Uzma Farooq - Higher Education...

Isolation and Characterization of Plant Growth

Promoting Rhizobacteria and their Role in

Biocontrol of Fusarium Stalk Rot of Maize

(Zea Mays L.)

Uzma Farooq

Department of Plant Sciences

Faculty of Biological Sciences

Quaid-i-Azam University Islamabad, Pakistan

2014

Isolation and Characterization of Plant Growth

Promoting Rhizobacteria and their Role in Biocontrol

of Fusarium Stalk Rot of Maize (Zea mays L.)

A dissertation submitted in the partial fulfillment of the

requirements for the degree of Doctor of Philosophy

In

Plant Sciences

(Plant Physiology)

By

Uzma Farooq


Faculty of Biological Sciences

Quaid-i-Azam University Islamabad, Pakistan

2014

DEDICATION

To

My loving Mother Rukhsana shaheen

whose selfless love will always be cherished but can never be repaid

My beloved Father

Muhammad Farooq who gave me the courage to dream high

& opened up avenues for me to explore my abilities

My incredibly wonderful husband Dr. Muhammad Ishtiaq Ali

& My beautiful and precious Kids

Muhammad Arsal Ali Haniya Ali

whose loving support in every aspect enabled me to achieve

what my parents had inspired me to dream!

DECLARATION

I hereby declare that the work presented in the following thesis is

my own effort, except where otherwise acknowledged and the

thesis is my own composition. No part of this thesis has been

previously presented for any other degree.

Uzma Farooq

CERTIFICATION

This is to certify that this PhD dissertation entitled “Isolation and characterization

of plant growth promoting rhizobacteria and their role in biocontrol of Fusarium

stalk rot of maize (Zea mays L.)” submitted by Mrs. Uzma Farooq is accepted in its

present form by the Department of Plant Sciences, Faculty of Biological sciences,

Quaid-i-Azam University, Islamabad as satisfying the thesis requirement for the

Degree of Doctor of Philosophy (Ph.D.) in Plant Physiology.

SUPERVISOR

Prof. Dr. Asghari Bano

EXTERNAL EXAMINER

EXTERNAL EXAMINER __________________________

CHAIRPERSON

Chairperson


Date:

TABLE OF CONTENTS

S. No Title Page No

1 Acknowledgments ……………………………………....... i

2 List of abbreviations………………………………….......... ii

3 List of tables………………………………………….......... v

4 List of figures…………………………………………........ vii

5 List of appendices…………………………………………. xi

6 Abstract………………………………………………........ xiii

Chapter # 1 GENERAL INTRODUCTION AND REVIEW OF

LITERATURE

1.1 Maize………………………………………………………. 1

1.2 Maize taxonomy…………………………………………… 1

1.3 Maize in Pakistan……………………………...................... 1

1.4 Maize growing areas in Pakistan……………....................... 3

1.5 Fungal diseases of maize in Pakistan……………………… 3

1.6 Stalk rots in maize………………………............................. 5

1.7 Fusarium species causing stalk rot in Maize........................ 5

1.8 Fusarium moniliforme……………………………………... 6

1.8.1 Symptoms of Fusarium stalk rot……………………........... 6

1.8.2 Host range…………………………………………………. 6

1.8.3 Life cycle of Fusarium moniliforme ……………………… 6

1.8.4 Factors effecting the infection of maize with Fusarium 7

1.9 Control of maize diseases ……………………..................... 7

1.10 Biological Control …............................................................ 9

1.10.1 Plant growth promoting rhizobacteria (PGPR) …………… 10

1.11 Mechanisms of action undertaken By PGPR in biological

control ………………….......................................................

11

1.11.1 Competition…………………………................................... 11

1.11.2 Parasitism/mycophagy………………................................. 12

1.11.3 Hydrolytic enzymes production………................................ 15

1.11.4 HCN production/ Cyanogenesis………................................ 15

1.11.5 Plant induced resistance........................................................ 15

1.11.6 Antibiosis……..................................................................... 18

1.11.6.1 2,4 Diacetylphloroglucinol (DAPG)……............................ 18

1.11.6.2 Pyrrolnitrin……………………………............................... 22

1.11.6.3 Phenazine……...................................................................... 24

1.11.6.4 Zwittermicin A………………………………..................... 30

Aims and scope of present research work………………. 30

Chapter # 2 Isolation and screening of rhizobacteria for

antagonistic activity

2.1 Introduction…………………………………………........ 32

Aims and Objective………………………………………. 33

2.2 Material and Methods…………………………………… 35

2.2.1 Collection of soil samples………........................................ 35

2.2.2 Soil Analyses………………................................................. 35

2..2.2.1 Moisture Content of Soil………………............................... 35

2.2.2.2 Soil pH and Electrical Conductivity (EC)............................. 35

2.2.2.3 Soil Nutrients Analyses......................................................... 37

2.2.2.4 Soil Texture……………………………………………….. 37

2.2.3 Isolation of rhizobacteria from soil………………………. 37

2.2.4 Tests for antagonism……..................................................... 39

2.2.4.1 Cell free supernatant preparation …………….................... 39

2.2.5 Phenotypic characterization of rhizobacteria…………........ 39

2.2.6 Detection of the phosphate solubilizing activity............... 39

2.2.7 Production of hydrogen cyanide........................................... 40

2.2.8 Siderophore production…………......................................... 40

2.2.9 Ammonia production…………..……................................... 40

2.2.10 Production of fungal cell wall degrading enzymes………... 40

2.2.10.1 Protease activity…………………………………………… 40

2.2.10.2 Chitinase Activity……......................................................... 40

2.2.10.3 Cellulase Activity………………………………………….. 41

2.2.11 Production of other beneficial enzymes…………………… 41

2.2.11.1 Catalase activity…………………………………………… 41

2.2.11.2 Oxidase activity…………………………………………… 41

2.2.12 Production of Plant growth promoting hormone (IAA) 41

2.2.13 Screening of rhizobacteria for plant growth promotion

activities……………………………………………………

42

2.2.13.1 Preparation of inoculum…………………………………… 42

2.2.13.2 Seed treatment…………………………………………….. 42

2.2.13.3 Pot culture study………………………………………….. 42

2.2.14 Morphological and biochemical characterization of

selected rhizobacteria………………………………………

45

2.2.14.1 Miniaturized Identification System-QTS 24………………. 45

2.2.15 Molecular identification …………………………………... 45

3.2.15.1 Primers and PCR conditions………………………………. 45

2.2.15.2 Sequencing and sequence analysis………………………… 45

1.1.15.3 Phylogenetic trees…………………………………………. 47

2.16 Statistical analysis ………………………………………… 47

Exp # 1

In vitro screening of rhizobacteria

2.3 Results…………………………………………………….. 49

2.3.1 Isolation of Bacterial isolates……………………………… 49

2.3.2 Antagonism assay against phytopathogenic fungi………… 49

2.3.3 Phosphate solubilization…………………………………… 61

2.3.4 Production of plant growth promoting hormone (IAA) …... 64

2.3.5 Production of siderophore…………………………………. 68

2.3.6 Production of HCN………………………………………… 68

2.3.7 Production of Ammonia…………………………………… 68

2.3.8 Catalase and oxidase enzymes activity………………… 72

2.3.9 Fungal cell wall degrading enzymes (protease, chitinase

and cellulase) activity……………………………………..

72

2.4 Discussion………………………………………………… 76

2.4.1 Isolation of rhizobacteria………………………………….. 76

2.4.2 Antifungal activity of isolated rhizobacteria………………. 76

2.4.3 Morphological Characteristics……………………………. 77

2.4.4 Phosphate solubilization…………………………………… 78

2.4.5 Production of IAA………………………………………… 78

2.4.6 Siderophore production……………………………………. 78

2.4.7 Production of enzymes…………………………………….. 79

2.4.8 HCN Production…………………………………………… 80

Exp # 2

In vivo screening of rhizobacteria

2.5 Introduction………………………………………………. 82

2.6 Results…………………………………………………….. 83

2.6.1 Survival efficiency (CFU) of antagonistic rhizobacteria….. 83

2.6.2 Shoot and root length……………………………………… 83

2.6.3 Root to shoot ratio………………………………………… 85

2.6.4 Shoot and root Fresh weight……………………………… 88

2.6.5 Leaf area………………………………………………….. 88

2.6.6 Identification of selected Rhizobacteria …………………. 92

2.6.6.1 Morphological and biochemical characterization of

bacterial antagonists………………………………………..

92

2.6.7 Identification at molecular level…………………………… 94

2.7 Discussion………………………………………………… 99

Chapter # 3 Induction of systemic resistance by antagonistic PGPR

against stalk rot in maize

3.1 Introduction……………………………………..…........... 102


3.2 Material and Methods……………………………..…....... 105

3.2.1 Plant Material used………………………………………… 105

3.2.2 Physiochemical Characteristics of Soil……………………. 105

3.2.2.1 Soil pH and EC…………………………………………….. 105

3.2.2.2 Soil Nutrients Analysis……………………………………. 105

3.2.3 PGPR strains used in the study……………………………. 105

3.2.4 Seed sterilization and inoculation of antagonistic PGPR…. 106

3.2.5 Method of application of PGPR strains……………………. 106

3.2.6 Preparation of F.moniliforme inoculum…………………… 106

3.2.7 Application of Chemical Fungicide (Ridomil Gold)……… 106

3.2.8 Treatments………………………………………………… 106

3.2.9 Greenhouse experiment…………………………………… 108

3.2.10 Application of F. moniliforme Inocula…………………… 108

3.2.11 Disease Scoring……………………………………………. 108

3.2.12 Sample Collection…………………………………………. 108

3.2.13 Superoxide dismutase (SOD) activity…………................... 109

3.2.14 Peroxidase (POD) activity………......................................... 109

3.2.15 Polyphenol peroxidase (PPO) activity…………………….. 109

3.2.16 Ascorbate peroxidase activity ……………………………. 109

3.2.17 Catalase activity…………………………………………… 110

3.2.18 Total soluble phenol content………..................................... 110

3.2.19 Malondialdehyde (MDA) activity ….................................... 110

3.2.20 Proline Content of Leaves………………………………… 111

3.2.21 Chlorophyll and Carotenoid Content……………………… 111

3.2.22 Protein Content……………………………………………. 111

3.2.23 Assay for PR protein ……………………………………… 111

3.2.23.1 Chitinase activity assay……………………………………. 111

3.2.24 Statistical analysis…………………………………………. 112

3.3 Results…………………………………………………….. 114

3.3.1 Disease reduction/ disease severity……………………….. 114

3.3.2 Superoxidase dismutase (SOD) activity…………………… 116

3.3.3 Peroxidase (PO) activity…………………………………… 116

3.3.4 Polyphenol peroxidase (PPO) activity……………………. 118

3.3.5 Ascorbate peroxidase activity……………………………... 119

3.3.6 Total soluble phenol content……………………………... 119


3.3.8 Protein content………..……………..……………….......... 123

3.3.9 Chitinase activity………………………………………….. 123

3.3.10 Malondialdehyde (MDA)…………………………………. 125

3.3.11 Proline content…………………………………………….. 126

3.3.11 Photosynthetic pigments…………………………………... 128

3.3.12.1 Chlorophyll content………………………………………... 128

3.3.12.2 Carotenoid content………………………………………… 128

3.4 Discussion………………………………………………… 131

3.4.1 Induced systemic resistance by chemical fungicide

(Ridomil Gold)……………………………………………..

131

3.4.2 Induced systemic resistance by antagonistic PGPR………. 132

Chapter # 4 Biological control of Fusarium stalk rot in maize under

field conditions by antagonistic PGPR

4.1 Introduction……………………………………………… 138



4.2.1 Collection of host plant seeds and pathogen………………. 140

4.2.2 Selected PGPR strains…………………………………….. 140

4.2.3 Inoculation with antagonistic PGPR ……………………… 140

4.2.4 Inocula preparation................................................................ 140

4.2.5 Field experiment…………………………………………… 140

4.2.6 Inoculation of the pathogen………………………………. 142

4.2.7 Disease scoring……………………………………………. 142

4.2.8 Sample Collection…………………………………………. 142

4.2.9 Superoxidase dismutase (SOD) activity………………….. 142

4.2.10 Peroxidase (POD) activity................................................... 142

4.2.11 Polyphenol oxidase (PPO) activity...................................... 142

4.2.12 Ascorbate peroxidase activity……………………………... 142


4.2.14 Total soluble phenol content............................................... 143

4.2.15 Chitinase activity ….……………………………………… 143

4.2.16 Extraction and Purification of IAA and ABA…………….. 143

4.2.17 Yield parameters………………………………………….. 144

4.2.18 Statistical analysis…………………………………………. 144

4.3 Results…………………………………………………….. 146

4.3.1 Disease severity/ disease reduction………………………... 146

4.3.2 Phytohormones production in maize leaves………………. 148

4.3.2.1 Indole acetic acid (IAA) content…………………………... 148

4.3.2.2 Absicsic acid (ABA) content………………………………. 150

4.3.3 Superoxidase dismutase (SOD) activity…………………… 152

4.3.4 Peroxidase (PO) activity………………………………….. 154

4.3.5 Polyphenol oxidase (PPO) activity……………………….. 154

4.3.6 Ascorbate peroxidase activity…………………………….. 154

4.3.7 Total soluble phenol content…………………………….. 156


4.3.9 Effect of antagonistic rhizobacteria on PR proteins 159

4.3.9.1 Chitinase activity…………………………………………. 159

4.3.10 Yield parameters………………………………………….. 162

4.4 Discussion………………………………………………… 167

Chapter #5 Molecular detection of antibiotics biosynthetic genes in

antagonistic PGPR

5.1 Introduction………………………………………………. 172



5.2.1 Antibiotic production by agar well diffusion method….. 175

5.2.2 Screening of putative antibiotic producing strains by

polymerase chain reaction…………………………………

175

5.2.2.1 Design of oligonucleotide primers for molecular detection

of antibiotic Gene Fragments ……………………………..

175

5.2.2.2 Amplification and Identification of the phenazine gene

Fragments …………………………………………………

175

5.2.3 Quantification of antibiotics by HPLC…………………….. 180

5.2.3.1 Culture Extracts of phz Positive strains ………………….. 180

5.2.3.2 HPLC analyses ……………………………………………. 180

5.2.4 Well plate assay………………………………………….. 180

5.2.5 RNA Isolation for RT-PCR………………………………. 182

5.2.6 Quantitative RT-PCR …………………………………….. 182

Nucleotide Sequence Accession Numbers ……………… 182

5.3 Results……………………………………………………. 184

5.3.1 Antibiotic production by agar well diffusion method…….. 184

5.3.2 Detection of DAPG, PRN gene by Polymerase chain

reaction (PCR) …………………………………………….

184

5.3.3 Detection of zwittermicin A self-resistance gene by

Polymerase chain reaction (PCR) …………………………

184

5.3.4 Detection of Phenazine genes by Polymerase chain

reaction (PCR) ……………………………………………..

188

5.3.5 Quantitative determination of antifungal metabolites……... 188

5.3.6 Well plate assay……………………………………………. 188

5.3.7 Gene expression of Phenazine……………………………... 188

5.4 Discussion…………………………………………………. 192

Chapter # 6 Concluding Chapter

6.1 Concluding Chapter……………………………………….. 196

6.2 Recommendations and Future perspective………………… 202

List of significant Publications……………………………. 204

Chapter # 7 REFERENCES…………………………………………… 205

Chapter # 8 APPENDICES…………………………………………… 252

i

ACKNOWLEDGEMENTS

All praises to “Almighty Allah”, the most beneficent, who is the source of all the wisdom and knowledge and bestowed me with potential and ability to complete the present work. Countless salutation upon the Holy Prophet (Peace Be Upon Him), source of knowledge and blessings for entire creation, who has guided his Ummah to seek knowledge from cradle to grave and enabled me to win honour of life.

The work in this manuscript was accomplished under the sympathetic attitude, expert guidance, scholarly criticism, constant passion, motherly behaviour and enlightened supervision of Prof. Dr. Asghari Bano, Dean, Faculty of biological Sciences, Quaid-i-Azam University, Islamabad. I feel so richly blessed to have her as my supervisor. Her efforts tower the inculcation of spirit of hard work and maintenance of professional integrity besides other valuable suggestions will serve as beacon of light through the course of my life.

My heartfelt and profound gratitude would aptly sum up my feelings toward, Higher Education Commission, Pakistan, I feel so richly blessed for having such a mile stone in our country promoting the higher education throughout the country. I would like to extend my deepest thanks to HEC for providing me indigenous Ph.D. scholarship and for all its financial assistance without which this work would not have been possible. Legitimately I feel highly privileged and obliged to express my sincere thanks to Dr. Torsten Thomas, Associate Professor, School of Biotechnology and Biomolecular Sciences and Centre for Marine Bio-Innovation, University of New South Wales, for extending the research facilities of his lab under his supervision, technical guidance and encouragement throughout my stay at the Sydney, Australia.

Words are lacking to express my thanks to Miss Rabia Naz, Asia Nosheen and Humera

Yasmin for their endearment, support, cooperation and consolatory behaviour during the whole

time period of this study. Any attribute will be less for them. Its my good luck to have true friends

Dr. Noshin ilyas, Dr. Sumera Iqbal and Noreen Sadiq and I am thankful to them for all their care,

prayers and love. I would like to thank my all lab fellows for their help and encouragement.

I am also grateful to my husband Dr. Ishtiaq Ali, for all his patience and cooperative behaviour during all the time during my Ph.D. Everlasting and heartfelt thanks to him for all his emotional backing, encouragement, concerns, love and care. His companionship is an asset for my life.

No acknowledgement could ever adequately express my feelings to my affectionate and adorable family without whom I feel myself incomplete. My Parents and mother in law deserve special mention for their inseparable support and prayers. From the start till the accomplishment of this manuscript, they have been my source of strength and love. I am honoured to have them as my parents.

Words fail to express my appreciation to my dearest brothers Imran, Kamran, Irfan and Abdul Rahman for their support, care and persistent confidence in me. Bundles of thanks to my sister, Amina, for being there always to take the loads off my shoulder with all her love, care and support. I am also thankful to my sisters in law Tahira, Saira, Shazia and brother in law Iffitkhar, Dr.Mumtaz, Zulfikar for their encouragement, support and prayers. In the end I want to present my unbending thanks to all those hands who prayed for my betterment and serenity.

(Uzma Farooq)

ii

LIST OF ABBREVIATION

Abbreviations Full name

% Percentage

@ At the rate of

°C Degree Centigrade

µL Microlitre

ABA Abscisic acid

ADIC Amino-2-deoxyisochorismic

BHT Butylated Hydroxy Toluene

BSA Bovine Serum Albumin

BTH Benzothiadiazole

Ca Calcium

CAS Chrome Azurol S

cfu Colony forming unit

CHR hydrometer reading(Corrected after temperature

adjustment)

cm centimetre

CRD Completely Randomized Design

d days

DAHP 3-deoxy-D-arabino hepullllosonate-7-phosphate

DAPG Diacetyl phlouroglucinol

DAS Days after sowing

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DNS Dinitrosalicylate

dS/m Deci Siemens per meter

EC Electrical conduvtivity

g Gram

GLU Glucanase

GOP Government of Pakistan

h Hours

H2O2 Hydrogen peroxide

HCL Hydrogen chloric acid

iii

HCN Hydrogen Cyanide

HPLC High performance liquid chromatography

IAA Indole acetic acid

IR Induced resistance

ISR Induced systemic resistance

IUPAC International Union of Pure and Applied Chemistry

K Potassium

KB kings medium B

Kg Killogram

LB Luria-Bertani media

LMCS Longitudinally modulated cryogenic system

LSD Least Significant Difference

M Molar

MDA Malondialdehyde

Mg Magnesium

mg Milligram

min Minute

mL Milliliters

mM Millimolar

MT Metric ton

N Normal

Na Sodium

NADH Nicotinamide dinucleotide

NaOCl Sodium hypochloride

NaOH Sodium hydroxide

NARC National Agricultural Research Centre

NBT Nitroblue tetrazolium

NCBI National Centre for Biotechnology Information

ng Nanogram

NJ Neighbour-joining

O.D. Optical density

O2− Superoxides

P Phosphorous

iv

PAGE Polyacrylamide Gel Electrophoresis

PAL Phenylalanine ammonia lyase

PBS Phosphate buffered saline

PCA Phenazine carboxylic acid

PCN Phenazine-1-caboxamide

PCR Polymerase chain reaction

PDB Potato dextrose broth

PGPR Plant growth promoting rhizobacteria

PMSF phenylmethanesulfonylfluoride

POD Peroxidase

ppm Parts per million

PPO Polyphenol oxidase

PR Pathogenesis-related

PRN Pyrronnitrin

PYO Pycocyanin

QTS Quick test system

RCBD Randomized Complete Block Design

RFE Rotary film evaporator

ROS Reactive oxygen species

rpm Revolution per minute

RT-PCR Real Time-Polymerase Chain Reaction

SA Salicylic acid

SAR Systemic Acquired Resistance

SDA Sabouraud dextrose agar

SOD Superoxide dismutase

TBA Thiobarbituric acid

TCA Trichloroacetic acid

UV Ultraviolet

v

LIST OF TABLES

Table No. Title Page No.

1.1 List of fungal pathogens suppressed by antagonistic

PGPR……………………………………………………… 14

1.2 Important antibiotics produced by antagonistic

PGPR……………………………………………………….. 19

2.1 Summary of field attributes and climatic characteristics of

sampling locations………………………………………….. 36

2.2 Physicochemical properties of soil used for isolation of

rhizobacteria………………………………………………… 36

2.3 Groups of indigenous rhizobacteria ………………………... 38

2.4 Treatments made for pot experiment……………………….. 43

2.5 PCR condition for Amplification of 16s rRNA…………….. 46

2.6 Phosphate solubilization by Group 1 rhizobacteria isolated

from the rhizosphere of maize fields of arid, semi-arid and

irrigated regions ……………………………………………. 62

2.7 Phosphate solubilization by Group 2 rhizobacteria isolated

from the rhizosphere of maize fields grown at arid, semi-

arid and irrigated regions ……………… 63

2.8 Production of Siderophore by rhizobacteria isolated from

rhizosphere of maize fields of irrigated region…………….. 69

2.9 HCN Production by rhizobacteria isolated from rhizosphere

of maize fields of irrigated region.......................................... 69

2.10 Ammonia production by rhizobacteria isolated from

rhizosphere of maize fields of irrigated region……………….. 69


rhizosphere of maize fields of Arid region…………………. 70


of maize fields of arid region……………………….……… 70

2.13 Ammonia Production by rhizobacteria isolated from

rhizosphere of maize fields of arid region………………….. 70


rhizosphere of maize fields of semi-arid region………….. 71


of maize fields of semi-arid region…………. 71

2.16 Ammonia Production by rhizobacteria isolated from

rhizosphere of maize fields of semi-arid region……………. 71

2.17 Catalase and oxidase enzymes activity by rhizobacteria

isolated from the rhizosphere maize fields of irrigated

region ……………………………………………………… 73


isolated from the rhizosphere maize fields of arid region…. 73


isolated from the rhizosphere maize fields of semi-arid

region……………………………………………………….. 73

vi

2.20 Hydrolytic enzymes activity (protease, chitinase and

cellulase) by rhizobacteria isolated from the rhizosphere

maize fields of irrigated region ……………………….

74

2.21 Hydrolytic enzymes activity (Protease, Chitinase and


maize fields of arid region.…………………………………...

74

Hydrolytic enzymes activity (Protease, Chitinase and


maize fields of semi-arid region………………………………

74

2.22 Morphological and biochemical characteristics (as determined

by QTS) of selected rhizobacteria isolated from rhizospheric

soil of maize plants …………………………………………..

93

2.23 Identified rhizobacteria and their Accession numbers………... 95

3.1 Treatment details for greenhouse experiment………………… 107

4.1 Treatments made for field experiments ……………………… 141

4.2 Effect of antagonistic PGPR on the disease severity (%) in

maize plants……………………………………………………… 147

4.3 Effect of antagonistic PGPR on the IAA (ug/g) content in

leaves of maize plants.............................................................. 149

4.4 Effect of antagonistic PGPR on the SOD activity in maize

leaves ………………………………………………………………... 151

4.5 Effect of antagonistic PGPR on the POD activity in leaves of

maize plants …………………………………………………. 153

4.6 Effect of antagonistic PGPR on the PPO activity in leaves of

maize plants …………………………………. 155

4.7 Effect of antagonistic PGPR on the ascorbate peroxidase in

maize leaves…………………………………………………... 157

4.8 Effect of antagonistic PGPR on the total soluble phenol in

maize leaves………………………………………………….. 158

4.9 Effect of antagonistic PGPR on the catalase activity in maize

leaves…………………………………………………………. 160

4.10 Effect of antagonistic PGPR on the chitinase activity in maize

leaves…………………………………………………. 161

4.11 Effect of antagonistic PGPR on the Yield of maize

plants…………………………………………………………. 163

4.12 Effect of antagonistic PGPR on 1000 seed weight of maize

plants……………………………………………………… 165

4.13 Effect of antagonistic PGPR on number of seeds/cob in

maize……………………………………………………….. 166

5.1 Primers of antibiotics biosynthetic genes used in this study

………………………………………….. 177

5.2 Conditions of PCR for amplification of antibiotic biosynthetic

genes …………………………………………. 178

vii

LIST OF FIGURES

Figure No. Title Page No. 1.1 Trends in Area, production and Yield of Maize in Pakistan…. 2

1.2 Pakistan share in world maize production............................. 2

1.3 Maize production in different parts of the world including

Pakistan ……………………………………………………. 4

1.4 Pakistan major maize growing areas………………………… 4

1.5 The parts of world with Fusarium moniliforme syn.

Verticillioides infection of maize……….. 8

1.6 Disease cycle of F. moniliforme on maize showing various

infection pathways…………………………………………. 8

1.7 Diagrammatic sketch of the mechanism of action of plant

growth promoting rhizobacteria……………………………. 12

1.8 A possible model for the mechanisms of action of 2,4-

DAPG……………………………………………………… 21

1.9 Genes involved in DAPG biosynthesis…………………….. 21

1.10 Schematic illustration of Pyrrolnitrin Biosynthesis ……….. 23

1.11 Mode of action of Phenazine derivatives…………………... 27

1.12 Biosynthesis of Phenazine ………….................................... 29

2.1 Scheme of study used for screening and characterization of

potential antagonistic PGPR………………………………. 44

2.2 Schematic illustration for molecular identification of

antagonistic rhizobacteria…………………………………... 48

2.3 Effect of Group 1 rhizobacteria of arid region on antifungal

activity against F.moniliforme……………………….……. 50


activity against H. sativum…………………………………. 50


activity against A. flavus……………………………………. 51


activity against F.moniliforme ……………………….……. 51


activity against H. sativum………………………………… 53


activity against A. flavus……………………………….. 53

2.9 Effect of Group 1 rhizobacteria of semi-arid region on

antifungal activity against F.moniliforme ……………….. 55


antifungal activity against H. sativum ………………….. 55


antifungal activity against A. flavus ……………………... 56


antifungal activity against F .moniliforme ……………. 56


antifungal activity against H. sativum …………………… 57


antifungal activity against A. flavus ………………………. 57

2.15 Effect of Group 1 rhizobacteria of irrigated region on

antifungal activity against F.moniliforme …………….. 58

viii




antifungal activity against A. flavus ………………………. 59


antifungal activity against F.moniliforme………………… 59




antifungal activity against A. flavus ……………………. 60

2.21 IAA production by Group 1 rhizobacteria of arid region…. 65

2.22 IAA production by Group 2 rhizobacteria of arid region…. 65

2.23 IAA production by Group 1 rhizobacteria of semi-Arid

region……………………………………………………….. 66

2.24 IAA production by Group2 rhizobacteria of semi-arid

region ………………………………………………………. 66

2.25 IAA production by Group 1 rhizobacteria of Irrigated region

………………………………………………………………. 67

2.26 IAA production by Group 2 rhizobacteria of Irrigated region

………………………………………………........................ 67

2.28 CFU of Group 1 rhizobacteria inoculated to maize seeds…. 84

2.29 CFU of Group 2 rhizobacteria inoculated to maize seeds …. 84

2.30 Effect of Group1 rhizobacteria on shoots and root length of

maize seedlings...................................................................... 86

2.31 Effect Group 2 rhizobacteria on shoot and root length of

maize seedlings..................................................................... 86

2.32 Effect of Group1 rhizobacteria on root to shoot ratio of

maize seedlings..................................................................... 87

2.33 Effect of Group 2 rhizobacteria on root to shoot ratio of

maize seedlings..................................................................... 87

2.34 Effect of Group1 rhizobacteria on shoot and root fresh

weight of Maize seedlings ………………………………… 89

2.35 Effect of Group2 rhizobacteria on shoot and root Fresh

weight of Maize seedlings…...……………………………. 89

2.36 Effect of Group1 rhizobacteria on leaf area of maize

seedlings …………………………………………………… 90

2.37 Effect of Group 2 rhizobacteria on leaf area of maize

seedlings……………………………………………………. 90

2.38 Polymerase Chain reaction for I6sRNA……………………. 95

2.39 Phylogenetic tree of Pseudomonas aeruginosa 4nm and

Pseudomonas sp. NDY………………………………………….. 96

2.40 Phylogenetic tree of Pseudomonas aeruginosa JYR and

B.firmus PTWz…………………………………………………… 97

2.41 Phylogenetic tree of Bacillus endophyticus Y5 and Bacillus

firmus Yio…………………………………………………………. 98

3.1 Schematic Illustration for the layout of Greenhouse

Experiment............................................................................. 113

3.2 Effect of antagonistic PGPR on disease severity (%) in 115

ix

maize under axenic condition of greenhouse in pots……….

3.3 Effect of antagonistic PGPR on disease reduction (%) in

maize under axenic condition of greenhouse in pots………. 115

3.4 Effect of antagonistic PGPR on SOD activity in maize

leaves under axenic condition of greenhouse………………. 117

3.5 Effect of antagonistic PGPR on POD activity of maize

leaves in pots under axenic condition of greenhouse…….... 117

3.6 Effect of antagonistic PGPR on polyphenol (PPO) activity

of maize leaves in pots under axenic conditions of

greenhouse…………………………………………………. 120

3.7 Effect of antagonistic PGPR on ascorbate peroxidase

activity of maize leaves in pots under axenic conditions of

greenhouse……...………………………………………….. 120

3.8 Effect of antagonistic PGPR on total soluble phenol activity

of maize leaves in pots under axenic conditions of

greenhouse……...………………………………………….. 122

3.9 Effect of antagonistic PGPR on catalase activity of maize

leaves in pots under axenic conditions of greenhouse…….. 122

3.10 Effect of antagonistic PGPR on protein activity of maize

leaves in pots under axenic conditions of

greenhouse………………………………………………….. 124

3.11 Effect of antagonistic PGPR on chitinase activity of maize


greenhouse………………………………………………….. 124

3.12 Effect of antagonistic PGPR on MDA content of maize


greenhouse…………………………………………………. 127

3.13 Effect of antagonistic PGPR on proline content of maize

leaves in pots under axenic conditions of greenhouse……… 127

3.14 Effect of antagonistic PGPR on chlorophyll content of

maize leaves in pots under axenic conditions of

greenhouse………………………………………………….. 129

3.15 Effect of antagonistic PGPR on carotenoid content of maize


greenhouse………………………………………………….. 129

4.1 Lay out of field

experiment…………………………………………………... 145

4.2 Effect of antagonistic PGPR on disease severity/ reduction

under field conditions…………………………………. 147

4.3 Effect of antagonistic PGPR on the IAA/ABA (µg/g) in

maize leaves under field conditions…………..…………….. 149

4.4 Effect of antagonistic PGPR on the SOD activity in maize

leaves under field conditions…………………….. 151

4.5 Effect of antagonistic PGPR on the POD activity in maize

leaves under field conditions …………………………….. 153

Effect of antagonistic PGPR on the PPO activity in maize

leaves under field conditions …………………………….. 155

4.6 Effect of antagonistic PGPR on the ascorbate peroxidase

activity in maize leaves under field conditions …………… 157

x

4.7 Effect of antagonistic PGPR on the total soluble phenol

activity in maize leaves under field conditions……………. 158

4.8 Effect of antagonistic PGPR on the catalase activity in

maize leaves under field conditions 160

4.9 Effect of antagonistic PGPR on the chitinase activity in

maize leaves under field conditions 161

4.10 Effect of antagonistic PGPR on the yield activity of maize

plants under field conditions 163

4.11 Effect of antagonistic PGPR on the 1000 seed weight of

maize plants under field conditions ……... 165

4.12 Effect of antagonistic PGPR on the number of seeds/cob of

maize plants under field conditions 166

5.1 Schematic presentation of steps followed for the detection

of antibiotic biosynthesis genes by using gene specific

primers…............................................................................... 179

5.2 Schematic illustration for the quantitative analysis of

antibiotics through HPLC…………………………………. 181

5.3 Schematic illustration of steps followed for gene expression

studies using Real time PCR………………………………. 183

5.4 Development of inhibition zone by antagonistic PGPR……. 185

5.5 Agarose gel electrophoresis of the PCR products of Pseudomonas

strains with PrnC primer…………………….. 185

5.6 Agarose gel electrophoresis of the PCR products of Bacillus

strains with zamR primer…………………………………….. 186

5.7 Agarose gel electrophoresis of PCR products of

Pseudomonas strains with PhzD/PhzF primers……………. 186

5.8 Agarose gel electrophoresis of the PCR products from

genomic DNA of selected Pseudomonas strains with Phz D

primers………………………………………………….. 187

5.9 Agarose gel electrophoresis of the PCR products from

genomic DNA of selected Pseudomonas strains with Phz F

primers. 187

5.10 Phenazine production by antagonistic PGPR………………. 189

5.11 Inhibition of fungal growth crude phenazine extract of

antagonistic PGPR………………………………………… 190

5.12 Development of inhibition zone by crude phenazine

antibiotic extracted from antagonistic PGPR (Pseudomonas

strains)………………………………………………………. 190

5.13 Agarose gel electrophoresis of the RNA isolated from the

Phz D PCR product of selected Pseudomonas strains……… 191

5.14 Phenazine gene expression in antagonistic PGPR of

Pseudomonas strains……………….……………………….. 191

6.1 Summary of induction of induced systemic resistance by the

application of antagonistic PGPR under axenic conditions of

greenhouse ……… 197

6.2 Summary of experiment conducted for the evaluation of

isolated PGPR as biocontrol agents under field

conditions……………………..…….……………………… 198

xi

LIST OF APPENDICES

Appendix

No.

Title Page

No.

1 Soil Analysis Reagents …………………………………… 252

2 Gram staining....................................................................... 253

3 LB (Lubria-Bertani) Medium………………………........... 254

4 Chitin agar Medium. ……………………………………… 254

5 Pikovskaya’s agar …………………………………………. 255

6 King's medium B…………………………………………… 255

7 CAS agar…………………………………………………… 257

8 Peptone water……………………………………………….. 257

9 Skim Milk Agar…………………………………………….. 257

10 Minimal Agar media (g/L)………………………………….. 258

11 Protein analysis reagents 259

12 Morphological characteristics of 3 day old colonies of

selected rhizobacteria isolated from the rhizosphere soil of

maize grown in fields………………………………………..

259


rhizobacteria isolated from rhizosphere of non-infected

maize fields from irrigated region

260


rhizobacteria isolated from rhizosphere of infected maize

fields from irrigated region

261



maize fields from arid region

262


rhizobacteria isolated from rhizosphere of infected maize

fields from arid region

263



maize fields from semiarid region

264



maize fields from semiarid region

265

19 Development of halo zone by rhizobacteria for the

production of (a and b) siderophore, (c and d}) protease, (e

and f) solubilization of phosphate

265

20 Change in colour from yellow to orange indicating HCN

production 265

21 Quick Test system (QTS) kits for the determination of

carbon/nitrogen utilization pattern 266

22 Development of halo zone by antagonistic PGPR

indicating the production of antibiotics 266

23 Effect of rhizobacteria of different regions on antifungal

activity against F.moniliforme 267

xii

24 Effect of rhizobacteria on plant growth 268

25 Effect of antagonistic PGPR on maize plant infected with

F.moniliforme 268

26 Symptoms of Fusarium stalk rot 268

xiii

ABSTRACT

Biocontrol using plant growth promoting rhizobacteria is an eco-friendly,

sustainable alternative to chemical pesticides. The present investigation was aimed (i)

to isolate and characterize the indigenous antagonistic rhizobacteria which inhibit the

infection and proliferation of Fusarium moniliforme, the casual organism for stalk rot,

(ii) to evaluate their potential as bio-inoculant in pot experiment under axenic

condition as well as under natural conditions of field (iii) to determine the mechanism

of action of the PGPR with particular emphasis on antibiotic production.

In the first experiment, characterization of 117 rhizobacteria, isolated from the

rhizosphere of non-infected and stalk rot infected maize plants grown from Jhang,

Yousafwalla and Islamabad territory, were made. The antifungal potential of the

PGPR were determined against Fusarium moniliforme, Helminthosporium sativum

and Aspergillus flavus. Out of 117 rhizobacteria, 50 rhizobacteria have shown the

potential to inhibit the growth of F. moniliforme, Helminthosporium sativum and

Aspergillus flavus. These rhizobacteria were further tested for the production of

siderophores, antimicrobial secondary metabolites (antibiotics and HCN), production

of hydrolytic enzymes (chitinases, proteases, cellulases) and phytohormone

production (IAA). On the basis of their efficacy, 18 rhizobacteria were selected as

potent biocontrol agent. These selected rhizobacteria were also used as bio-inoculant

on maize in an experiment conducted under axenic conditions. Out of these, six

rhizobacteria codes as 4nm, NDY, JYR, PTWz, Y5 and Yio have shown higher

survival efficiency in soil and significantly improved the growth of maize seedlings.

These rhizobacteria were identified by 16S rRNA gene sequencing and two

antagonistic rhizobacteria JYR, 4nm, NDY PTWz, Y5 and Yio were identified as

Pseudomonas aeruginosa, Pseudomonas sp., Bacillus firmus, Bacillus endophyticus,

and Bacillus pumilus, respectively.

In the second experiment, the efficacy of antagonistic rhizobacteria was

evaluated alone and in combination with fungicide against stalk rot in maize. The

experiment was conducted under axenic conditions in pots. All the antagonistic

rhizobacteria significantly reduced (up to 61%) stalk rot disease in maize plants. The

antioxidant enzymes like superoxidase dismutase, peroxidase, polyphenol oxidase,

ascorbate peroxidase, proteases and chitinases were enhanced significantly in the

rhizobacteria inoculated maize plants. The combined applications of B.endophyticus,

xiv

P.aeruginosa JYR and P.aeruginosa 4nm were at par with the full dose (0.2%) of

chemical fungicide for controlling the growth of F.moniliforme in maize plants.

In the third experiment, the selected rhizobacteria were evaluated as bio-

inoculant on maize under natural conditions of field. Four antagonistic rhizobacteria

including P. aeruginosa JYR, B .endophyticus Y5, P. aeruginosa 4nm and

Pseudomonas sp. NDY exhibited significant decrease (up to 56%) against stalk rot in

field. The percentage decrease in disease severity was higher under axenic conditions

in pots as compared to that of the field experiment. There were significant increase in

enzymes activities, PR proteins and endogenous IAA level in maize leaves. Low

concentration (half dose, 0.1%) of fungicide applied in combination with antagonistic

rhizobacteria augmented the effect of antagonistic rhizobacteria by 1.36 folds.

In fourth experiment, the antagonistic rhizobacteria were characterized for the

production of antibiotics 2, 4, diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN),

Phenazine (Phz), and Zwittermicin A and the genes involved in the biosynthesis of

antibiotics were detected by PCR. The phenazine and pyrrolnitrin biosynthestic genes

were found in three Pseudomonas strains P. aeruginosa JYR, P. aeruginosa 4nm and

Pseudomonas sp. NDY while, zwittermicin A biosynthetic gene was found in Bacillus

endophyticus. The production of phenazine and the expression of its biosynthesis

genes by Pseudomonas strains wee quantified by high performance liquid

chromatography (HPLC) and RT-PCR, respectively.

It is inferred from the results that P. aeruginosa JYR, B. endophyticus and P.

aeruginosa 4nm are the most efficient and consistent antagonist PGPR. Three

Pseudomonas strains produce antibiotic and their expression of genes possibly

correlate with their activity as biocontrol agent.

i

ABSTRACT

Biocontrol using plant growth promoting rhizobacteria is an eco-friendly,

sustainable alternative to chemical pesticides. The present investigation was aimed (i)

to isolate and characterize the indigenous antagonistic rhizobacteria which inhibit the

infection and proliferation of Fusarium moniliforme, the casual organism for stalk rot,

(ii) to evaluate their potential as bio-inoculant in pot experiment under axenic

condition as well as under natural conditions of field (iii) to determine the mechanism

of action of the PGPR with particular emphasis on antibiotic production.

