits (including P-solubilization, N-fixation, IAA-production, biocontrol activity)

and metabolic versatility. Many reports supported the bacterial phosphate solubilization

ability to enhance plant growth by increased P-uptake from the soil (Chen et al., 2006;

Shirmardi et al., 2010). On the other hand, plant metabolic processes, responsible for

increase in vegetative and reproductive growth, are mainly dependent on N supply (Lawlor,

2002). Diazotrophic bacterial strains are believed to be responsible for biological fixation

of N2 (Franche et al., 2009, Naqqash et al., 2016). Phytohormone production ability is

widely distributed among rhizospheric bacteria (Souza et al., 2013) and in endophytic

bacterial strains (Costa et al., 2014). Plant growth promoting rhizobacterial strains are well

reported to utilize sugars from root exudates to produce organic acid by metabolizing these

surges. These organic acids act as Ca2+ chelators, resulting in release of phosphates from

bound phosphatic compounds (Goswami, et al., 2014).


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The inoculation experiment’s results of present study confirmed the earlier reports of plant

growth promotion by sunflower associated PGPR under different agro-ecological zones

(Shahid et al., 2014). Many reports attributed the pronounced effect of PGPR in

augmenting the growth and yield attributes including plant height, fresh and dry weight,

leaf area, chlorophyll content, plant root length, number and weight of nodules, number

and weight of seeds, seed oil contents and consequently total biomass of several crop

(DeKokalis-Burelle et al., 2006; Van Loon, 2007; Mia et al., 2010, Hussain et al., 2015;

Imran et al., 2015; Ayyaz et al., 2016). Bacterial inoculation plays a key role in the

improvement of crop nutrition (Afzal and Bano, 2008; Shahid et al., 2014) increasing

growth and yield of cereal crops (Biari, et al., 2008; Baig et al., 2014; Ali et al., 2016) and

sustaining the productivity of soil and reduce costs of crop production by reducing level of

chemical fertilizers and fungicides (Yasmin et al., 2013, Isam et al., 2016). Namvar et al.,

(2012) reported significantly enhanced plant height, head diameter, number and weight of

achenes, achene yield, and achene oil content over all biological yield of sunflower in

response to integrated use of PGPR strains and chemical fertilizers. Moreover, Yazdani et

al. (2011) claimed 50 % decreased P-fertilizer demand with the application of PGPR along

with significant increase in grain yield, harvest index, biological yield and nutrient uptake.

On the other hand, one of the main reasons for effectiveness of these potential strains could

be their root colonization potential which was confirmed by several techniques coupled

with confocal laser scanning and transmission electron microscopy. A positive and

consistent benefit of any PGPR with plant is only materialized when bacteria enters in a

close association with plant roots (Kamilova et al., 2005). Moreover, successful biofilm

formation of these bacterial strains well supports the colonization potential of the most


204

effective bacterial strains along with maintenance of their population in rhizosphere

checked in terms of CFU g-1 in present study. Etesami and Hosseini (2015) reported that

the PGPR residing in rhizosphere, rhizoplane, and endophytic niches can produce IAA and

support plant growth colonization. Additional to externally associated root colonizers,

many bacterial species like Azospirillum brasilense was demonstrated to be able to

penetrate the outer root layer and to establish itself in intercellular spaces of the root cortex

(Schloter and Hartmann, 1998).

Among the sampling sites, the PGPR obtained from Diyar Gali were more efficient and

diverse as compared with the other sites. Pseudomonas sp. AF-54 and Citrobacter freundii

AF-56 were indigenous to Diyar Gali and were the most efficient sunflower plant growth

promoters both in vitro and in vivo. It was followed by the isolated obtained from Arja and

Bandi areas, from where four of the potential strains (two from each site) Pseudomonas

brassicacearum AF-6, Bacillus subtilis AF-9, and Citrobacter braakii AF-21, Azospirillum

brasilense AF-22, respectively were isolated. Azospirillum brasilense AF-22 remained as

one of the most efficient bacterial strains from this study. Although, there was a great

variation in the temperature of soil and air, altitude, heat index and soil physico-chemical

properties of sampling sites, but no correlation could be established among the bacterial

diversity, PGPR efficiency and site.