In the first experiment, characterization of 117 rhizobacteria, isolated from the

rhizosphere of non-infected and stalk rot infected maize plants grown from Jhang,

Yousafwalla and Islamabad territory, were made. The antifungal potential of the

PGPR were determined against Fusarium moniliforme, Helminthosporium sativum

and Aspergillus flavus. Out of 117 rhizobacteria, 50 rhizobacteria have shown the

potential to inhibit the growth of F. moniliforme, Helminthosporium sativum and

Aspergillus flavus. These rhizobacteria were further tested for the production of

siderophores, antimicrobial secondary metabolites (antibiotics and HCN), production

of hydrolytic enzymes (chitinases, proteases, cellulases) and phytohormone

production (IAA). On the basis of their efficacy, 18 rhizobacteria were selected as

potent biocontrol agent. These selected rhizobacteria were also used as bio-inoculant

on maize in an experiment conducted under axenic conditions. Out of these, six

rhizobacteria codes as 4nm, NDY, JYR, PTWz, Y5 and Yio have shown higher

survival efficiency in soil and significantly improved the growth of maize seedlings.

These rhizobacteria were identified by 16S rRNA gene sequencing and two

antagonistic rhizobacteria JYR, 4nm, NDY PTWz, Y5 and Yio were identified as

Pseudomonas aeruginosa, Pseudomonas sp., Bacillus firmus, Bacillus endophyticus,

and Bacillus pumilus, respectively.

In the second experiment, the efficacy of antagonistic rhizobacteria was

evaluated alone and in combination with fungicide against stalk rot in maize. The

experiment was conducted under axenic conditions in pots. All the antagonistic

rhizobacteria significantly reduced (up to 61%) stalk rot disease in maize plants. The

antioxidant enzymes like superoxidase dismutase, peroxidase, polyphenol oxidase,

ascorbate peroxidase, proteases and chitinases were enhanced significantly in the

rhizobacteria inoculated maize plants. The combined applications of B.endophyticus,

ii

P.aeruginosa JYR and P.aeruginosa 4nm were at par with the full dose (0.2%) of

chemical fungicide for controlling the growth of F.moniliforme in maize plants.

In the third experiment, the selected rhizobacteria were evaluated as bio-

inoculant on maize under natural conditions of field. Four antagonistic rhizobacteria

including P. aeruginosa JYR, B .endophyticus Y5, P. aeruginosa 4nm and

Pseudomonas sp. NDY exhibited significant decrease (up to 56%) against stalk rot in

field. The percentage decrease in disease severity was higher under axenic conditions

in pots as compared to that of the field experiment. There were significant increase in

enzymes activities, PR proteins and endogenous IAA level in maize leaves. Low

concentration (half dose, 0.1%) of fungicide applied in combination with antagonistic

rhizobacteria augmented the effect of antagonistic rhizobacteria by 1.36 folds.

In fourth experiment, the antagonistic rhizobacteria were characterized for the

production of antibiotics 2, 4, diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN),

Phenazine (Phz), and Zwittermicin A and the genes involved in the biosynthesis of

antibiotics were detected by PCR. The phenazine and pyrrolnitrin biosynthestic genes

were found in three Pseudomonas strains P. aeruginosa JYR, P. aeruginosa 4nm and

Pseudomonas sp. NDY while, zwittermicin A biosynthetic gene was found in Bacillus

endophyticus. The production of phenazine and the expression of its biosynthesis

genes by Pseudomonas strains wee quantified by high performance liquid

chromatography (HPLC) and RT-PCR, respectively.

It is inferred from the results that P. aeruginosa JYR, B. endophyticus and P.

aeruginosa 4nm are the most efficient and consistent antagonist PGPR. Three

Pseudomonas strains produce antibiotic and their expression of genes possibly

correlate with their activity as biocontrol agent.

General Introduction and Review of Literature Chapter 1

1

1.1 Maize

Maize is an important cereal crop being utilized in formulating human food, animal

feed and raw material for various agri-products. It is transformed into number of products

including starch, corn, maltodextrins, corn syrup, corn oil, substrate for fermentation and

distillation products (Bibi et al., 2010). Beside, being a major source of human food, it has

now been employed in the preparation of biofuel.

Product versatility and environmental compatibility are two important features of the

maize crop; make its cultivation possible in broad range of agro-climatic regions (Ferdu et al.,

2002). It is grown from below sea level (from 58 ºN to 40 ºS) to higher altitudes (more than

3000 m), and in areas with minimum 250 mm to maximum 5000 mm of rainfall per annum

(Dowswell et al.,1996) and with a growth cycle ranging from 3 to 13 months (CIMMYT

2000). The edible portion of maize (dry) contains protein (11.1 g), fat (3.6 g), carbohydrates

(66.2 g), vitamins C (0.12 mg), Iron (2.3 mg) , minerals (1.5 g) and calories (342) (Chaudhry,

1994; Gopalan et al., 2007). Due to its excellent nutritional profile, the demand of maize and

related product is continue to increase globally. However, the major limitation in meeting

ever increasing demand of maize is productivity loss as a result of microbial infections.

Therefore, there is a need to find out effective counter measurements against microbial

pathogens.

1.2 Maize Taxonomy

Maize belongs to the grass family Poaceaeandthe tribe Maydeae. “Zea” or “zela” has

been derived from old Greek name means food grass. The genus Zea contains four species

and Zea mays L. is one of the economically important specie (Doeblay, 1990). The

chromosomes number of Zea mays is 2 n = 20.

1.3 Maize in Pakistan

The history of maize in Pakistan is controversial and difficult to track down. It is

generally said that the Portuguese are the introducers of maize in Indian western coast during

16th century. The word “makki” is local name of maize that has been originated from Arabic,

suggests that maize was introduced in sub-continents through Arab African sources. In Pakistan,

maize is an essential cereal crop after wheat and rice (Shah et al., 2011) and it has shown a

steady increase in productivity since 1947 from 0.38 million tons to 3.59 million tons during

2009 (MINFAL, 2009).


2

Fig 1.1: Trends in Area, production and Yield of Maize in Pakistan (FAO, 2013)

Fig 1.2: Pakistan share in world maize production

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

20

01

20

02

20

03

20

04

20

05

20

06

20

07

20

08

20

09

20

10

20

11

20

12

Year

Yie

ld (

Hg/

Ha)

Pro

du

ctio

n (

ton

ne

s)

Area Harvested (Ha) Production (tonnes) Yield (Hg/Ha)

Pakistan 0.5%

Rest of world

Pakistan share in world maize production


3

Currently, maize is being grown on an area of 910000 hectares with annual

production of 3536000 tones and average grain yield of 38857 kg/ha (FAOSTAT, 2013).

Maize production in Pakistan has increased from 487,680 tons in 1961 to 3,536,000 tons

during 2012 (Fig 1.1). Pakistan is at number 25 with 3706910 MT in the world (FAOSTAT,

2012) and have 0.5% share in world total maize production (FAOSTAT, 2012) (Fig 1.2).

A large gap exists between the potential and actual productivity of maize in Pakistan

(UNO, 2000) and major factors responsible for that accounts for this low productivity are the

inappropriate crop nutrition management, insects and diseases and poor agricultural practice.

1.4 Maize growing areas in Pakistan

The major portion of maize production (99%) in Pakistan comes from two provinces,

Khyber-Pakhtoonkhwa (51%) and Punjab (48%). While only 1.0% is produced in the Sindh and

Balochistan (Fig 1.4). Maize is an important crop of AJK with about 0.122 million hectare of

maize being planted during kharif. In Pakistan nearly 66% of the maize has access to irrigation

while, the rest of it is cultivated in rain-fed regions. Maize is cultivated in two main geographic

clusters in Pakistan i.e. eleven districts of AJK and Northern Punjab and nine districts of central

Punjab (Tariq and Habib, 2010)

1.5 Fungal disease of maize in Pakistan

Maize is a considered as one of the most productive crops in the world however, in

Pakistan its production is comparatively lower than expectations (Fig 1.3). The major

obstacle in this productivity loss is microbial diseases. Sitara and Akhtar, (2007) reported that

maize crop is infected more than sixty invading pathogens. Moreover, much of the losses are

directly related to attack of pathogenic fungi. The most common fungal genera that infect

maize crops includes; Fusarium, Aspergillus, Penicillium, and certain xerophytic strains.

Most of these fungi produce toxins in the maize plants (Castellarie et al., 2010). Proliferation

of these fungi is mostly favoured by the moisture content (Gtorni et al., 2009).

There are 11 different fungal species identified from six maize producing districts of

Khyber-Pakhtoonkhwa and Punjab, Pakistan.

The isolated species were A. niger, Aspergillus flavus, A. fumigatus, Alternaria

alternata, Fusarium moniliforme, Macrophomina phaseolina, Cladosporium sp., Drechslera

sp., Penicillium oxalicum, Penicillium sp. and Rhizopus arrhizus. Maximum number (10) of

fungal species were isolated from Okara, Sahiwal and Nowshera, nine species from

Mansehra, while eight were verified from Pakpatan and Mardan (Saleem et al., 2012).


4

Fig 1.3: Maize production in different parts of the world including Pakistan

Fig 1.4: Pakistan major maize growing areas (Tariq and Habib, 2010)


5

F. moniliforme, Aspergillus flavus and A. Niger, attack more than of 50% maize grain

prior to harvest and produce myco-toxins (Bakan et al., 2002). F. moniliforme is one of the

most ubiquitous fungi in maize as described by Fandohan et al., (2003), Bhuttaet al., (2004)

and Aksun (2006). The highest disease occurrence was recorded by Saleem et al., (2012) for

A. flavus from Sahiwal (94%) followed by A. Nigerand F. moniliforme i.e. 62% and 43%,

respectively from Okara. Sitara and Akhtar, (2007) have also observed several fungal species

as pathogen to maize i.e. A. niger, A. fumigatus, A. flavus, Drechslera sp., F. moniliforme and

Rhizopus sp. These findings demonstrate a large spectrum of fungal species that may be a risk

for maize seeds during storage and in the field.

1.6 Stalk rots in Maize

Stalk rot is the most severe and prevalent maize disease, usually caused by intricate

activity of both bacteria and fungi. Stalk rots causes great loss in yield and production due to

lodging of premature plants. Stalk rot is aggravated by stress during grain filling stage of

maize crop (Munkvold et al., 2000). Fusarium stalk rot is induced by the environmental

stresses as Dodd, (1980) observed the reduction in disease severity by reducing the tillage

practice to mitigate the drought stress. Most of the species were seed-borne and sources of

systemic infections that spread from the stalks into the ears (Munkvold et al., 1997). Stalk rot

is normally predictable after harvest as it is essential for the nutrients and organic matter

recycling however; if started prior to the physiological development, it results in yield loss

due to the poor development of ears.

1.7 Fusarium species causing stalk rot in Maize

Fusarium species are universal in soil and act as field fungi which attack more than

50% of maize grains prior to harvest (Robledo-Robledo, 1991). The pathogenic Fusarium

species has been known as one of the major causes of low yield in many crops like maize,

barley and wheat (Schisler et al., 2002). Numerous phytopathogenic Fusarium species are

related with maize including F. proliferatum, F.moniliforme, F. Anthophilum and F.

graminearum (Scott, 1993; Munkvold and Desjardins, 1997).In Pakistan, Fusarium spp.,

followed by Macrophomina phaseolina (Tassi) have been established to cause stalk rot in

maize crop (Ahmad et al., 1995 and 1996) specifically the Islamabad territory and northern

areas of Pakistan (Ahmed et al., 1997).


6

1.8 Fusarium moniliforme

Fusarium verticillioides syn. Fusarium moniliforme (Teleomorph: Gibberella

fujikuroi) is the most important species isolated from diseased plants of maize (Munkvold

and Desjardins, 1997) and from different parts of the world (CIMMYT, 2004) (Fig 1.5). The

central concern related to F. moniliforme is the fact that it produces myco-toxin (fumonisins)

that is a prominent inducer of cancer in human being and disease syndromes in animals

(Nayaka et al., 2008). The Fusarium moniliforme also cause massive losses in grain quality

and yield (Fandohan et al., 2003). Although limited information is available in Pakistan,

certain surveys and reports has shown that F. moniliforme is widespread in different parts of

Pakistan on maize crop and causes economic losses to maize crop in Pakistan (Sitara and

Akhtar 2007; Saleem et al., 2012).

1.8.1 Symptoms of Fusarium stalk rot

Most of the stalk rots show numerous related symptoms, but certain symptomsare

specific to specific fungi.

The common symptoms according to Muppa, (2009) are

1. Disintegration and discoloration of pith tissues/nodes

2. Lodging of stalks

3. Scorching and wilting of leaves

4. Infected maize seedlings show stunted growth, pale or purple leaves and poor root

system.

In mature plants Symptoms of Fusarium stalk rot are hard to differentiate from then

on-infected stalks, the internal tissues of infected stalks are get rotten and turn get reddish

brown. The discolouration may be visible on the stalk surface near nodal region. Stalks are

very weak and lodge easily.

1.8.2 Host range

Fusarium moniliforme syn. verticillioides infects a large range of crops including

sorghum, maize, sugarcane, wheat, banana cotton, tomato and pineapple (CIMMYT, 2004).

1.8.3 Life cycle of Fusarium moniliforme

The endophytic nature of Fusarium moniliforme cause long duration, symptomless

associations with the maize plant (Pitt and Hocking, 1999). They can infect and proliferate in

plant (stems, leaves, roots and grains) but does not show the observable damage to plant. F.

moniliforme can cause infection at all developmental stages of maize plant through various


7

channels including infected seeds, silk channels and wounds, resulting in grain deterioration

during pre and post-harvest of maize crop (Munkvold and Desjardins, 1997).

Fusarium moniliforme remain in debirs or soil overwinter as mycelium or

chlamydospores. Stalk rot fungi infect stalks via colonization of root system, wounds caused

by insects (Gatch and Munkvold, 2002) and natural openings (silk, stomata etc.) The

Fusarium species that infect the plant through roots can also infect the stalks and ears

resulting in stalk rot and ear rot (Logrieco et al., 2002). Many of the pathogenic Fusarium

species enter through the roots, causing root rot prior to pollination and stalk rot later in the

season (Munkvold, 1996). A diagrammatic presentation of the F.moniliforme disease cycle in

maize was presented in Fig 1.6 (Munkvold and Desjardins, 1997).

1. Infection from root to grain via stalk

2. Infection by air-borne or water flapped conidia to silk later on promoted to grain

3. Infection through insect wounds acting as vectors of fungus

1.8.4 Factors effecting the infection of maize with Fusarium

A number of factors favouring infection in maize caused by Fusarium species

according to Fandohan et al., (2003) are as follows

1. Environmental conditions (climate, humidity and temperature)

2. Interactions among fungi and host plant

3. Damage caused by insects before and after harvesting

4. Agricultural practices

5. Maize characteristics

6. Post-harvest operations

Severity to Fusarium stalk rot and reduction in grain yield of maize occurs in both spring and

summer maize crops. During summer, Fusarium moniliforme became more destructive

fungus in causing disease and decreasing the grain yield due to the accessibility of

appropriate hot and humid conditions in Pakistan (Ahmed et al., 1997).

1.9 Control of maize diseases

The control of maize disease is imperative not only for the productivity but also for

reducing the disease induced toxins production which is hazardous to humans and animals.


8

Fig 1.5: The parts of world with Fusarium moniliforme syn. Verticillioides infection of

maize (CIMMYT, 2004)

Fig 1.6: Disease cycle of F. moniliforme on maize showing various infection pathways

(modified from Munkvold and Desjardins, 1997)


9

Tagne et al., (2008) reported different approaches to control the maize diseases, these include

breeding resistance varieties as well as chemical control of diseases, fumigants are also used

to control soil borne pathogens but in turn they cause toxicity to a number of living organisms

in soil (Hasan, 2010).

The most frequently used method is the use of synthetic chemicals for the control of

fungal diseases. Limited product have been registered to control the diseases caused by

Fusarium sp. on maize and other crops, these products also have shown variability in their

efficacy against diseases. Javaid et al., (2006) have reported Metalaxyl+mancozeb and

Dithane M-45 (Mancozeb) as the most operative chemical fungicide in cereals. Sitara and

Akhtar, (2007) also reported the Aliette 80 WP (Aluminium fosetyl) and Ridomil Gold

(Metalaxyl plus mancozeb) as more efficient for maize seed treatment. Other recommended

fungicides include Thiophanate methyl (Bowen et al., 2000), Benlate (Bhutta et al., 2004),

Thiram (Thomas and Sweetinghum, 1999) and Carbendazim (Singh et al., 2006). The use of

chemical pesticide has enhanced the quantity and quality of food but, their use has also

increased the concerns related to environment and non-target organisms (Hasan, 2010).

Pimentel, (2005) have listed some of the adverse effects caused by the

implementation of pesticides for the control of fungal pathogens. These may include hazards

to human and animal health, water and soil pollution and injurious to valuable organisms

(decomposers, natural enemies and pollinators). Synthetic pesticides often stimulate the

development of resistance in pathogen strains as they execute selection pressure on the

pathogen populations (Richardson, 2005).Moreover, Stark, (2008) have reported that the

strict regulatory processes result in withdrawal of a large number of formulations from the

agricultural market (EPA, 2010a; European Commission, 2010). This has generated many

difficulties due to insufficiency of effective pesticides to encounter the pest problems and

need of new formulations to be registered for crop protection (Richardson, 2005; EPA,

2010b).

1.10 Biological control

Biological control has received much attention as one of the most

promisingalternatives to chemical control of phytopathogens i.e. Pythium, Rhizoctoniaand

Fusarium (Khan et al., 2008; Walters, 2009; Hasan, 2010). Generally, the biological control

agents bring about the control of pathogenic microorganisms through antagonism between

pathogen and themselves. Useful microorganisms implemented as biological control agents


10

in integrated management can be an alternative and efficient mean to reduce the use of

synthetic chemical pesticides (Chincholkar and Mukerji, 2007).

Biological control is implemented by two different approaches;

1. Conventional biological control

2. Improved biological control

In the conventional bio-control, an exogenic microbial agent is employed to inhibit particular

pest while augmentation biological control exploits the native populations which are

increased by mass culture.

Biological control is a safe approach to manage the phytopathogens as it is eco-

friendly and has no hazardous effects on animal and human health. A potent biocontrol agent

must have following features:

1. Better rate of survival in soil for a longer time

2. Maximum probability to proliferation of pathogens

3. Environment friendly and has no toxic effect on human and animals

4. Mass multiplication must be economical and easy

1.10.1 Plant growth promoting rhizobacteria (PGPR)

The competing microorganisms specifically those releasing various antagonistic

chemicals favours the growth of plants. Mostly, rhizospheric microorganisms have dominant

role in this context. These bacteria are technically referred as plant growth promoting

rhizobacteria (PGPR) (Bashan and Holguin, 1998). Generally, these microbes impart positive

effect on plant growth by two methods proposed by Glick (1995):

1. Direct method by the stimulation of plant growth and development

2. Indirect method by the biocontrol of soil-borne diseases

PGPR has the capability to control a large array of plant pathogens including viruses,

bacteria, fungi and nematodes; which are the causative agents of various diseases in plants

(Vidhyasekaran et al., 2001; Viswanathan and Samiyappan, 2002). Protection and

stimulation of various crop plants by PGPR has been reviewed by Reed and Glick, (2004)

under both controlled conditions and field trials.

PGPR act as front line defence against the pathogens and antagonise them prior to and

during the primary infection. The first report for the utilization of PGPR as seed treatment

which results in improved growth and biocontrol of pathogens was provided by Burr et al.,


11

(1978) and Kloepper et al., (1980). Furthermore they reported the plant growth promoting

characteristics of Pseudomonas strains which have the antagonistic effect of varied number of

phytopathogens in vitro. Plant growth promoting rhizobacteria suppress the plant pathogens

by direct antagonism (Ryder and McClure, 1997)

The antagonistic bacteria isolated from the rhizosphere of a particular crop well

adapted to that crop and may provide efficient diseases suppression than the rhizobacteria

isolated from the rhizosphere of other plant species. Such plant growth promoting

rhizobacteria (PGPR) act as potent biological control agents, due of their close association

and adaptation to the rhizosphere and specific environmental condition which is main site of

their activity (Cook and Baker, 1983).

1.11 Mechanisms of action undertaken by PGPR in biological control

The antagonists PGPR have shown potential to suppress the growth of fungal

pathogens. Gupta et al., (1999) reported P. aeruginosa as a strong antagonist against the

fungal pathogens (Macrophomina phaseolina and Fusarium oxysporum). Pseudomonas

fluorescent inhibits the growth of Rhizoctonia solani in maize (Tripathi and Johri, 2002).

Ahmadzadeh et al., (2004) has reported the antagonistic activity of fluorescent

Pseudomonads and Bacillus species against fungal and bacterial diseases. P. aeruginosa has

shown the antifungal activity against three fungal pathogens (F.moniliforme,

Helminthosporium halodes and Altenaria solani) due to the production of secondary

metabolites (Sharma et al., 2007).

The antagonistic activity of antagonistic PGPR is usually accompanied by the

production of secondary metabolites (Silva et al., 2001). Most common way for the

antagonistic activity is the direct physical contact between the phytopathogens and biocontrol

agent (Chincholkar and Mukerji, 2007; Hasan, 2010). The mechanisms of biological control

comprise the competition, parasitism, antibiosis, hydrolytic enzymes, and induced systemic

resistance (Haggag and Mohamed, 2007).

1.11.1 Competition

Competition is a synergistic interaction between non-pathogenic and pathogenic

microorganisms for survival and food acquisition (Lorito et al., 1994a). This phenomenon

displays enormous opportunity to control pathogens using non- pathogenic competitors as

biocontrol agent.


12

Fig 1.7: Diagrammatic sketch of mechanism of action used by plant growth promoting

rhizobacteria


13

Generally, nutritional competition believed to have an immense role in combating infectious

diseases, nevertheless, the exact mechanism underlying this phenomenon is remained

conclusive. Biocontrol by nutrient competition can occur when the bio-control agent suppress

the availability of a particular nutrient thereby, limiting the growth of pathogen species. It has

been suggested that biocontrol agents possess superior nutritional uptake or utilizing systems

than pathogens (Harman and Nelson, 1994). For instance, biological control of Pseudomonas

syringae causing leaf frost damage is a proof positive of niche exclusion phenomenon (Chin-

A-Woeng et al., 2003).

Bio-control agent has a competitive advantage over other probably by their ability to

striving better in rhizosphere. They have much developed membrane transport systems for

the assimilation of particular nutrient then other microorganisms (Lugtenberg et al., 2001).

Better micronutrient acquisition is another important parameter that enables

biocontrol agents to act more effectively against pathogenic fungal growth. For instance, iron

has established role in promoting the growth of various fungal pathogens. It has been

demonstrated that rhizobacteria have excellent iron chelating activity which subsequently

scavenges iron supply from other competitors specifically deleterious plant fungal pathogens

(Whipps, 2001).

1.11.2 Parasitism/mycophagy

Parasitism is a well-known microbial interaction through which bacteria gets

nutritional support through fungi and vice versa. Myco-parasitism has been very well

documented in various bacterial and fungal species (Jacobs et al., 2005). However,

biochemical basis of these interactions vary greatly among different fungal species. For

example, Trichoderma elicit necrotrophic effect and produce hydrolytic enzyme to penetrate

the cell wall of host and get required nutrition from it (Zeilinger et al., 1999). Other fungal

parasites obtain the food nutrients by implementing the bio-trophic strategy.

Certain bacteria also get their nutrition by growing on the fungal species and this

phenomenon is known as bacterial mycophagy (Fritsche et al., 2006). Various such bacterial

strains have been reported in literature e.g. Paenibacillus sp. is a physiologically active

bacterial strain inside various fungal species like Fusarium oxysporum. Furthermore other

bacterial strains including P-proteobacteria, Actinomycetes, Myxobactcria and Bacilli also

cause the fungal hyphae lysis by the production of fungicidal compounds (Boer et al., 2005).


14

Table 1.1: List of fungal pathogens suppressed by antagonistic PGPR

Sr. No. Antagonistic PGPR Pathogen suppressed Reference

1. Bacillus subtilis R. solani Merriman et al., 1974

2. Arthrobacter Fusarium oxysporum Sneh et al., 1984

3. Alcaligenes Fusarium oxysporumf.sp.

dianthi

Yuen and Schroth

(1986)

4. B. cepacia F. moniliforme Hebbar et al. , 1992

5. B. subtilisMR 112 R. solani Rosales et al., 1995

6. B. subtilis Macrophomina phaseolina Pal et al., 1996

7. Psedomonas aeruginosa R. solani Rosales et al., 1995

8. Bacillus coagulans MRI F. moniliforme Pal et al., 1996

9. P.fluorescens7-14

Pyricularia

, R. solani Chatterjee et al., 1996

10. P. glumaeEM85 P. ultimum Pal et al., 1996

11. P. fluorescens S. rolfsii Jagadeesh et al., 1998


15

1.11.3 Hydrolytic enzymes production

PGPR produce various extracellular hydrolytic enzymes which play an important role

in disease suppression infected by fungal species. The major constituent of fungal wall are

chitin and β-1,3-glucans (Lam and Gaffney, 1993) which are lysed by hydrolytic enzymes

chitinase and β-1,3-glucanase (Lorito et al., 1993; Lorito et al., 1994a; Lorito et al.,

1994b).The antagonistic PGPR produce these enzymes to suppress the fungal pathogens.

Palumbo et al., (2005) demonstrated biocontrolling activity of Lysobacterenzy mogenes

was associated with the production of β-1, 3-glucanase. In another case, chitinases produced

by Bacillus subtilis AF 1 (Manjula et al., 2004), Bacillus megaterium (Bertagnolli et al.,

1996) and Pseudomonas aeruginosa (Kishore et al., 2005) have antifungal activity.

Fridlender et al., (1993) reported the inhibition of several fungal pathogens by the

Pseudomonas cepacia which has the ability to produce β-1,3-glucanase. The combine action

of chitinases and β-1,3-glucanases have been found to be more efficient in inhibition of

phyto-pathogens (Tanaka and Watanabe, 1995). As described by Laville et al., (1998)

chitinase, β-1, 3 glucanase and cellulase intricate the antagonism against fungal

phytopathogens.

1.11.4 HCN production/ Cyanogenesis

Cyanide has toxic effect on a variety of pathogens by the inhibition of cytochrome C

oxidase and various metallo-enzymes. The cyanogenesis or the production of cyanide takes

place in both plants and bacteria. In bacteria its production takes place by the oxidation of

glycine through HCN synthase. There are numerous reports that support the involvement of

bacterial cyanogenesis in suppression of disease. The HCN producing recombinant P.

PutidaBK8661 strains inhibit the pathogens in wheat crop (Flaishman et al., 1996).

Cyanogenesis acts as defence mechanism in plants where cyanogenic glycosides are

produced as precursor of cyanide and which is compartmentalized in the vacuole where

tonoplast keeps the enzymes glycosides away from the auto-toxicity. It is released when plant

is attacked by pathogens. Bacterial strains may also provide protection to plants from fungal

pathogens by HCN production (Ahmad et al., 2008). P. fluorescence CHAO improves the

root growth and brings about the suppression of black root rot (Defago et al., 1990).

1.11.5 Plant induced resistance

Induced resistance is recognized as influential factor to strengthen defensive ability of

the plants. This is an adoptive response strongly link with certain extrinsic factors including


16

their interaction with microorganisms and stress environmental conditions (Vallad and

Goodman, 2004). These cues led to change in gene expression thereby, plants develop novel

defence strategies against viruses, bacteria, fungus and nematodes (Kessler and Baldwin,

2002).

It has been postulated that induced resistance in plants can be activated in three different

ways:

1. Control of infection with a necrotizing pathogen

2. Usage of various chemicals, like dichloroisonicotinic acid or salicylic acid

3. Colonization of beneficial PGP microorganisms in the rhizosphere (Van Peer et

al., 1991; Metraux et al., 1991).

Now the induction of plant resistance is the subject of extensive interest of many

researchers all over the world. Plant resistance can be categorized into two types (Bakker et

al., 2003).

1. Systemic acquired resistance (SAR): This type of resistance can be elicited by the

exposure to pathogens or by certain chemical (Ross, 196lb).

2. Induced systemic resistance (ISR): The resistance induced by PGPR is broadly termed

as induced systemic resistance (Kloepper et al., 1992).

Systemic acquired resistance (SAR) commonly prevail throughout the life in the plant

body while the Induced systemic resistance (ISR) is diminished with time. Both the

mechanisms use different signal transduction pathways and have been broadly employed for

the integrated disease management in plants (Vallad and Goodman, 2004).

These defence responses may be associated with physical fortification of cells by

lignification and callose deposition, increasing the level of antimicrobial substances

(phytoalexins), synthesis of pathogenesisrelated (PR) proteins (chitinasesand glucanases),

synthesis and accumulation of peroxidases (POD, SOD, PPO, Ascorbate peroxidases)

(Sticher et al., 1997).

a. Systemic acquired resistance

Systemic acquired resistance (SAR) is established in case of attack by necrotizing

pathogen to the plant (Conrath, 2006; Goellner and Conrath, 2008). In the first place when

pathogenic or non-pathogenic organisms are exposed to the plant, a hypersensitive response

is induced that triggers the synthesis and accumulation of pathogenesis related factors (Van

Loon et al., 1998). This phenomenon has been extensively studied in tobacco, cucumber and


17

explained with reference to Arabidopsis (Uknes et al., 1993; Cameron et al., 1994; Mauch-

Mani and Slusarenko, 1994).

The SAR signalling pathway is associated with an increase level of endogenous SA

(Malamy et al., 1990). Gaffney et al., (1993) reported that transgenic plants are unable to

accumulate SA and SAR. Furthermore, SAR genes are expressed by increase in SA (Ward et

al., 1991) which encodes the PR proteins (Van Loon, 1985). These PR proteins exhibit the

antifungal activity bothin vitro (Vigers et al., 1991) and in vivoconditions (Liu et al., 1994).

Exogenous application of SA induced SAR to suppress the similar spectra of pathogens by

following the expression of SAR genes (Uknes et al., 1992 and 1993).

Chitinase and β-1, 3-glucanases are two important pathogenesis related proteins that

show synergistic antimycotic activity. They are associated with SAR pathway regulates the

biosynthesis of salicylic acid. Subsequently, salicylic acid acts as signalling chemical that is

activated by necrotizing pathogens and chemical inducers. These enzymes are also cause

release of certain other molecular species that eventually help in induction of resistance,

release of phytoalexins and synthesis of phenolic compounds by the plants (Mauch and

Stachelin, 1989).

b. Induced systemic resistance

The PGPR induced the resistance in the plant body is known as induced systemic

resistance (ISR). Nanda et al., (2010) defined ISR as the state of PGPR enhanced defence

capability in plants by the activation of latent resistance prior to pathogenic attack. A number

of PGPR strains have been observed to control the plant diseases effectively by the induction

of systemic resistance (Alstrom, 1995).

ISR results in increased biosynthesis and accumulation of various enzymes like

peroxidases (Rajinimala et al., 2003), acid soluble proteins (Zdor and Anderson, 1992),

Phenylalanine ammonia lyase and phytoalexins in the plant tissues (Van peer et al., 1991).

Peroxidase may fortify the cell wall by cross linking the pre-existing wall constituents

(pectin, hemicellulose, callose) and scavenge the reactive oxygen species (hydrogen peroxide

superoxide anion and hydroxyl radical) produced as a result of pathogen attack, by the

production of peroxidases including superoxide dismutase peroxidase dismutase, catalase,

ascorbate peroxidase, and glutathione peroxidase (Singh et al., 2009).

Studies on ISR specified the role of phenylalanine ammonia lyase (PAL) in induction

of systemic resistance (Jetiyanon, 2007). Phenylalanineammonia-lyase (PAL) results in

production of lignin and phytoalexins through the phenylpropanoid pathway. The first step

which involves the convertion of phenylalanine to cinnamic acidis catalysed by PAL.


18

Cinnamic acid acts as the precursor of lignin, salicylic acid, some pigments (anthocyanidins,

condensed tannins, and phytoalexin phenylpropanoids or phenol) (Lamb and Dixon, 1997).

The PR proteins, chitinase and β-1,3-glucanase mostly detected at the fungal

penetration site where these enzymes degrade the cell wall and contents of cell (Benhamou et

al., 1996). These enzymes may be released in response to PGPR elicitors (Ren and West,

1992) which subsequently inhibit the growth of fungal pathogen including Fusarium

oxysporum (Singh et al., 1999) and Botrytis cinerea (Frankowski et al., 2001). Numerous

Bacillus and Pseudomonas strains suppress the phytopathogenic fungi by the production of

chitinases and β-1,3-glucanase (Bressan and Figueiredo, 2010).

1.11.6 Antibiosis

Antibiosis referred to a natural pathogen controlling process by which elimination of

pathogens is carried out by producing low molecular weight antagonistic metabolites by

PGPR. These biochemicals show excellent antimicrobial activity at relatively lower

concentrations (Fravel, 1988).

Several studies demonstrated the role of antibiosis in biological control of soil borne

pathogens (Getha and Vikineswary, 2002). Production of different antibiotics by various

microorganisms that are actively involved in biocontrol of pathogens indicated strong

antagonistic activity. Table 1.2 presents list of broad range of antibiotics produced by these

microorganisms.

1.11.6.1 2,4-Diacetylphloroglucinol (DAPG)

2,4-DAPG is a frequently reported phenolic compound showing excellent antiviral,

antifungal, antibacterial, antitumor activities and phytotoxic properties (Haas and Keel,

2003). Various microorganisms have metabolic capacity to produce 2, 4-DAPG. For instance,

different plant associated Pseudomonads strains that exhibited biocontrolling characteristics

are known to produce this metabolite (Ramette et al., 2006).

2,4-DAPG producing Pseudomonads strains have been isolated from wide range of

ecological habitats from the rhizosphere as well as endosphere of maize, pea, and wheat

plants (Raaijmakers and Weller, 2001; Landa et al., 2002; Bergsma-Vlamiet al., 2005).

It has been reported that mutations in gene cluster of DAPG led to a multifold

decrease in biocontrol activity of antagonistic bacteria (Nowak-Thompson et al., 1994).

Moreover, DAPG producing microbial community and antibiotic production are the two main

causes for pathogen elimination from different soils.


19

Table 1.2: Important antibiotics produced by antagonistic PGPR

Sr. No. Antibiotics Reference

1. 2, 4- Diacetylphloroglucinol (2, 4-DAPG) Ramette et al., 2003

2. Phenazine-1 -carboxylic acid Delaney et al., 2001

3. Phenazine-1-carboxamide Chin-A-Woeng et al, 2001

4. Pyoluteorin (PLT) Souza and Raaijmakers, 2003

5. Pyrrolnitrin (PRN) Brodhagen et al., 2004

6. Zwittermicin A Raffel et al., 1996

7. Kanosamine Milner et al., 1996

8. Butyrolactones Gamard et al., 1997

9. Oligomycin A Kim et al., 1999

10. Oomycin A Howie and Suslow, 1991

11. Mycosubtilin Leclere et al., 2005

12. Herbicohn Sandra et al., 2001

13. Bacillomycin D Moyne et al. 2001

14. Surfactin Ajlani et al.,2007

15. Iturin A Kloepper et al., 2004


20

Association of different DAPG producers in the rhizosphere of crop plants was responsible

for disease suppression (Raaijmakers et al., 1999).

Mode of action of DAPG

It is believed that DAPG targets a vital cellular component or metabolic processes of

the fungal cell. Kwak et al., (2011) have highlighted most probable mode of action of 2,4-

DAPG in combating the fungal pathogens. The proposed mechanism of 2,4-DAPG are as

follows;

1. 2,4-DAPG is believed to alter membrane structure and function thereby, decrease

their growth rates

2. 2,4-DAPG interferes the homeostasis of cell by triggering accumulation of

superoxide radical and hydrogen peroxide.

3. Being a phenolic species, 2,4-DAPG has excellent antimicrobial activity mediated via

manipulation at cellular surfaces (http://pubchem.ncbi.nlm.nih.gov/).

4. 2,4-DAPG can cause impairment of mitochondrial function in fungal cell (Gleeson et

al., 2010).

Genes involved in 2,4-DAPG biosynthesis and regulation

The biosynthetic operon responsible for 2, 4 diacetylphloroglucinol synthesis has

been well characterized in P. Fluorescens Q2-87 (Bangera and Thomashow, 1999). It

consists of genomic region of about 6.5 kb long in which six open reading frames has been

identified, four of which phlACBD comprise an operon and are indispensable for the

production of DAPG as well as its precursor monoacetyl phloroglucinol (MAPG). Three

genes phlACB are found to be conserved between eubacteria and archaebacteria while phlD

gene encodes a typeIIIpolyketide synthase homologous to the chalcone and stilbene synthase

from plants (Austin and Noel, 2003).

The remaining two genes phlE and phlF encoding putative efflux protein and

regulatory repressor protein respectively are transcribed divergently and occupies position on

either side of the biosynthetic loci. In addition to these, two new open reading frames phIG

and phlH present at downstream of phlF have also been reported by Schnider-Keel et al.,

(2000). The arrangement of genes encoding various products to synthesize 2, 4- diacetyl

phloroglucinol is shown in the Fig 1.9.

http://pubchem.ncbi.nlm.nih.gov/


21

Fig 1.8: A possible model for the mechanisms of action of 2,4-DAPG

(Kwak et al., 2011)

Fig 1.9: Genes involved in DAPG biosynthesis (Fernando et al., 2005)


22

Factors affecting DAPG production

Biotic and abiotic factors associated with the crop and environment affect the

performance of fluorescent Pseudomonads (Duffy and Defago, 1997; Notz et al., 2002) for

the production of DAPG. Biotic factors such as plant species, plant age, cultivar and

pathogens alter the expression of the gene phlA (Notz et al., 2001).

The DAPG production is influenced by abiotic factors such as carbon sources and

various minerals. Fe3+

and sucrose increased DAPG production in P. fluorescensF113, while

glucose increased DAPG production in P. fluorescens Pf-5 and CHA0 (Nowak- Thompson et

al., 1994; Duffy and Defago, 1999). In P. fluorescens strain highest DAPG yield was

obtained with ethanol as the sole source of carbon. Micronutrients Zn2+

, Cu2+

and Mo2+

stimulated DAPG production in P. fluorescensCHA0 (Notz et al., 2001).

1.11.6.2 Pyrrolnitrin

A chlorinated phenylpyrrole antibiotic produced by different PGPR is Pyrrolnitrin [3-

chloro-4-(3-chloro-2-nitrophenyl) pyrrole] which is used to suppress a wide number of

Pyhtopathogens. It was isolated from P. aeruginosa for the first time by Takeda, (1958),

afterwards it was isolated from the strains of P. fluoresens (Pf-5 and CHAO) (Bender et al.,

1999). It is reported to be produced by a number of Pseudomonads (fluorescent and non-

fluorescent) and also by many other genera including Enterobacter agglomerans,

Burkholderia pyrrocinia, Serratia and Myxococcus fulvus (Hammer et al., 1999).

Pyrrolnitrin (PRN) doesn’t readily diffuse and persist for a long time in soil even

more than one month. It releases slowly after the lysis of bacterial cell. The capability of

PRN produced by rhizobacteria to control the fungal diseases is well studied area. For

example, Hill et al., (1994) reported the inability of P. fluorescensBL915 (mutant strain

deficient in PRN production) to inhibit the growth of R. Solani in cotton. Likewisethe cloning

of PRN biosynthesis genes in non-antagonistic and pyrrolnitrin deficient strain enable the

strain to control the disease both under in vitro and in vivo conditions. Furthermore, P.

Fluorescens strains producing the pyrrolnitrin decreased the prevalence of all disease in

wheat (Tazawa et al., 2000). It has a wide range of activity against fungi comprising

ascomycetes, basidiomycetes, and deuteromycetes (Ligon et al., 2000).


23

Fig 1.10: Schematic illustration of Pyrrolnitrin Biosynthesis (Fernando et al., 2005)


24

Mode of action

Mechanism of action of Pyrrolnitrin was described by Tripathi and Gottlieb, (1969).

The electron transport chain at it terminal end at succinate or reduced nicotinamide adenine

dinucleotide (NADH) is main site of action observed in S. cerevisiae. It also restricts the

coenzyme Q activity and inhibits the respiration. This antibiotic has no effect on the activity

of cytochrome oxidase but it inhibit the working of mitochondrial enzymes involve in its

preparations i.e. NADH oxidase, succinate oxidase, NADH cytochrome C reductase,

succinate-coenzyme Q reductase and succinate cytochrome C reductase. In addition,

pyrrolnitrinalso prevent the reduction of dichlorophenolindophenol, tetrazolium dye and 2-

iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium.

Biosynthesis and genes involved

Tryptophan acts as a precursor for the synthesis of pyrrolnitrin (Chang, 1981). The

prnABCD operon is responsible for biosynthesis of pyrrolnitrin. The prn operon is located in

5.8 kb DNA and consists of four ORFs (open reading frames) prnA, prnB, prnC and prnD.

All ORFs transcribed as a single unit. L-tryptophan is converted to 7-chloro-trp by it

chlorination and further converted to amino pyrrolnitrin which is oxidized to from

pyrrolnitrin (Hammer et al., 1997). These biosynthesis reactions of pyrrolnitrin are catalyzed

by prnA and PrnDgene respectively (Nakats et al., 1995). Biosynthesis of pyrrolnitrin is

illustrated in schematic form in Fig 1.10. The prn operon is regulated by the regulatory gene,

gacAas described by Souza and Raaijmakers (2003).

1.11.6.3 Phenazine

Phenazine is nitrogen containing low molecular weight, heterocyclic antibiotic

comprising of bright coloured pigment (Kavitha et al., 2005). It is produced by a large array

of bacteria belonging to genera Pseudomonas, Brevibacterium, Streptomyces and

Burkholderia (Gealy et al., 1996; Anjaiah et al., 1998; Tambong and Hofte, 2001).

Phenazines embrace a diverse group of more than 100 different structural derivatives

of phenazine in nature and above 6,000 compounds has been synthesized which contain

phenazine as a main moiety (Mavrodi et al., 2006). The sole source of natural phenazines is

bacteria including Pseudomonas, Sorangium, Nocardia, Brevibacterium, Erwinia,

Burkholderia, Pantoea agglomerans, Pelagiobacte rand ibrio V (Mentel et al., 2009).

The important phenazine derivatives produced by antagonistic PGPR are pyocyanin

(PYO), phenazine-1-carboxylic acid (PCA) phenazine-1-carboxamide (PCN) and 1-


25

hydoxyphenzine (Chin-A-Woeng et al., 2001; Haas and Defago, 2005). Some PGPR strains

are able to produce 10 different derivatives of phenazine at one time (Smirnov and

Kiprianova, 1990). Many strains synthesize more than one Phz derivatives simultaneously.

For example, concurrent production of PCA and PCN was observed in P. aeruginosa; P.

aureofaciens (Pierson et al., 1995) and P. chlororaphis (Chin-A- Woeng et al., 1998).

Derivatives of phenazine

The Phenazine produced by different rhizobacteria has shown the control of diverse

range phytopathogenic fungi (Mavrodi et al., 2006). P. chlororaphis strain 30-84 produces

three phenazine derivatives (Pierson and Thomashow, 1992), P. fluorescens strain 2-79

produces PCA, while P. chlororaphis strain PCL1391 produces PCN and PCA (Chin-A-

Woeng et al., 1998). The PCN exhibited fungal growth inhibition of Fusarium oxysporum,

PCA and PCN control root rot caused by Pythium (Tambong and Hofte, 2001) while PYO

inhibited Septoriatritici of wheat (Flaishman et al., 1990). The capability of phenazine

production is also correlated with bacterial persistence in rhizosphere in the vicinity of the

native microbial community (Schoonbeek et al., 2002).


26

Mode of action

The mechanism for the action of phenazines is not well studied but it is presumed that

they diffuse across the membrane and bring about the uncoupling of oxidative

phosphorylation and ROS production (superoxide radicals and hydrogen peroxide) harmful to

host plant (Mavrodi et al., 2001) by acting as a reducing agent (Hassett et al., 1992; Mahajan

et al, 1999). The enhanced level of antioxidant enzyme (SOD) in the plant inoculated with

pyocyanin producing P. aeruginosa also supports the role of phenazine in inducing the

resistance against diseases (Hassett et al., 1995). Hence, show their active role in induction of

systemic resistance (ISR) in plants. Important functions of phenazines include (Fig. 1.11).

1. Induction of plant defence pathway

2. Play important role in electron shuttling

3. Iron chelator

4. Biofilm formation

5. Signal transduction to modulate the gene expression

P. aeruginosa elicited the ISR in rice, by reducing the development of rice blast

pathogen Magneporthe grisea. PYO is found to be critical for ISR induction as

Vleesschauwer et al., (2006) reported the loss of ISR against M. grisea in the absence of PYO

production. It is commercially marketed as Cedomon (BioAgri AB, Uppsala, Sweden).

The ecological competence and persistence of rhizobacteria is accredited to phenazine

(Mazzola et al.,1992).The terminal electron acceptors unavailability usually limits growth

and survival of bacteria in rhizosphere due to generation of low energy. This condition results

in a state of reduced intracellular redox has a high ratio of NADH/NAD+ (Graef et al., 1999).

P. aeruginosa is maintain the ratio of NADH/ NAD+ by the production of PYO, by acting as

alternate electron acceptor which bring about the oxidation NADH to NAD+

(Price-Whelan et

al., 2007). Wang et al., (2009) also reported the PYO role in survival of bacteria under

anaerobic conditions. The P. chlororaphis PCL1391is able to convert ferric hydroxides to

ferrous under acidic conditions by producing phenazine-1-carboxamide (PCN).The incapable

iron-reducing bacterium Shewanella oneidensis MR1 mineralize Fe3+

hydroxides by utilizing

exogenously applied PCN, and enable it to develop under electron acceptors limited

condition (Hernandez et al., 2004).


27

Fig 1.11: Mode of action of Phenazine derivatives


28

Phenazine biosynthesis

Phenazine biosynthetic precursor is shikimate acid pathway which also acts as

precursor for the synthesis of many other aromatic amino acids like tryptophan,

phenylalanine and tyrosine.

Firstly a condensation reaction takes place catalyzed by DAHP synthase and brings

about the conversion of primary metabolites phosphoenol pyruvate and erythrose-4-

phosphate in to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) (Hermann, 1995a;

1995b). DAHP is further pass through a series of conversions resulting in the formation of

shikimic acid and finally chorismate, which in turns formed the amino-2-deoxyisochorismic

acid (ADIC). It has been reported that in shikimate biosynthetic pathway, ADIC is the point

from which divergence occur for the formation of tryptophan or phenazine (Mcdonald et al.,

2001). L-tryptophan also acts as precursor for pyrrolnitrin, proposing an evolutionary link

between biosynthesis of pyrrolnitrin and phenazine.

Genes involved in Phenazine biosynthesis

The phenazine-1- carboxylic acid (PCA) and its derivative is well studied in different

strains of Pseudomonas. The production of PCA implicates 5 genes cluster including

phzFABCD in P. aureofaciens (Pierson et al., 1995). The biosynthetic operon of phenazine

consist of seven gene core in P. chlororaphis PCL1391 (Stover et al., 2000) and

P.fluorescens2-79 (Mavrodi et al., 2004).

The biosynthesis of phenazine (PCA) is accomplished by a set of seven genes

containing phzABCDEFG. PhzC encodes DAHP synthase, is the enzyme that ensures the

movement of primary metabolites to chorismic acid in shikimate pathway. Chorismic acid is

the point where the PCA biosynthesis is catalyzed by phzABDEFG. These seven core genes

of phenazine biosynthesis have been identified in almost all the bacterial strains which have

the ability to produce phenazine derivatives. Other genes have also detected which regulate

the biosynthesis of phenazine.

The gene phzH is found downstream of phenazine operon in P. chlororaphis

PCL1391 is amino-transferase gene involved in the conversion phenazine-1-carboxamide

(PCN) from PCA (Chin-A-Woeng et al., 1998).


29

Fig 1.12: Biosynthesis of Phenazine (Pierson and Pierson, 2010)


30

1.11.6.4 Zwittermicin A

Zwittermicin A is a novel antifungal metabolite secreted by several Bacillus sp. It is

widely distributed such that the minimum population of Zwittermicin A producers is 104

CFU/g of the soil throughout the world (Raffel et al., 1996). The antibiotic is responsible for

the inhibition and control plant diseases caused by oomycetes. Zwittermicin is the only

known aminopolyol antibiotic and has structural features in common with peptide and

polyketide antibiotics.

The alternating hydroxyl groups on the carbon backbone are similar to partially reduced

polyketide structure; the nitrogen rich end of zwittermicin may be derived from an amino

acid, possibly citrulline, similar to peptide antibiotic. Zwittermicin A biosynthesis takes place

by a non-ribosomal peptide synthetic pathway. The genes responsible for zwittermicin A

production are located on 16 kb cluster which have nine orfs and a self-resistant gene zmaR

(Stohl et al., 1999).

Mode of action

Spontaneous zwittermicin A-resistant mutants of Escherichia coli are affected in

genes encoding subunits of RNA polymerase; however, zwittermicin A does not appear to

inhibit RNA transcription in vivo, suggesting that zwittermicin A has an unusual mode of

action.

It is possible that zmaR could be transferred to these target organisms, short-circuiting

the ability of B. cereus to control plant disease. It has long been suggested that antibiotic-

producing organisms are the source of the antibiotic resistance genes found in clinical isolates

because the biochemical mechanisms of antibiotic resistance from antibiotic-producing

organisms and target organisms are similar.

The prevalence of zmaR in an agricultural setting concomitant with selection pressure

from zwittermicin A, may represent an analogous situation for plant pathogenic oomycetes

and other soil microflora. Further studies of zmaR will contribute to our understanding of

zwittermicin A resistance and may aid in the development of strategies to reduce the rate of

appearance of resistance in target organisms.

Aims and scope of present research work

Fusarium moniliforme is a fungal pathogen which causes stalk rot disease on maize

plant and significantly limits its production worldwide. Various management practices like

developing disease resistant varieties, use of pathogen free seed and different fungicides have

been adopted to combat the disease but these approaches have limitations e.g. the newly

http://en.wikipedia.org/wiki/Biosynthesis

http://en.wikipedia.org/wiki/Gene


31

developed resistant varieties become susceptible to new races of the pathogen which develop

due to excessive use of pesticide. The use of pathogen free seed is effective but the pathogen

usually penetrates into field through irrigation. Few fungicides are effective in controlling

disease but they are banned due to health hazardous effects. Hence, there is urgent need to

develop any alternate management strategies to control the disease and minimize yield losses

of maize.

Plant growth promoting rhizobacteria belonging to various genera like Pseudomonas,

Burkholderia and Bacillus are being used to suppress the soil borne pathogens causing

diseases in different crops (Cazorla et al., 2007; Romero et al., 2007). Control of several

diseases on different crops such as bean, carnation, rice, and cucumber using PGPR strains is

well documented (Wei et al., 1991; Nandakumar et al., 2001). Application of the

Pseudomonas strains reduced red rot incidences ignificantly on sugarcane crop under field

conditions (Viswanathan and Samiyappan, 1999).

The present investigation was aimed at the determining the antifungal metabolite

produced by the antagonistic PGPR and to have a comparative account of the antagonistic

PGPR with that of Ridomil Gold (synthetic fungicide). The efficacy was checked both in pot

under axenic conditions and in field under natural conditions.

The objectives were met with

Screening of PGPR antagonistic to F.moniliforme

1. Isolation of bacteria from maize rhizosphere

2. Elucidation of biocontrol mechanisms of the antagonistic bacteria

Characterization of potent antagonistic bacterial strains

1. In vitro selection of antagonistic isolates

2. In vivo evaluation of bio-antagonists in pot and field conditions

3. Identification of potent antagonistic strains by sequencing 16S rRNA gene

4. Detection of antibiotics produced by the antagonistic PGPR

5. Expression of genes involved in antibiotic biosynthesis by the antagonistic PGPR

Isolation and screening of rhizobacteria for antagonistic activity Chapter 2

32

2.1 INTRODUCTION

The integrated management of diseases all over the world is usually

accomplished by the application of fungicides. This method has serious limitation due

to the concerns related to health of human being, environmental pollution,

development of resistance against fungal strains and negative effect on the beneficiary

microbes present in ecosystem. Therefore it is imperative to search out the

environment friendly alternative methods to combat the fungal pathogens

(Ghisalberti, 2000).

The use of beneficial microorganisms could be an environmentally sound

option to increase crop yields and reduce disease incidence. One of the best strategies

of this crop disease management program is the isolation of bacteria from the natural

resources like soil, water and plants that exhibit antifungal property (Blondelle, 1992).

The isolation and screening of these biological antagonistic agents from natural

resources may exhibit certain novel characteristics that can be effectively used against

certain phytopathogens.

Rhizosphere and the surrounding regions inhabiting the roots are occupied by

a large number of microbial communities which exert favourable, neutral or harmful

effects to plant growth (Whipps, 2001). Among the existing micro flora, rhizobacteria

have been an important consideration, not only for their wide distribution and

diversity of the population, but also for its ability to provide a wide range of bioactive

metabolites with antimicrobial properties as well as their potential for plant growth

(Lange et al., 1996). These rhizobacteria can be a perfect choice to be used as

biocontrol agents, as rhizosphere delivers the front line defense to plant roots against

pathogen attack. Pathogens come across antagonism with rhizobacteria before and

during the development of primary infection and during their secondary spread in the

plant roots.

The extent to which the antagonistic microbes suppress the pathogen depends

on production of secondary metabolites and their metabolism in soil. The greater the

microbiological activities, the more carbon, nutrients and energy are used, thus,

rendering the pathogen weak (Sullivan, 2004). The microbial diversity in the fertile

soil is usually higher than the infertile soil. Therefore, the fertile soil produces more

biomass and microbial activities that inhibit the activities of soil borne pathogens.


33

The microbes in the fertile soil tend to lower the intensity of the disease resulting

from the soil borne pathogen (Garbeva et al., 2004). The more diverse the soil

microbial population, the greater the possibility to find antagonistic microbes which

potentially can be used to control the soil borne pathogens

Antagonistic rhizobacteria suppress the soil borne pathogens by direct and

indirect method. Direct effects involves the interaction of rhizobacteria with the

pathogen which include competition for niches (for colonization or infection

establishment) and nutrients (carbon and nitrogen sources as nutrients and signals),

production of phytohormones (IAA), competition for iron through the production of

iron-chelating compounds (siderophores), by the production of antimicrobial

secondary metabolites (antibiotics and HCN), production of hydrolytic enzymes and

parasitism (degradation of pathogen germination factors or pathogenicity factors).

These methods are accompanied with indirect mechanisms by inducing the systemic

resistance. A potential biological control agent has the ability to suppress the soil

borne pathogens by using the combination of different mechanisms (Whipps, 2001).

The ability of rhizobacteria to obtain iron via siderophores may results in

efficient root colonization and inhibition of phytopathogens by competition (Crowley,

2006) and help to improve the growth of plant. The biocontrol abilities essentially

depend on aggressive colonization of roots of host plant, induction of systemic

resistance and production of antifungal metabolites including antibiotics (Haas and

Keel, 2003). The production of antibiotics is normally associated with the capability

of plant growth promoting rhizobacteria to inhibit the growth of phytopathogens

(Glick et al., 2007). The antibiotic secretions kill or decrease the growth of the target

pathogen (Lugtenberg and Kamilova, 2009). Lytic enzymes (β -1, 3-glucanase,

cellulases, chitinases and proteases) produced by microorganisms act on fungal cell

wall which is made up of chitin, cellulose and hemicellulose etc. and results in

inhibition of phytopathogens (Chin-A-Wing et al., 2003). The interactive effect of

antibiotics, hydrolytic enzymes, hydrogen cyanide and siderophore production results

in effective biological control.

Aims and objective

The present work was designed to isolate the rhizobacteria antagonistic against

selected fungal pathogens from different areas of Pakistan. For this purpose soil

samples from rhizosphere of maize infected with and without stalk rot disease were

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571425/#b13-gmb-35-1044







34

collected from three different regions (NARC Islamabad, Jhang and Sahiwal

(Yousafwala) differing in soil moisture content. Furthermore, the indigenous

rhizobacteria which possess better antagonistic characteristics and multiple plant

growth promoting activities were screened and selected as potent biocontrol agents for

substituting the conventional use of commonly applied fungicide.


35

2.2 MATERIAL AND METHODS

A systematic study was undertaken to isolate indigenous antagonistic

rhizobacteria from the farmer fields of maize growing at three different agro-

environments of Pakistan, in order to evaluate their potential against different

pathogens infecting maize crop, their mechanisms of biocontrol and plant growth

promotion was assessed. The rhizosphere soil samples of maize crop were collected

from semi-arid areas of Yousafwalla, Sahiwal (15% soil moisture) and arid areas of

Jhang (9% soil moisture). Samples collected from irrigated area of National

Agricultural Research Centre, Islamabad (26% soil moisture) were taken as control.

Summary of field attributes and climatic characteristics of experimental locations is

given in Table 2.1.

2.2.1 Collection of soil samples

Samples of soil were collected from the rhizosphere of maize fields infected

with stalk rot at a depth of 6 cm from the vicinity of the roots of maize plants. In

parallel, soil samples were collected from non-infected rhizosphere of maize fields.

Sampling was done at reproductive stage (tasseling stage, 60 DAS). Each sample was

taken in separate polythene bag, labelled and stored at 4oC for further processing.

2.2.2 Soil Analyses

2.2.2.1. Moisture Content of Soil

At the time of sampling, moisture content of soil was determined

gravimetrically. Soil (20 g) sample was taken at a uniform depth of 6 cm from the

surface of soil. Fresh weight of the samples was recorded. Dry weight was determined

after drying the soil in oven for 72 h at 7oC till constant weight.

Soil moisture (%) = (Weight of wet soil (g)-Weight of dry soil (g) x 100

Weight of dry soil (g)

2.2.2.2. Soil pH and Electrical Conductivity (EC)

Fresh soil (20 g) was sieved and placed in a beaker. Sterilized water (40 mL)

was added and stirred vigorously for 30 min on a magnetic stirrer afterwards pH

(Horiba pH meter) and EC (Milkaukee Smar EC system, SM 301 make USA) was

determined (Radojevic and Bashkin, 1999).


36

Table 2.1: Summary of field attributes and climatic characteristics of

sampling locations

location Climatic zone Latitude

Nº

Longitude

Eº

Altitude

(m)

Islamabad

Irrigated region

(humid subtropical climate ) 33° 42' 0" 72° 10' 0" 457 to 610

Jhang

Arid 31° 17' 06 72° 23' 18 158

Sahiwal

(Yousafwala) Wet Semi-Arid 30

o 40´ 73

o 06´ 172

Table 2.2: Physicochemical properties of soil used for isolation of rhizobacteria

Soil samples

Soil moisture

content Soil texture pH EC Ca

2+

Mg2+

K+

Na+

(%) dS/m (µg/g)

Group

1

Irrigated area 26 Sandy clay

loam 7.9 0.61 40.2 5.163 15.1 22.12

Semi-arid area 15 Clay loam 6.8 1.16 36.01 4.85 11.87 18.90

Arid area 9 Sandy loam 7.8 0.55 35.87 3.42 12.7 17.98

Group

2

Irrigated area 26 Sandy clay

loam 7.8 0.68 41.68 6.172 15.2 21.87

Semi-arid area 15 Clay loam 7.1 1.12 35.12 4.22 12.28 18.67

Arid area 9 Sandy loam 7.6 0.52 36.01 3.32 12.9 9.65


37

2.2.2.3. Soil Nutrients Analyses

The analyses of macronutrients including Na+, Ca

2+, Mg

2+, K

+ of soil samples

were done by ammonium bicarbonate-DTPA method as described by Soltanpour and

Schwab (1977). The methods for the preparation of stock solutions, reagents,

standards solutions and working solution are given in appendix 1.

2.2.2.4. Soil Texture

Soil texture was assayed by air drying the soil samples which were further

sieved (2 mm sieve) and 100 g of it was added in to 500 mL beaker as described by

the method of Brady, (1990). Hydrogen peroxide (5 mL) was dropped down until the

effervescence diminished. At that time, 15 mL sodium oxalate solution (0.5 N

dispersing agent) and 200 mL distilled water was added, stirred for a little while and

left for 24 h. Afterwards, soil solution was shaken for 20 min and transferred to 1000

mL cylinder to make volume of solution upto1000 mL by using sterile water make

and made homogeneous mixture by vigorous shaking. Two readings were recorded

after 5 min. and after 5 h of sedimentation. Suspension temperature was also recorded

both at the start and end of the experiment.

Percentage of sand, clay and silt = CHR X 100

Weight of soil taken

CHR= hydrometer reading (Corrected after temperature adjustment)

Textural class of soil was determined by means of textural triangle (US Department of

Agriculture Classification System).

2.2.3 Isolation of rhizobacteria from soil

Soil (1 g) was properly mixed in autoclave distilled water (90 mL) for the

isolation of indigenous rhizobacteria. Decimal dilution was made and an aliquot (100

μl) from three dilutions (10-2

, 10-4

and 10-8

) was inoculated on Luria-Bertani (LB)

medium in triplicate and incubated at 28°C for 2 d (Aneja, 2002). The colonies which

have been well isolated were re-streaked on fresh LB plates by using four way

streaking method. The purified strains were maintained and stored in 80% glycerol at

-20°C. The isolated rhizobacteria were divided in to two groups on the basis of

weather they were isolated from the rhizosphere of maize fields infected without or

with the stalk rot from each region (Table 2.3).


38

Table 2.3: Groups of indigenous rhizobacteria

Groups Rhizobacteria

Group 1

Rhizobacteria isolated from

maize fields without stalk rot

Group 2 Rhizobacteria isolated from

maize fields infected with stalk rot


39

2.2.4 Tests for antagonism

Antagonistic activity of rhizobacteria was evaluated following the agar tube

dilution method as described by Washington and Sutter, (1980) by using cell free

supernatant of rhizobacteria.

2.2.4.1 Preparation of cell free supernatant

The rhizobacteria were inoculated in LB broth and incubated in a shaker

(Excella E24, New Brunswick Scientific, USA) at 125 rpm for 48 h. The

rhizobacteria were centrifuged and filtered through Millipore filters (0.22 µm). The

subsequent cell free supernatant was used for the assay of antifungal activity.

Cell free supernatant (67 µl) of each rhizobacteria was inoculated in sabouraud

dextrose agar (Sigma-Aldrich) autoclave in screw capped or cotton plugged test tubes

after cooling it at 50°C. The test tubes were placed in inclined position to prepare the

slants in triplicate. Afterwards, fungal plug (4 mm diameter) from seven days old

culture of fungus tubes was inoculated in each slant and incubated for 7 d at 28°C.

Reading was taken by measuring the linear length (cm) of fungal growth. Percentage

inhibition (%) of fungal growth for cell free supernatant of each rhizobacteria was

calculated by the following formula

Percentage inhibition (%) = 100 - Linear growth in test tubes (cm) x 100

Linear growth in control (cm)

2.2.5 Phenotypic characterization of rhizobacteria

Morphological characteristics of rhizobacterial colonies were observed on LB

agar plates. The three old cultures were used to determine the following

characteristics including size, shape, elevation, margin, surface, colour, odour and

pigmentation.

Rhizobacteria were grown over night on KB (kings medium B) agar medium

and visualized under 366mm UV light to evaluate fluorescence of the strains.

2.2.6 Determination of phosphate solubilizing activity

Phosphate solubilizing activity was evaluated by inoculating the rhizobacteria

on Pikovskaya agar plates. The development of halo zone sfter incubation for 7 d at

28°C, was taken as positive for phosphate solubilization (Katznelson et al., 1959).

Solubilization Index = (Colony diameter (mm) + halo zone diameter(mm)

Colony diameter (mm)


40

2.2.7 Production of hydrogen cyanide

Hydrogen cyanide (HCN) production was assayed by spreading rhizobacteria

on LB agar supplemented with 4.4 g L-1

glycine. Filter paper strip was flooded with

the solution containing picric acid (0.5%) and sodium carbonate (2%) and placed in

the upper lid of each petri plate. Petri plates were properly sealed with parafilm to

avoid the gas discharge. After 2-3 days of incubation at 28°C, yellow to orange-brown

colour change of the filter paper piece was observed (Gajbhiye et al., 2010).

2.2.8 Siderophore production

Production of siderophore determined according to the methodology described

by Clark and Bavoil, (1994). Rhizobacteria was streaked on Chrome Azurol S (CAS)

agar plates and placed in the incubator for 5 d at 28°C. Siderophore production was

indicated by assaying the orange halos around the colonies.

2.2.9 Ammonia production

Rhizobacterial strains were assayed for ammonia by using peptone water

following the method of Cappuccino and Sherman, (2005). Fresh rhizobacterial

cultures were inoculated in test tubes containing 10 mL peptone water and incubated

for 2 d at 28°C. Afterwards, 0.5 ml Nessler’s reagent was loaded in each test tube and

appearance of brown to orange colour was the indication of ammonia production by

the respective rhizobacteria.

2.2.10 Fungal cell wall degrading enzymes

2.2.10.1 Protease activity

Skim milk agar medium was used to evaluate the protease activity by

observing the clear halo zone around the rhizobacterial colony (Maurhofer et al.,

1995). The rhizobacteria was spot inoculated on skim milk agar plate and placed in

incubator for 24 h. Appearance of halo zone around rhizobacterial colony was taken

as positive for protease activity.

2.2.10.2 Chitinase Activity

Chitinase production was evaluated following the method of Gohel et al.,

(2004). Each rhizobacteria was spot inoculated on chitin agar plates and incubated for

7 d at 30°C and observed for the development of inhibition zone around the colonies.


41

2.2.10.3 Cellulase Activity

Bacterial strains were also tested for cellulase activity by spot inoculation

onto LB agar supplemented with cellulose (10 g) and incubated for 8 d at 28°C. The

colonies of rhizobacteria surrounded by halo zones were taken as positive for the

cellulase activity (Cattelan et al., 1999).

2.2.11 Production of other beneficial enzymes

2.2.11.1 Catalase activity

The 24 h old rhizobacteria culture was used for the evaluation of catalase

activity by adding some drops of 30% H2O2 to culture placed on glass slide.

Appearance and intensity of gas bubbles were observed for catalase activity (Schaad,

1992).

2.2.11.2 Oxidase activity

The filter paper spot method of Gerhardt et al., (1981) was used to determine

the oxidase activity. Rhizobacterial culture (24 h) was added onto a small strip of

filter paper. One or two drops of Kovacs oxidase (1%) reagent was added on the

culture. Change in colour to dark purple within 60 to 90 s was taken as positive for

oxidase activity.

2.2.12 Plant growth promoting hormone (IAA) production

Rhizobacteria were analyzed for indole 3-acetic acid (IAA) production in pure

culture. The LB growth media (100 mL) in 250 mL flask were inoculated with 24 h

old rhizobacterial cultures and incubated in shaker (EXCELLA E24, New Brunswick

Scientific USA) for 7 d, at 100 rpm. Subsequently, centrifugation of cultures was

done at 10,000 rpm for 15 min and supernatant was used for phytohormone extraction

after adjusting the pH to 2.8 with 1N HCl by using equal volumes of ethyl acetate

(Tien et al., 1979). The ethyl acetate extract was dried at 35oC and residue was re-

dissolved in 1500 μl methanol (Sigma Chemical Co). Thereafter, analysis for

hormones was done on HPLC (Agilent 1100) with C18 column (39 x 300 mm) and

UV detector. The 100 μl of sample was filtered through Millipore filter (0.45) and

injected into column. Pure IAA (Sigma Chemical Co. USA) dissolved in methanol

was run as standard and growth hormones were analyzed on the basis of peak area and

retention time of standard. Methanol, acetic acid and water were used in 29:1:70 ratios


42

as mobile phase with 1 mL/min flow rate and an average run time of 20 min/sample.

The wavelength 280 nm was used for IAA detection (Sarwar et al., 1992).

2.2.13 Screening of rhizobacteria for plant growth promotion activities

2.2.13.1 Preparation of inoculum

Rhizobacteria isolated from farmer fields used as inoculant were divided into

two groups on the basis of their isolation made from fields infected with and without

stalk rot. Group 1 consisted of rhizobacteria isolated from plants infected with stalk

rot and group 2 comprised of rhizobacteria isolated from non-infected plants.

Rhizobacteria were inoculated in 100 mL of LB broth and incubated on a

rotary shaker for 48 h at 28°C. The growth cultures were centrifuged at 10,000 x g at

4°C for 10 min and cells pellet was re-suspended in sterile distilled water (100 mL).

The optical density (O.D) was determined at 660nm and adjusted to 1.

2.2.13.2 Seed treatment

Maize seeds (Islamabad Gold) obtained from Crop Research Institute,

National Agricultural Research Centre, Islamabad (NARC) were surface sterilized

with ethanol (95%) followed by shaking for 2–3 min in 10% chlorox and

subsequently rinsed with distilled sterile water. Thereafter, seeds were soaked in

rhizobacterial inocula for 2 to 4 h. The residual rhizobacterial suspension was drained

and used for sowing after shade drying (Nandakumar et al., 2001). Seeds soaked in

sterilized water were used for control treatment.

2.2.13.3 Pot culture study

Sterilized pots measuring (11x 8cm2) were filled with autoclaved soil (soil and

sand in 3:1) and maize seeds were sown at 6 seeds per pot. Pots were arranged in

completely randomized design in the green house of Quaid-i-Azam University,

Islamabad. Sterilized water was used for watering and harvested after 15d and data

was recorded for leaf area, length, dry and fresh weight of shoot and root.


43

Table 2.4: Treatments made for pot experiment

Abbreviation Treatments

Ir1+8nm

Rhizobacteria isolated from non-infected

maize fields (irrigated region) Ir1+17nm

Ir1+4nm

A1+JY1


maize fields (arid region) A1+JYG

A1+JYR

Sa1+Yys


maize fields (semi-arid region) Sa1+YDYs

Sa1+Y5

Ir2+NP

Rhizobacteria isolated from infected

maize fields (irrigated region) Ir2+NFY

Ir2+NDY

A2+PO


maize fields (arid region) A2+PY1a

A2+PTWz

Sa2+YiPe


maize fields (semi-arid region) Sa2+YiH

Sa2+Yio


44

Fig 2.1: Scheme of study used for screening and characterization of potential

antagonistic PGPR


45

2.2.14 Morphological and biochemical characterization of selected rhizobacteria

2.2.14.1 Miniaturized Identification System-QTS 24

The (QTS) 24 miniaturized identification system (DESTO Laboratories

Karachi, Pakistan) was used for determining the C/N source utilization pattern of the

PGPR isolates.

2.2.15 Molecular identification

The genomic DNA of all the selected rhizobacteria was isolated by the

procedure followed by Khan et al., (2004).

2.2.15.1. Primers and PCR conditions

Amplification of 16S rRNA gene was done for the genomic DNA of bacterial

strains by using the universal primers 27f (5‘-GAGTTTGATCCTGGCTCAG-3‘) and

1492R (5‘-GGTTACCTTGTTACGACTT-3‘) (James, 2010). Total 50 µl PCR

mixture contained primers (50 pM), genomic DNA (50 ng) , 1 U of Taq DNA

polymerase (Promega, Madison, WI, USA), 1× Taq DNA polymerase buffer, all four

dNTPs (0.2 mM), and MgCl2 (1.5 mM). DNA Amplification was executed in a

thermo cycler ((Biometra, Germany). The PCR conditions are given in table 2.5. A 5

μl of PCR product was electrophoresed on a 1.5% (w/v) agarose gel in 1× TAE buffer

for 45 min at 80 V, stained with 0.01 g/mL ethidium bromide and the PCR products

were visualized with a UV illuminator (Bio RAD, Italy).

2.2.15.2. Sequencing and sequence analysis

The PCR products were purified with PCR purification Kit (QIAGEN) with

micro centrifuge. PCR products were sequenced with automated DNA sequencer by

using the facility at Macrogen Inc. (Seoul, Korea). All the Sequences were aligned

with bio edit 7.1.7. The nucleotide-nucleotide BLAST (blastn) of the NCBI (National

Centre for Biotechnology Information) database was used for the identification of

highly similar 16S rRNA gene sequences. Afterwards sequences were submitted by

using SEQUIN to Gene bank.


46

Table 2.5: PCR condition for Amplification of 16s rRNA

Conditions Amplification of 16s rRNA

Temperature Time

Denaturation 94°C 3 min


Denaturation 96°C 20 s

Annealing 52°C 20 s

Extension 72°C 2 min

Final extension 72°C 20 min


47

2.2.15.3. Phylogenetic trees

For the construction of phylogenetic tree reference sequences were

downloaded from the website http:// www.ncbi.nlm.nih.gov/Genbank and further

processed for comparison. All the sequences of 16S rRNA were aligned using the

multiple sequence alignment programs (bioedit 7.1.7 and Mafft). The aligned

sequences were further analyzed manually for gaps and made 600 bp blocks in each

row (Ayyadurai et al., 2007). These sort-out sequences were saved as molecular

evolutionary genetics analysis (MEGA) format in software MEGA v4.0. Bootstrap

analysis method was used to get the confidence values by using the 1000 times

resampled original data set. Multiple distance matrixes were calculated by using

MEGA 4.0 through direct use of the bootstrapped data set (Kumar et al., 2004). The

multiple distance matrix were further utilize to construct phylogenetic trees with the

help of neighbour-joining (NJ) method (Chen et al., 2011).

2.2.16 Statistical analysis

Completely Randomized Design (CRD) was used for pot experiment; one pot

containing 4 plants was considered as experimental unit and was repeated three times.

The data were analyzed statistically by analysis of variance technique and comparison

among means was made by the least significant difference (LSD) at P < 0.05 (Gomez

and Gomez 1984) using statistix 1.8.

http://www.ncbi.nlm.nih.gov/Genbank


48

Fig 2.2: Schematic illustration for molecular identification of

antagonistic rhizobacteria


49

2.3 RESULTS

The present investigation was carried out for the isolation, screening and

selection of efficient strains of rhizobacteria effective against species of Fusarium

moniliforme, Aspergillus flavus and Helminthosporium sativum. An attempt was also

made to reveal the mechanism of biocontrol and plant growth promotion of potent

antagonistic strains. The results of the experiments conducted are presented under

the following headings

2.3.1 Isolation of Bacterial isolates

During the present investigation, 117 rhizobacteria were efficaciously isolated

from rhizosphere soil of maize fields located at three different regions differing in soil

moisture content of Pakistan including semi-arid areas of Sahiwal (15% moisture

content) and arid areas of Jhang (9% moisture content) and irrigated area (26%

moisture content) of National Agricultural Research Centre, Islamabad.

Morphological characteristics of each rhizobacterial colony were examined on LB

agar plates. Colonies of all the isolates greatly vary in size, shape, colour and

pigmentation (Appendix 12-17).

2.3.2 Antagonism assay against phytopathogenic fungi

All the rhizobacteria were assayed for inhibition of mycelial growth of

Fusarium moniliforme, Aspergillus flavus and Helminthosporium sativum. All of

them were tested by comparing fungal growth co-cultured with isolated rhizobacteria

(relative to controls inoculated with fungi) for probable antifungal activities against

three different fungal phytopathogens. The data were presented in Fig 2.3 to 2.20 for

antagonistic activity of bacterial isolates against selected fungal pathogens. The fungal

growth was inhibited by a number of rhizobacteria by varying extents ranging from 0

to 94%. The rhizobacteria isolated from the rhizosphere soil of non-infected maize

fields were placed in group 1 and rhizobacteria isolated from infected maize fields in

group 2.

The results in Fig 2.3 revealed that among the arid zone (9% moisture

content) isolates of group1, JYR showed the highest inhibition (77.03%) against F.

moniliforme while Jshi, JWIR and JYG also inhibited the growth of F. moniliforme by

more than 60%. JY, JLPO and JY1 suppressed the mycelial growth with more than

40%.

http://www.google.com.au/search?hl=en&biw=1280&bih=656&sa=X&ei=vnZJUOzoLsjZigedyoGgDg&ved=0CBsQvwUoAQ&q=Helminthosporium+sativum&spell=1

http://www.google.com.au/search?hl=en&biw=1280&bih=656&sa=X&ei=vnZJUOzoLsjZigedyoGgDg&ved=0CBsQvwUoAQ&q=Helminthosporium+sativum&spell=1


50

Fig 2.3: Effect of Group 1 rhizobacteria of arid region on antifungal

activity against F. moniliforme

Group 1: Rhizobacteria isolated from maize fields rhizosphere having non-infected plants, Terbinafine:

synthetic antifungal compound, Fungicide: commercial chemical fungicide (Ridomil Gold). Each bar

represents the average of three independent measurements. All means sharing the common letter differ

non-significantly as (P ≤ 0.05), LSD: 1.706.

Fig 2.4: Effect of Group 1 rhizobacteria of arid region on antifungal activity

against H. sativum

Detail of treatment as given in figure 2.3. Each bar represents the average of three independent

measurements. All means sharing the common letter differ non-significantly as (P ≤ 0.05) LSD: 1.767.

n

l

n

k

o

j

o p

i

m

p

d

f

h g

f g

f

b c

o

a

e

0102030405060708090

100

JWIR

JWIR

2

JWIR

1

JDW

JFW

JYD

Jpe

JTz

Jsh

i

JMT

JYT

JY1 JP JY

JSIR

JSIR

2

JWC

H1

JLP

O

JYR

JYG

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)

Group 1 rhizobacteria of arid region

F.moniliforme

i h

i

f

l m

k

0

j kl

m

g

k

h

e d

g h

c

f

m

a b

0102030405060708090

100

JWIR

JWIR

2

JWIR

1

JDW

JFW

JYD

Jpe

JTz

Jsh

i

JMT

JYT

JY1 JP JY

JSIR

JSIR

2

JWC

H1

JLP

O

JYR

JYG

con

tro

l

Terb

inaf

ine

Fun

gici

dePe

rce

nta

ge In

hib

itio

n (

%)


H.stavium


51

Fig 2.5: Effect of Group 1 rhizobacteria of arid region on

antifungal activity against A. flavus




against F. moniliforme

Group 2: Rhizobacteria isolated from maize field rhizosphere infect with stalk rot. Terbinafine:



non-significantly as (P ≤ 0.05) LSD: 3.561.

d

l

g

i j

kl

h

j

d

jk l

e

i

e

l

f

l

e

b

d

l

a

c

0102030405060708090

100

JWIR

JWIR

2

JWIR

1

JDW

JFW

JYD

Jpe

JTz

Jsh

i

JMT

JYT

JY1 JP JY

JSIR

JSIR

2

JWC

H1

JLP

O

JYR

JYG

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)


A.flavus

j

g

j i

h h g

i

j

f

i

cd d e

cd

f f ef

b

j

a

bc

0102030405060708090

100

PW Pyz

PYT

2b

PB

PFW

PC

IR

PYD Pp

e

PTz

Psh

i

Pw

a

PO

PY1

a

PYT

2a

PC

P

PTW

PC

WIR

PTW

1

PTW

z

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge in

hib

itio

n (

%)


F.moniliforme


52

The arid zone isolates including JYR, JSIR2, JSIR, JDW, JYG, JY1, JWCH1,

JWIR2 and JLPO exhibited the significant inhibition of H. sativum ranging from 40 to

74% respectively (Fig 2.4). In case of A. flavus growth inhibition, rhizobacteria

including JYR, Jshi, JWIR, JYG, JY, JY1, JLPO, JSIR2 and JWIR1 inhibited the

mycelial growth ranging from 30 to 77% (Fig 2.5). Maximum inhibition was shown

by JYR by 77%.

Group2 rhizobacteria of arid region inhibited the mycelial growth of F.

moniliforme by varying percentage ranging from 10 to 80%, maximum inhibition

(80%) was observed by PTWz followed by PCP (72%) and PO (70%) (Fig 2.6). Two

of them (PYT2a, PY1a) showed more than 60% inhibition, three rhizobacteria

including (PTW1, PTW, PCWIR) exhibited more than 50% inhibition while Pshi

exhibited more than 40% inhibition against F. moniliforme. These rhizobacteria did

not show strong inhibition against the fungal growth of H. sativum as maximum

inhibition (> 60%) was detected for PTW and PYD. Two rhizobacteria including

PTWz and PY suppress its growth by more than 50%, three rhizobacteria PCWIR,

PYT2a and Pyz more than 40%, one rhizobacteria PY1a by 38%, two rhizobacteria

PTW1 and Pshi, by more than 20% (Fig 2.7). As indicated in Fig 2.8 very few

rhizobacteria of group2 from arid region inhibited the growth of A. flavus including

PTWz (61%), PTW (58%), )PO (45%) and PCWIR, PYT2a and Pyz (more than

30%).