Summary

Sunflower is an important oil seed crop that has the potential to bridge up the huge gap

between oil production and consumption in Pakistan. Unfortunately, oil seed crops are high

fertilizer-demanding crops and excessive fertilization not only destroys the ecosystem but

pollutes the environment. The use of biofertilizer is rather environment-friendly and cost-

effective, hence, is greatly needed for sustainable agriculture. Limited knowledge about

sunflower associated bacterial diversity is available, and Azad Jammu and Kashmir is

completely unexplored area regarding beneficial micro-fauna associated to this crop.

Present study was designed to explore 16 sites of subdivision Dhirkot, AKJ to identify

beneficial PGPR strains with the aim to develop indigenous bio-fertilizer for sunflower

crop. Total 163 isolates were obtained from rhizosphere (97) and root interior (66 putative

endophytes) of sunflower to evaluate the potential of beneficial root associated bacteria

and their root colonization potential with ultimate objective to improve sunflower growth,

nutrient uptake, yield and oil contents. Out of 163 screened isolates 40 % were found to be

positive for phosphate solubilization (9.51 to 59 µg mL-1), 24 % for IAA production (1.13-

24.6 µg mL-1), 20% for nitrogen fixation (28.68-137.84 nmoles mg-1 protein h-1) and 12%

were biocontrol agents against Fusarium oxysporum detected by using standard

microbiological and biochemical procedures. Most of the phosphate solubilizing isolates

were able to produce a variety of organic acids dominated by gluconic acid (G.A) ranged

between 2.17 µg mL-1 to 15.44 µg mL-1. After bio-chemical and molecular

characterization and plant inoculation experiments on sunflower in controlled and field

conditions, 11 potential PGPR strains were screened that showed improvement in growth

and yield of sunflower. These PGPR were Pseudomonas thivervalensis strain AF-6,

Bacillus safensis AF-9, Citrobacter braakii AF-21, Azospirillum brasilense AF-22,

Enterobacter cloacae AF-31, Bacillus cereus AF-48, Pseudomonas sp. strain AF-54,

Citrobacter freundii AF-56, Pseudomonas brassicacearum AF-95, Lysinibacillus sp.

strain AF-146 and Arthrobacter sp. AF-163. Out of these 11 strains, A. brasilense AF-22,

E. cloacae AF-31, Pseudomonas sp. strain AF-54 and C. freundii AF-56 showed

comparatively better response in augmenting sunflower growth, yield and oil contents as

compared to other bacterial strain as well as non-inoculated control treatments (½ nitrogen

and phosphorus fertilizers). These four PGPR strains also increased nitrogen and

phosphorus uptake in plants and were good colonizers of sunflower root and rhizosphere.

These PGPR showed potential to attach on glass surface and develop biofilm in vitro

confirming their colonization potential. Transmission electron microscopy, FAs staining,

FISH and confocal microscopy confirmed the endophytic nature of A. brasilense AF-22,

and C. freundii AF-56, while rhizospheric association/colonization of E. cloacae AF-31,

Pseudomonas sp. strain AF-54. These 4 PGPR strains performed best at two agro-

ecologically different locations; Rawalakot and Faisalabad, confirming their adaptability

and effectiveness on a wide range of soil and environmental conditions. Additionally, all

these rhizobacterial strains showed divers metabolic potential with variable antibiotic

resistance and biocontrol abilities which make them suitable candidates for developing

biofertilizer.

Based on all these facts, A. brasilense AF-22, E. cloacae AF-31, Pseudomonas sp. strain

AF-54 and C. freundii AF-56 it is concluded that these PGPR can be used to develop

biofertilizer for sunflower crop. It is further recommended, that the proposed inoculum

should be checked along with the cross inoculation potential of this inoculum on other oil

seed crops in field.