Among the isolates from the non-infected maize field of semi-arid region

(15% moisture content), one rhizobacteria Y5 strongly inhibit the mycelial growth of

F. moniliforme by 81.8% (Fig 2.9). Three rhizobacteria i.e. Yys, YDYs and Y3

exhibited highest (> 70%) inhibition against F. moniliforme while YCC1, Y2 and

YDY showed more than 50% inhibition against F. moniliforme. Yw, Y4 and YCC

inhibited the mycelia growth of F. moniliforme by more than 40%. The rhizobacteria

of semiarid region were less effective for the mycelial inhibition of H. sativum (Fig

2.10). Maximum inhibition had exhibited by Y5 and YLY i.e. >60%. While H.

sativum mycelial growth was also suppressed by YWD (51.48%), Y1a, Y4, YDY

(48.5, 46.6 and 41.8%) and Yys, YDYs (more than 30%). The inhibition of mycelial

growth was even more less in case of A. flavus by the group1 rhizobacteria of semi-

arid region as compared to inhibition of F. moniliforme and H. sativum (Fig 2.11).


53


against H. sativum




against A. flavus



k

f

k jk

h

k

de

i k

g

ij

c

f e

18

c

e

g

d

k

a b

0

20

40

60

80

100

PW Pyz

PYT

2b

PB

PFW PC

IR

PYD Pp

e

PTz

Psh

i

Pw

a

PO

PY1

a

PYT

2a

PC

P

PTW

PC

WIR

PTW

1

PTW

z

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge in

hib

itio

n (

%)


H. sativum

m

g

m m

j

m

i

k m

h

l

e

l

g

h

d

f

m

c

m

a

b

0102030405060708090

100

PW Pyz

PYT

2b

PB

PFW PC

IR

PYD Pp

e

PTz

Psh

i

Pw

a

PO

PY1

a

PYT

2a

PC

P

PTW

PC

WIR

PTW

1

PTW

z

con

tro

l

Terb

inaf

ine

Fun

gici

deP

erc

en

tage

inh

ibit

ion

(%

)


A.flavus


54

A total number of 19 rhizobacteria were isolated from maize field infected

with stalk rot from semi-arid region two rhizobacteria including Yio and YiBa

suppresses the mycelial growth of F. moniliforme by more than 70% (Fig 2.12). Three

rhizobacteria YiH, YiBs and YiPe significantly inhibited (> 60%) the F. moniliforme

growth while only one isolate inhibit its growth by more than 50%. Isolates Yi16,

YiLy and Yiy exhibited more than 40% inhibition of F. moniliforme. This group of

rhizobacteria had shown the less inhibition of H. sativum and A. flavus as that of the

rhizobacteria of arid region. Maximum inhibition was shown by Yipy (71%) against

H. sativum and 64% by Yiy against A. flavus (Fig 2.13 and 2.14).

The result in Fig 2.15 indicated that the rhizobacteria isolated from the

irrigated region (26% moisture content) of group 1 showed significantly higher

inhibition against F. moniliforme i.e. 90% by 4nm and more than 80% by three

rhizobacteria (8nm, 12nm, 18nm). Other isolates also inhibit the mycelial growth by a

higher percentage ranging from 40 to 70%. This rhizobacteria group also showed high

inhibition potential against H. sativum i.e. more than 87.4% by 4nm, 72.2% by 8nm,

more than 62.5% by 3nm, more than 50% by 9nm and 15nm and more than 40% by

11nm, 7nm and 16nm (Fig 2.16). Inhibition against A. flavus was shown by very few

rhizobacteria including 4nm (73.3%), 9nm (72.9%), 15nm (50%), 8nm (49.2%) and

1nm (30%) (Fig 2.17).

A number of group2 rhizobacteria of irrigated region inhibited the mycelial

growth of F. moniliforme by more than 50% including Nwp, Ny, N91, Nwsm, Nwce,

Npe (Fig 2.18). Three of them showed more than 70% inhibition, one rhizobacteria

NDY exhibited 77% inhibition while Nwce2, Nws and Nst exhibited more than 40%

inhibition of F. moniliforme. These rhizobacteria also shown high inhibition of H.

sativum as one rhizobacteria Nwp suppress its growth by 94%, two rhizobacteria

NLY and Np more than 80%, two rhizobacteria Nwcir, NWsm by 71%, four

rhizobacteria NPe, NFY, Nsp, NWce2 by more than 60% and Ny, Nws, NDY by

more than 50% (Fig 2.19). As indicated in Fig 2.20 very few rhizobacteria of group2

from irrigated region inhibited the growth of A. flavus including NLY (72.59 %),

NFY, NDY, NWce, Npe (more than 50%) and Np (40.7%).


55

Fig 2.9: Effect of Group 1 rhizobacteria of semi-arid region on antifungal


Group 1: Rhizobacteria isolated from maize fields rhizosphere having non-infected plants, Terbinafine:





activity against H. sativum



ghi ij

efgh

j j j

hi

de

j

def

ghi

de cd cde

fghi

defg

bc

cd

ab

j

a

bc

0102030405060708090

100

YLY

YTC

YCC

YCH

1

YLB Y1 Y1a

Y2 Y8 Yw

YWD

YDY

YDYs

YCC

1

YCH

1 Y4 Yys

Y3 Y5

con

tro

l

Terbinafi…

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)

Group 1 Rhizobacteria of Semi arid-region

F.moniliforme

c

hi hi

j

hi

j

de

j j j

d ef

fg gh i

de ef

j

c

j

a

b

0

10

20

30

40

50

60

70

80

90

100

YLY

YTC

YCC

YCH

1

YLB Y1 Y1a

Y2 Y8 Yw

YWD

YDY

YDYs

YCC

1

YCH

1 Y4 Yys

Y3 Y5

con

tro

l

Terbinaf…

Fun

gici

de

Pe

rce

nta

ge in

hib

itio

n (

%)

Group1 Rhizobacteria of semi arid region

H.sativum


56


activity against A. flavus





Group 2: Rhizobacteria isolated from rhizosphere of maize field infected with stalk rot. Terbinafine:




de

f f f

e de de

c

f f

bc

f

de de

e d

e e

c

f

a

b

0

10

20

30

40

50

60

70

80

90

100

YLY

YTC

YCC

YCH

1

YLB Y1 Y1a

Y2 Y8 Yw

YWD

YDY

YDYs

YCC

1

YCH

1 Y4 Yys

Y3 Y5

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)

Group1 Rhizobacteria of semi-arid region

A.flavus

f

h

k

g

k

hi

j i

j

k

j

d

c

f e

d d

f

b

k

a

bc

0

10

20

30

40

50

60

70

80

90

100

Yiy

yiys

YicL

Yiw

Yip

s

Yiw

s

Yiw

p

Yics

t

Yip

y

YiC

1

Yi1

a

YiP

e

YiB

a

YiLy YiC

YiH

YiB

s

Yi1

6

Yio

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge in

hib

itio

n (

%)

Group 2 Rhizobacteria of semi-arid region

F.moniliforme


57









fg

d

m

i

m

d e

k

c

m m

hi j

l

g f

h g

e

m

a

b

0102030405060708090

100

Yiy

yiys

YicL

Yiw

Yip

s

Yiw

s

Yiw

p

Yics

t

Yip

y

YiC

1

Yi1

a

YiP

e

YiB

a

YiLy YiC

YiH

YiB

s

Yi1

6

Yio

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)


H.sativum

c

g

0

de

i i i i

h i

h

e f

c de

de de

i

d

i

a

b

0

10

20

30

40

50

60

70

80

90

100

Yiy

yiys

YicL

Yiw

Yip

s

Yiw

s

Yiw

p

Yics

t

Yip

y

YiC

1

Yi1

a

YiP

e

YiB

a

YiLy YiC

YiH

YiB

s

Yi1

6

Yio

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)

Group2 Rhizobacteria of semi-arid region

A.flavus


58

Fig 2.15: Effect of Group 1 rhizobacteria of irrigated region on antifungal


Group 1: Rhizobacteria isolated from rhizosphere of maize field having non-infected plants.

Terbinafine: synthetic antifungal compound, Fungicide: commercial chemical fungicide (Ridomil

Gold). Each bar represents the average of three independent measurements. All means sharing the

common letter differ non-significantly as (P ≤ 0.05) LSD: 1.265.




measurements. All means sharing the common letter differ non-significantly as (P ≤ 0.05), LSD: 2.631.

i

j

k l

g

k

l

h i

ij

l

hg g

bc

f

cd f

d b

l

a

e

0102030405060708090

100

1n

m

3n

m

6n

m

7n

m

11

nm

13

nm

14

nm

15

nm

16

nm

19

nm

20

nm

2n

m

5n

m

8n

m

9n

m

12

nm

17

nm

18

nm

4n

m

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)

Group 1 rhizobacteria of irrigated region

F.moniliforme

k

e

k

h

fg

jk

m

fg

h

j

l

i i

d

f

m

j

m

b

m

a

c

0

20

40

60

80

100

1n

m

3n

m

6n

m

7n

m

11

nm

13

nm

14

nm

15

nm

16

nm

19

nm

20

nm

2n

m

5n

m

8n

m

9n

m

12

nm

17

nm

18

nm

4n

m

con

tro

l

Terbinafi…

Fun

gici

de

Pe

rce

nta

ge in

hib

itio

n (

%)


H.sativum


59




measurements. All means sharing the common letter differ non-significantly as (P ≤ 0.05), LSD: 1.055.



Group 2: Rhizobacteria isolated from rhizosphere of maize field infected with stalk rot. Each bar



d

e d de

h h h

c

h g

e e

g

c

b

h

d

h

b

h

a

b

0102030405060708090

100

1n

m

3n

m

6n

m

7n

m

11

nm

13

nm

14

nm

15

nm

16

nm

19

nm

20

nm

2n

m

5n

m

8n

m

9n

m

12

nm

17

nm

18

nm

4n

m

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)


A.flavus

i

fg g

ij

h

de

j k

j

c

k

f

c

f d

ef

bc

f f

b

k

a

bc

0102030405060708090

100

NLY

Nw

s

Nst

N2

C

NSP N

Y

Np

Y

NLW

NW

R1

Nw

cir

Nw

p2

Np

e

Np

NW

sm

Nw

p

N9

1

NFY

Nw

ce

Nw

ce2

ND

Y

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)

Group 2 rhizaobacteria of irrigated region

F. moniliforme


60






activity of against A. flavus


measurements and means with same letter are non-significantly different according to least significant

difference (P ≤ 0.05) LSD: 2.137.

ab

gh

i hi

ef fg

l m

lm

cd

m

ef

b

d

a

k

ef

j

de

gh

m

a

ab

0102030405060708090

100

NLY

Nw

s

Nst

N2

C

NSP N

Y

Np

Y

NLW

NW

R1

Nw

cir

Nw

p2

Np

e

Np

NW

sm

Nw

p

N9

1

NFY

Nw

ce

Nw

ce2

ND

Y

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)


H. sativum

b

efg

0

j i

fg

kl l k

i

0

cde

f h

g gh

c de

fg

cd

0

a

b

0102030405060708090

100

NLY

Nw

s

Nst

N2

C

NSP N

Y

Np

Y

NLW

NW

R1

Nw

cir

Nw

p2

Np

e

Np

NW

sm

Nw

p

N9

1

NFY

Nw

ce

Nw

ce2

ND

Y

con

tro

l

Terb

inaf

ine

Fun

gici

de

Pe

rce

nta

ge In

hib

itio

n (

%)


A.flavus


61

The inhibition was 60% by YWD, more than 50% by Y2, Y5 and Y4 while all the

remaining rhizobacteria shown inhibition less than 20%.

2.3.3 Phosphate solubilization

The results of present study revealed that among group 1 rhizobacteria of

irrigated region, 9 rhizobacteria (3nm,14nm, 15nm, 16nm, 5nm, 8nm, 17nm, 18nm

and 4nm) were able to solubilize phosphorous while, seven rhizobacteria including

NLY, N2C, NSP, NP, Nwp, NFY and NDY of group 2 exhibited the phosphate

solubilization ability (Table 2.6 and 2.7). It was confirmed by the development of

halos surrounding those colonies.

Eight group 1 rhizobacteria (Pyz, PFW, PYD, Pwa, Py1a, PTW, PCWIR and

PTWz) out of nineteen isolated from the rhizosphere of maize fields of arid region has

shown the phosphate solubilizing ability whereas, 10 of group 2 rhizobacteria

(JWIR1, JDW, JTz, Jshi, JMT, JY1, JSIR2, JLPO, JYR and JYG) from arid region

solubilized phosphorous (Table 2.6 and 2.7).

Among group 2 rhizobacteria of semi-arid region eight rhizobacteria (Yiy,

YicL, Yicst, Yipe, YiLy, YiH, Yi16 and Yio) had shown the ability to solubilize

phosphorous (Table 2.6 and 2.7) and only six of group 2 rhizobacteria (YCH1, YWD,

YDYs, Yys, Y3 and Y5) isolated from maize fields of semi-arid region exhibited the

ability to solubilize phosphorous.

The rhizobacteria isolated from all three different regions were selected for

further analyses based on the following criteria

1. Rhizobacteria that exhibited high mycelial of F. moniliforme by using the

cell free culture supernatant of rhizobacteria.

2. Rhizobacteria having higher inhibition potential against H. sativum and A.

flavus along with significantly high inhibition of F. moniliforme.

3. Rhizobacteria which show the Phosphate solubilization ability along with

their higher antifungal activity against three fungal pathogens.

On the basis of above mentioned criterion, among group 2 nine rhizobacteria

including Npe, Np, Nwsm, Nwp, N91, NFY, Nwce, Nwce2 and NDY were selected

from irrigated region, eight (PO, PY1, PYT2a, PCP, PTW, PCWIR, PTW1, PTW2)

rhizobacteria from arid region and eight (YiPe, YiBa, YiLy, YiC, YiH, YiBs, Yi16,

Yio) from semi-arid region.


62

Table 2.6: Phosphate solubilization by Group 1 rhizobacteria isolated from the

rhizosphere of maize fields of arid, semi-arid and irrigated regions

Rhizobacteria

(irrigated

region)

Solubilization

index

Rhizobacteria

(arid region)

Solubilization

index

Rhizobacteria

(semi-arid

region)

Solubilization

index

1nm - JP -

YLY -

3nm 1.4±0 JY -

YTC -

6nm - JWIR1 1.42±0

YCC -

7nm - JDW 1.70±0.02

YCH1 1.32±0.01

11nm - JFW -

YLB -

13nm - JYD -

Y1 -

14nm 2.4±1.11 Jpe -

Y1a -

15nm 1.62±0 JTz 1.44±0

Y2 -

16nm 1.66±0 Jshi 1.6±0

Y8 -

19nm - JMT 1.39±0.01

Yw -

20nm - JYT -

YWD 1.56±0

2nm - JY1 1.60±0.03

YDY -

5nm 1.5±0 JWIR -

YDYs 1.46±0.008

8nm 1.47±0.02 JWIR2 -

YCC1 -

9nm - JSIR -

YCH1 -

12nm - JSIR2 1.66±0.02

Y4 -

17nm 1.7±0 JWCH1 -

Yys 1.14±0.007

18nm 1.37±0 JLPO 1.66±0

Y3 1.46±0

4nm 1.36±0.04 JYR 1.62±0.01

Y5 1.38±0.01

- - JYG 1.43±0.002 - -

Group 1: Rhizobacteria isolated from rhizosphere of maize field having non-infected plants. The

symbol - indicate negative in test and + indicate positive in test


63

Table 2.7: Phosphate solubilization by Group 2 rhizobacteria isolated from the

rhizosphere of maize fields grown at arid, semi-arid and irrigated regions

Rhizobacteria

(irrigated region)

Solubilization

index

Rhizobacteria

(arid region)

Solubilization

index

Rhizobacteria

(semi-arid

region)

Solubilizati

on index

NLY 1.62±0.05 PW 0

Yiy 1.34±0.04

Nws - Pyz 1.66±0

yiys -

Nst - PYT2b 0

YicL 1.48±0.03

N2C 1.5±0.01 PFW 1.44±0

Yiw -

NSP 1.37±0 PCIR 0

Yips -

NY - PYD 1.56±0.01

Yiws -

NpY - Ppe -

Yiwp -

NLW - PTz -

Yicst 1.5±0.03

NWR1 - Pshi -

Yipy -

Nwcir - Pwa 1.45±0

YiC1 -

Nwp2 - PO -

Yi1a -

Npe - PY1a 1.38±0

YiPe 1.34±0.007

Np 1.03±0.006 PYT2a -

YiBa -

NWsm - PCP -

YiLy 1.58±0.02

Nwp 2.42±0.01 PTW 1.44±0

YiC -

N91 - PCWIR 1.31±0.006

YiH 1.27±0.02

NFY 1.01±0.03 PTW1 -

YiBs -

Nwce - PTWz 1.15±0.007

Yi16 1.81±0

Nwce2 - - -

Yio 1.36±0

NDY 1.63±0.01 JYG 1.43±0.002 - -

Group 2: Rhizobacteria isolated from rhizosphere of maize field infected with stalk rot. The symbol -

indicate negative in test and + indicate positive in test


64

Among Group1, eight rhizobacteria (2nm, 5nm, 8nm, 9nm, 12nm, 17nm,

18nm, 4nm) were selected from irrigated region whereas, nine rhizobacteria (JY1, JP,

JY, JSIR, JSIR2, JWCH1, JLPO, JYR, JYG) from arid zone and eight rhizobacteria

(YDY, YDYs, YCC1, YCH1, Y4, Yys, Y3 and Y5) from semi-arid region were

selected for further analyses.

2.3.4 Production of plant growth promoting hormone (IAA) Among the bacterial antagonists of arid region, five bacterial antagonists

(JSIR2, JWCH1, JLPO, JYR, JYG) of group1 and all the selected antagonistic

bacteria of group 2 exhibited the production of IAA. The results presented in Fig 2.21

revealed that, in group 2 maximum amount of IAA (1.79 μg/mL) was produced by

JYG. Other rhizobacteria producing IAA were ranked as JYR (1.44 μg/mL)>JWCH1

(1.12µg/mL) > JSIR2 (0.85 µg/mL) > JLPO (0.59 µg/mL). In group 1, PTW has

produced the significantly high amount (1.32 µg/mL) of IAA, while PYT2a, PTWz,

PO, PCP, PY1a, PCWIR and PTW1 produced 1.27, 1.10, 0.96, 0.89, 0.88, 0.85

µg/mL IAA as indicated in Fig 2.22).

The entire group 1 rhizobacteria of irrigated region exhibited the production of

IAA as indicated in Fig 2.25. Maximum amount was produced by 4nm of group 1

(1.88 µg/mL) while minimum amount was produced by rhizobacteria 4nm and 17nm

(0.21 and 0.12 µg/mL). The group 2 rhizobacteria of irrigated region produced

significantly higher amount of IAA (Fig 2.26). Highest amount of IAA was produced

by rhizobacteria NP (1.28 µg/mL) and minimum amount was produced by NFY (0.84

µg/mL). Among the antagonistic rhizobacteria of semiarid region only four group 2

rhizobacteria and five rhizobacteria of group 1 produced IAA (Fig 2.23 and 2.24). The

group1 rhizobacteria YCH1 produced significantly higher amount i.e. 1.70 µg/mL of

IAA. Other group 1 rhizobacteria produced IAA in the following order Y5 (1.63

µg/mL) > Yys and YDY (1.46 µg/mL) > YDYs (1.08 µg/mL). Among group 2, Yio

produced maximum amount of IAA 1.e. 1.93 µg/mL and minimum amount (0.76

µg/mL) was produced by YiLy.

All the rhizobacteria isolated from irrigated region produced IAA while the

isolates of arid region have also shown the production of IAA except JSIR. All

isolates of semi-arid region didn’t produced IAA while, those produced only one

group 2 rhizobacteria (Yio) produced significantly high amount (1.93 µg/mL) of IAA.


65

Fig 2.21: IAA production by Group 1 rhizobacteria of arid region

Group 1: Rhizobacteria isolated from maize fields rhizosphere having non-infected plants. Means with

same letter are non-significantly different according to least significant difference (P ≤ 0.05), LSD:

0.0931.

Fig 2.22: IAA production by Group 2 rhizobacteria of arid region

Group 1: Rhizobacteria isolated from maize fields rhizosphere with stalk rot infected plants. All means

sharing the common letter differ non-significantly as (P ≤ 0.05) LSD: 0.085.

0

0.5

1

1.5

2

JY1 JSIR JSIR2 JWCH1 JLPO JYG JYR

d

f

d

c

e

b

a

IAA

(µ

g/m

L)

Group 1 Rhizobaceria of Arid region

0

0.5

1

1.5

2

PO PY1a PYT2a PCP PTW PCWIR PTW1 PTWz

cd d

ab

d

a

d d

bc

IAA

(µ

g/m

L)

Group 2 Rhizobacteria of Arid region


66

Fig 2.23: IAA production by Group 1 rhizobacteria of semi-Arid region

Group 1: Rhizobacteria isolated from maize fields rhizosphere with non-infected plants. All means

sharing the common letter differ non-significantly as (P ≤ 0.05), LSD: 0.031.

Fig 2.24: IAA production by Group2 rhizobacteria of semi-arid region

Group 2: Rhizobacteria isolated from maize fields rhizosphere infected with stalk rot. All means


0

0.5

1

1.5

2

YDY Y4 YCC1 YCH1 YDYs Yys Y3 Y5

b

d d

a

c

b

d

a

IAA

(µ

g/m

L)

Group 1 Rhizobacteria of Semi-arid region

0

0.5

1

1.5

2

YiPe YiBa YiLy YiC YiH YiBs Yi16 Yio

e e

d

0

c b

e

a

IAA

(µ

g/m

L)



67

Fig 2.25: IAA production by Group 1 rhizobacteria of Irrigated region

Group 2: Rhizobacteria isolated from maize fields rhizosphere having non-infected plants. All means


Fig 2.26: IAA production by Group 2 rhizobacteria of Irrigated region

Group 2: Rhizobacteria isolated from maize fields rhizosphere infected with stalk rot. All means


0

0.5

1

1.5

2

2nm 5nm 8nm 9nm 12nm 17nm 18nm 4nm

b

e e

d

c cd

d

a

IAA

(µ

g/m

L)

Group 1 Rizobacteria of irrigated region

0

0.5

1

1.5

2

de

a

bcd e de

cde bc cde

ab

IAA

(µ

g/m

L)

Group 2 Rhizobacteria of Irrigated region


68

2.3.5 Production of siderophore

All the selected rhizobacteria of arid zone were tested for the production of

siderophore. Three of group 1 rhizobacteria (JSIR2, JYR, JYG) and two of group 2

rhizobacteria (PY1a, PTWz) isolated from maize fields of arid region, showed the

production of siderophore indicated by exhibiting the change in colour of the CAS

medium from blue to orange Among the selected bacterial antagonists of semiarid

region YDYs, Yys and Y5 of group1 and YiBs and Yi16 of group 2 indicated the

production of siderophore (Table 2.9). Two rhizobacteria NP and NFY of group 2 and

four rhizobacteria of group 1(8nm, 12nm, 17nm, 4nm) were tested positive for

siderophore production among the selected bacteria of Irrigated region (Table 2.8).

2.3.6 Production of HCN

Among the selected rhizobacteria isolated from the rhizosphere of maize fields

of irrigated region, four rhizobacteria from group 1 including 2nm, 4nm, 8nm, 18nm

and two from group 2 rhizobacteria (NP, NFY) exhibited the production of HCN

(Table 2.9). Three group 2 rhizobacteria (PO, PYT2a, and PCP) and four of group 1

rhizobacteria (JYR, JYG, JSIR, JSIR2) of arid region also showed the change in

colour of filter paper strip placed on the upper of petri plate indicated the production

of HCN (Table 2.12). Similarly in case of rhizobacteria isolated from semi-arid region

four of group 1 rhizobacteria i.e. YDYs, Yys, Y3 and Y5 and four of group 2

rhizobacteria (YiPe, YiO, YiH, YiBs) has shown the production of HCN (Table 2.15)

2.3.7 Production of Ammonia

All the selected rhizobacteria isolated from the rhizosphere of maize fields

from irrigated region of both groups indicated ammonia production except group 1

rhizobacteria 5nm and group 2 rhizobacteria Nwsm, Nwp and Nwce2 (Table 2.10).

Two rhizobacteria from each group (JWIR, JSIR2 of group 1 and PCP,

PCWIR of group 2) of arid region did not show the production of ammonia (Table

2.13). Group 2 rhizobacteria including YiO, Yi16, YiH, YiC, YiLy, YiPe and group 1

rhizobacteria YDYs, YCH1, Y3, Y5and Yys of semiarid region has exhibited the

ammonia production (Table 2.16).


69

Table 2.8: Production of Siderophore by rhizobacteria isolated from rhizosphere

of maize fields of irrigated region

Group 1

Rhizobacteria

Siderophore

production

Group 2

Rhizobacteria

Siderophore

production

4nm + Np +

5nm - NWsm -

8nm + Nwp -

9nm - N91 -

12nm + NFY +

17nm + Nwce -

18nm - Nwce2 -

- - NDY -

Table 2.9: HCN Production by rhizobacteria isolated from rhizosphere of maize

fields of irrigated region

Group 1

Rhizobacteria

HCN production Group 2

Rhizobacteria

HCN production

2nm + Npe -

4nm + Np +

5nm - NWsm -

8nm + Nwp +

9nm - N91 -

12nm - NFY +

17nm - Nwce -

18nm + Nwce2 -

- - NDY +

Table 2.10: Ammonia production by rhizobacteria isolated from rhizosphere of

maize fields of irrigated region

Group 1

Rhizobacteria

Ammonia production Group 2

Rhizobacteria

Ammonia production

2nm + Npe +

4nm + Np +

5nm - NWsm -

8nm + Nwp -

9nm + N91 +

12nm + NFY +

17nm + Nwce +

18nm + Nwce2 -

- - NDY +

Group 1: Rhizobacteria isolated from maize non-infected fields, Group 2: Rhizobacteria

isolated from maize fields infected with stalk rot, the symbol - indicate negative in test and +

indicate positive in test


70

Table 2.11: Production of Siderophore by rhizobacteria isolated from

rhizosphere of maize fields of arid region

Group 1

Rhizobacteria

Siderophore

production

Group 2

Rhizobacteria

Siderophore

production

JY1 - PO -

JWIR - PY1a +

JWIR2 - PYT2a -

JSIR - PCP -

JSIR2 + PTW -

JWCH1 - PCWIR -

JLPO - PTW1 -

JYR + PTWz +

JYG + - -


fields of arid region

Group 1

Rhizobacteria HCN production

Group 2


JY1 - PO +

JWIR - PY1a -

JWIR2 - PYT2a +

JSIR + PCP +

JSIR2 + PTW -

JWCH1 - PCWIR -

JLPO - PTW1 -

JYR ++ PTWz -

JYG +++ - -

Table 2.13: Ammonia Production by rhizobacteria isolated from rhizosphere of

maize fields of arid region

Group 1

Rhizobacteria Ammonia production

Group 2


JY1 + PO +

JWIR - PY1a +

JWIR2 + PYT2a +

JSIR + PCP -

JSIR2 - PTW +

JWCH1 + PCWIR -

JLPO + PTW1 +

JYR + PTWz +





71

Table 2.14: Production of siderophore by rhizobacteria isolated from

rhizosphere of maize fields of semi-arid region

Group 1

Rhizobacteria

Siderophore

production

Group 2

Rhizobacteria

Siderophore

production

YDY - YiPe -

YDYs + YiBa -

YCC1 - YiLy -

YCH1 - YiC -

Y3 - YiH +

Y4 - YiBs +

Y5 + Yi16 -

Yys + Yio -


fields of semi-arid region

Group 1


Group 2


YDY - YiPe ++

YDYs + YiBa -

YCC1 - YiLy -

YCH1 - YiC -

Y3 ++ YiH +

Y4 - YiBs -

Y5 ++ Yi16 -

Yys ++ Yio ++

Table 2.16: Ammonia Production by rhizobacteria isolated from rhizosphere of

maize fields of semi-arid region

Group 1


Group 2


YDY - YiPe +

YDYs + YiBa -

YCC1 - YiLy +

YCH1 + YiC +

Y3 + YiH +

Y4 - YiBs -

Y5 + Yi16 +

Yys + Yio +





72

2.3.8 Production catalase and oxidase enzymes

All the selected antagonistic rhizobacteria of all regions showed the catalase

activity except group 2 rhizobacteria PCWIR and group 1 rhizobacteria JWIR and

JWIR2 of arid region (Table 2.17, 2.18, 2.19). Rhizobacteria isolated from the

rhizosphere of arid and semiarid region maize plants exhibited higher reaction

intensity in catalase activity as compared to that from the rhizosphere of irrigated

region.

The oxidase activity was also shown by all the rhizobacteria isolated from

irrigated region. Among the rhizobacteria isolated from the rhizospheric soil of arid

region, all group 2 rhizobacteria exhibited the oxidase activity except PCWIR. The

group 1 rhizobacteria including JY1, JSIR, JSIR2, JWCH1 JLPO, JYG and JYR

indicated the oxidase activity. The group 1 rhizobacteria i.e. Y5, Y3, Yys, YCH1,

YCC1, YDYs and group 2 rhizobacteria i.e. Yio, Yi16, YiBs, YiLy, YiH and YiPe,

isolated from the maize field rhizosphere of semi-arid region, have shown the oxidase

activity by changing the colour of rhizobacterial culture on addition of Kovacs

oxidase reagent (Table 2.17, 2.18, 2.19).

2.3.9 Production of fungal cell wall degrading enzymes (Protease, Chitinase

and Cellulase

The results presented in table 2.20 revealed that three of group1 rhizobacteria

(8nm, 18nm, 4nm) and two of group 2 rhizobacteria (NWP, NFY, NDY) from

irrigated region showed protease activity as indicated by the development of clear

zones around the colonies on skim milk agar. Among arid zone selected rhizobacteria

four of the antagonists (PO, PY1a, PTW1, PTWz) of group 2 were tested positive for

protease activity while four rhizobacteria of group 1 (JY1, JSIR2, JYG, JYR) have

shown protease activity. Four out of 8 selected rhizobacteria of group 1 including

YDYs, YCH1, Y5, Yys and four group2 rhizobacteria i.e. YiPe, YiLY, YiH, YiBs

isolated from the semi-arid region has shown the production of protease activity.

Among irrigated region rhizobacteria two rhizobacteria from each group

(17nm, 4nm of group 1 and NFY, NDY of group 2) showed chitinase activity on

chitin agar medium by the development of halos around their colonies (Table 2.20).

While, four rhizobacteria of group 1 (JY1, JSIR2, JYR, JYG) and two rhizobacteria

of group 2 (PTW1, PTWz) belonging to arid region exhibited the chitinase

production.


73

Table 2.17: Catalase and oxidase enzymes activity by rhizobacteria isolated from

the rhizosphere maize fields of irrigated region

Rhizobacteria

(Group 1)

Catalase oxidase Rhizobacteria

(Group 2)

Catalase oxidase

2nm + + Np ++ +

5nm + - NWsm + +

8nm +++ + Nwp + +

9nm + - N91 + +

12nm + + NFY +++ +

17nm +++ + Nwce + +

18nm + + Nwce2 + +

4nm +++ + NDY ++ +


the rhizosphere maize fields of arid region

Rhizobacteria

(Group 1)


(Group 2)

Catalase oxidase

JY1 ++ + PO ++ +

JWIR - - PY1a + +

JWIR2 - - PYT2a ++ +

JSIR + + PCP + +

JSIR2 +++ + PTW ++ +

JWCH1 + + PCWIR - -

JLPO ++ + PTW1 ++ +

JYR +++ + PTWz +++ +

JYG ++ + - - -


the rhizosphere maize fields of semi-arid region

Rhizobacteria

(Group 1)


(Group 2)

Catalase oxidase

YDY ++ - YiPe ++ +

YDYs + + YiBa + -

YCC1 +++ - YiLy ++ +

YCH1 + + YiC + -

Y4 ++ - YiH +++ +

Yys +++ + YiBs + +

Y3 + + Yi16 + +

Y5 +++ + Yio +++ +





74

Table 2.20: Hydrolytic enzymes activity (Protease, Chitinase and cellulase) by

rhizobacteria isolated from the rhizosphere maize fields of irrigated region

Rhizobacteria (Group 1)

Protease Chitinase Cellulase Rhizobacteria (Group 2)

Protease Chitinase Cellulase

2nm - - - Np - -` -

5nm - - + NWsm - - -

8nm + - - Nwp + - -

9nm - - - N91 - - -

12nm - - + NFY + + +

17nm - + - Nwce - - -

18nm + - - Nwce2 - - -

4nm + + + NDY + + -


rhizobacteria isolated from the rhizosphere maize fields of arid region




JY1 + + - PO + - -

JWIR - - - PY1a + - -

JWIR2 - - - PYT2a - - -

JSIR - - - PCP - - -

JSIR2 + + - PTW - - -

JWCH1 - - - PCWIR - - -

JLPO - - - PTW1 + + -

JYR + + + PTWz + + -

JYG + + - - - - -


rhizobacteria isolated from the rhizosphere maize fields of semi -arid region




YDY - - - YiPe + + -

YDYs + + - YiBa - - -

YCC1 - - - YiLy + - -

YCH1 + - - YiC - - -

Y4 - - - YiH + + -

Yys + + - YiBs + + -

Y3 - - - Yi16 - - -

Y5 + + - Yio - - -





75

Three rhizobacteria of both groups (YDYs, Yys, Y5 and YiPe, YiH, YiBs) of semi-

arid region were indicated positive for chitinase activity (Table 2.21 and 2.22).

Very few rhizobacteria tested positive for cellulase production from all regions

and groups (Table 2.20 to 2.22). As among group 2 rhizobacteria only NFY and three

of group 2 rhizobacteria including 5nm, 12nm and 4nm of irrigated region showed the

cellulase activity whereas, among group1 rhizobacteria only JYR of arid region

exhibited the production of cellulase. All the rhizobacteria of semi-arid region of both

groups and all rhizobacteria of group 2 from arid region didn’t show the cellulase

activity.

At this step, Antagonistic rhizobacteria was screened on the basis of following

criteria

1. The antagonistic rhizobacteria which exhibit siderophore, HCN and Ammonia

production.

2. The rhizobacteria responsible for the production of cell wall degrading

enzymes.

3. The antagonistic rhizobacteria showing the ability to produce high IAA

content.

On the basis of above criterion, three rhizobacteria were selected from each region

and group including the 9 rhizobacteria from non-infected maize field (4nm,

18nm, 17nm of irrigated region, JY1, JYG, JYR of arid region, Yys, YDYs, Y5 of

semi-arid region) and 9 rhizobacteria from infected maize fields (NP, NFY, NDY

of irrigated region, PO, PY1a, PTWz of arid region and YiPe, YiH, YiO of semi-

arid region).


76

2.4 DISCUSSION

The rhizobacteria have unique capability to inhibit the infection of pathogens

through the enhancement of host plant growth (Sullivan and Gara, 1992) along with

inhibiting the proliferation of pathogen by using several different mechanisms (Hass

and Defago, 2005). Hence the present the investigation was carried out on the

biocontrol potential and plant growth promotional activities of rhizobacteria isolated

from the rhizosphere of maize of infected with stalk rot and uninfected maize grown

in field under varying moisture regimes.

2.4.1 Isolation of rhizobacteria

Earlier researchers have found that rhizobacteria obtained from the

rhizosphere of non-infected plants growing in non-infected soil and the rhizosphere of

non-infected plants present around the infected plants is a favourable habitat for the

antagonistic rhizobacteria (Cazorla et al., 2006). So, it could be inferred that almost all

kinds of agricultural soils induce suppressive effect on various soil borne

phytopathogens that is the result of antagonistic activities of these rhizobacteria

inhabiting in rhizosphere (Weller et al., 2002).

Fertile non-infected soil is commonly dominated by the antagonistic microbes

which produce a number of antibiotics, siderophores, fungicidal compounds, and

which enable them to compete with microbes, and induce plant resistance against

pathogenic microbes (Singh and Singh, 2008). So, in the present investigation,

rhizobacteria isolated from different regions of varying moisture regimes were

divided in 2 groups on the basis of their relation with maize isolation infected with

and without stalk rot disease. Group 1 consisted of rhizobacteria from the soil of

maize field without any infection and group 2 comprised of rhizobacteria obtained

from the maize filed infected with stalk rot.

2.4.2 Antifungal activity of isolated rhizobacteria

In the present investigation 117 rhizobacteria were isolated from the

rhizosphere of maize field, were tested for their in vitro antifungal potential against

three fungal pathogens (F.moniliforme, Helminthosporium sativum, Aspergillus

flavus). It was found that a number of rhizobacteria demonstrated strong antifungal

activity against F. moniliforme, out of them the rhizobacteria which showed 50-90%

inhibition of F. moniliforme in agar tube dilution method was selected for the further


77

study. Similar findings were observed by Charles et al., (2001) for the inhibition of F.

moniliforme by rhizobacteria. It was reported by Pal et al., (2001) that the antagonistic

activity of various secondary metabolites produced by fluorescent Pseudomonas and

Bacillus sp. involved in inhibition against F. moniliforme. Other authors also found

the in vitro inhibition of F. moniliforme by P. fluorescens (Hebbar et al., 1992;

Rajappan and Ramaraj, 1999).

The selected rhizobacteria isolated in this study, not only inhibited the fungal

growth of F. moniliforme but also suppressed the growth of H. sativum and A. flavus

by a range of percentage inhibition. Shalini and Srivastava, (2008) determined the

antifungal activity of Pseudomonas strains against various plant fungal pathogens

(Srivastava and Shalni 2008). A number of other studies reported the successful

biological control of A. flavus by different antagonistic rhizobacteria (Jeffrey et al.,

2006; Palumbo et al., 2007; Mushtaq et al., 2010). Antifungal potential of

Streptomyces and Bacillus species has also been reported for the suppression of fungal

growth of several of Fusarium and Aspergillus species (Munimbazi and Bullerman,

1998; Nourozian et al., 2006). A number of fluorescent Pseudomonas isolated from

the rhizosphere wheat proved to be antagonistic to H. sativum (Gaur et al., 2004).

The rhizobacteria isolated from the rhizosphere of maize field with non-

infected plants have inhibited the growth of fungal pathogens with high percentage

inhibition and more number of rhizobacteria isolated from rhizosphere of non-infected

plants has exhibited the antifungal activity as compared to the rhizobacteria isolated

from the rhizosphere of infected maize fields. This finding is in accordance with Peng

et al., (1999) who reported that the soil without pathogen infection contains more

antagonistic bacteria and fungus than the soil infected with diseases. In the non-

infected soil, the antagonistic microbes like Bacillus, Trichoderma, Pseudomonas,

Actinomycetes and non-pathogenic fungal agents effectively protect plants from soil

borne fungal pathogens (Weller et al., 2002; Garbeva et al., 2004).

2.4.3 Morphological Characteristics

All the bacterial isolates were found to be different in their morphological

characteristics from each other. The difference in phenotypic characteristics explains

that bacterial morphology is generally related to the adaptation of bacterial strains to

different ecological aspects of particular studied areas (Bochner, 2009).

http://www.ncbi.nlm.nih.gov/pubmed?term=Pal%20KK%5BAuthor%5D&cauthor=true&cauthor_uid=11716210


78

2.4.4 Phosphate solubilization

The second criterion used for the selection of antagonistic rhizobacteria was

phosphate solubilization, a plant growth promoting activity in order to isolate the

potent rhizobacteria which not only have the capability to suppress the proliferation of

fungal pathogens but also promote the growth of plants. Phosphorous solubilizing

strains of rhizobacteria have an effective role in uptake of phosphate and plants

growth promotion by converting the inorganic insoluble phosphate to available form

of phosphorus to the plants (Hafeez et al., 2006; Supraja et al., 2011).

Phosphate solubilizing bacteria are considered among the potential group of

rhizobacteria in agriculture for enhancing the plant growth (Chaiharn et al., 2008).

The results of present study show that among the rhizobacteria which show the

antagonistic activity against fungal pathogens, 30 rhizobacteria were found the

efficient solubilizes of inorganic phosphorus might have better interaction and

adaptation within the rhizosphere. These results are in accordance with earlier studies

of Igual, (2001) and Chen et al., (2006). Tilak et al., (2005) founded maximum

occurrence of phosphate solubilizing bacteria in rhizoplane followed by rhizosphere

and then root free soil. The phosphate solubilising bacteria can effectively be

inoculated to improve the plant growth by enhancing the uptake of phosphorous

(Barraquio, 2000). Similarly, B. megaterium isolated from the tea rhizosphere

promote the plant growth by its ability to solubilize phosphate (Chakraborty et al.,

2006).

2.4.5 Production of IAA

A total number of 48 selected rhizobacteria were used to elucidate the possible

mechanism for the antagonistic activity and plant growth promoting potential. These

rhizobacteria were tested for plant growth promoting activity like IAA production. In

the present investigation IAA production was recorded by majority of the

rhizobacteria. The IAA hormone has the potential to enhance the plant growth and as

well as involved in the biological control by suppressing the spore germination and

fungal growth by triggering the glutathione transferases in defense related pathways

(Brown and Hamilton, 1993; Strittmatte, 1994). Noel et al., (2001) demonstrated that

exogenous application of IAA decreased the disease severity. The rhizobacteria of

isolated from irrigated region have shown higher production of IAA as compared to


79

arid and semi-arid regions. The low amount of IAA can be attributed to the lack of

tryptophane deaminase activity in arid region selected rhizobacteria as observed in

QTS tests. These results are in accordance with the finding of Ilyas, (2009).

2.4.6 Siderophore production

Siderophore production is an important attribute of rhizobacteria for survival

and growth in competitive ecosystem having iron as a limiting factor (Khan et al.,

2006; Dimkpa et al., 2009). According to the results of presents study, 30

rhizobacteria of both groups exhibited the production of siderophore. Siderophore

production by rhizobacteria has also been sown in various previous studies (Raval and

Desai, 2012). Siderophore producing rhizobacteria have strong antifungal activity

against fungal pathogens by the acquisition of iron (Scher and Baker, 2006; Idris et

al., 2010; Glick, 2012) as well as for the better growth of plants (Robin et al., 2008).

2.4.7 Production of enzymes

The role of enzymes in biological control is usually associated with the

mechanisms referred as parasitism and antibiosis. Particularly, the hydrolytic enzymes

including β-1,3-glucanases, chitinases, proteases and cellulases are not only important

for mycoparasitism but, also involved in the host fungi colonization by exhibiting

antifungal activity (Hermosa, et al., 2000). Kamala and Devi, (2012) also documented

that cell wall degrading enzymes have prominent role in inhibition of fungal

phytopathogens.

The production of protease enzyme has been detected by twenty one

rhizobacteria. Earlier studies reported that microorganisms secrete the extra cellular

enzyme including proteases which inhibit various bacterial (Johansen et al., 2002)

and fungal communities (Girlanda et al., 2001). A number of reporters

demonstrated that rhizobacteria that exhibit the protease activity help in the biological

control of pathogens (Marcia et al., 2006; Rakh et al., 2011). Expression and secretion

of protease enzymes by rhizobacteria result in the suppression of plant pathogen

activities directly by degradation of fungal cell wall (Howell, 2003). These bacterial

strains contribute significantly in inhibition of fungal phytopahtogens with a

significant increase in root colonization and plant development (Gray and Smith,

2005). Golzary, (2011) demonstrated that protease enzyme is effective for

biocontrol of fungal pathogens directly or indirectly.

Other enzymatic activities such as chitinase, cellulase, oxidase and catalase of

the bacterial isolates were also determined in vitro condition. Most of the


80

rhizobacteria showed oxidase and catalase activity isolated from arid, semi-arid and

irrigated regions. These results are in accordance with the finding of other researchers

(Joseph et al., 2007; Shobha and Kumudini 2012). It is one of the important ability of

a biological control agents (Samuel and Muthukkaruppa, 2011; Ramyasmruthi et al.,

2012). According to Joseph et al., (2007) catalase activity of rhizobacteria enables

them extremely resistant to different types of stresses and renders them to act as

potential biological control agents against plant diseases. The intensity of catalase

activity was higher in the rhizobacteria isolated from arid and semi -arid regions than

the rhizobacteria of irrigated region this is in accordance with the finding of Ilyas,

(2009).

The cell wall of fungal pathogens consists of chitin, glucan and the b-1, 3-

glucanase and chitinase produced by antagonistic rhizobacteria are major enzymes

involve in degradation of fungal cell wall (Kucuk and Kivanc, 2004). Chitinase

induce swelling by in fungal mycelium and results in vacuole formation. It is also

accompanied by the cell walls degradation of fungi and the release of intracellular

constituents (Melentev et al., 2001). The increase in chitinase and β-1,3-glucanase is

essential for the retardation of fungal growth. The enzyme involved in fungal cell

wall degradation may also release general elicitors (Ham et al., 1991; Ren and West,

1992) which elicits several defense reactions in plants and induce systemic resistance.

A strong relationship was reported by Nagarajkumar et al., (2004) between the

antagonistic capability of P. fluoresecens and its potential to produce extracellular

chitinase. More rhizobacteria isolated from the non-infected rhizosphere have shown

the activity of chitinase and protease as compared to rhizobacteria isolated from the

infected rhizosphere. More number of rhizobacteria isolated from arid and semi-arid

region has shown the chitinase and protease activity as compared to irrigated region.

2.4.8 HCN Production

Bacterial strains producing the hydrogen cyanide are good biocontrol agents as

it induces the resistance in plants (Berg et al., 2002). Ramette et al., (2003) have

demonstrated that HCN is involved in biocontrol of plant pathogens. It has been

suggested that HCN may constitute a stress condition in the plants and aggravate

resistance to fungal diseases (Defago et al., 1990). Rhizobacteria inhibit the

phytopathogenic fungi by the production of volatile HCN (Ahmad et al., 2008;

Blumer and Hass, 2000). The intensity of HCN production was more in the


81

rhizobacteria isolated from arid and semi-arid region as compared to irrigated region.

While no considerable difference was observed in case of rhizobacteria isolated from

infected and non-infected maize field for the production of HCN.

It has been well documented by a number of researchers that rhizobacterial

strains affect the plant health indirectly by the production of secondary metabolites

including cell wall degrading enzymes, antibiotics, siderophores and HCN metabolite

and prevent the damaging effects of pathogens (Gupta et al., 2000; Tenuta 2003).

Similarly, the antagonistic bacteria selected in present study exhibited various

mechanisms for the inhibition of fungal pathogens. The efficient antagonistic strains

produced the hydrolytic enzymes, HCN, siderophores and antibiotics.

Production of more than one metabolite was detected in different strains. On

the basis of above analysis 18 rhizobacteria were selected, all of them exhibited

several desirable characteristics which may suppress the fungal pathogens and

promote the plant growth directly or indirectly or synergistically.


82

2.5 INTRODUCTION

A successful biocontrol agent is generally equipped with several attributes

which often promote the plant growth as efficiently as it inhibit the fungal growth by

efficient root colonization, phytohormone production and nutrient competition may be

related to the ability of PGPR for plant growth. They rhizobacteria has the ability to

improve the plant growth in different ways and enhance the vegetative and

reproductive growth as determined in several crops like cereals, vegetables, pulses,

and some trees. The application with PGPR results in improvement of germination

percentage (%), seedling vigour and emergence, shoot and root growth, total plant

biomass (Van Loon et al., 1998; Ramamoorthy et al., 2001).

Various mechanisms are involved in plant growth promotion including the

increase in the production of phytohormone, solubilization of phosphorous,

siderophore production, increases in root permeability, better capability to survive in

strict competitive niche and root sites and suppression of harmful microorganisms.

(Pal et al., 1999; Enebak and Carey, 2000). It is suggested that rhizobacteria mediated

phytohormone production is the one of the important mechanism used by PGPR to

enhance the plant growth, while siderophore production is another important

mechanism for stimulation of plant growth in the presence of deleterious rhizospheric

microorganisms (Bossier et al., 1988).


83

2.6 RESULTS

2.6.1 Survival efficiency (CFU) of antagonistic rhizobacteria

The selected rhizobacteria isolated from different agro-climatic region and

from the rhizosphere of maize fields infected with (Group 2) and without stalk rot

(Group 1) were further used for the assessment of their effect on growth of maize (Zea

mays L.) seedlings. The number of cfu/g soil tested ranged between 1.9 and 4.8 log

cfu/g soil for treatments inoculated with group 1 rhizobacteria (Fig 2.28).

The treatments Sa1+Y5 and A1+JYR exhibited high cfu count (4.83 log

cfu/g) among group1 rhizobacterial treatments. It was followed by treatments

Ir1+4nm (4.03cfu/g) >Ir1+17nm (3.49cfu/g) > A1+JY1 (3.25cfu/g) > Sa1+Yys (2.42)

> A1+JYG (2.03 log cfu/g) > Sa1+YDYs (1.92 log cfu/g) > Ir1+8nm (1.9cfu/g).

Among group 2 rhizobacterial treatments, Ir2+NDY, A2+PTWz and Sa2+Yio were

most effective showing the high cfu count (4.36, 3.8, 4.24 log cfu/g soil). Minimum

CFU count, among group 2 rhizobacterial treatments, was exhibited by Sa2+YiPe i.e.

1.44 log cfu/g of soil. The rhizobacteria of irrigated region had better survival

efficiency as compared to the rhizobacteria of arid and semi-arid region. The survival

efficiency of group 1 rhizobacteria was higher as compared to rhizobacteria of group

2 irrespective to the difference in moisture content of different regions.

2.6.2 Shoot and root length

The results (Fig 2.30) revealed that inoculation of rhizobacteria showed

significant increase in root and shoot length of maize as compared to un-inoculated

control. Among group1 isolates maximum increase in root length of maize seedling

was observed for A1+JYG i.e. 72% as compared to un-inoculated control. Next to this

all other treatments were ranked for the increase in root length as Sa1+Y5 (69%),

>Ir1+17nm (62%) > Ir1+8nm (51%) > Ir1+17nm (47%) > Sa1+Yys (40%) >

A1+JYG (33%) > Sa1+YDYs (22%). The treatment A1+JY1 has shown no

significant increase in root length as compared with that of un-inoculated control. This

increase by group 2 rhizobacterial treatments was not that as pronounced as group1

rhizobacterial treatment. Group 2 treatment Ir2+NDY significantly increased (58%)

the root length as compared to un inoculated control while, this increase in root length

was followed by A2+PTWz (54%), A2+PO and Ir2+NP (48%), Sa2+Yio (42%),

A2+PY1a and (15%) as presented in Fig 2.31. The treatments Ir2+NP and Sa2+YiH

exhibited non-significant effect on root length of maize seedlings.


84

Fig 2.28: CFU of Group 1 rhizobacteria inoculated to maize seeds

Group 1: Rhizobacteria isolated from the rhizosphere of maize non-infected fields. Ir1+8nm: isolated

from irrigated region, Ir1+17nm: isolated from irrigated region, Ir1+4nm: isolated from irrigated

region, A1+JY1: isolated from arid region; A1+JYG: isolated from arid region, A1+JYR: isolated from

arid region, Sa1+Yys: isolated from semi-arid region; Sa1+YDYs: isolated from semi-arid region;

Sa1+Y5: isolated from semi-arid region. Each bar represents the average of three independent

measurements and means with same letter are non-significantly different according to least significant

difference (P ≤ 0.05). LSD: 0.200.

Fig 2.29: CFU of Group 2 rhizobacteria

Group 2: Rhizobacteria isolated from the rhizosphere of maize infected fields. Ir2+NP: isolated from

irrigated region, Ir2+NFY: isolated from irrigated region, Ir2+NDY: isolated from irrigated region,

A2+PO: isolated from arid region; A2+PTWZ: isolated from arid region, A2+PY1a: isolated from arid

region, Sa2+YiPe: isolated from semi-arid region; Sa2+YiH: isolated from semi-arid region; Sa2+YiO

: isolated from semi-arid region. Each bar represents the average of three independent measurements

and means with same letter are non-significantly different according to least significant difference (P ≤

0.05) LSD: 0.277.

c c

a

e de

b

d e

a

0

1

2

3

4

5

6

log

CFU

/g s

oil

Treatments

CFU/g soil

cd bcd

a

de ef

ab

g

f

bc

00.5

11.5

22.5

33.5

44.5

5

Log

CFU

/g s

oil

Treatments


85

All the rhizobacteria treatments of both groups have significantly increased the shoot

length of maize seedlings as compared to un-inoculated control whereas much

increase was not observed among the treatments. In case of Group 1 rhizobacterial

treatments, maximum increase in shoot length was recorded for treatment Ir1+4nm

(29%). Other treatments for increase in shoot length were ranked as follow A1+JYR

(18%) > A1+JY1 (16%) > Sa1+Y5 and Ir1+17nm (15%). Among the group2

rhizobacterial treatments the increase in shoot length showed a large range from 1.3-

54%. Maximum (54%) increase was exhibited by A2+PTWz. The ranking of other

treatments was as follow Sa2+Yio and Ir2+NDY (24 %) > Ir2+NP (20%) > Sa2+YiPe

(19%) > A2+PY1a (17%) > Ir2+NFY (11%). The treatments Sa2+YiH, A2+PO didn’t

show significant increase in shoot length of maize seedlings.

2.6.3 Root to shoot ratio

Most of the rhizobacterial treatments has shown significantly high root to

shoot ratio of maize seedlings in both of the groups. The data presented in Fig 2.34

revealed that all rhizobacterial treatments of group1 showed stimulatory effect on root

to shoot ratio of maize seedlings as compared to un-inoculated control. Among

group1 isolates significantly high root to shoot ratio of maize seedling was observed

for Sa1+Y5 (80%) as compared to un-inoculated control. Next to this all other

treatments were ranked for root to shoot ratio as follows A1+JYR and Ir1+4nm

(75%)> Sa1+YDYs (50%) > A1+JYG (49%) > Sa1+Yys (48%) > Ir1+17nm (39%) >

A1+JY1 (33%).

The treatment Ir1+8nm has shown no significant effect on root to shoot ratio

when compared to that of un-inoculated control. In case of Group2, treatment

Ir2+NDY exhibited significantly high (77%) root to shoot ratio as compared to un

inoculated control while, this trend was followed by other treatments as A2+PTWz (

65%), Sa2+Yio (59%) ,A2+PO (47%), Sa2+YiPe (32%), A2+PY1a (30%) ,

Ir2+NFY(31%) , Ir2+NP (14%) , Sa2+YiH (11% ) (Fig 2.35).


86

Fig 2.30: Effect of Group1 rhizobacteria on shoots and root length of maize

seedlings

Group 1: Rhizobacteria isolated from the rhizosphere of maize non-infected fields. Detail of treatments

is given in 2.28. Each bar represents the average of three independent measurements. All means sharing

the common letter differ non-significantly as (P ≤ 0.05) Root length LSD: 0.741, Shoot length LSD:

0.3788.

Fig 2.31: Effect Group 2 rhizobacteria on shoot and root length of maize

seedlings

Group 2: Rhizobacteria isolated from the rhizosphere of maize infected fields. Detail of treatments is

given in 2.29. Each bar represents the average of three independent measurements. Means with same

letter are not significantly different according to least significant difference (P ≤ 0.05), Root length

LSD: 0.375, Shoot length LSD: 0.277.

h de c b de c a c d b

h

h d

c

h f

a

e g

b

0

5

10

15

20

25

30

35

40

0

5

10

15

20

25

Ro

ot

len

gth

(cm

)

sho

ot

Len

gth

(cm

)

Treatments

shoot length root length

e b d a bc e bc e e cd

h c

d

a

d

f

b

fg gh

e

0

5

10

15

20

25

0

5

10

15

20

25

Ro

ot

len

gth

(cm

)

Sho

ot

Len

gth

(cm

)

Treatments

Shoot length Root length


87

Fig 2.32: Effect of Group1 rhizobacteria on root to shoot ratio of maize seedlings



the common letter differ non-significantly as (P ≤ 0.05), LSD: 0.0723.

Fig 2.33: Effect of Group 2 rhizobacteria on root to shoot ratio of maize seedlings


given in 2.28. Each bar represents the average of three independent measurements. All means sharing

the common letter differ non-significantly as (P ≤ 0.05), LSD: 0.055.

f f de

b

e c

b

cd c

a

0

0.5

1

1.5

2

2.5

3

Ro

ot

to s

ho

ot

rati

o

Treatments

f f e

b

d e

a

e f

c

0

0.5

1

1.5

2

2.5

roo

t to

sh

oo

t ra

tio

Treatments


88

2.6.4 Shoot and root Fresh weight

The results (Fig 2.32) revealed that inoculation of rhizobacteria showed

significant increase in root and shoot fresh weight of maize seedlings as compared to

un-inoculated control. Among group1 isolates maximum increase in root weight of

maize seedling was observed for A1+JYG i.e. 94% as compared to un-inoculated

control. Next to this all other treatments ranked for the increase in root fresh weight as

Sa1+Y5 (93%) > Ir1+4nm (75%) > Sa1+Yys (52%) > Ir1+17nm > A1+JYG,

Sa1+YDYs > (45%) > A1+JY1 (40%). The treatment Ir1+8nm has shown no

significant increase in root fresh weight as compared with un-inoculated control. This

increase in root weight by group2 rhizobacterial treatments was not that as much

pronounced as group1 rhizobacterial treatment. Group 2 treatment Sa2+Yio

significantly increased (65%) the root weight as compared to un inoculated control

while, this increasing trend in root fresh weight was followed by A2+PTWz (63%),

Ir2+NDY(56%) > Ir2+NFY (51%) > Ir2+NP (41%) > Sa2+YiPe (29%) > A2+PO

(28%) > Sa2+YiH (13%) as presented in Fig 2.33.

All inoculated rhizobacterial treatments showed that increase in shoot fresh

weight was statistically non-significant with each other while they exhibited

significant increase as compared to control. The increase in shoot fresh weight as

compared to control ranged from 18-65% for group1 rhizobacterial treatments (Fig

2.32). Maximum increase was exhibited by treatment Ir2+NDY (65%) while, other

treatments ranked as follows A2+PTWz (54%) > Sa2+Yio (48%) > Ir2+NP (27%) >

Ir2+NFY (18%). In case of Group 2 rhizobacterial treatments the maximum increase

in shoot fresh weight was recorded for treatment A1+JYR (51%). Other treatments for

increase in shoot fresh weight were ranked as followed Ir1+4nm (48%) > Sa1+Y5

(38%) > Ir1+17nm (33%) > A1+JYG (32%) > Sa1+Yys (15%), A1+JY1 (12%).

2.6.5 Leaf area

The data presented in Fig 2.36 and 2.37 indicated that all rhizobacterial

treatments of both groups showed stimulatory effect on leaf area of maize seedlings as

compared to un-inoculated control The increase in leaf area with different group1

treatments ranged from 10-65%, the treatments A1+JYR isolated from arid region

was the most effective treatment which produced 65% increase in leaf area when

compared to that of un-inoculated control.


89

Fig 2.34: Effect of Group1 rhizobacteria on shoots and root fresh weight of

Maize seedlings



the common letter differ non-significantly as (P ≤ 0.05) LSD: 0.054.

Fig 2.35: Effect of Group2 rhizobacteria on shoot and root Fresh weight of

Maize seedlings




e e

d b

d d

c

d d

a

d d

c b

d c

a

d d

ab

0

0.5

1

1.5

2

2.5

0

0.2

0.4

0.6

0.8

1

1.2

Ro

ot

fre

sh w

eig

ht

(g)

Sho

ot

fre

sh w

eig

ht

(g)

Treatments

shoot fresh weight Root fresh weight

e

c cd

a

de e

ab

de de

b g

cd c a

de ef

b

de f

b

0

0.5

1

1.5

2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Ro

ot

fre

sh w

eig

ht(

g)

Sho

ot

Fre

sh w

eig

ht

(g)

Treatments

shoot fresh weight Root fresh weight


90

Fig 3.36: Effect of Group1 rhizobacteria on Leaf area of maize seedlings




Fig 2.37: Effect of Group 2 rhizobacteria on Leaf area of maize seedlings




h gh

c

b

fg de

a

cd ef

ab

0

5

10

15

20

25

Leaf

are

a (c

m)

Treatments

leaf area

f

c

d

a

c

e

a

e ef

b

0

5

10

15

20

25

Leaf

are

a (c

m)

Treatments


91

Among the treatments inoculated with group 2 rhizobacteria, treatment

Ir2+NDY showed significant increase (61%) in leaf area as compared to control. All

other treatments ranked for increase in leaf area as follows A2+PTWz (59%), Ir2+NP

(52%) > Sa2+Yio (47%) > A2+PO (41%) > Ir2+NFY (34%) > Sa2+YiPe (19%) and

A2+PY1a (17%).

On account of selected rhizobacterial treatments effect on the plant growth

promotion of maize seedlings, three rhizobacteria were selected from each group for

molecular characterization and detection of antibiotic biosynthetic genes including

4nm and NDY from irrigated region, JYR and PTWz from arid region and Y5 and

Yio from semi-arid region rhizobacteria.


92

2.6.6 Identification of selected Rhizobacteria

2.6.6.1 Morphological and biochemical characterization of bacterial antagonists

Colonies of all the isolates were rod shaped but vary in size, shape, colour and

pigmentation. Fresh culture of bacteria was used for the evaluation of the biochemical

characteristics by using microbial identification kits QTS-24 (based on utilization of

carbon/nitrogen source). All rhizobacteria showed activity in the utilization of lysine

decarboxylase, Ortho-nitro phenyl β-D-galactopyranoside, glucose, raffinose and

malonate (Table 2.22). These bacterial antagonist 4nm, JYR, NDY were Gram-

negative and Yio, Y5, PTWz were gram positive. All bacterial antagonists utilized

mannose, melibiose, arabinose, mannitol except PTWz but exhibited the utilization of

adonitol.

Among the rhizobacteria belonging to group 1 were detected positive for the

utilization of mannitol, arabinose, melibiose and arginine dihydrolase. Among group

2 rhizobacteria all isolates were negative for lysine decarboxylase and maltose while,

PTWz from arid region and Yio isolated from semi-arid region were negative for the

utilization of orthinine decarboxylase, trytophane deaminase and Indole production.

All the rhizobacteria of group 2 were positive for sucrose and gelatine hydrolysis. The

group 2 rhizobacteria NDY and group 1 rhizobacteria 4nm exhibited better utilization

of carbohydrates as compared to rhizobacteria isolated from the rhizosphere of arid

and semi-arid region. Along with this the group 1 rhizobacteria also showed better

utilization of carbohydrates as compared to group 2 rhizobacteria.


93

Table 2.22: Morphological and biochemical characteristics (as determined by

QTS) of selected rhizobacteria isolated from rhizospheric soil of maize plants

Tests

Reactions Group 1 Group2

4nm JYR Y5 NDY PTWz Yio

OPNG Ortho nitro phenyl β-

D-galactopyranoside + + + + + +

CIT Sodium citrate + + + + + +

MALO Sodium malonate + + + + + +

LDC Lysine decarboxylase + + - - - -

ADH Arginine dihydrolase + + + - + -

ODC Orthinine

decarboxylase + + - + - -

TDA Trytophane deaminase + - + + - -

IND Indole - - - + - -

VP

Voger

Proskaur(acetion) + + - + - +

GEL

Gelatin hydrolysis + + - + + +

GLU

Acid from glucose + + + + + +

MAL

Acid from maltose + - + - - -

SUC

Acid from sucrose + - + + + +

MAN Acid from mannitol

+ + + + - +

ARA Acid from arabinose

+ + + + - +

RHA Acid from rhammose

+ - + + - +

SOR Acid from sorbitol

+ - - + + -

INO l Acid from inositol

- - + - - +

ADON Acid from adonitol

- - - - + -

MEL Acid from melibiose

+ + + + - +

RAF Acid from raffinose

+ + + + + +

Organism

identified

Pseudo

monas

Pseudo

monas

Bacillus Pseudo

monas

Bacillus Bacillus

The symbol - indicate negative in test and + indicate positive in test


94

2.6.7 Identification at molecular level

The 16S rRNA sequences from isolates showed higher levels of similarity

between 91 to 99% with bacterial sequences from Gene Bank. The strains were

identified by comparing the evolutionary distance to neighbouring strains present in

Gene bank database. Bootstrap test of phylogeny by neighbour-joining phylogenetic

tree was performed on the basis of the obtained distance matrix data.

The phylogenetic trees for 4nm rhizobacteria (Gene bank accession number:

JQ792037) and NDY (Gene bank accession number: JQ792039) rhizobacteria falls in

the Pseudomonas genus cluster. The 4nm showed 99% similarity with pseudomonas

aeruginosa (Fig 2.39 a) and NDY maximum similarity is shown for Pseudomonas sp.

(Fig 2.39 b).

The rhizobacteria JYR (Gene bank accession number: JQ792038) was

included in the big cluster of genus Pseudomonas, having 99% similarity with

Pseudomonas aeruginosa (Fig 2.40 a). Rhizobacteria PTWz (Gene bank accession

number: JQ792036) was included in the big cluster of genus Bacillus, having 99%

similarity with Bacillus pumilus (Fig 2.40 b).

The phylogenetic trees for Y5 rhizobacteria (Gene bank accession number:

JQ792035) and Yio (Gene bank accession number: JQ792034) rhizobacteria falls in

the Bacillus genus cluster. Y5 showed 99% similarity with Bacillus endophyticus (Fig

2.41 a) and Yio maximum similarity is shown for Bacillus firmus (Fig 2.41 b).


95

Fig 2.38 Polymerase Chain reaction for I6sRNA

Lane 1: Marker Lamda hind3 and Lane 2 – 6 are DNA of given samples with 16s 27F and 1492R

primer mix. Lane 2 (Y5), Lane 3 (YiO), Lane 4 (NDY), Lane 5 (PTWz), Lane 6 (4nm), Lane 7 (JYR)

Table 2.23: Identified rhizobacteria and their Accession numbers

Bacterial isolates Identified bacteria Accession number

JYR Pseudomonas aeruginosa JQ792038

PTWz Bacillus firmus JQ792036

Y5 Bacillus endophyticus JQ792035

Yio Bacillus pumilus JQ792034

NDY Pseudomonas sp. JQ792039

4nm Pseudomonas aeruginosa JQ792037


96

Fig 2.39: Phylogenetic tree of Pseudomonas aeruginosa 4nm and

Pseudomonas sp. NDY

(a): The phylogenetic tree of Pseudomonas aeruginosa 4nm indicate the position of rhizobacteria in

the established related species of the genus. The tree is drawn to scale, with branch lengths in the same

units as those of the evolutionary distances used to infer the phylogenetic tree. Numbers at branch

points are bootstrap percentages based on 1000 replicates. (b) The neighbour-joining phylogenetic tree

of Pseudomonas sp. NDY indicating the position of strain in the established related species of the

genus. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary

distances used to infer the phylogenetic tree. Numbers at branch points are bootstrap percentages based

on 1000 replicates. The scale bar represents the expected number of substitutions.

P.aeruginosa-S164S-Ind(JF513146)

P.aerogenosa-JCM6119-Jpn(AB594760)

Uncultured-16sps16-3h07.p1k-UK(FM996038)

Uncultured.16sps19-3g01.p1k-UK(FM996540)

P.aerogenosa-DQ8-Ch(GU269267)

P.aerogenosa-1-15-Ch(HQ434555)

Pseudomonas.sp-1d-11-Ch(FJ598142)

P.aerogenosa-AS2-Ind(GU447238)

Uncultured-16sps15-1a08.p1k-UK(FM995757)

Uncultured-16sps20-2g02.q1ka-(FM996736)

Uncultured.16sps17-3a07.p1k-UK(FM996166)

Pseudomonas.sp-DG1b-Itly(HQ609597)

Uncultured-16sps20-1a10.p1ka-UK(FM996...


P.aerogenosa-MZA-85-Pk(HQ023428)

Uncultured-16sps16-3a12.w2k-UK(FM995985)


P.aerogenosa-nsm-Ind(HQ658761)

Uncultured.16sps17-1h04.w2k-UK(FM996082)

Pseudomonas.sp-EMB23-Ind(JF281099)

Uncultured.16sps17-2d06.w2k-UK(FM996116)

Pseudomonas.sp-pseudo-EJB5-Ch(GU966670)

P.aerogenosa-10-Ch(FJ907193)


Uncultured.16sps19-1e02.p1k-UK(FM996424)

P.aerogenosa-HS9-Ch(GU323371)

P.aerogenosa-C24-Ch(HM036358)

Uncultured-16sps16-3g01.w2k-UK(FM996023)

Uncultured.16sps17-3e08.w2k-UK(FM996205)

Uncultured-16sps24-1h09.w2ka-UK(FM997...

P.aeruginosa-G6-Ch(GQ221872)

Uncultured.16sps17-2a04.w2k-UK(FM996089)

P.aerogenosa-ANSC-Ch(GU296674)

Uncultured-16sps23-1e08.w2ka-UK(FM997...

P.aeruginosa-IRMD-Irn(JF708942)

Uncultured.16sps16-2h11.p1k-UK(FM995975)

P.aerogenosa-BSF-g-Ch(GU121439)

Pseudomonas.sp-EJB3-Ch(GU966668)

P.aerogenosa-1-8-Ch(GU586139)

Uncultured-16sps17-1d10.p1k-UK(FM996063)

Pseudomonas.sp-AGP-01-Ind(HM587311)

P.aerogenosa-Ind(FJ665510)

P.aerogenosa-Ind(HM637743)

Pseudomonas.sp-TBP-Y-Ch(FJ804740)

P.aerogenosa-JCM5516-Jpn(AB594761)

P.aeruginosa-FJAT346-Ch(JN572122)

Uncultured-16sps20-1g11.p1ka-UK(FM996...

Uncultured-16sps22-1a09.w2ka-UK(FM996...

P.aerogenosa-B295-Ch(JF833630)

Pseudomonas.sp-DG2b-itly(HQ609593)

Uncultured-16sps23-2b10.p1k-UK(FM997057)

Uncultured-16sps15-2e01.p1k-UK(FM995843)



P.aerogenosa-NO5-Korea(FJ972533)

Uncultured-16sps23-2c01.p1k-UK(FM997059)


Uncultured-16sps22-1c05.p1ka-UK(FM996...

P.aerogenosa-JCM-2776-Jpn(AB594757)

P.aeruginosa-F1-Ch(JN412064)

Uncultured-16sps21-2c11.p1kb-UK(FM996...


Pseudomonas.sp-CIFRI.D-TSB-6-Ind(JF78...

Uncultured-16sps21-1c08.p1ka-UK(FM996...



P.aeruginosa-BS8-Ind(JN003625)

Uncultured.16sps19-2d04.p1k-UK(FM996461)

Uncultured.16sps17-2d08.w2k-UK(FM996117)

Uncultured-16sps24-2b02.p1k-UK(FM997200)


Uncultured-16sps21-2h09.w2ka-UK(FM996...

P.aerogenosa-ZAQ222-(GQ375800)

Uncultured-16sps16-3f04.w2k-UK(FM996018)

uncultured.16sps18-1a11.p1k-UK(FM996250)

Uncultured-16sps16-3d01.w2k-UK(FM996000)

pseudomonas.sp-IHB-B4040-Ind(HM234002)

pseudomonas.sp-3-Ch(EU784954)


Pseudomonas.sp-NR2-Ind(GU566322)


Uncultured-16sps22-1b02.w2ka-UK(FM996...

P.aerogenosa-GPSD-59-Ind(HQ270549)


P.aerogenosa-EH8-Ind(GU339238)

P.aerogenosa-9-Ch(FJ907192)

Uncultured.16sps15-2c12.p1k-UK(FM995835)

Uncultured.16sps18-1g05.p1k-UK(FM996309)

Uncultured-16sps20-1e04.p1ka-UK(FM996...


Pseudomonas.sp-OU67-Ind(FN663622)

P.aeruginosa-G14-N.land(HQ288928)

4nm-Pk-2011

P.aeruginosa-PW09-Ind(JN020962)

Pseudomonas.sp-G16-Ch(GU086454)

Burkholderia.sp-IBUN-S1602-Clmbia(DQ8...

Burkholderia.sp-CCBAU23014-Ch(AY839565)

P.fluorescens-HXQ-N33-Ch(HM439651)

Pseudomonadaceae-KVD-1959-04-USA(DQ49...

Pseudomonas.sp-B2-8-Ch(JF900038)

P.moraviensis-PSB32-Ch(HQ242745)

Pseudomonas.sp-MR14-Ind(JN082731)

Pseudomonas.sp-JA03-Korea(DQ365568)

Uncultured-D5-Ch(JF833750)

P.fluorescens-PFD11-Ind(GQ900589)

Pseudomonas.sp-bB10-Ch(JF772541)

Pseudomonas.sp-G22-Ch(GU086449)

Pseudomonas.sp-AR29-Tiwn(HM027909)

Pseudomonadaceae-KVD-1959-01-Ch(DQ490...

P.koreensis-EA2-7-Ch(JF496406)

Pseudomonas.sp-GH07-Korea(DQ365562)

Uncultured-PmeaMucE10-USA(EU249971)

Pseudomonadaceae-KVD-unk-58-USA(DQ490...

P.fluorescens-RHH45-Ch(HQ143617)


Pseudomonas.sp-W15Feb9-Blgm(EU680988)

Uncultured-WHIb14-Cnda(HQ230197)

Pseudomonas.sp-NZ017-N.Z(AY014805)

P.fluorescens-Mc07-Korea(EF672049)


Pseudomonadaceae-KVD-unk-76-USA(DQ490...

Pseudomonas.sp-TS2-Ind(HM747951)

Pseudomonas.sp-OT6-Ind(HM747953)

Uncultured-EDW07B003-USA(HM066462)

Pseudomonas.sp-3-28-Ch(HM489948)

P.fluorescens-FE1326-Ch(GU177878)

Proteobacterium-CLi21-USA(AF529319)


P.fluorescens-B58-Ch(EU169156)





NDY-Pak-2011

Proteobacterium-NAB24-USA(AY395028)

Pseudomonas.sp-MW6-Ch(HQ231962)

Pseudomonas.sp-SF4c-Arg(AY880843)

P.koreensis-PSB33-Ch(HQ242746)

Bacterium-PYP2-Ch(EF462379)

Burkholderia.cepacia-BCC933-Ch(GQ149776)

Pseudomonas.sp-II4X-Ch(HQ727967)

Burkholderia.cepacia-ATCC53795-USA(AY...

Uncultured-16slp114-1a06.q1k-UK(GQ157...

P.fluorescens-1408-V.nam(GU726880)

P.fluorescens-d3-Ch(HQ166099)

Uncultured-16slp108-1g06.w2k-UK(GQ157...

Pseudomonas.sp-III4X-Ch(HQ727968)

Uncultured-16slp101-3h03.w2k-UK(GQ157...

P.fluorescens-HNR16-Korea(EU373377)

Pseudomonas.sp-SMCC-B0333-USA(AY029759)

Uncultured-16slp124-4a05.p1k-UK(GQ157...

Pseudomonas.sp-NZ096-N.Z(AY014817)

P.teessidea-PT65-UK(AM419154)

P.fluorescens-JF04-Ch(HQ123477)

P.fluorescens-LMG-14673-Blgm(GU198123)

Uncultired-HSM-SS-019-Jpn(AB2387820

P.koreensis-SSG5-Korea(HM367599)

Pseudomonas.sp-W15Feb9B-Blgm(EU680989)

Bacterium.TH-1-Ch(JN416110)

Uncultured-HSM050P-B-4-Jpn(AB262715)

Pseudomonas.sp-CYEB-13-Ch(FJ422392)

P.fluorescens-166-Korea(EU730928)

Uncultured-HSM-SS-001-Jpn(AB238764)

Uncultured-HSM-SS-001-Jpn(AB238774)

P.putida-CM5002-Brzl(EF529517)



Uncultured-SZS-0 96-Ch(HM049711)


Pseudomonas.sp-NR25-Ind(JN082749)

P.koreensis-JDM-2-Ch(GQ368179)

P.moraviensis-Hd7-Ch(JF899299)

P.putida-Tg-Peru(EU275363)

P.putida-DSQ4-Ch(HM217118)

Pseudomonas.sp-AZ22L5-Spn(AY308048)

Uncultured-Phe56-USA(AF534214)

P.koreensis-MS200-Swdn(HQ589332)

Pseudomonas.sp-TB2-8-I-Itlay(AY599703)

Uncultured-Glu3-USA(AF534197)

P.koreensis-WA1-9-Ch(JF496459)

Pseudomonas.sp-ITP30-Spn(FR667178)


Pseudomonas.sp-AEBL3-Ch(AY247063)

P.koreensis-SSG6-Korea(HM367600)

Pseudomonas.sp-RN-B-Ind(HQ222612)

Pseudomonas.sp-AUTH-28-Greece(FR725962)


Uncultured-Phe10-USA(AF534205)

P.fluorescens-1582-Russia(JN679853)

Pseudomonas.sp-3-11-Ch(HM057102)


Uncultured-B331-Ch(JF833644)

Pseudomonas.sp-CNE-28-Arg(FR749872)

Pseudomonas.sp-CPC20-USA(DQ013850)

a:

:

(b)


97

Fig 2.40: Phylogenetic tree of Pseudomonas aeruginosa JYR and B.firmus PTWz

(a): The phylogenetic tree of Pseudomonas aeruginosa JYR indicating the position of strain in the

established related species of the genus. The tree is drawn to scale, with branch lengths in the same

units as those of the evolutionary distances used to infer the phylogenetic tree. Numbers at branch

points are bootstrap percentages based on 1000 replicates.

(b) The neighbour-joining phylogenetic tree of Bacillus firmus PTWz indicating the position of strain

in the established related species of the genus. The tree is drawn to scale, with branch lengths in the

same units as those of the evolutionary distances used to infer the phylogenetic tree. Numbers at branch

points are bootstrap percentages based on 1000 replicates. The scale bar represents the expected

number of substitutions.

Pseudomonas.sp-1d-11-ChFJ598142

Uncultured-16sps17-1d10.p1k-UKFM996063

Uncultured-16sps24-1h09.w2ka-UKFM997...

Uncultured-16sps19-3e03.p1k-UKFM996518


P.aerogenosa-10-ChFJ907193

P.aerogenosa-nsm-IndHQ658761

Uncultured-16sps16-3a12.w2k-UKFM995985

Uncultured-16sps16-3g01.w2k-UKFM996023

Uncultured.16sps16-2h11.p1k-UKFM995975

Uncultured.16sps19-1e02.p1k-UKFM996424


Uncultured-16sps20-2g02.q1ka-FM996736

Uncultured-16sps23-1e08.w2ka-UKFM997...




P.aerogenosa-HS9-ChGU323371

Uncultured-16sps20-1a10.p1ka-UKFM996...

P.aeruginosa-G6-ChGQ221872


Uncultured-16sps22-1c05.p1ka-UKFM996...

P.aerogenosa-DQ8-ChGU269267

P.aerogenosa-JCM5516-JpnAB594761

P.aerogenosa-ANSC-ChGU296674

P.aerogenosa-9-ChFJ907192

Uncultured.16sps17-3a07.p1k-UKFM996166

Uncultured-16sps23-2b10.p1k-UKFM997057

Uncultured-16sps21-2c11.p1kb-UKFM996...

Uncultured-16sps24-2b02.p1k-UKFM997200

Uncultured.16sps17-1h04.w2k-UKFM996082

JYR-Pk-2011

P.aerogenosa-EH8-IndGU339238

P.aeruginosa-FJAT346-ChJN572122

Uncultured-16sps16-3h07.p1k-UKFM996038

P.aerogenosa-B295-ChJF833630

Uncultured-16sps16-3d01.w2k-UKFM996000


Uncultured.16sps19-3g01.p1k-UKFM996540


P.aerogenosa-JCM6119-JpnAB594760

Pseudomonas.sp-DG2b-itlyHQ609593

Uncultured-16sps21-1c08.p1ka-UKFM996...

pseudomonas.sp-IHB-B4040-IndHM234002

Pseudomonas.sp-EJB3-ChGU966668

Uncultured-16sps27-3a09.p1k-UKFM997544

uncultured.16sps18-1a11.p1k-UKFM996250

Uncultured.16sps17-2a04.w2k-UKFM996089

P.aerogenosa-BSF-g-ChGU121439


P.aeruginosa-G14-N.landHQ288928

P.aerogenosa-NO5-KoreaFJ972533

P.aerogenosa-ZAQ222-GQ375800

P.aerogenosa-1-8-ChGU586139

P.aeruginosa-BS8-IndJN003625

P.aerogenosa-IndHM637743

Uncultured-16sps23-2c01.p1k-UKFM997059

P.aerogenosa-MZA-85-PkHQ023428

Pseudomonas.sp-NR2-IndGU566322

Pseudomonas.sp-CIFRI.D-TSB-6-IndJF78...