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Appendix- 1

LURIA BERTANI (L.B) MEDIUM

Tryptone 1 %

Yeast extract 0.5 %

NaCl 0.5 %

D.Water 1000 mL

pH 7.5

Appendix- 2

GRAM STAINING.

Crystal Violet Solution

Crystal Violet 10.0g

Ammonium oxalate 4.0g

Ethanol 100mL

D. Water 400mL

Iodine Solution

Iodine 1.0g

K2I 2.0

Ethanol 25mL

Distilled Water 100mL

Counter stain

Safranine 2.5g

Ethanol 10mL

Distilled Water 100mL

Appendix- 3

NITROGEN FREE MEDIUM (NFM)

Malic acid or Na malate 0.5g/L

MgSO4.7H2O 0.2g/L

CaCl2 0.02g/L

NaCl 0.1g/L

NaMoO4.2H2O 0.002g/L

BTB (0.5%) 5mL

KOH (in case of malic acid) 4.5g/L

Biotin 10μg

pH 7.0

Agar 2% (for plates)

D. Water 1000 mL

Appendix- 4

IAA ESTIMATION

Solution – A

FeH2SO4 for IAA estimation

0.5 M Fe Cl3 1 ml

D.Water 50 ml

Conc. H2SO4 30 ml

Standard Solution of IAA

IAA 1 mg/ml

Dissolved in ethanol (70%)

Make the solution 50 ppm

Appendix- 5

PIKOVSKAIA MEDIUM

Ca3 (PO4)2 3.0g/L

Sucrose 10g/L

(NH4)2SO4 0.5g/L

NaCl 0.2g/L

MgSO4.7H2O 0.1g/L

KCl 0.2g/L

Yeast extract 0.5g/L

MnSO4 0.004/L

FeSO4 (Fe-EDTA) 0.002g/L

CaCO3 0.3g/L

Agar 15g/L

D. Water 1000 ml

pH 7±0.2

[12 g ammonium paramolybdate (NH4)6Mo7O24.4H2O)

dissolved in 250 mL distilled water, 0.2908 g potassium antimony tartrate

{K2Sb2(C4H2O6)2} dissolved in 100 mL distilled water, both reagents were dissolved

in 5N H2SO4 and volume was made up to 2 L and the whole reagent was designated

as reagent A, 1.056 g ascorbic acid was dissolved in 200 mL of reagent A to make

reagent B as standard reagent (Murphy and Riley, 1962)]

Appendix- 6

FISH BUFFERS

Hybridization buffer

5MNaCl 360µl

1M TrisHCl 40µl

10% SDS 2µl

pH 7.0

D. Water 1600µl

Washing buffer

5MNaCl 900µl

1M TrisHCl 1mL

10% SDS 2µl

pH 8

D. Water to make vol. up to 50ml

Appendix- 7

TEM

IGL buffer

PIPES buffer 0. 5 mM

BSA 0.5%

Tween 20 0.5%

Sodium azide 0.02%

Appendix- 8

HOAGLAND NUTRIENT SOLUTION

Ca (NO3) 10 ml / L

1 M KNO3 10 ml / L

1 M MgSO4 . 7H2O 4 ml / L

1.64 % Fe. EDTA 2 ml / L

Micronutrients 2 mL

1 M CaCl2 10 ml / L

1 M KCl 10 ml / L

Hoagland Micronutrients Trace Elements

Boric Acid 0.31 g/L

MnSO4 1.115 g/L

ZnSO4 . 7 H2O 0.430 g/L

Na2 MoO4. 2H2O 0.0125 g/L

CuSO4. 5H2O 0.00125g/L

KI 0.0375g/L

CoCl2 0.00125g/L

Total volume 500 ml

(For prepared micronutrient solution 2ml/L)

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