Pseudomonas.sp-pseudo-EJB5-ChGU966670

P.aerogenosa-AS2-IndGU447238

Pseudomonas.sp-EMB23-IndJF281099

P.aerogenosa-JCM-2776-JpnAB594757

Uncultured.16sps17-2d06.w2k-UKFM996116

Uncultured-16sps21-2h09.w2ka-UKFM996...


Uncultured-16sps16-3f04.w2k-UKFM996018

Pseudomonas.sp-AGP-01-IndHM587311

Uncultured-16sps22-1a09.w2ka-UKFM996...

Uncultured.16sps17-3e08.w2k-UKFM996205


Uncultured-16sps20-1g11.p1ka-UKFM996...

P.aeruginosa-S164S-IndJF513146

Uncultured-16sps22-1b02.w2ka-UKFM996...

Pseudomonas.sp-OU67-IndFN663622

P.aeruginosa-IRMD-IrnJF708942

Pseudomonas.sp-DG1b-ItlyHQ609597


P.aerogenosa-IndFJ665510


P.aerogenosa-1-15-ChHQ434555


P.aerogenosa-C24-ChHM036358

Uncultured.16sps18-1g05.p1k-UKFM996309

Uncultured.16sps19-2d04.p1k-UKFM996461

Uncultured.16sps17-2d08.w2k-UKFM996117

P.aeruginosa-F1-ChJN412064

P.aerogenosa-GPSD-59-IndHQ270549

Uncultured.16sps15-2c12.p1k-UKFM995835

Uncultured-16sps20-1e04.p1ka-UKFM996...

Pseudomonas.sp-TBP-Y-ChFJ804740

pseudomonas.sp-3-ChEU784954

(a)

(b)


98

Fig 2.41: Phylogenetic tree of Bacillus pumilus Yio and

Bacillus endophyticus Y5

(a): The phylogenetic tree of Bacillus pumilus Yio indicating the position of strain in the established

related species of the genus. The tree is drawn to scale, with branch lengths in the same units as those

of the evolutionary distances used to infer the phylogenetic tree. Numbers at branch points are

bootstrap percentages based on 1000 replicates.

(b): The neighbour-joining phylogenetic tree of Bacillus endophyticus Y5 indicating the position of

strain in the established related species of the genus. The tree is drawn to scale, with branch lengths in

the same units as those of the evolutionary distances used to infer the phylogenetic tree. Numbers at

branch points are bootstrap percentages based on 1000 replicates. The scale bar represents the expected

number of substitutions.

Bacillus.sp-MiB1-Ind(JF910018)

B.pumilus-sk-1-Ch(HQ164542)

Bacillus.spp-DBTMGS2-Ind(FJ842658)

Bacillus.spp-TSSAS2-44-Ind(GQ284505)

Bacillus.spp-TSWCS4-Ind(GQ284397)

B.pumilus-MS28-UK(FN997624)

B.pumilus-XJSL5-4-Ch(GQ903423)

Bacillus.sp-H6-Ch(HQ222345)

B.subtilis-FQ06-Ch(GQ360038)

Bacillus.spp-L149-Ger(AM913919)

Bacillus.sp-3429ABRRJ-Brzl(JF309245)

Bacillus.sp-34386BBRRJ-Brzl(JF309252)

Bacillus.spp-By253Ydz-fq-Ch(EU070406)

Bacillus.sp-FP303-Jpn(AB374299)

Bacillus.sp-Rb-ZD-1-Ch(HM246684)

Bacillus.spp-cp-h23-Ch(EU558973)

Bacillus.sp-RHH58-Ch(HQ202550)

B.altitudinis-41KF2b-Ind(NR 042337)

Bacillus.sp-TZQ28-Ch(HQ202562)

Bacillus.sp-Bg-2-Ch(HQ916742)

B.pumilus-S68-Ch(FJ763649)

Bacillus.sp-28KZ-Ind(FJ615521)

YiO-Pak 2011

Bacillus.spp-WPCB093-Korea(FJ006891)

B.pumilus-DZH2-Ch(GQ375785)

B.altitudinis-AsdM5-2B-Ind(FM955870)

B.pumilus-IMAU80221-Ch(GU125637)

Bacillus.spp-BM3-Ch(EU869279)

Bacillus.sp-3429BBRRJ-Brzl(JF309246)

Bacillus.spp-B351-Ch(EU070368)

Bact.fjat-scb-Ch(HQ873709)

Bacillus.sp-PPB8-Mala(HM771663)

Bacillus.spp-TSSAS2-12-Ind(GQ284500)

B.pumilus-14a-Ch(FJ478440)

Bacterium-Kye2-Ch(GQ289128)

Bacillus.sp-C-Ch(JF495461)

Bacillus.spp-41KBZ-Ind(FJ615523)

B.altitudinis-DYJK5-Ch(HQ843846)

Bacillus.sp-BT1-2-Ch(GU332605)

B.pumilus-DYJL55-Ch(HQ317196)

Bacillus.sp-KZ-Frnce(GU726185)

Bacillus.sp-NIB6-Pk(JF313264)

B.pumilus-13635D-C.rica(EU741079)

B.pumilus-CT13-Ind(EU660365)

Bacillus.sp-3429DBRRJ-Brzl(JF309248)

Bacillus.spp-BM1-b-Ch(EU940370)

B.pumilus-SYBC-W-Ch(GU084168)

B.pumilus-b0-Ch(EU869282)

B.altitudinis-AP-MSU-Ch(HM582688)

B.safensis-HNS004-Ch(JN128238)

B.altitudinis-GCH-2-Ch(FJ611965)

B.pumilus-KD3-Ch(EU500930)

B.pumilus-st9-Ch(FJ544367)

Bacillus.spp-L11-Ch(HQ222333)

B.altitudinis-DYJK5-Ch(HQ843847)

Bacillus.sp-34386DBRRJ-Brzl(JF309254)

B.pumilus-MS10-UK(FN997610)

B.altitudinis-IMAU80219-Ch(GU125636)

B.stratosphericus-GD65-Ch(HQ857755)

B.pumilus-YQQ10-Ch(GQ375796)

B.pumilus-AUCAB16-Ind(JN315777)

B.pumilus-VB6-Ind(JN215511)

Bacillus.spp-3a-Ch(FJ478433)

B.pumilus-DYJL54-Ch(HQ317195)

Bacillus.spp-RHH1-Ch(HQ143613)

Bacillus.spp-41KAZ-Ind(FJ615522)

B.pumilus-PRE14-Ch(EU880532)

Bacillus.sp-34386ABRRJ-Brzl(JF309251)

Bacillus.spp-BSFA18-3-Ch(FJ495143)

Bacillusspp-DZQ11-Ch(GQ375792)

Bacillus.spp-LD125-Ger(AM913918)

B.subtilis-N43-Ch(GQ465935)

V.parahaemolyticus-MS27-UK(FN997623)

B.pumilus-BMI-Ch(EU869273)

Bacillus.spp-210 50-Ch(GQ199752)

B.stratosphericus-41KF2a-Ind(NR 042336)

B.aerophilus-28K-Ind(NR 042339)

Bacillus.spp-S244-Ch(HQ704706)

Bacillus.spp-SFA10-Ch(EU878266)

Bacillus.spp-23a-Ch(FJ478447)

Bacillus.spp-L74-Ger(AM913938)


B.pumilus-S105-Ch(HQ704707)

Bacillus.sp-HaiN-1-Ch(HM242289)

B.pumilus-SDB-01-Ch(EU874254)

Bacillus.spp-BSFA18-2-Ch(FJ495142)

B.pumilus-DYJL-C-Ch(HQ317176)

Bacillus.spp-ZY011-Ch(EU652879)

B.pumilus-JK-SX001-Ch(GQ169785)

Bacillus.sp-34386CBRRJ-Brzl(JF309253)


B.pumilus-DZH1-Ch(GQ375784)

Bacillus.sp-L4-Ch(HQ222326)

B.subtilis-YT2-Ch(HQ143571)

Bacillus.sp-NIB4-Pk(JF313263)

Bacillus.sp-3LF.48P-Ind(FN666893)

B.altitudinis-Ind(N668692)

Bacillus.spp-ljh-2-Ch(GU217692)

Bacillus.spp-R-25542-Blgm(AM944032)

Bacterium.sp-SE5-Ch(EU520340)

Bacillus.spp-PSM2-Ch(JF738142)

(a)

(b)


99

2.7 Discussion

Biocontrol agents have the ability to suppress the pathogen attack by

manipulating their growth and survival. The rhizobacteria isolated during the present

investigation has the ability to inhibit the growth of pathogens but potent biological

control agents also has the ability to promote the growth of plant by acting as plant

growth promoting rhizobacteria. A number of researchers reported the plant growth

promoting traits among antagonistic rhizobacteria (Yasmin et al., 2010; Noori and

Saud, 2012) by using a number of mechanisms (Glick et al., 2007) including

secondary metabolite production such as hydrogen cyanide (HCN), antibiotics (Duffy

et al., 2004; Chakraborty et al., 2009); and volatile compounds that stimulate growth

of plants (Ryu et al., 2003).

Rhizobacteria may also act as PGPR by promoting the plant growth through

the production of siderophores, which assist in delayed senescence, biological control

(Buyer et al., 1993) and produce plant hormones such as auxins (IAA), which at low

concentrations influence plant physiological functions. They also help the plant to

improve growth by phosphate solubilization in order to increase the uptake of

nutrients (Richardson et al., 2009).

Among 18, potent rhizobacteria selected during the present investigation

which were found to solubilize phosphate and produce siderophore and IAA. Besides

they were antagonist of fungal pathogens (F. moniliforme, H. sativum and A. flavus)

and produce lytic enzymes including protease, protease, chitinase, catalase and

oxidase. Similar results were observed in earlier studies (Hadad et al., 2010; Suresh et

al., 2010; Dastager et al., 2011). From this study it is obvious that these selected

rhizobacteria had the ability to effect the plant growth. As reported earlier

rhizobacteria can be used as efficient PGPR to increase the growth and yield of the

crop and reduce the disease intensity (Chakraborty et al., 2006; Munees and

Mohammad, 2009). The selected antagonistic rhizobacteria were further applied as

seed treatment for the assessment of their effect on growth of maize (Zea mays L.)

seedlings.

The inoculation of beneficial rhizobacteria was done by a number of different

methods while, seed treatment is one of the most common method. Seed inoculation

of common bean by Pseudomonas strains resulted in improvement of root and shoot

biomass (Egamberdieva, 2011). Inoculation of maize and wheat seeds with


100

Azotobacter and Azospirillum also resulted in plant growth promotion (Dobbelaere et

al., 2001)

Root colonization was a major factor for the favourable interactions involve in

biological control of phytopathogens (Thomas et al., 2008) and plant growth

promotion (Barea et al., 2005). Antagonistic PGPR can colonize the root surface,

rhizosphere, intercellular spaces of roots (McCully, 2001). The ability to colonize the

rhizosphere for an extended period was characterized by resilient microbial

competition and their role in plant growth promotion using both direct and indirect

method (Whipps, 2001). Root colonization of rhizobacteria is usually influenced by a

number of factors including biotic (the genetic traits of host plant and the colonizing

rhizobacteria) and abiotic (growth substrate, rhizosphere pH, humidity and

temperature). The rhizobacteria which had already better colonizing ability in the

environment in which they are employed get better results of their potential abilities.

The rhizobacteria isolated from the rhizosphere of non-infected maize fields

have higher cfu as compared to the rhizobacteria isolated from infected maize fields.

Furthermore, the survival efficiency of rhizobacteria isolated from irrigated region

was higher as compared to arid and semi-arid regions.

During in vivo evaluation of the antagonistic bacteria under greenhouse

conditions, all isolates showed significant increase in dry mass of maize plants as

compared to un-inoculated control. The findings were supportive to the earlier

observations that bacteria with in vitro plant growth promoting activities have

capability to promote plant growth in vivo (Ran et al., 2005). All isolates showed

significant increase in shoot and root length of maize as compared to un-inoculated

control. Ashrafi and Seiedi, (2011) observed significant increase in plant height as a

result of rhizobacteria inoculation as compared to un-inoculated control. Yasmin et

al., (2012) also reported the improvement in root length of maize as compared to

control. The application of rhizobacteria brings about considerable increase in root

length (Erturk et al., 2010).

During the present study it was found that the effect of inoculation with

rhizobacteria 4nm, NDY, PTWz, JYR, Y5 and Yio in increasing shoot and root fresh

weight of maize was found more pronounced as compared with all other treatments.

Yasmin et al., (2012) found that the rhizobacteria inoculation results in significant

increase in root and shoot fresh weight. Similar results were reported by (Shaharoona


101

et al., 2007). Bhromsiri and Bhromsiri, (2010) observed that the PGPR inoculation

increased the shoot dry weight. Egamberdiyeva et al., (2002) reported the effect of

Pseudomonas fluorescens PsIA12 and Pantoea agglomerans on the growth of maize

and rhizobacteria were found to significantly enhance the root and shoot development

of maize. The higher root to shoot ratio shown by the rhizobacteria isolated from arid

and semi-arid region isolates indicated their natural potential to increase the root

growth under low moisture content. The rhizobacteria isolated from non-infected

maize fields have shown pronounced effect on the growth of maize seedlings as

compared to rhizobacteria isolated from infected maize field of all the regions. This

difference may be attributed to the difference in the phytohormone produced by these

rhizobacteria.

The antagonistic rhizobacteria acting as PGPR have a twofold benefit in the

rhizosphere by inhibiting the disease incidence and enhancing the plant growth.

Therefore, the rhizobacteria can act as ideal supplement for the management of plant

disease and improving the plant growth. In this study, the efficient antagonistic

rhizobacteria were 4nm, JYR, Y5 of group 1 and NDY, PTWz, Yio of group 2.

Identification of Rhizobacteria

In the present study, our effective antagonistic strains were found to belong to

genus Pseudomonas (4nm, JYR, JYG, NDY) and Bacillus (Y5, PTWz, Yio) on the

basis of 16S rRNA gene sequencing and successive molecular phylogeny. The

antagonistic capabilities of these two genera are well recognized in literature (Schisler

et al., 2004). It is reported that Bacillus and Pseudomonas are the predominate

rhizobacteria that release a range of hydrolytic enzymes (Foldes et al., 2000; Shoda,

2000; Ping and Boland, 2004).

Therefore, it is obvious from the present investigation that rhizobacteria are

capable of promoting the plant growth along with antagonism against phytopathogens

through mixed type mechanism.

Induction of systemic resistance by antagonistic PGPR against stalk rot in maize Chapter 3

102

3.1 INTRODUCTION

Stalk rot casused by F. moniliforme is an intricate chronic disease of maize

which has a strong connection with the host senescene. F. moniliforme being a week

parasitic fungus that infect the host when its host is passing through the aging stress.

Pathogens for stalk rots are universaly present in each and every field where maize is

grown. Under favorable conditions the development of disease is very fast and the

host perished before the ears get mature. This lead to poorly filled ears and chaffy

kernel follwed by secondary losses by breakage and lodging of stalk and root.

Another important impact of F. moniliforme is that it produces numerous toxins that

cause possible toxicity to humans and animals. The most substantial toxin produced

by F. moniliforme is the fumonisins (Desjardins et al., 1995).

F. moniliforme infects the maize host endophytically by the systemic

infection of it seeds soon after the seed germination (Bacon and. Hinton, 1996). The

systemic infection of maize seeds produces vertical transmission of disease generation

after generation. In addition to the vertical transmission phase, maize is subjected to

infection throughout the growing season (Leslie, 1996; Desjardins et al., 1998), As

this fungus is saprophytic so its spores are also produced in soil and dead maize debris

that can attack the crop from outside (maize silks). In this way F. moniliforme is

vertically transferred to the subsequent generations. The horizontal phase of infection

can be controlled by fungicide application but the vertical transmission is crucial as it

provides the infection reservoir for each next generation, so during this phase

fungicides application is unable to control the fungus. Secondly these chemical

fungicides render tolerance in the pathogen strains (Gupta and Shyam, 1996) and

require frequent sprays to control the disease efficiently. This raise the crop

cultivation cost along with its ecological and environmental concerns. These problems

encourage the search for an alternative approach to plant protection (Schoenbeck,

1996).

Plant can get resistance through various biological control agents against a

number of bacterial and fungal pathogens (Van loon et al., 1998). Antagonistic

rhizobacterial treatment prior to the development of fungal infection with virulent

pathogen initiates the quick and efficient defence related responses and results in

effective control of disease (Van der Ent et al., 2008). This phenomenon is known as

induced resistance (Hammerschmidt and Kuc, 1995). Antagonistic rhizobacteria


103

affect indirectly by the induction of host defence response that check the invasion of

fungal pathogen by root and modify course of fungal pathogenicity. It is reported by

Choudhary et al., (2007) that plants are able to get enhanced resistance against

pathogens by a number PGPRs by rhizobacteria mediated ISR (De vleesschauwer and

Hofte, 2009). Among the antagonistic PGPR, Pseudomonas strains are predominant

PGPR which have the ability to induce systemic resistance in plants including P.

fluorescens (Maurhofer et al., 1994), P. aeruginosa (Bigirimana and Hofte, 2002 ; De

Vleesschauwer et al., 2006), P. syringae (Wei et al., 1991) .Besides Pseudomonas,

numerous Bacillus strains including B. pumilus, B. amyloliquefaciens, B. pasteuri, B.

subtilis, B. cereus, B. sphaericus and B. mycoides are known as potent ISR elicitors

and have the ability to exhibit significant reduction in disease severity on various

hosts (Choudhary and Johri 2008). ISR elicited by antagonistic rhizobacteria has been

shown in maize by Bacillus species (Van Wees et al., 1997) and in other crops like

wheat and rice (De Vleesschauwer et al., 2008; Shoresh et al., 2010).

The ISR (Induced systemic resistance) is the systematic protection of plants by

increasing the plant defensive aptitude against a wide range of pathogens. The ISR is

different fundamentally from other disease control mechanisms. As it is established

by the defence mechanisms triggered by the biocontrol agents and after it has been

expressed, it stimulates several other defence mechanisms. Another essential feature

of ISR is the broad spectrum pathogens that can be efficiently controlled by the

application of a single biocontrol agent (Hoffland et al., 1996) Hence, ISR activate a

number of defence mechanisms which are effective against an extensive array of

phytopathogens. The ISR also have an additional eco-friendly advantage, as it

controls the pathogen indirectly by preventing it’s proliferation in host following the

optimization of host defence system rather than direct killing of pathogen by toxin

production.

The ISR is associated with re-mobilization of defence related enzymes

including pathogenesis-related (PR) proteins such as β-1,3-glucanases, chitinases

peroxidase (PO), superoxide dismutase (SOD), phenylalanine ammonia-lyase (PAL),

polyphenol peroxidase (PPO) (Chen et al., 2000; Magnin-Robert et al., 2007),

increased levels of certain acid soluble proteins (Zdor and Anderson, 1992) and the

accumulation of phytoalexins in the induced plant tissue (Vanpeer et al., 1991). The

mobilization and enhancement of these defence enzymes is the collective


104

consequence of inducing agent and physiological condition of host and pathogen

(Tuzun, 2001).

Maize (Zea mays L.) is an important crop in temperate and semi-arid regions

and it is one of the three main staple crops in the world. In developing countries maize

holds great importance, as these countries are facing serious food crisis due to fast

growing population. It has prominent position after wheat and rice in Pakistan for its

grain production and contributes an important share in economic development of the

country (Saleem et al., 2012). The crop production of maize in Pakistan is fairly low

as compared to other maize growing countries due to the manifestation of diseases.

Among different disease the Fusarium moniliforme is most prevalent disease in

different areas of Pakistan (Saleem et al., 2012) It is important to find the alternative

eco-friendly ways to control the fungal disease of maize in a developing country like

Pakistan to decrease the economic losses both in terms of productivity as well as to

protect the ecosystem and human being form the adverse effect of chemical fungicide.

Aims and objective

During the present study, the mechanism of disease control in maize plants

following the treatments of previously selected Pseudomonas and Bacillus

rhizobacteria was investigated with an objective to reveal the involvement of ISR

based biocontrol of Fusarium stalk rot. Furthermore their effect has been compared

with that of the commercial fungicide (Ridomil Gold) alone and in combined

application, with the aim to protect the environment from harmful fungicidal effect as

well as to economize the process of disease control.


105


The present investigation was carried out at the Department of Plant Sciences,

Quaid-i-Azam University, Islamabad (latitude, 33° 42' 0" N, longitude 72° 10' 0" E

and altitude 457 to 610 m) during the maize growing season of 2010 and aim was to

evaluate the selected PGPR strains for their biocontrol potential against Fusarium

stalk rot in maize and track down the possible mechanism of action. The materials and

methods employed in this investigation are outlined below.

3.2.1 Plant Material used

Seeds of maize cv. Islamabad Gold (susceptible to stalk rot) were obtained

from Crop Research Institute, National Agriculture Research Centre (NARC),

Islamabad.

3.2.2 Physiochemical characteristics of soil

3.3.1 Moisture content of soil

Soil moisture content was determined by the method described in Chapter 2.

3.3.2 Soil pH and EC

The pH and EC was determined following the method given by Radojevic and

Bashkin, 1999) described in Chapter 2

3.2.3 Soil Nutrients Analyses

Rhizospheric soil was analyzed for nutrients (P, K, Ca and Mg) following the

Ammonium Bicarbonate-DTPA method developed by Soltanpour and Schwab,

(1977). Details of the method are given in Chapter 2.

3.2.4 PGPR strains used in the study

Six PGPR strains were screened on the basis of their antifungal activity,

biochemical characteristics and plant growth promoting ability as described in chapter

2. These antagonistic PGPRs include three Pseudomonas strains (Pseudomonas

aeruginosa 4nm, Pseudomonas aeruginosa JYR, Pseudomonas Sp. NDY) and three

Bacillus strains including Bacillus pumilus Yio, Bacillus firmus and Bacillus

endophyticus Y5.


106

3.2.5 Seed sterilization and inoculation of antagonistic PGPR

Seeds of maize (cv. Islamabad gold) were sterilized with 95% ethanol

followed by shaking in 10% chlorox for 2–3 min, afterwards; seeds were carefully

rinsed 3 times with sterile water.

The PGPR strains were grown in100 mL of LB broth on a rotary shaker

(EXCELLA E24, New Brunswick Scientific USA) for 48 h at 28 °C. The broth was

centrifuged at 10,000 g for 10 min at 4°C to get the pellet which was re-suspended in

sterile distilled water. The optical density (O.D) as measured by spectrophotometer at

660nm was maintained 1.

Seeds were soaked for 2-4 h in the inocula of Pseudomonas and Bacillus

strains. Later on the bacterial suspension was drained carefully and seeds were sown

after air drying for 30 min (Nandakumar et al., 2001). For control treatment soaking

was done in autoclaved distilled water.

3.2.6 Preparation of F. moniliforme inoculum

The pure culture of F. moniliforme collected from National Agriculture

Research Centre (NARC), Islamabad, was maintained on Potato dextrose agar (PDA)

agar plates. The fungal inoculum was prepared following the method of Tesso et al.,

(2009) with slight modifications. Inoculum suspension was prepared in potato

dextrose broth (PDB-DIFCO) by inoculating the fresh culture of Fusarium

moniliforme. The inoculated broth was incubated on a rotary shaker at room

temperature till the development of fungal microconidia.

This broth culture containing microconidia was strained through four layers of

cheese cloth to separate the mycelial mass. The concentration of suspension was

adjusted to the required dose (1 × 106 conidia mL

-1) with phosphate buffered saline

(PBS) solution and lower concentrations were obtained by serial dilution

3.2.7 Application of Chemical Fungicide (Ridomil Gold)

The chemical fungicide Ridomil Gold (full dose: 0.2% and half dose: 0.1%)

was applied 72 h after the inoculation of disease

3.2.8 Treatments

The experiment was conducted in pots under greenhouse condition with 16

treatments and 3 replications.


107

Table 3.1: Treatment details for pot experiment in greenhouse

Symbols

used

Treatments

Without fungicide With Fungicide

Control non-infected plants Non-infected plant + fungicide (0.2%)

In-C Infected with F. moniliforme F. moniliforme + fungicide (0.2%)

PA1 F. moniliforme +Pseudomonas

aeruginosa 4nm

F. moniliforme + Pseudomonas

aeruginosa 4nm+ fungicide (0.1%)

Ps F. moniliforme + Pseudomonas sp.

NDY

F. moniliforme + Pseudomonas sp.

NDY + fungicide (0.1%)


aeruginosa JYR

F.moniliforme + Pseudomonas

aeruginosa JYR + fungicide (0.1%)

BF F. moniliforme + Bacillus firmus

PTWz

F. moniliforme + Bacillus firmus PTWz

+ fungicide (0.1%)

BP F. moniliforme + Bacillus pumilus

Yio

F. moniliforme + Bacillus pumilus Yio

+ fungicide (0.1%)

BE F. moniliforme + inoculated with

Bacillus endophyticus Y5

F. moniliforme + Bacillus endophyticus

Y5 + fungicide (0.1%)


108

The treatments involved inoculation with selected fungus (F. moniliforme),

antagonistic PGPR application and fungicide application in various combinations.

Detail of treatments given in Table 3.1.

3.2.9 Pot experiment in greenhouse

Seeds of maize (cv. Islamabad Gold) were sown in earthen pots (25x40 cm2)

containing sterilized soil and sand in a ratio of 3:1. The soil had pH 7.8, EC 0.68

dS/m, soil moisture content 16-17% and the available amount of nutrients Na++

, K+,

P+, Mg

+ and Ca

++ were 23, 15, 9, 1.2 and 41 µg/g, respectively. Pots (3 replicates for

each treatment) were arranged in completely randomized design (CRD) with the

average temperature of 25–30ºC and day length ranging from 10–13 h.

3.2.10 Application of F.moniliforme Inocula

At tasseling stage (60 days after sowing), maize plants were inoculated with F.

moniliforme. The inoculum (I mL) was applied through syringe to second node of

each plant stem (Tesso et al., 2009). Afterwards, the area of stem where inoculation

was done for disease induction was covered with tape to conserve the humidity for

fungal growth. Control plants were inoculated with a similar volume of PBS buffer.

3.2.11 Disease Scoring

Disease scoring was done 21 d after inoculation of fungus. Inoculated plants

(three for each treatment) from each pot were harvested and the stalk was splitted

longitudinally to measure the length of visible necrotic lesion (cm). Disease data was

determined on 0-5 scale (Hooker, 1957) for the calculation the disease index and

disease severity the following formula (Tesso et al., 2009) was used

Disease index = Sum of lesion length

Total no of plants examined

Disease severity = Disease index x100

5

3.2.12 Sample Collection

Plant leaves were collected at three different times (silk stage, blister stage and

dough stage) after the inoculation with pathogen (Fusarium moniliforme). Sampling

was done from each replication and treatments were maintained for physiological and

biochemical analyses.


109

3.2.13 Superoxide dismutase (SOD) activity

Extraction of enzyme

For enzyme extraction, flag leaves (0.5 g) were ground in 5 mL of 50 mM

phosphate buffer placed in ice bath at 4˚C. The homogenate was centrifuged at 13000

x g at 4ºC for 20 min. The supernatant was used for assay of the activities of enzymes.

Superoxide dismutase activity (SOD) was determined following the method of

Beauchamp and Fridovich (1971) which is based on measurement of inhibitory action

involving nitroblue tetrazolium (NBT) photochemical reduction. The reaction mixture

(3 mL) contained 0.075 mM NBT, 13 mM methionine, 0.002 mM riboflavin,0.1 mM

EDTA and 0.1 mL of enzyme extract in phosphate buffer (50 mM : pH 7.8). The

reaction mixture was shifted to light box for 15 min and the lights was turned off to

stop the reaction. A reaction mixture without irradiation was taken as blank. The

absorbance was read on a spectrophotometer at 560 nm. One unit of SOD activity was

defined as total amount of enzyme, required to reduce 50% absorbance as compared

to control (lacking enzyme) and expressed as units/g fresh weight of leaves

3.2.14 Peroxidase (POD) activity

POD activity was determined following the method of Vetter et al., (1958)

with the modifications adapted by Gorin and Heidema (1976). The enzyme mixture

contained enzyme extract, 1.35 mL, 0.1 mL MES buffer (100 mM: pH 5.5), 0.1 ρ-

phenylenediamine and 0.05% H2O2. Absorbance changes were documented at 485 nm

for three min with the help of spectrophotometer. One unit of POD activity was taken

as change in OD 485 nm per minute. The activity of POD was presented as units/g

fresh weight.

3.2.15 Polyphenol oxidase activity (PPO)

To measure the PPO activity a reaction mixture (3 mL), containing 0.1 mM

pyrogallol, 25 mM phosphate buffer (pH 6.8), 0.1 mL enzyme extract was prepared

by the method of Kar and Mishra, (1976) with slight modification. The blank

contained the enzyme mixture except pyrogallol. The resultant compound

purpurogallin formed and its absorbance was recorded at 420 nm.

3.2.16 Ascorbate peroxidase

Ascorbate peroxidase activity was measured following the method described

by Asada and Takahashi, (1987). The enzyme activity was expressed in U mg -1


110

protein where, U is taken as change of 0.1 absorbance per minute per milligram of

protein.

3.2.17 Catalase Activity

Catalase activity was analyzed by measuring the H2O2 disappearance

(Teranishi et al., 1974). Phosphate buffer (50 mM; pH 7.5), 20 mM H2O2 and 0.1 mL

enzyme extract was added to prepare the reaction mixture. Titanium reagent (2 mL)

was added to stop the reaction after five minutes which was indicated by the

formation of coloured complex with H2O2 residue. This resultant mixture was

centrifuged at 10000 rpm for 10 min and the supernatant was used to measure the

absorbance at 410 nm.

3.2.18 Total soluble phenol content

Total soluble phenol from maize leaves was estimated as described by Hsu et

al., (2003) with some modifications. Plant leaves (0.625 g) were grounded in 10 mL

of methanol and kept overnight. The suspension was filtered and diluted with sterile

water up to 100 mL, and used as a stock solution. As described by Slinkard and

Singleton, (1997), 50% Folin-Ciocalteu phenol reagent (0.1 mL) and distilled water

(1.4 mL) to 200 μL of stock solution was and placed for 3 min. Afterwards sodium

carbonate (20% w/v) was added and allowed to stand for 2 h and vortex the

suspension before the measurement at 765 nm. Total soluble phenol content was

standardized against gallic acid.

3.2.19 Malondialdehyde (MDA) activity

Lipid peroxidation level was assayed in terms of TBARS content by following

the method of Prochazkova et al, (2001). Fresh leaves (0.1 g) were grounded in 2 mL

trichloroacetic acid (0.1% TCA). It was further centrifuged for 15 min at 15000×g.

Thiobarbituric acid (4 mL of 0.5% TBA) and 20% TCA was added to 1 mL of

supernatant. The mixture was heated for 30 min at 95°C and cooled in an ice bath.

The absorbance was recoded at 440, 532 and 600 nm after centrifugation for 10 min at

10000×g. Malondialdehyde equivalents were calculated in the following formula (Du

and Bramlage, 1992):

MDA = 6.45 (A532–A600) – 0.56 A450.


111

3.2.20 Proline Content of Leaves

Proline content of flag leaves was determined following the method of Bates

et al., (1973). Fresh plant material (0.1 g) was homogenized with 4 mL sulfosalicylic

acid (3.0%) and placed overnight at 5°C. Suspension was centrifuged at 3000 rpm for

5 min. Acetic-ninhydrin reagent (4 mL) was added to the supernatant and shaken

mechanically. The reaction mixture was placed in boiling water bath for 1 h. Then

after cooling the extraction was done with 4 mL of toluene in a separating funnel.

Afterwards absorbance of toluene layer was measured at 520 nm. The concentration

of sample was measured with reference to the standard curve.

3.2.21 Chlorophyll and Carotenoid Content

Chlorophyll and carotenoid contents were estimated by the extraction from

leaf material (0.05 g) in dimethylsulfoxide (DMSO) in accordance with Hiscox and

Israelstam, (1979). The samples were placed in water bath at 65°C for 4 h and

absorbance of extract was documented at 665 and 645 nm. Chlorophyll content was

estimated according to standard method described by Arnon, (1949). Carotenoid

content was determined by following the method of Lichtenthaler and Wellburn

(1983).

3.2.22 Protein Content

Protein content of leaves was determined according to the method of Lowry et

al., (1951) by using Bovine Serum Albumen (BSA) as standard. Plant material (0.1 g)

was homogenized in 1 mL of sodium phosphate buffer (pH 7.5) and centrifuged for

10 min at 3000 rpm. Distilled water was added to supernatant (0.1 mL) and volume

was made up to 1 mL. Subsequently, 1mL of reagent C (Appendix 11) was added.

The whole mixture was shaken for 10 min and 0.1 mL of reagent D (Appendix 11)

was added. The absorbance of all the samples was taken after 30 min of incubation at

650 nm. The concentration of protein was calculated with reference to standard curve

of standard Bovine Serum Albumen (BSA).

3.2.23 Assay for PR protein

3.2.23.1 Chitinase assay

Chitinase activity was assayed by using the purple dye labelled biopolymeric

substrate, CM chitin- RBV (Loewe Biochemical, Germany). Two hundred microliters

of CM-chitin-RBV (2 mg/mL) was mixed in 300 mL of protein extract (under non-

denaturing conditions) and 300 mL of Tris-HCl 10 mM, pH 7.5; Triton 1%. The


112

mixture was incubated for 3 h at 37°C and 2 M HCl (200 mL) was added to stop the

reaction. Samples were placed on ice for 15 min and centrifuged at 20,000 x g. The

supernatant was collected and the measurement was made spectrophotometrically at

550 nm. Chitinase activity was expressed as unit/mg protein/h. One unit of chitinase

activity is equal to an increase of absorbance of 0.1 (Ramirez et al., 2004; Lopes et al.,

2008).


The data were analyzed by analysis of variance technique and the comparison

among treatments means was made by the least significant difference (LSD) at P <

0.05 (Gomez and Gomez 1984) using Statistix 1.8.


113

Fig 3.1: Schematic presentation for the layout of pot experiment in greenhouse


114

3.3 RESULTS

The pot experiment was conducted in growing season of maize during 2010.

The leaves samples were collected at three different physiological stages and all the

biochemical analyses were done. We observed the gradual increase/decrease in

different physiological and biochemical analyses for first two sampling stages and

maximum decline for third sampling stage. Therefore, over here we presented only

the results of 2nd

sampling stage as it showed more clear effect of different

inoculations on physiological and biochemical parameters.

3.3.1 Disease reduction/ disease severity

The results presented (Fig 3.2) revealed that all the treatments inoculated with

PGPR antagonists significantly reduced the stalk rot disease in maize by 38-61% in

pot experiment against infected control. Maximum reduction (61%) was observed

with treatment BE (Bacillus endophyticus Y5) whereas the effect of other treatments

ranked as PA2 (P. aeruginosa JYR) > PA1 (P. aeruginosa 4nm) > Ps (Pseudomonas

sp. NDY) > BF (B. firmus PTWz) > BP (B. pumilus Yio).

The application of fungicide improved the efficacy of antagonistic PGPR.

Hence, the combined application with half dose of fungicide along with antagonistic

rhizobacteria was found to be more effective in reducing the disease (46-76%)

incidence in comparison to single application of antagonistic rhizobacteria. Among

the PGPR treatments the effect of combined treatment PA2 inoculated with P.

aeruginosa JYR was at par with chemical fungicide.

The inoculation with antagonistic PGPR significantly decreased the disease

severity which is in the range of 33 to 53% when applied alone (fig 3.3). When

applied in combination with half-dose of chemical fungicide their efficiency was

enhanced and the disease severity was significantly decreased. Maximum reduction in

disease severity (20%) for combined application was observed for two treatments BE

and PA2 inoculated with B. endophyticus Y5 and P. aeruginosa JYR isolated from

non-infected maize fields of semi-arid and arid regions. The effect of these two

treatments was at par with chemical fungicide.


115

Fig 3.2: Effect of antagonistic PGPR on disease severity (%) in maize under

axenic condition of greenhouse in pots In-C: Infected maize plants, PA1: inoculated with P. aeruginosa 4nm, PA2: inoculated with P.

aeruginosa JYR, Ps: inoculated with Pseudomonas sp., BF: inoculated with B. firmus PTWz, BP:

inoculated with B. pumilus Yio; BE: inoculated with B. endophyticus Y5. Results are presented as

means of three replicate and the vertical bars specify the means standard deviations.

Fig 3.3: Effect of antagonistic PGPR on disease reduction (%) in maize under

axenic condition of greenhouse in pots

Treatment details as described in Fig 3.2. Results are presented as means of three replicate and the

vertical bars specify the means standard deviations.

0

10

20

30

40

50

60

70

80

90

100

In-C PA1 Ps PA2 BF BP BE

Dis

eas

e s

eve

rity

(%

)

Treatments

without fungicide with fungicide

0

10

20

30

40

50

60

70

80

In-C PA1 Ps PA2 BF BP BE

Dis

eas

e r

ed

uct

ion

(%

)

Treatments



116


The results presented in fig 3.4 revealed an increase in SOD activity following

PGPR inoculation in maize leaves. The disease inoculation resulted in an increase

(1.66 folds) in SOD activity as compared to non-infected control. The application of

chemical fungicide to infected plants resulted in significant increase in SOD activity

i.e. 1.9 and 3.9 folds as compared to diseases inoculated control and non-infected

maize plants.

The antagonistic PGPR inoculation in disease inoculated maize plants resulted

in significant increase in SOD activity of leaves, this increase ranges from 1.30 to

1.90 folds as compared to infected control and 2.1 to 3.1 folds as compared to non-

infected control. Maximum increase was observed for the treatment BE inoculated

with B. endophyticus Y5 (1.90 folds as compared to disease inoculated control).

Whereas, the effect of other treatments was ranked as follows PA1> PA2> BF> Ps

and BP.

SOD activity was higher in the combined application of antagonistic PGPR +

half dose of fungicide as compared to disease infected control and non-infected

control. The SOD activity was found to be significantly higher for combined

treatment BE inoculated with B. endophyticus Y5 i.e. 2.18 and 3.16 fold as compared

to disease infected control and non-infected control, respectively. The combined

treatments showing increased SOD activity ranked as follows PA1> PA2> BF> Ps>

BF. The effect of treatment BE is 13.7% more as compared to the treatment of

chemical fungicide (0.2%) while combined treatments PA2 and PA1 (with 0.1%

fungicide) were at par with full dose of chemical fungicide.

3.3.3 Peroxidase activity (POD)

The POD activity was significantly increased in all the treatments with PGPR

application as compared to infected control as well as non-infected control. The

infection increased the POD activity in maize leaves by 51% as compared to non-

infected control (Fig 3.5). In all the inoculated treatments fungicide augmented the

efficiency of PGPR by enhancing the POD activity in maize leaves infected with stalk

rot. The maximum increase in POD activity was exhibited by BE treatment (B.

endophyticus Y5) which was 35% as compared to disease infected control when used

alone. The effect of other treatments was ranked as PA1>PA2>Ps>BF>BP for

increase in POD activity.


117

Fig 3.4: Effect of antagonistic PGPR on SOD activity of maize leaves in pots

under axenic condition of greenhouse Control: non-inoculated plants, In-C: Infected maize plants, PA1: inoculated with P.aeruginosa 4nm,

PA2: inoculated with P.aeruginosa JYR, Ps: inoculated with Pseudomonas sp., BF : inoculated with

BP. firmus PTWz, BP: inoculated with B. pumilus Yio, BE: inoculated with B. endophyticus Y5.

Results are presented as means of three replicate and the vertical bars specify the means standard

deviations. All means sharing the common letter differ non-significantly as P< 0.05. LSD: 0.428.

Fig 3.5: Effect of antagonistic PGPR on POD activity of maize leaves in pots

under axenic condition of greenhouse Treatments detail as indicated in Fig 3.4. Results are presented as means of three replicate and the

vertical bars specify the means standard deviations. All means sharing the common letter differ non-

significantly as P< 0.05 LSD: 0.0913.

i

g

d

f

c

d

f

bc

h

bc c

d

ab

d

e

a

0

2

4

6

8

10

12

14

16

18

Control In-C PA1 Ps PA2 BF BP BE

SOD

(un

its/

g F.

wt

of

leav

es)

Treatments


h

g

cd e de e

f

c

h

cd

b

c

b

cd

ef

a

0

0.5

1

1.5

2

2.5

3

PO

D (

un

it/m

in/g

F. w

t o

f le

ave

s)

Treatments



118

When applied in combination with half-dose of fungicide (0.1%) the increase was

68% for BE treatment as compared to infected control that is 24% higher as compared

to BE treatment alone.

The combined treatments of PA1 (P. aeruginosa 4nm) and PA2 (P.

aeruginosa JYR) also performed well showing 53 and 52% increase respectively, as

compared to infected control. The increase in POD activity was 32, 20 and 19%

higher as compared to the full dose of fungicide application for the combined

treatments BE (B. endophyticus Y5), PA1 (P. aeruginosa 4nm) and PA2 (P.

aeruginosa JYR).

Other three treatments including BP (B. pumilus PTWz), BF (B. firmus Yio)

and Ps (Pseudomonas sp. NDY), has significantly enhanced the POD activity by 18,

12, 10% respectively, as compared to infected control when applied alone while in

combined application, the chemical fertilizer enhanced their efficacy and the increase

in POD activity was 26, 23 and 22% as compared to infected control.

3.3.4 Polyphenol oxidase (PPO)

The disease inoculation with fungal pathogen (F. moniliforme) increased 40%

in PPO activity in infected plants as compared to non-infected control. Upon the

application of chemical fungicide (0.2%) on the disease infected plants, PPO activity

was significantly enhanced by 1.95 and 2.73 fold as compared to pathogen inoculated

and non-infected control plants, respectively (Fig 3.6).

The antagonistic PGPR inoculation in disease inoculated plants results in

significant increase in PPO activity, this increase ranges from 13 to 70% as compared

to infected control. Maximum increase was observed for the treatment BE inoculated

with B. endophyticus Y5 (70% as compared to disease inoculated control) while the

effect of other treatments was ranked as PA1 (P. aeruginosa 4nm) > PA2 > BF > Ps >

BP as compared to disease infected control.

The PPO activity reached to a higher level in the combined application of

antagonistic PGPR + half dose of fungicide as compared to disease infected and non-

infected control. The PPO activity was found to be significantly higher for the

combined treatment PA2 inoculated with P. aeruginosa JYR i.e. 98% as compared to

infected control.


119

The combined treatments of (BE) B. endophyticusY5 and (PA1) P. aeruginosa

4nm + half dose of fungicide also significantly increased the PPO activity by 91 and

86% as compared to infected control while this increase was 2.67 and 2.60 folds as

compared to non-infected control. The increase in PPO activity by the combined

treatments PA2, BE, PA1+half dose of fungicide was nearly or a little higher than that

of the treatment with chemical fungicide.

3.3.5 Ascorbate peroxidase activity

The results in Fig 3.7 revealed that ascorbate peroxidase activity has been

increased in all the treatment inoculated with antagonistic rhizobacteria. This increase

ranges from 21 to 70% as compared to disease infected control. Maximum increase

was observed for the treatment BE inoculated with B.endophyticus Y5 i.e. 70%. The

ranking of other treatments was as PA2 (66%)> BF (50%)> PA1 (49%) Ps (42%) >

BP (21%) as compared to disease infected control. The least amount of ascorbate

activity was observed in non-infected control.

The chemical fungicide application significantly increased (86%) the

ascorbate peroxidase activity when compared with disease infected control. Similarly

the combined treatments of antagonistic rhizobacteria with half dose of fungicide had

also showed an enhancing trend on the ascorbate activity as compared to infected and

non-infected control. The increase was higher for the treatment PA2 i.e. 2.01 and 2.73

folds as compared to disease infected and non-infected control respectively. This

enhancing trend was followed by the treatments BE> PA1> Ps> BF>BP i.e. 1.97,

1.77, 1.63, 1.60 and 1.30 folds as compared to disease infected control. When

compared with chemical fungicide treatment (0.2%), the combine treatments BE and

PA2 had an enhancing effect on ascorbate peroxidase activity by a percentage of 8.7%

while BE and PA1 were at par to the treatment with full dose of fungicide.

3.3.6 Total soluble Phenol

The accumulation of phenol was significantly increased in all the treatment

with antagonistic PGPR inoculation in alone and combined application with half dose

of fungicide (Fig 3.8). Phenol content in infected plants was increased by 3.09 folds

as compared to non-infected control but much less when compared to all the

treatments inoculated with rhizobacteria and chemical fungicide.


120

Fig 3.6: Effect of antagonistic PGPR on polyphenol (PPO) activity of maize

leaves in pots under axenic condition of greenhouse

Treatments detail as indicated in Fig 3.4. Results are presented as means of three replicate and the



Fig 3.7: Effect of antagonistic PGPR on ascorbate peroxidase of maize leaves in

pots under axenic condition of greenhouse Treatments detail as indicated in Fig 3.4. Results are presented as means of three replicate and the



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The treatment (In-C) with chemical fungicide application in full dose (0.2%)

significantly increased the phenol content as compared the disease infected and non-

infected control by 1.95 and 2.73 folds.

Maximum accumulation of phenols was recorded for the treatment PA2

inoculated with P. aeruginosa JYR i.e. 55 and 87% in alone and combined application

with half dose of fungicide respectively, as compared to diseases infected control. The

accumulation of phenol was increased by 45%, 39, 12, 11 and 8% for rhizobacteria

inoculated treatments BE, PA1, Ps BF and BP respectively, when applied alone.

The enzyme activity was significantly increased with the combined application

of antagonistic rhizobacteria + half dose of fungicide. This increase ranges from 15 to

87% as compared to infected control. A higher level of phenol was recoded for PA2

(87%), BE (82%) and PA1 (62%) as compared to disease infected control. When

compared with the chemical fungicide the combined treatment PA1and BE inoculated

with P. aeruginosa 4nm and B. endophyticus Y5 +half dose of fungicide has showed

the significant increase (17.5 and 14.5%) in the phenol content of maize leaves.

3.3.7 Catalase activity (CAT)

Minimum CAT activities was recorded in the non-infected control plants as

compared to the treatments inoculated with antagonistic PGPR inoculation in alone

and combine application with half dose of fungicide (Fig 3.9). Catalase activity in

disease inoculated plants was increased by 2.36 folds as compared to non-infected

control but much less when compared to all the treatments inoculated with

rhizobacteria and chemical fungicide.

The chemical fungicide application in full dose considerably increases the

CAT activity as compared the disease infected and non-infected control. Maximum

catalase activity was recorded for the treatment BE inoculated with B. endophyticus

Y5 i.e. 1.97 and 2.19 folds in alone and combine application with half dose of

fungicide respectively, as compared to diseases infected control. Minimum catalase

activity was recorded for the treatment BP inoculated with B. pumilus Yio i.e. 1.20

and 1.29 folds (in alone and combine application with half dose of fungicide) as

compared to diseases infected control.

The enzyme activity was significantly increase with the combine application

of antagonistic rhizobacteria + half dose of fungicide.


122

Fig 3.8: Effect of antagonistic PGPR on total soluble phenol of maize leaves in

pots under axenic condition of greenhouse Treatments detail as indicated in Fig 3.4. Results are presented as means of three replicate and the



Fig 3.9: Effect of antagonistic PGPR on catalase activity of maize leaves in pots

under axenic condition of greenhouse




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This increase was 2.20, 1.31, 1.91, 1.39, 1.29 and 2.20 folds for treatment PA1, Ps,

PA2, BP, BF, BE respectively as compared to disease inoculated control. When

compared with the chemical fungicide the treatment PA1 inoculated P. aeruginosa

4nm has showed the maximum increase in the catalase activity i.e. 2.20 fold as

compared to disease infected control.

3.3.8 Protein content

The protein content has been significantly increased in all the treatments

inoculated with antagonistic rhizobacteria as indicated in fig 3.10. This increase

ranges from 65 to 136% as compared to disease infected control. Maximum increase

was observed for the treatment BE inoculated with B. endophyticus Y5 i.e. 136%

whereas other treatment ranked as PA2 (108%), PA1 (99%), BF (93%), Ps (72%) and

BP (65%) as compare to disease infected control. The plants inoculated with disease

have shown a significant decrease (23%) in protein as compare to non-infected

control.

The chemical fungicide application significantly increased (139%) the protein

content as compared to disease infected control. Antagonistic rhizobacteria with half

dose of fungicide showed an increasing trend on the protein content as compared to

infected and non-infected control. This increase was maximum for the treatment BE

i.e. 2.6 and 1.9 folds as compared to disease infected and non-infected control

respectively. This trend was followed by the treatment PA2> PA1> Ps> BF> BP i.e.

2.08> 1.99> 1.93> 1.72> 1.65 folds as compared to disease infected control. When

compared with chemical fungicide (0.2%) treatment the combine treatment BE had an

enhancing effect on total protein content by 11%.

3.3.9 Chitinase activity

The chitinase activity has been increased in all the treatment inoculated with

antagonistic rhizobacteria (Fig 3.11). This increase ranges from 38 to 76.2% as

compared to disease infected control. Maximum increase was observed for the

treatment BE inoculated with B. endophyticus Y5 i.e. 76.2%. Other treatments was

ranked as follows PA1 (57%)> PA2 (67%)> Ps (38%)> BP (42%) as compared to

disease infected control.


124

Fig 3.10: Effect of antagonistic PGPR on protein content of maize leaves in pots





Fig 3.11: Effect of antagonistic PGPR on chitinase activity of maize leaves in pots





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The treatment BP inoculated with Bacillus pumilus Yio did not significantly

increase the chitinase activity as compared to infected control but it showed a

significantly higher chitinase activity (3 folds) as compared to the non-infected

control.

The chemical fungicide application significantly increased the chitinase

activity as compared to disease infected control. Similarly the combine treatments of

antagonistic rhizobacteria and half dose of fungicide (0.1%) had showed an enhancing

trend on the chitinase activity as compared to infected and non-infected control. The

increase was maximum for the treatment BE i.e. 1.16 and 8.9 folds as compared to

disease infected and non-infected control, respectively. This trend was followed by

the treatments PA1>Ps> PA2>BF i.e. 1.18, 1.16, 1.15 and 1 folds as compared to

disease infected control. The treatment BP showed a declining trend in chitinase

activity as compared to disease infected control.

3.3.10 Malondialdehyde (MDA)

MDA content was significantly decreased by the PGPR inoculation in maize

plants (Fig 3.12). The disease inoculation results in an increase (4.80 folds) in MDA

content as compared to non-infected control.

The application of chemical fungicide (0.2%) on infected plants brings about

significant decrease in MDA content i.e. 70% as compared to diseases inoculated

control. The antagonistic PGPR inoculation in disease inoculated plants also resulted

in significant decrease in MDA content; this decrease ranges from 34 to 65% as

compared to infected control. Maximum decrease was observed for the treatment PA2

i.e. 65% whereas the ranking of other treatments for decrease in MDA content was as

follows BE (56%)> PA1 (50%)> BF (50%)> Ps (35%)> BP (34%) as compared to

disease infected control.

MDA content was significantly low for the combined application of

antagonistic PGPR + half dose of fungicide (0.1%) as compared to both of the disease

infected control and alone application of antagonistic PGPR. Minimum amount of

MDA was recorded for the combined treatment PA2 and BE i.e. 79 and 72% as

compared to disease inoculated control.

The combined treatments including PA1, BF (B. firmus PTWz), BP(P. pumilus

Yio) and Ps (Pseudomonas sp. NDY) +half dose of fungicide also significantly


126

decreased the MDA content by 70, 66, 56 and 54% respectively, as compared to

disease infected control. The decreasing trend in MDA content by the combined

application of B. endophyticusY5 and P. aeruginosa 4nm +half dose of fungicide was

at par to the treatment with full dose of chemical fungicide while, the effect of

treatment PA2 was decreased even more (28%) as compared to full dose of chemical

fungicide treatment and at par to non-infected control.

3.3.11 Proline content

The results presented in Fig. 3.13 revealed that increase in proline content was

significantly increased upon treatment with antagonistic rhizobacteria and chemical

fungicide in maize leaves. The disease inoculation 15.9% increased the proline

content in infected control as compared to non-infected control. The proline content

was further enhanced i.e. 99 and 131% as compared to diseases infected and non-

infected control plants, respectively by the application of chemical fungicide.

The antagonistic PGPR inoculation in disease inoculated plants resulted in

significant increase in proline, this increase ranges from 28 to 96% as compared to

infected control. Maximum increase was observed for the treatment BE inoculated

with B. endophyticus Y5 (96% as compared to disease inoculated control) while the

effect of other treatments was as follows (74%)> PA2 (70%) > BF (47%) > Ps (29%)

> BP (28%) as compared to disease infected control.

Proline content reached to a higher level in the combined application of

antagonistic PGPR + half dose of fungicide (0.1%) as compared to both of the disease

infected and non-infected control. The proline content was found to be significantly

higher for the combined treatment BE inoculated with B. endophyticus Y5 i.e. 110%

as compared to disease inoculated control. The combined treatments of P.aeruginosa

4nm (PA1) and P. aeruginosa JYR (PA2) +half dose of fungicide also significantly

increased the proline content by 97 and 96% as compared to disease infected control

while this increase was 2.0 and 1.9 folds as compared to non-infected control. The

increase in proline content by the combined treatments PA2, BE, PA1 +half dose of

fungicide was at par with chemical fungicide (0.2%).


127

Fig 3.12: Effect of antagonistic PGPR on MDA content of maize leaves in pots





Fig 3.13: Effect of antagonistic PGPR on proline content of maize leaves in pots





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3.3.12 Photosynthetic pigments

3.3.12.1 Chlorophyll

As indicated in Fig 3.14, the synthesis of chlorophyll content was significantly

increased in all treatments with PGPR application as compared to infected maize

plants as well as non-infected control. The infection resulted in the significant

decrease in the chlorophyll content as the chlorophyll content was reduced to 13.5%

in infected control over that of non-infected control. In all the inoculated treatments

fungicide augmented the efficiency of PGPR for increased synthesis of chlorophyll.

The maximum increase in chlorophyll content was due to BE treatment (B.

endophyticus Y5) which was 73% as compared to disease infected control when used

alone. When applied in combination with half dose of fungicide the increase was 97%

for BE treatment as compared to infected control, which is 14% higher as compared to

BE treatment alone. The other combined treatments were ranked as PA1 (70%), PA2

(69%) > BF (41%) Ps (21.1%) and BP (2.6%) for increase in chlorophyll content as

compared to infected control. In the combined application, chemical fertilizer

enhanced the efficacy of rhizobacteria. When compared with the chemical fungicide

the effect of combined treatments BF, PA1 and PA2 was more than then the chemical

fungicide treatment by 41%, 21% and 21% respectively.

3.3.12.2 Carotenoid content

The results presented in fig 3.15 have shown that amount of carotenoid content

was significantly increased in all the treatment with antagonistic PGPR inoculation in

alone and combined application with half dose of fungicide. Carotenoid content was

decreased in disease inoculated plants by 40% as compared to non-infected control.

The treatment In-C (chemical fungicide application in full dose) considerably

increased the carotenoids as compared the disease infected and non-infected control

by 2.94 and 1.62 folds.


129

Fig 3.14: Effect of antagonistic PGPR on chlorophyll content of maize leaves in

pots under axenic condition of greenhouse




Fig 3.15: Effect of antagonistic PGPR on carotenoids of maize leaves

in pots under axenic condition of greenhouse



significantly as P< 0.05, LSD = 0.67.

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Maximum accumulation of carotenoid content was recorded for the treatment

BE (B. endophyticus Y5) which showed increase in carotenoids by 2.89 folds as

compared to infected control. Followed by PA2 and PA1 inoculated with P.

aeruginosa JYR and P. aeruginosa 4nm i.e. 2.61 and 2.55% in alone application

respectively, as compared to infected control. The carotenoids accumulation was also

increased by 2.28, 2.27 and 2.11 folds for rhizobacteria inoculated treatments, Ps

(Pseudomonas sp. NDY) BF (B.firmus PTWz) and BP (B.pumilus Yio) respectively,

in alone application.

The carotenoid content was improved even more when half dose of chemical

fungicide (0.1%) was applied along with antagonistic rhizobacteria. This increase

ranges from 2.83 to 3.57 folds as compared to infected control. A higher level of

carotenoid content was recoded for BE (3.57 folds)> PA1 and PA2 (3.35) > BF

(69%)> BP and BF (3.03 folds) Ps (2.83 folds) as compared to disease inoculated

control. When compared with the chemical fungicide the combine treatment BE, PA1

and PA2 inoculated with B. endophyticus Y5, P. aeruginosa 4nm and P. aeruginosa

JYR was at par with the alone application of chemical fungicide. While the combined

application had increased the carotenoid content 1.21 folds when inoculated with B.

endophyticus Y5 + half dose of fungicide and 1.13 folds on the application by P.

aeruginosa 4nm and P.aeruginosa JYR + half dose of fungicide.


131

3.4 Discussion

Disease development is the outcome of interaction between plant and

pathogen by the production of reactive oxygen species (ROS). Basically main focus of

ROS production is the induction of defence mechanisms such as formation of lignin,

direct antifungal action, initiating of systemic acquired and hypersensitive response

(Xu et al., 2008; Shoresh et al., 2010). However, over production of ROS brings about

the oxidative damage followed by lipid peroxidation, macromolecules destruction

(nucleic acids, proteins, carbohydrates) reduction of photosynthetic pigments (Singh

et al., 2009 and 2010). All these reaction resulted in localized death of plant cells at

the infection site (Nanda et al., 2010). Generally, there is an intracellular balance

between generation of ROS and their scavenging action in all types of cells. ROS

homeostasis requires an efficient coordination of various chemical reactions among

cell organelles which is started by particular signal transduction pathways. These

scavengers contain glutathione, ascorbate, hydrophobic molecules (carotenoids,

xanthophylls and tocopherols) and detoxifying enzymes like POD, SOD, and CAT.

All of these scavengers work together with the enzymes of ascorbate–glutathione

cycle to stimulate the ROS scavenging (Hernandez et al., 2001).

Plant defence mechanisms can be triggered by the former application of

antagonistic rhizobacteria and it is one of the unique and eco-friendly approaches of

plant protection (Shoresh and Harman, 2008; Van Loon et al., 2008) through cellular

responses known as ISR. It involves earlier oxidative burst and an active up-

regulation of defence genes (Ahn et al., 2007). It has been reported by Corne at al.,

(1996) that PGPR antagonists protect plants against fungal pathogen infection by ISR

(induction of systemic resistance).

3.4.1 Resistance by chemical fungicide (Ridomil Gold)

Among the various mode of action used by chemical fungicides to protect the

plants, one of the mechanisms is induction of systemic resistance in host plant used by

some fungicides (Singh et al., 2011). The present study revealed that chemical

fungicide when applied in full dose significantly decreased the disease severity in

plants infected with Fusarium stalk rot and brought about increase in POD, SOD,

PPO, Ascorbate, chitinase, catalase and total phenols. These results are in accordance

with Sendhil, (2003), who reported that fungicide treated grapevine plants have high

amount of the PPO , PO, PAL, chitinase, ß-1,3-glucanase and total phenols. An


132

increase in the activity of PO and PPO in plants raised from fungicide (carbendazim)

treated seeds as compared with untreated seeds have been reported by Kalim et al.,

(2000). In another study, Hewitt (1988) found that the chemical fungicide increase the

levels of PPO, POD, PAL and tyrosine ammonia lyase in the leaves, which showed

that the chemical fungicides (azoxystrobin and probenazole) control the disease by

host-mediated reaction. The production of pathogenesis related (PR) proteins by the

fungicide were correlated with their ability to supress the leaf blight disease

(Lalithakumari and Dhakshinamoorthy, 1995).

Similarly Oostendorp et al., (1996) reported triazole compounds like

propiconazole and epoxiconazole which induce systemic resistance in cucumber

plants. BTH (benzothiadiazole) showed higher levels and enhanced activity of

peroxidases in plants inoculated with disease (Kaur and Kolte, 2001). Therefore the

enhances activity of POD, SOD and PPO, along with the higher amount of total

phenols and PR proteins by the application of fungicide is one of the possible

mechanism of disease control in maize plants as found in the present study following

the application of chemical fungicide (ridomil gold) on maize plants infected with

Fusarium moniliforme.

3.4.2 Induced systemic resistance by antagonistic PGPR

Plants have a range of defence genes and it is well-known that these defence

related genes are activated by the prior application of the biocontrol agent and this is

an environment friendly and novel approach to control the plant diseases (Archana et

al., 2011). A number of earlier studies have shown that P. aeruginosa and P.

fluorescens induce systemic resistance in plants (Zehnder, 2000; Bigirimana and

Hofte 2002). Seed inoculation with P. fluorescens inhibited the pathogen growth

(anthracnose and mildews) by the induction of systemic resistance (Wei et al., 1996).

Choudhary and Johri (2008) have reported induced systemic resistance by a number

of Bacillus sp. in crop plants and demonstrated their role in ISR.

In the present study, three strains of Pseudomonas (Pseudomonas aeruginosa

4nm, Pseudomonas aeruginosa JYR, Pseudomonas sp. NDY) and three strains of

Bacillus (B. endophyticus Y5, B. pumilus Yio and B. firmus PTWz) had been

inoculated in maize plants infected with Fusarium stalk rot. The application of these

antagonistic PGPR control the Fusarium stalk rot development and prevalence along

with the synthesis and accumulation of POD, PPO, SOD, chitinase, catalase, total

phenols, proline, protein and total soluble sugar. Similar findings were reported by


133

Sendhil Vel (2003) in grapevine plants inoculated with P. fuorescene. A number of

worker reported that former application of P. fluorescens fortifies the host cell wall,

make barrier to restrict the invading pathogen (Chen et al., 2000). P. aeruginosa

7NSK2 have found as an important elicitor of ISR to M. grisea and R. solani in rice

(De Vleesschauwer et al., 2006). A number of Bacillus sp. have been reported to be

involved in induction of systemic resistance as B. pumilus strain INR-7 provide

systemic protection (Liu et al., 1995b; Wei et al., 1996). Similar observations were

made by Fernando et al., (2007), they showed that bacterial antagonists P.

chlororaphis PA-23 and B. amyloliquefaciens BS6 significantly reduced the stem rot

caused by S. sclerotiorum.

The results presented in this study revealed that the application with

antagonistic PGPR significantly increases the peroxidase activity alone as well as in

combined application with half dose of fungicide. These results are in accordance

with the earlier study which reported that Bacillus strains induce high SOD and POD

activities as compared to untreated control (Jetiyanon, 2007). Ramamoorthy and

Samiyappan (2001) presented the similar increase of POD in chilli plants. Shoresh

and Harman (2008) found the increased levels of antioxidant and other detoxifying

enzymes in maize. These reports confirm the role of the antagonistic rhizobacteria in

eliciting POD and SOD activities in plants and support the results of the present

study.

Peroxidases are involved in the synthesis of cell wall polymers (lignin and

suberin), which act as the physical barriers to biotic and abiotic stresses (Quiroga et

al., 2000) and confer the plant with high rigidity. POD is among the important defence

elements that get stimulated in response to pathogen infection like F. oxysporum

(Morkunas and Gemerek, 2007). It is present in cell wall and catalyze the oxidation of

numerous organic compounds including lignin, suberin, and phenolics and contribute

in the reinforcement of host cell against pathogens. It also has an important role in the

inhibition of fungal spore germination and growth of mycelia of certain fungi (Joseph

et al., 1998). The enzymes SOD and POD work together with other enzymes of

ascorbate-glutathione cycle, to promote the scavenging of free radicals (Hernandez et

al., 2001). SOD protects the cells from oxidative stress which is considered as the first

line of defence against ROS (Singh et al., 2009).

Polyphenol oxidases have a key role in plant defence by catalyzing the

oxidation of phenolic compounds into o-quinones. These quinone radicals are toxic to


134

invading pathogens (Mohammadi and Kazemi, 2002) that results in their effective

antimicrobial activity. Therefore, PPO has a direct action in stopping the pathogen

development and prevalence by accelerating the cellular death occur at the infection

site in toxic environment (Bi and Felton, 1995). Maximum PPO activity in the present

investigation was detected in maize plants having the application of antagonistic

rhizobacteria B. endophyticus Y5 and P. aeruginosa 4nm. Our results are also in

agreement with Harish et al., (2009), who reported the increased activities of POD

and PPO in plants, treated with P. fluorescens Pf1 and Bacillus sp. EPB22. Similarly,

in this study increase in phenols has been observes by prior application of

rhizobacteria. This finding is in consistency with the finding of Anand et al., (2007)

and Jain et al., (2012).

The antioxidant catalase (CAT) also works with other enzymes including

SOD, POD to stimulate the ROS scavenging (Singh et al., 2010). Catalases are

present in the peroxisomes of plant cells and provide protection by catalyzing the

decomposition of free radicals into stable compounds (Nanda et al., 2010). In this

study, inoculation with Bacillus and Pseudomonas strains to stalk rot infected maize

plants significantly stimulated the activities of antioxidant enzymes CAT. These

results are in agreement with previous findings, which reported the increase in CAT

level during pathogen infection by the inoculation with rhizobacteria (Anand et al.,

2007).

It is reported by a number of researchers that the activity of antioxidant

enzymes in leaves under pathogen infection increased and act as an effective tool in

scavenging mechanism to eliminate the H2O2 and O2 produced in leaves

(Subramaniam et al., 2006 ; El- Khallal, 2007). Therefore it can be conferred that the

greater activity of POD, SOD and PPO, along with the higher amount of total phenols,

may enhance host resistance. These results are inconsistence with the finding of Kaur

and Kolte, (2001).

We found in the present study that total soluble protein content in infected

maize leaves increased significantly by the inoculation with antagonist PGPR. Plant

pathogens stimulate the synthesis of host proteins which help in controlling the

development and spread of pathogens in the non-infected plant tissue (Datta et al.,

1999). It also has been shown by Gnieszka and Iwona, (2003) that the pathogenesis

related (PR) proteins including chitinase, -1, 3-glucanase or thaumatin in the tissue

infected by pathogens is associated with the plant resistance to pathogens. PR and


135

host coded proteins are elicited by various biotic and abiotic stresses (Van Loon et al.,

1998). PR proteins synthesis and accumulation play a key role in plant protection

against fungal pathogens (Sendhil Vel, 2003). Systemic resistance by the inoculation

of P. fluorescens is associated with the increased levels of chitinase and ß-1,3-

glucanase activity (Maurhofer et al., 1994). These enzymes accelerate the degradation

of fungal cell wall and various cell-organelles (Benhamou et al., 1996). In our study,

higher level of chitinase was induced by antagonistic rhizobacteria against F.

moniliforme. The results are in consistence with Singh et al., (2012) they reported the

involvement of chitinase in the defence against Sclerotinia sclerotiorum pathogens in

leaves of pea by the inoculation of B. subtilis. Inoculation with P. fluorescens also

showed the enhanced level of phenols and chitinase and b-1,3-glucanase activities in

coconut palm (Karthikeyan et al., 2006). Chitinase are among the completely

characterized pathogenesis related proteins and have the ability to hydrolyze the

fungal cell wall’s constituents. Hence it is suggested that they induce strong

resistance against the fungal pathogens (Gnieszka and Iwona, 2003).

In order to further verify whether antagonistic rhizobacteria influence the

oxidative damage in maize plants, the contents of lipid oxidation should significantly

decreased as compared to infected plants and results are expressed in the terms of

malondialdehyde (MDA) contents. As reported by Singh et al., (2010) that both biotic

and abiotic stresses results in peroxidation of lipid membranes by over production of

ROS and it is indicated by the formation of MDA. The results of present study

showed that the MDA in treated plants were significantly lower as compared to

infected plant. Among all rhizobacteria B. endophyticus Y5 and P. aeruginosa 4nm

had potent efficacy in alleviating pathogen induced oxidative damages in maize

plants. Similarly, decrease in MDA content was determined in plants inoculated with

antagonistic PGPR as compared to untreated infected plants and decrease in MDA

content indicate the decrease in lipid peroxidation is correlated with the increased

activities antioxidant enzymes such as SOD, POD etc. (Singh et al., 2013). It was

reported that B. subtilis SY1 also help in improving the resistance of plants by

enhancing the activities of antioxidant enzymes and reducing the MDA content

(Zongzheng et al., 2009).

The accumulation of free proline in plants represents a strong stress response

to the invading pathogens (Grote et al., 2006; Arie et al., 2007). The amino acid

proline is an efficient scavenger and this ability of proline prevents the induction of


136

localized cell death by ROS (Srinivas and Balasubraman, 1995). Besides, it also

regulates the acidity of cytosol (Fabro et al., 2004). In the present study, the F.

moniliforme infected maize plants significantly raised the level of proline. Berber and

Onlu, (2012) have reported a linear relationship between the accumulation of proline

and the decrease in plant infection. Perhaps the increased proline accumulation in the

Pseudomonas and Bacillus in infected maize plants in the present investigation may

demonstrate a correlation to an early response of maize plants to the inoculated

pathogen (F. moniliforme) for the control of oxidative stress.

The inoculation of F. moniliforme in maize plants resulted in decreased

chlorophyll and carotenoid contents as compared to non-infected plants. Similar

results were reported by Srobarova et al., (2003) who found the decrease in

chlorophyll content when the maize plants were infected with F. verticillioides syn. F.

moniliforme as compared to non-infected plants. Agamy et al., (2013) also showed

that, chlorophyll a, b and carotenoids were significantly reduced in plants infected

with A. tenuissima as compared to non-infected control. This reduction in chlorophyll

and carotenoids contents may be the result of toxins released by fungus followed by

the elicitation of ROS causing localized cell death (Howlett, 2006).

Carotenoids are required for the correct assembly of photosystems (Li et al.,

2009) and have the ability to scavenge ROS (Bailey and Grossman 2008; Alboresi et

al., 2011). Carotenoids are considered as one of the most effective non-enzymatic

quenchers of ROS in the cells. Recent studies have shown that the carotenoids serve

in a protective function against plant infection (Petrova et al., 2009). The

photosynthetic pigments including chlorophyll and carotenoid contents were

enhanced significantly in maize plants treated with Pseudomonas and Bacillus

antagonistic PGPRs in the present study. A large body of researchers reported that

chlorophyll and carotenoids are significantly increased in plants inoculated with

Pseudomonas strains (Hameed and Farhan 2007; Rakh et al., 2011). In another study

Srobarova et al., (2003) also reported that biocontrol agents reduce the decrease in

chlorophyll concentration in maize plants infected with F. verticillioides. The

chlorophyll and carotenoid concentration was increased by the inoculation with B.

subtilis SY1 (Zongzheng et al., 2009).

It is inferred that the prior treatment of maize plants with antagonistic PGPR

triggered a plant-defense mechanism to protect the plants infected with F.

moniliforme. The combined application of antagonistic PGPR along with the chemical


137

fungicide triggers more efficiently the defence response of plants as compared to

application of antagonistic PGPR alone.

Biological control of stalk rot in maize under field conditions by antagonistic PGPR Chapter 4

138

4.1 INTRODUCTION

Biological control is an attractive and efficient approach for the control of plants

fungal diseases. Moreover, it also provides a practical strategy for a sustainable

agricultural system (Lo et al., 1998). Biocontrol by the use of PGPR (plant growth

promoting rhizobacteria) presents an eco-friendly alternative for the management of

plant diseases besides improvement in the plant growth and yield (Jetiyanon and

Kloepper, 2002). Biological control of Fusarium stalks rot, caused by pathogenic

species of Fusarium moniliforme, through antagonistic PGPR is an effective

management of this disease in maize (Bressan and Figueiredo, 2010 ; Bacon et al.,

2001; Pal et al., 2001; Mishra et al., 2011). In the previous sections, we demonstrated

the efficacy of selected Pseudomonas and Bacillus strains isolated from the

rhizosphere of infected and non-infected maize. Strong antifungal activity was

observed against Fusarium moniliforme, Helminthosporium sativum and Aspergillus

flavus. These strains were equally effective in reducing the incidence of Fusarium

stalk rot of maize under axenic conditions of greenhouse experiment and the mode of

action these PGPR has also been evaluated. However, it is imperative to demonstrate

the performance of these antagonistic PGPR under field conditions where the effect is

modulated by the natural environmental and edaphic factors.

The PGPR inoculation increases the productivity of agronomical vital cereal crops

(Asghar et al., 2002; Bashan et al., 2004). Jagadish, (2006) and Kloepper and

Beauchamp, (1992) reported that yield of crops were increased by the inoculation of

Bacillus and Pseudomonas PGPR. Several rhizobacteria had promoted the growth and

yield along with the control of diseases by inducing the systemic resistance

(Earnapalli, 2005). It is demonstrated that PGPR enhanced the growth and yield by

greater production of phytohormones and nutrient uptake which may be utilized for

the control of diseases (Jagadish, 2006).

Bargabus et al., (2002) reported earlier that ISR is supposed to be most

probable mechanism for the disease control in greenhouse conditions that provides the

spatial inhibition of pathogen by PGPR but this effect is not usually retained in the

field conditions. Induced systemic disease resistance has been studied mainly in

laboratories and greenhouses. However, some reports also indicate that ISR can

protect crop plants under field conditions (Tuzun et al., 1992). Thus, it is


139

indispensable to evaluate the mode of action of the potent strains in environmental

conditions of field.

Aims and objective

The present investigation was aimed to evaluate the selected strains of

Pseudomonas and Bacillus against Fusarium stalk rot of maize under natural

environmental conditions of field. In the present study, attempt was made to control

Fusarium stalk rot with biocontrol agents under field conditions. PGPR may offer a

practical way of delivering 1SR to agriculture but the feasibility of this approach has

not been reported under field conditions.


140


The present investigation was aimed to evaluate the selected PGPR strains for

their ability to control Fusarium stalk rot in maize and the possible mechanism used

by the antagonistic PGPR under field conditions. The materials and methods

employed in this investigation are outlined below.

4.2.1 Collection of host plant seeds and pathogen

The seeds of maize cv. Islamabad gold, susceptible to stalk rot and the

associated fungal pathogen (Fusarium moniliforme) were obtained from National

Agriculture Research Centre (NARC), Islamabad, Pakistan.

4.2.2 Selected PGPR strains

Six PGPR strains were screened on the basis of their antifungal activity,

biochemical characteristics and plant growth promoting ability as described in chapter

2. These antagonistic PGPRs include three Pseudomonas strains (P.aeruginosa 4nm,

P.aeruginosa JYR, Pseudomonas sp. NDY) and three Bacillus strains including

Bacillus pumilus Yio, Bacillus firmus and Bacillus endophyticus Y5.

4.2.3 Inoculation with antagonistic PGPR

Seeds of maize (cv. Islamabad gold) were sterilized and inoculated with

antagonistic PGPR as the method described in chapter 1.

4.2.4 Inocula preparation

Inocula suspension for the Fusarium moniliforme culture was prepared following

the method of Tesso et al., (2009) with some modifications using potato dextrose

broth (PDB, DIFCO) as explained in chapter 3.

4.2.5 Field experiment

Field experiment was conducted in two consecutive years (2010 and 2011) at the

wire house of Quaid-i-Azam University, Islamabad (Pakistan) to evaluate the efficacy

of selected antagonistic PGPR against Fusarium stalk rot infection. The randomized

complete block design was used with three replications. The pH and EC of the field

soil was 7.9 and 0.66 dS/cm that contained the available amount of nutrients Na, K, P,

Mg and Ca as 11.4, 12.5, 16.3, 4.5 and 14.8 µg/g, respectively.


141

Table 4.1: Treatments made for field experiments

Symbols

used

Treatments

Symbols used

Treatments

Without fungicide With Fungicide

Control Non-infected plants Control+F

Non-infected plant +

fungicide (0.2%)

In-C Infected with F. moniliforme In-C+F

F. moniliforme + fungicide

(0.2%)


aeruginosa 4nm PA1+HF

F. moniliforme +

Pseudomonas aeruginosa

4nm+ fungicide (0.1%)

Ps F.moniliforme + Pseudomonas

sp. NDY Ps+HF

F. moniliforme +

Pseudomonas sp. NDY +

fungicide (0.1%)


aeruginosa JYR PA2+HF

F. moniliforme +

Pseudomonas aeruginosa

JYR + fungicide (0.1%)

BF F. moniliforme + Bacillus

firmus PTWz BF+Hf

F. moniliforme + Bacillus

firmus PTWz + fungicide

(0.1%)

BP F. moniliforme + Bacillus

pumilus Yio BP+HF


pumilus Yio + fungicide

(0.1%)

BE F. moniliforme + inoculated

with Bacillus endophyticus Y5 BE+HF


endophyticus Y5 + fungicide

(0.1%)


142

The maize seeds treated with the PGPR were sown in the field plot (size 1x1m2)

and the row to row distance was 45cm.

4.2.6 Inoculation of the pathogen

After 60 d of sowing (tasseling stage), plants were tagged with different colour

tapes and the pathogen (F. moniliforme) was introduced with the help of 1 mL syringe

to the second node of plant stem as described by Tesso, et al., (2009). The site of

inoculation was covered with tape to facilitate humidity for the proliferation of

fungus. Control plants were inoculated with equal volume of PBS buffer.

4.2.7 Disease scoring

After 21 d of inoculation (dough stage), five plants were harvested from each

treatment and scored for the disease severity following the method of Tesso et al.,

(2009) as described in chapter 3. The data was transformed to percentages on 0-5

scale for the statistical analysis by using the formula given in chapter 3.

4.2.8 Sample Collection

Plant leaf tissues were collected at three different stages (silk stage, blister stage

and dough stage) after the inoculation of Fusarium moniliforme.

4.2.9 Superoxide Dismutase (SOD) activity

The SOD was determined following the method of Beauchamp and Frodovich (1971)

as described in Chapter 3.

4.2.10 Peroxidase activity

The POD activity was determined following the method of Vetter et al., (1958) as

modified by Gorin and Heidema, (1976). The detailed method was explained in chapter 3.

4.2.11 Polyphenol oxidase (PPO)

The activity of polyphenol oxidase was determined by method described by Kar

and Mishra, (1976) with some modification. The detailed protocol was given in

chapter 3.

4.2.12 Ascorbate peroxidase activity

The POD activity was determined following the method of Asada and Takahashi,

(1987) with some modifications. The detailed method was explained in chapter 3.


143

4.2.13 Catalase activity

Catalase activity was measured following the method of Teranish et al., (1974)

by analysing the disappearance of H2O2 as described in chapter 3.

4.2.14 Chitinase assay

The colorimetric assays was used for the measurement of chitinase activity with

CM chitin-RBV (Loewe Biochemical, Germany) (Stangarlin et al., 2000) as described

in chapter 3.

4.2.15 Total soluble phenol content

The total soluble phenol content in maize leaves was determined as described by

Hsu, et al., (2003) with some modifications as explained in chapter 3.

4.2.16 Extraction and Purification of IAA and ABA

The extraction and purification of phytohormones was done according to the

method described by Kettner and Doerffling, (1995).

Fresh maize leaves (1 g) were homogenized in methanol (80%) at 4oC by adding

the butylated hydroxy toluene (BHT) as antioxidant at the rate of 10 mg/mL. furthermore,

extraction was done for 72 h at 4oC with successive change in solvent after each 24 h. The

leaf extract was centrifuged and the supernatant evaporated to aqueous phase by using the

rotary film evaporator (RFE) and partitioned four times with ½ volume of ethyl acetate

after adjusting the pH 2.5–3.0. The ethyl acetate was completely dried by using RFE and

subsequently the residue was re-dissolved in 100% methanol (1 mL) and stored at -20oC

till the further analyses.

Analysis of IAA and ABA through HPLC

The samples prepared were analyzed on HPLC (Agilent 1100) with U.V. detector

and C-18 column. Hormones identification was made after filtration of samples

through Millipore filters (0.45 µ) and injected into column. Identification of hormones

was done the basis of peak area and retention of standards. Methanol, acetic acid and

water (30:1:70) were used as mobile phase and flow rate was adjusted to 1 ml/min

with an average run time of 20 min/sample. Detection of IAA was made at

wavelength 280 nm (Sarwar et al., 1992), whereas, for ABA wavelength 254 nm was

used (Li et al., 1994).


144

4.2.17 Yield parameters

Plants were harvested and yield parameters were determined including

1. 1000 seed weight

2. Number of seed/cob

3. Grain yield (kg ha-1

)


The data generated from field experiments was subjected to ANOVA and statistical

analysis by using Statistix 8.1 and the comparison among mean values of treatments was

made by least significant difference (LSD) at P< 0.05 (Gomez and Gomez, 1984).


145

Fig 4.1: Lay out of field experiment


146

4.3 RESULTS

The field experiment was conducted for two consecutive years (2010 and 2011)

during growing season of maize (June -September) the results were presented as the

mean of two years. Furthermore, the sampling was done at 7, 14 and 21 days after

disease inoculation. The gradual increase/decrease for first two samplings (7 and 14

days after pathogen inoculation) and decline for the third sampling stage, in all

parameters was observed. Therefore, in the proceeding section the results of second

stage are presented.

4.3.1 Disease severity/ disease reduction

The results presented in Fig 4.2 revealed that all the treatments inoculated with

PGPR antagonists significantly reduced the stalk rot disease in maize plants by 33-

59% in field experiment as compared to infected control. Maximum significant

reduction (59%) exhibited by treatment PA2 (P. aeruginosa JYR), whereas, the effects

of other treatments was ranked as BE (56%) >PA1 (55%) >Ps (33%). Two treatment

including BF (B. firmus PTWz) and BP (B. pumilus Yio) did not exhibited any

significant reduction in the disease incidence and only 4.6 and 8.3% decrease was

recorded, respectively under natural conditions of field.

The application of fungicide improved the efficacy of the antagonistic PGPR.

It was observed that the combined application with half dose of fungicide along with

antagonistic rhizobacteria was more effective in reducing the disease (52-77%)

occurrence in comparison to single application of antagonistic rhizobacteria. Among

the PGPR treatments the effect of treatment PA2+HF, BE+HF and PA1+HF was at

par with chemical fungicide.

The inoculation with antagonistic PGPR when applied alone significantly

decreased the disease severity which was in the range of 40 to 80% (fig 4.2). But in

combined application with half-dose of chemical fungicide the efficiency of most of

the rhizobacteria was enhanced and they significantly decreased the disease severity.

Maximum reduction in disease severity (20%) for combined application was observed

for treatment PA2+HF. The effect of this treatment was at par with chemical

fungicide. The treatment BF+HF and BP+HF in combined application with chemical

fungicide has not significantly decreased the disease prevalence.


147

Table 4.2: Effect of antagonistic PGPR on the disease severity (%)

in maize plants

Treatments Disease severity (%)

Mean of two years 2010 2011

In-C 80 100 90 a

PA1 40 40 40 f

Ps 60 60 60 e

PA2 33.3 40 36.7 f

BF 73.3 80 76.7 cd

BP 80 80 80 b

BE 33.3 46.7 40 f

In-C+ F 20 20 20 g

PA1 +HF 20 26.7 23.3 g

Ps +HF 40 46.7 43.3 ef

PA2 +HF 20 20 20 g

BF+HF 73.3 73.3 73.3 de

BP +HF 73.3 73.3 73.3 bc

BE +HF 20 33.3 26.7 g

Fig 4.2: Effect of antagonistic PGPR on disease severity/reduction

under field conditions

In-C: Infected maize plants, PA1: P. aeruginosa 4nm, PA2 : P. aeruginosa JYR, Ps: Pseudomonas sp,

BF : B. firmus PTWz, BP : B. pumilus Yio, BE : B. endophyticus Y5, In-C+F: Infected maize plants+

full dose of fungicide, PA1+HF: P. aeruginosa 4nm+Half dose of fungicide, PA2+HF: P. aeruginosa

JYR+ Half dose of fungicide, Ps+HF: Pseudomonas sp.+ Half dose of fungicide, BF +HF: B. firmus

PTWz+ Half dose of fungicide, BP +HF: B. pumilus Yio+ Half dose of fungicide, BE+HF: B.

endophyticus Y5+ Half dose of fungicide. Results are expressed as means of three replicate, and

vertical bars indicate the standard deviations of means. All means sharing the common letter differ non-

significantly at P< 0.05. LSD: 4.70.

a

f

e f

cd b

f

g g

ef

g

de bc

g

-100102030405060708090

0

20

40

60

80

100

120

Dis

eas

e r

ed

uct

ion

(%

)

Dis

eas

e s

eve

rity

(%

)

Treatments

Disease severity (%) Disease reduction (%)


148

4.3.2 Phytohormones production in maize leaves

4.3.2.1 Indole acetic acid (IAA) content

The results presented in Fig 4.3 revealed that endogenous IAA content in maize

leaves was significantly higher upon treatment with most of the antagonistic

rhizobacteria and chemical fungicide in maize plants. The pathogen inoculation

significantly decreased (46%) the endogenous IAA content in disease infected plant

(In-C) as compared to non-infected control. Upon the application of chemical

fungicide on the disease inoculated plants, IAA was increased significantly (63%) as

compared to diseases inoculated plants.

The antagonistic PGPR inoculation in infected plants also resulted in significant

increase in endogenous IAA content in maize leaves (22 to 42%) as compared to

infected control. Maximum increase (42%) was observed for the treatments BE and

PA1 (inoculated with B. endophyticus and P. aeruginosa) as compared to disease

inoculated control, while the effects of other treatments ranked as PA2 (35%)> Ps

(22%). The treatments BF and BP did not affect the amount of endogenous IAA in

leaves of maize plants infected with Fusarium stalk rot.

Endogenous IAA content was recoded higher in the combined application of

antagonistic PGPR + half dose of fungicide as compared to the disease infected

control. The endogenous IAA content was found to be significantly higher (68%) in

maize leaves for the combined treatment PA2+ HF as compared to disease infected

control. The combined treatments with B. endophyticus Y5 (BE+HF) , P. aeruginosa

4nm (PA2+HF) and Pseudomonas sp. NDY (Ps+HF) also significantly increased

endogenous IAA content by 67, 61 and 44%, respectively as compared to disease

infected control. The increase in IAA content by the combined treatments PA2+HF

and BE+HF was at par with chemical fungicide. In contrast, combined treatments

BF+HF and BP+HF exhibited no effect on the amount of endogenous IAA in the

leaves of maize infected with stalk rot.

4.3.2.2 Absicsic acid (ABA) content

Endogenous ABA content was significantly decreased in leaves following the

PGPR inoculation in maize plants. The disease infection resulted in an increase

(127%) in ABA content as compared to non-infected control (Fig 4.3).


149

Table 4.3: Effect of antagonistic PGPR on the IAA (ug/g) content in maize leaves

Treatments IAA (ug/g)


Control 401.81 396.54 399.18 b

In-C 226.03 202.65 214.34 h

PA1 309.45 273.42 291.44 ef

Ps 287.55 236.52 262.03 fg

PA2 319.28 288.31 303.80 de

BF 231.43 220.89 226.16 gh

BP 226.79 216.25 221.52 gh

BE 321.87 286.41 304.14 de

Control + F 454.53 511.04 482.79 a

In-C+ F 364.52 335.34 349.93 c

PA1 +HF 364.81 325.64 345.22 cd

Ps +HF 334.47 262.88 298.67 ef

PA2 +HF 379.91 339.05 359.48 bc

BF+HF 210.34 205.07 207.71 h

BP +HF 213.61 187.25 200.43 h

BE +HF 371.32 341.64 356.48 ef

Fig 4.3: Effect of antagonistic PGPR on the IAA/ABA (µg/g) in maize leaves


In-C: Infected maize plants, PA1: P. aeruginosa 4nm, PA2 : P. aeruginosa JYR, Ps: Pseudomonas sp.,

BF : B. firmus PTWz, BP : B. pumilus Yio, BE : B. endophyticus Y5, In-C+F: Infected maize plants+

Full dose of fungicide, PA1+HF: P. aeruginosa 4nm+Half dose of fungicide, PA2+HF P. aeruginosa

JYR+ Half dose of fungicide, Ps+HF: Pseudomonas sp.+ Half dose of fungicide, BF +HF: B. firmus

PTWz+ Half dose of fungicide, BP +HF: Bacillus pumilus Yio+ Half dose of fungicide, BE+HF : B.

endophyticus Y5+ Half dose of fungicide. Results are expressed as means of three replicate, and



b h ef fg de gh gh de a c cd ef bc h h c

f

a

b b cd

a a

cd de cde cd c f

a a

ef

0

50

100

150

200

250

0

100

200

300

400

500

600

AB

A (

µg/

g)

IAA

(µ

g/g)

Treatments

IAA (ug/g) ABA (ug/g)


150

The application of chemical fungicide to infected plants significantly decreased

(40%) endogenous ABA content as compared to diseases infected control, but the

value was higher over that of non-infected control. The antagonistic PGPR

inoculation in disease induced plants also resulted significant decrease in the

endogenous ABA content; ranging from 20 to 34% as compared to infected control.

Maximum decrease was observed for the treatment BE (35%) whereas, other ranked

as PA2 (34%), PA1 (20%) > Ps (21%) as compared to disease infected control.

Endogenous ABA content was significantly decreased for the combined

application of antagonistic PGPR + half dose of fungicide as compared to both of the

disease infected control and single application of antagonistic PGPR. Minimum

amount of endogenous ABA was recorded for the combined treatment PA2+HF i.e.

53% as compared to disease inoculated control. The combined treatments including

BE+HF, PA1+HF and Ps+HF also significantly decreased the endogenous ABA

content in maize leaves by 49, 36 and 31% respectively, as compared to disease

infected control. The increasing trend in ABA content by the combined application of

B. endophyticus and P. aeruginosa 4nm +half dose of fungicide was at par to the

treatment with full dose of chemical fungicide while the treatment PA2 had decreased

(22%) the ABA content even more than the full dose of chemical fungicide. The

inoculation treatments with B. firmus PTWz and B. Pumilus Yio had not expressed any

change in endogenous ABA content in maize leaves for either alone and in

combination with chemical fungicide as compared to infected control under field

natural conditions.


The disease induction resulted in increased (39%) SOD activity as compared to

non-infected control. The application of chemical fungicide to infected plants

significantly increased the SOD activity by 79% as compared to infected control

plants.

The results presented in fig 4.4 revealed an increase in SOD activity following

the application of PGPR inoculation in maize plants. Increase in SOD activity by the

inoculation of PGPR was recorded, ranging from 25-47% as compared to infected

control. Maximum increase (47%) was observed for the treatment PA2 inoculated

with P. aeruginosa JYR as compared to disease infected control.


151

Table 4.4: Effect of antagonistic PGPR on the SOD activity in maize leaves

Treatments SOD activity ( (units/min/g F. wt of leaves) Mean of two

years 2011 2012

Control 6.32 6.67 6.50 g

In-C 8.73 9.27 9.00 e

PA1 11.67 14.73 13.20 cd

Ps 10.86 12.02 11.44 d

PA2 12.19 14.22 13.20 bc

BF 7.98 8.92 8.45 ef

BP 8.06 9.01 8.53 ef

BE 12.62 12.82 12.72 c

Control + FF 7.43 7.51 7.47 fg

In-C+ FF 12.45 16.77 14.61 a

PA1 +HF 13.23 16.83 15.03 b

Ps +HF 12.97 13.36 13.17 bc

PA2 +HF 14.82 17.12 15.97 a

BF +HF 8.09 8.07 8.08 e

BP +HF 9.16 8.82 8.99 ef

BE +HF 16.85 17.48 17.16 a

Fig 4.4: Effect of antagonistic PGPR on the SOD activity in maize leaves under

field conditions

Treatments detail as indicated in Fig 4.3. Results are expressed as means of three replicate, and vertical

bars indicate the standard deviations of means. All means sharing the common letter differ non-

significantly at P< 0.0. LSD: 0.662

g e

cd

d

bc

ef ef

c

fg

a b

bc

a

e ef

a

0

5

10

15

20

SOD

(u

nit

/g F

. Wt

of

leav

es)

Treatments

SOD(unit/min/g F.wt


152

This was followed by treatment PA1, BE and Ps showing significant increase in SOD

activity in infected maize plants by 41, 32 and 25%, respectively.

SOD activity was much higher with the combined application of antagonistic

PGPR + half dose of fungicide as compared to infected control. The SOD activity was

found to be significantly higher (91%) for the combined treatment BE+HF inoculated

with B. endophyticus Y5+ half dose of fungicide (0.1%). The combined treatments

showing enhanced SOD activity ranked as PA2+HF (83%) >PA1+HF (62%) >Ps +HF

(46%). The effect of combined treatment BE+HF and PA2+HF was 17.5 and 9.3%

higher as compared to the treatment In-C+F with full dose of chemical fungicide

(0.2%).

The treatments BF and BP had no significant effect on the SOD activity in the

leaves of infected plants when applied singly. The combined application of treatment

BF+HF has shown10% decline in SOD activity as compared to infected control.

4.3.4 Peroxidase activity (POD)

The POD activity was significantly increased in all the treatments with PGPR

application as compared to infected control as well as non-infected control (Fig 4.5).

The pathogen infection increased the POD activity by 28% as compared to non-

infected control. In the inoculated treatments fungicide augmented the efficiency of

PGPR by enhancing the POD activity. When applied alone, maximum increase in

POD activity was exhibited by PA2 treatment (P. aeruginosa JYR) which was 50%

higher as compared to disease infected control. The effect of other treatments in

increasing the POD activity was in the following manner BE (45%)>PA1 (41%)>Ps

(35%).

The application of fungicide significantly supplemented the effect of PGPR. The

combined treatment with half-dose of fungicide significantly increased the POD

activity (77%) for treatment PA2+HF as compared to infected control which is at par

to the treatment (In-C+F) with full dose of fungicide. The next best treatments were

BE+HF, PA1 +HF and Ps +HF showing 74, 69 and 58% increase as compared to

infected control.

Other two treatments including BP (Bacillus pumilus PTWz) and BF (Bacillus

firmus Yio) has not significantly enhanced the POD activity as compared to infected

control both when applied alone and in combination with chemical fertilizer.


153

Table 4.5: Effect of antagonistic PGPR on the POD activity in leaves

of maize plants

Treatments POD activity (units/min/g F. wt of leaves)


Control 0.52 0.65 0.58 i

In-C 0.7 0.8 0.75 gh

PA1 1.01 1.11 1.06 c

Ps 0.98 1.04 1.01 f

PA2 1.06 1.19 1.12 d

BF 0.71 0.75 0.73 h

BP 0.72 0.78 0.75 gh

BE 1.04 1.14 1.09 dc

Control + FF 0.58 0.68 0.63 i

In-C+ FF 1.28 1.37 1.32 a

PA1 +HF 1.21 1.32 1.26 b

Ps +HF 1.17 1.2 1.18 c

PA2 +HF 1.28 1.38 1.33 a

BF +HF 0.74 0.83 0.78 g

BP +HF 0.76 0.81 0.78 g

BE +HF 1.26 1.344 1.30 ab

Fig 4.5: Effect of antagonistic PGPR on the POD activity in maize leaves under

field conditions



significantly at P< 0.05. LSD: 0.021

j gh

e f d

h gh

de

i

a b c

a

g g

ab

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

PO

D (

un

its/

min

/g F

.wt)

Treatments


154

4.3.5 Polyphenol oxidase

The results presented in Fig 4.6 indicated that, the disease induction resulted in

46% increase in PPO activity in disease infected plants as compared to non-infected

control. By the application of chemical fungicide to infected plants PPO activity was

further enhanced by 73% as compared to infected maize plants.

The antagonistic PGPR inoculation to infected plants resulted in significant

increase in PPO activity, ranging from 18 to 41% as compared to infected control.

Maximum increase was observed for the treatment PA2 inoculated with P. aeruginosa

JYR (41% as compared to infected control) while the effect of other treatments ranked

as BE (39%) > PA1 (27%) >Ps (18%) when compared with infected control.

PPO activity reached to a higher level when PGPR was applied in combination with

half dose of fungicide as compared to both of the disease infected and healthy control.

The application of fungicide augmented the effect of antagonistic PGPR.

Maximum PPO activity (74%) was recorded for the combined treatment PA2 +HF

as compared to infected control. The combined treatments BE+HF, PA1+HF and

Ps+HF also significantly increased the PPO activity by 68, 63, 53% as compared to

infected control. The increase in PPO activity with the combined treatments PA2+HF

was at par with the treatment of full dose of chemical fungicide. The treatment BF

and BP inoculated with B. firmus PTWz and B. pumilus Yio exhibited no significant

effect on the PPO activity either applied singly or combined with chemical fungicide.

4.3.6 Ascorbate peroxidase

The results in Fig 4.7 revealed that ascorbate peroxidase activity has been

increased in most of the treatment inoculated with antagonistic rhizobacteria ranging

from 25 to 68% as compared to infected control. Maximum increase (49%) was

observed for the treatment PA2 inoculated with P. aeruginosa JYR. Other treatments

ranked as BE (54%)>PA1 (37%)>Ps (25%) as compared to infected control. The

treatments BP and BF inoculate with B. pumilus yio and B. firmus PTWz has not

significantly increase the level of the ascorbate peroxidase activity as compared to

infected control.

The full dose of chemical fungicide (0.2%) significantly increased (88%) the

ascorbate peroxidase activity when compared with infected control.


155

Table 4.6: Effect of antagonistic PGPR on the PPO activity in maize leaves

Treatments PPO activity (Units/mg protein/min)


Control 6.93 6.56 6.75 h

In-C 10.01 9.75 9.88 g

PA1 13.27 11.93 12.60 de

Ps 11.91 11.46 11.69 ef

PA2 14.92 13.02 13.97 cd

BF 10.46 9.32 9.89 g

BP 10.49 9.95 10.22 fg

BE 14.06 13.49 13.77 cd

Control+F 7.58 7.97 7.78 h

In-C+ F 16.70 17.52 17.11 a

PA1 +HF 15.41 16.73 16.07 ab

Ps +HF 14.35 15.81 15.08 bc

PA2 +HF 17.06 17.31 17.19 a

BF+HF 10.81 10.08 10.45 fg

BP +HF 10.66 10.15 10.41 fg

BE +HF 16.04 17.18 16.61 a

Fig 4.6: Effect of antagonistic PGPR on the PPO activity in maize leaves


Treatments detail as indicated in Fig 4.3.Results are expressed as means of three replicate, and vertical



h

g

de ef

cd

g fg

cd

h

a ab

bc

a

fg fg

a

0

2

4

6

8

10

12

14

16

18

20

PP

O (

un

its/

mg

pro

tein

/min

)

Treatments


156

This increase was maximum (93%) for the treatment PA2+HF as compared to

infected control. This trend was followed by the treatment BE+HF> PA1+HF > Ps

+HF i.e. 80, 73 and 55% as compared to infected control. When compared with

chemical fungicide the combined treatment PA2+HF was at par to that of the full dose

of chemical fungicide. In case of combined treatment BF+HF and BP+HF the low

dose of chemical fungicide (0.1%) and the respected rhizobacteria both exhibited no

significant effect on ascorbate peroxidase activity in the leaves of maize plants

infected with Fusarium stalk rot under natural conditions of field.

4.3.7 Total soluble phenol

The accumulation of phenol was significantly increased in all the treatments

with antagonistic PGPR inoculation alone and in combined application with half dose

of fungicide (Fig 4.8). Phenol content in infected plants increased by 58% as

compared to non-infected control but was much less when compared to all the

treatments inoculated with rhizobacteria and chemical fungicide.

The treatment In-C+F (chemical fungicide application in full dose) considerably

increased the phenol content as compared the disease infected plants by 128%.

Maximum accumulation (69%) of phenols was recorded for the treatment PA2

inoculated with P. aeruginosa JYR in single application. The accumulation of phenol

was increased by 68, 45 and 28% for rhizobacteria inoculated treatments BE, PA1 and

Ps respectively, in single application.

The level of phenol content was significantly increased with the combined

application of antagonistic rhizobacteria + half dose of fungicide ranging from 73 to

105% as compared to infected control. A higher level of phenol was recoded for

PA2+HF (105%), BE+HF (102%) and PA1 +HF (86%) as compared to infected

control. The combined treatments PA2+ HF and BE+HF were at par with chemical

fungicide. Treatment BP and BF inoculated with B. pumilus Yio and had not affected

the total soluble phenol content both in single and combined application with half

dose of fungicide.


157

Table 4.7: Effect of antagonistic PGPR on the ascorbate peroxidase

in maize leaves

Treatments Ascorbate peroxidase (Unit/g F. wt)


Control 0.80 0.77 0.78 h

In-C 1.30 1.14 1.22 f

PA1 1.78 1.56 1.67 e

Ps 1.60 1.44 1.52 e

PA2 2.09 2.00 2.04 bc

BF 1.12 1.22 1.17 f

BP 1.30 1.25 1.27 f

BE 1.95 1.81 1.88 d

Control+F 1.02 0.94 0.98 g

In-C+ F 2.36 2.22 2.29 a

PA1 +HF 2.20 2.03 2.11 b

Ps +HF 1.98 1.79 1.89 cd

PA2 +HF 2.49 2.21 2.35 a

BF+HF 1.17 1.28 1.23 f

BP +HF 1.24 1.06 1.15 f

BE +HF 2.29 2.11 2.20 ab

Fig 4.7: Effect of antagonistic PGPR on the Ascorbate Peroxidase in maize leaves





h

f

e e

bc

f f

d

g

a b

cd

a

f f

ab

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Asc

orb

ate

pe

roxi

das

e (

un

it/

g F.

wt)

Treatments


158

Table 4.8: Effect of antagonistic PGPR on the total soluble phenol

in maize leaves

Treatments Total soluble phenol Mean of two

years 2011 2012

Control 0.57 0.71 0.64 h

In-C 1.05 0.97 1.01 fg

PA1 1.37 1.57 1.47 de

Ps 1.32 1.25 1.29 ef

PA2 1.61 1.81 1.71 cd

BF 1.01 0.99 1.00 fg

BP 1.08 1.02 1.05 fg

BE 1.62 1.77 1.69 cd

Control+F 0.98 0.82 0.90 gh

In-C+ F 1.95 2.67 2.31 a

PA1 +HF 1.96 1.81 1.88 bc

Ps +HF 1.69 1.81 1.75 bcd

PA2 +HF 1.97 2.18 2.08 ab

BF+HF 1.16 1.03 1.09 fg

BP +HF 0.93 1.11 1.02 fg

BE +HF 1.99 2.09 2.04 ab

Fig 4.8: Effect of antagonistic PGPR on the total soluble phenol in maize leaves





h

fg de ef

cd

fg fg

cd

gh

a

bc bcd

ab

fg

fg

ab

0

0.5

1

1.5

2

2.5

3

Tota

l so

lub

le p

he

no

l (m

g/g

F. w

t)

Treatments


159

4.3.8 Catalase activity

The catalase activity has been significantly increased in all treatments inoculated

with antagonistic rhizobacteria as indicated in fig 4.9 ranging from 20 to 78% as

compared to infected control. Maximum increase (78%) was observed for the

treatment PA2 inoculated with P. aeruginosa JYR whereas, other treatments ranked

as BE (77%)> PA1 (69%)> BF (27%)> Ps (20%) as compared to infected control.

The effect of treatment BP is at par with the infected control (In-C).

The chemical fungicide application significantly increased (83%) the catalase

activity as compared to disease infected control. In the combined treatments of

antagonistic rhizobacteria the half dose of fungicide exhibited an increasing effect on

the catalase activity as compared to infected and non-infected control.

This increase was maximum (119%) for the treatment PA2+HF as compared to

infected control. This trend was followed by other treatments BE+HF> PA1+HF>

Ps+HF> BF+HF i.e. 105% > 195% > 57% >46% > 8% as compared to infected

control. When compared with chemical fungicide the combined treatment PA2+HF

had an enhancing effect on catalase activity by 19% and effect of combined treatment

BE+HF was at par to the treatment with full dose of fungicide (0.2%).

4.3.9 Effect of antagonistic rhizobacteria on PR proteins

4.3.9.1 Chitinase activity

The chitinase activity has been increased in most of the treatment inoculated with

antagonistic rhizobacteria (Fig 4.10) ranging from 24 to 61% as compared to disease

infected control. Maximum increase (61%) was observed for the treatment PA2

inoculated with P. aeruginosa JYR. Other treatments ranked as follows BE (56%)>

PA1 (40%) >Ps (24%) as compared to infected control. The treatment BP and BF

inoculated with Bacillus pumilus Yio and B. firmus PTWz had no significant effect on

chitinase activity as compared to infected control.

The chemical fungicide application significantly increased the chitinase

activity as compared to disease infected control when compared to other treatments.

Similarly, in the combined treatments with antagonistic rhizobacteria the half dose of

fungicide supplemented their effect on the chitinase activity as compared to infected

and non-infected control.


160

Table 4.9: Effect of antagonistic PGPR on the catalase activity in maize leaves

Treatments Catalase activity (units/min/g F.wt. of leaves)


Control 0.56 0.50 0.53 h

In-C 0.88 1.07 0.97 g

PA1 1.83 1.55 1.69 e

Ps 1.10 1.40 1.25 f

PA2 1.90 1.65 1.77 cd

BF 1.05 1.31 1.18 g

BP 1.01 0.91 0.96 g

BE 1.63 1.61 1.62 de

Control+F 0.76 0.67 0.72 h

In-C+ F 2.10 1.57 1.83 cd

PA1 +HF 2.34 1.78 2.06 bc

Ps +HF 1.69 1.44 1.56 e

PA2 +HF 2.45 1.93 2.19 a

BF+HF 1.31 1.16 1.24 g

BP +HF 1.30 0.97 1.13 g

BE +HF 1.94 1.96 1.95 ab

Fig 4.9: Effect of antagonistic PGPR on the catalase activity in maize leaves of

Treatments detail as indicated in Fig 4.3. Results are expressed as means of three replicate, and



h

g

e

f

cd

g

g

de

h

cd

bc

e

a

g

g

ab

0

0.5

1

1.5

2

2.5

Cat

alas

e (

un

it/m

in/g

F w

t)

Treatments


161

Table 4.10: Effect of antagonistic PGPR on the chitinase activity in maize leaves

Treatments Chitinase activity (units/mg F. wt.)


Control 4.36 4.72 4.54 i

In-C 7.11 6.42 6.76 gh

PA1 9.96 9.06 9.51 d

Ps 8.76 8.06 8.41 e

PA2 11.45 10.31 10.88 bc

BF 7.08 7.06 7.07 fgh

BP 6.31 6.64 6.47 f

BE 10.93 10.11 10.52 c

Control+F 7.58 7.92 7.75 ef

In-C+ F 13.07 12.08 12.58 a

PA1 +HF 12.00 11.17 11.58 b

Ps +HF 11.11 10.67 10.89 bc

PA2 +HF 13.67 12.28 12.97 a

BF+HF 7.81 6.89 7.35 fg

BP +HF 7.44 6.81 7.13 fgh

BE +HF 12.32 11.06 11.69 b

Fig 4.10: Effect of antagonistic PGPR on the chitinase activity in maize leaves





i

gh

d e

bc

fgh h

c

ef

a b

bc

a

fg fgh

b

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

Ch

itin

ase

(u

nit

/mg

F.w

t)

Treatments


162

This increase was maximum (96%) for the treatment PA2+HF as compare to disease

infected control. Other treatments ranked as BE+HF > PA1+HF > Ps +HF > BF+HF

i.e. 73, 71 and 61% as compared to infected control. The treatment BP+HF and

BF+HF showed no significant effect on the chitinase activity as compared to infected

control.

4.3.10 Yield Parameters

The yield parameters including grain yield (kg/ha), 1000 seed weight and number

of seeds/cob has been significantly increased in all treatments inoculated with

antagonistic rhizobacteria as indicated in fig 4.11,4.12 and 4.13.

The maximum increase (59%) in grain yield was recorded for treatment PA2

whereas, the treatments BE, PA1 and Ps also exhibited the significant increase in

grain yield by 55, 44 and 32% as compared to infected control. The treatments BP and

BF has not significantly affect the grain yield in maize under field conditions. Their

effect was similar to the pathogen infected plants which also had low (61%) grain

yield as compare to infected control.

The chemical fungicide application increased (98%) the grain yield as compared

to disease infected control. In the combined treatments the low concentration of

fungicide (half dose 0.1%) supplemented the enhancing effect of antagonistic

rhizobacteria. The increase was maximum (110%) for the treatment PA2+HF as

compared to infected control. This trend was followed by other treatments BE+HF>

PA1+HF > Ps+HF > BP+HF > BF+HF i.e. 106% > 75% > 60% as compared to

infected control. The combined treatment PA2+HF and BE+HF was at par to the

treatment with full dose of chemical fungicide.

The results in Fig 4.12 and 4.13 revealed that 1000 seed weight and no of

seeds/cob has been increased in most of the treatment inoculated with antagonistic

rhizobacteria under field conditions as compared to infected control. Maximum

increase (62%) in 1000 seed weight and no. of seeds/cob (75%) was observed for the

treatment PA2 inoculated with P. aeruginosa JYR as compared to infected control.

Other treatments ranked as PA1 (56%)> BE (47%) > Ps (26%) as compared to

infected control for 1000 seed weight of maize under field conditions. Same ranking

order (BE > PA1> Ps) was followed in case of number of seeds per cob.


163

Table 4.11: Effect of antagonistic PGPR on the Yield of maize plants

Treatments Yield (kg/ha) Mean of two

years 2010 2011

Control 1977 2060 2019 a

In-C 774 813 794 f

PA1 1108 1175 1141 de

Ps 1001 1097 1049 e

PA2 1236 1296 1266 cd

BF 805 714 759 f

BP 823 700 762 f

BE 1200 1260 1230 cd

Control+F 2004 2168 2086 a

In-C+ F 1527 1622 1575 b

PA1 +HF 1306 1472 1389 c

Ps +HF 1210 1338 1274 cd

PA2 +HF 1636 1702 1669 b

BF+HF 899 702 801 f

BP +HF 884 722 803 f

BE +HF 1593 1680 1637 b

Fig 4.11: Effect of antagonistic PGPR on the yield of maize plants





a

f

de e cd

f f

cd

a

b c

cd

b

f f

b

0

500

1000

1500

2000

2500

Yie

ld (

kg/h

a)

Treatments

Yield


164

The treatments BP and BF inoculate with B. pumilus yio and B. firmus PTWz has

not significantly affect the 1000 seed weight and number of seeds/cob in maize plants

as compared to infected control.

The full dose of chemical fungicide significantly increased the 1000 seed

weight and number of seeds/cob as compared to infected control by 94 and 90%.

Similarly, the combined treatments of antagonistic rhizobacteria with half dose of

fungicide have also shown an enhancing trend in the yield parameters as compared to

infected control.

Maximum increase (113%) was observed for the combined treatment PA2+HF as

compared to infected control in case of 1000 seed weight. This trend was followed by

the combined treatments BE+HF> PA2+HF > Ps +HF > BF+HF > BP+HF i.e. 94, 81

and 70% as compared to infected control. When compared with chemical fungicide

for 1000 seed weight, the combined treatment PA2+HF, BE+HF and PA1+HF was at

par with full dose of chemical fungicide.


165

Table 4.12: Effect of antagonistic PGPR on 1000 seed weight of maize plants

Treatments 1000 seed weight Mean of two

years 2010 2011

Control 237.0 269.0 253.0 a

In-C 104.3 109.0 106.7 i

PA1 146.0 167.7 156.8 g

Ps 121.7 147.3 134.5 h

PA2 167.3 183.3 175.3 efg

BF 78.7 111.3 95.0 i

BP 100.7 84.7 92.7 i

BE 160.7 176.7 168.7 fg

Control+F 266.7 287.0 276.8 a

In-C+ F 202.0 211.7 206.8 cd

PA1 +HF 182.3 204.0 193.2 de

Ps +HF 166.7 195.7 181.2 ef

PA2 +HF 212.7 241.3 227.0 c

BF+HF 104.0 113.7 108.8 i

BP +HF 114.3 102.7 108.5 i

BE +HF 201.7 212.0 206.8 cd

Fig 4.12: Effect of antagonistic PGPR on 1000 seed weight of maize





b

i

g h

efg

i i

fg

a

cd de ef

c

i i

cd

0.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0

10

00

se

ed

we

igh

t (g

)

Treatments

1000 seed weight


166

Table 4.13: Effect of antagonistic PGPR on number of seeds/cob in maize

Treatments No. of seeds/cob Mean of two

years 2010 2011

Control 514.3 542.7 528.5 a

In-C 210.0 248.7 229.3 h

PA1 341.0 406.7 373.8 e

Ps 304.3 365.3 334.8 f

PA2 355.0 451.3 403.2 de

BF 211.0 263.7 237.3 gh

BP 210.3 293.3 251.8 gh

BE 336.0 428.3 382.2 de

Control+F 530.0 580.3 555.2 a

In-C+ F 395.7 474.7 435.2 bc

PA1 +HF 361.3 454.3 407.8 cd

Ps +HF 358.3 395.0 376.7 e

PA2 +HF 415.7 490.3 453.0 b

BF+HF 230.3 299.7 265.0 g

BP +HF 234.3 288.7 261.5 g

BE +HF 405.7 466.0 435.8 bc

Fig 4.13: Effect of antagonistic PGPR on number of seeds/cob in maize





a

h

e

f

de

gh gh

de

a

bc cd

e

b

g g

bc

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0

Nu

mb

er

of

see

ds/

cob

Treatments


167

4.4 DISCUSSION

The results from the field experiments performed over a period of two years

indicated that antagonistic PGPR suppressed and control the fungal disease caused by

F. moniliforme, which was mediated by inducing ISR as a mechanism of action under

natural field conditions. These findings are in accordance with the findings of Wei et

al., (1991), Liu et al., (1995 a and b) and Wei et al., (1996). In the present study

protection was observed against Fusarium moniliforme i.e. the causal agents of stalk

rot in maize following the use of selected antagonistic PGPR strains. PGPR induced

defence against the inoculated pathogen was very consistent at a statistically

significant level (P< 0.05) with three Pseudomonas strains and one Bacillus strain.

While two stains of Bacillus didn’t exhibit the significant disease protection in maize

plants under field conditions. These results are in consistency with the previous

findings that Bacillus species are considered to have low competence in the

rhizosphere than Pseudomonas species (Weller, 1998). It is also reported that in spite

of environmental limitations and their interactions with other rhizospheric

microorganisms, some bacteria have the ability to colonize efficiently in the

rhizosphere than others (Kumar et al., 2011). Bargabus et al., (2002) reported ISR

based disease control in greenhouse experiment by the application of B. mycoides was

not maintained in the field experiments. There are also a number of reports indicating

the biocontrol Bacillus species with better root colonization (Zehnder et al., 2000;

Kloepper et al., 2004). Furthermore, the activity and establishment of biological

control agents may not be favoured by the environmental conditions prevailing in the

field. Many attempts of biological control have resulted in inconsistent or

unsatisfactory disease control under field conditions as compared to controlled

conditions of greenhouse experiments (Deacon, 1991; Osburn et al., 1995; Dik et al.,

1998; Schisler et al., 2000). Pseudomonads possess many traits that make them well

suited as biocontrol and growth-promoting agents even in field condition (Weller,

1988). This has been reviewed in a number of studies. Wei et al., (1996) reported that

the P. fluorescens 89B-61 significantly reduced the severity of angular leaf spot in

field experiments. The inhibition of soil bonre pathogens (F. graminearum, F.

moniliforme and M. phaseolina) by P. cepacea was also reported by Hebbar et al.,

(1992). Similarly Chen et al., (1999) reported that the application of Bacillus and

Pseudomonas sp. to maize seeds resulted in substantial control of seedling, stalk and


168

root rots of maize infected by F. graminearum. Muthamilan, (1994) also reported

excellent results by Pseudomonas and Bacillus inoculation against wilt disease under

field condition.

The biocontrol agents modulate levels of ABA and IAA that directly or

indirectly regulate the genes involved in the defence response. A higher level of IAA

was observed following the application of P. fluorescence in Fusarium infected plants

in response to invading pathogen and results in the disease reduction and related yield

loss (Petti et al., 2012). Smyth et al., (2011) also documented that Pseudomonas strain

ability to produce a higher level of IAA. As cited in literature (Kochar et al., 2011;

Kulkarni et al., 2011), the increased IAA was more possibly produced by inoculated

PGPR rather than plant tissues. The higher level of IAA might stimulate the plant to

trigger it defence mechanisms in order to response efficiently to the invading

pathogen hence increase the plant resistance. Similar results was also found in the

present study indicating the higher level of IAA results in decrease in disease severity

and increase in subsequent yield. Hasnain and Sabri, (1996) showed that

Pseudomonas inoculation improved the plant growth by enhancing the auxin content

of wheat grown in Pakistan.

In contrast to well-studied ABA role in plant response to various abiotic stresses

(Fujita et al., 2006) and its role in plant resistance to diseases is comparatively not

fully understood. However, a number of reports demonstrated the suppression of plant

pathogens by increased level of ABA. This explains that the ABA suppresses the

defence responses against biotic stress (Ton et al., 2009). There are also some reports

which have shown a positive contribution of ABA in induction of immunity in plants

(Adie et al., 2007; Vleesschauwer et al., 2010). Furthermore, it has been indicated that

ABA role in disease suppression depends on the pathogen way of development and

progressive conditions that indicate the complex mechanism of ABA in modulating

the plant defence response (Asselbergh et al., 2008; Cao et al., 2011). In the present

study, inoculation with antagonistic rhizobacteria significantly decreased the ABA

level by decreasing the disease severity of maize plants. This is in accordance with

previous studies showing that ABA negatively regulates the immunity of plants

against fungal pathogen (Jiang et al., 2010; Xu et al., 2013).

Inoculation of selected strains under field conditions (three, Pseudomonas and one

Bacillus strain) resulted in significant increase in yield of infected maize plants by


169

suppressing the Fusarium stalk rot. Similar results have been reported by Higa et al.,

(1999) indicating the beneficial effects of antagonistic rhizobacteria to enhance the

yield by protecting the plant against pathogen attack. A number of researchers showed

positive correlation of PGPR inoculation with improved growth and yield of wheat,

maize, soyaben and sugar beet in field experiments (Cattelan et al., 1999; Cakmakc et

al., 2006; Salantur et al., 2006; Egamberdiyeva, 2007). The increased effect on maize

yield by the seed inoculation with rhizobacteria was also reported by Shaharoona et

al., (2006).

During the present investigation two Bacillus species did not show any significant

increase in yield as compared to the maize plants infected with stalk rot although

these strains have shown better control of disease in the greenhouse experiment. This

inconsistency is related to various factors including interaction of antagonistic

rhizobacteria with host/pathogens, edaphic factors and soil environment in field

conditions (Rashid et al., 1997; Suprapta, 2012). According to Niranjan et al., (2005)

the phenomenon of PGPR and host interactions is well understood in field

environmental conditions.

The induction of ISR demonstrated as the predominate mechanism for the disease

suppression in several plants inoculated with antagonistic rhizobacteria (Raj et al.,

2003; Halfeld-Vieira et al., 2006). It has been reported that this mode of action to

provide protection operates both under both controlled axenic condition in potted

plants of greenhouse and field natural conditions (Bonaldo et al., 2005). During the

present investigation, ISR may act as an important mechanism by the prior application

of PGPR including P. aeruginosa JYR, P. aeruginosa 4nm, Pseudomonas sp. and B.

endophyticus, for the protection of maize against the challenged pathogen (F.

moniliforme). An increasing trend in the activities of antioxidants (POD, SOD, and

ascorbate peroxidase), catalases, total soluble phenol and induction of PR proteins has

been observed in maize leaves by the application of these Pseudomonas and Bacillus

strains as compared to the infected control in field experiment. These results are

consistent with the previous work which demonstrated that antagonistic rhizobacteria

can induce systemic resistance against fungal pathogen under field condition. Several

other studies have also demonstrated the PGPR induced ISR under diverse conditions

of field (Wei et al., 1996; Raupach and Kloepper, 2000; Murphy et al., 2000; Zehnder

et al., 2001; Jetiyanon et al., 2003). Quyet-Tien et al., (2010) had shown the


170

involvement of PGPR stain in induction of systemic resistance along with plant

growth promotion.

The integration of biological control of disease with the chemical control and

some cultural practices are the subject of interest to reduce the use of chemical

pesticides (Baker, 1990; Pierson and Weller, 1994; Mazzola et al., 1995; Wei et al.,

1996; Raupach and Kloepper, 1998; Jetiyanon et al., 2003). The present investigation

indicates the beneficial findings by the integrated application of PGPR along with

foliar applied chemical fungicides. Luz (2003a) reported significant reduction of

pathogens activity by the use of combined treatment of seeds with Paenibacillus

macerans and chemical product (difenoconazole) in field along with improved

germination and yield. Similarly, when maize seeds were treated with biocontrol

agent + chemical fungicide (fludioxonil + metalaxyl M) resulted in enhanced

production under field conditions (Luz 2003b). The inoculation of Bacillus along with

chemical seed treatments have also been successfully used to control the disease and

to improve growth and yield (Bugg et al., 2009). Conway, (1997) had demonstrated

the use of antagonistic PGPR and foliar spray of fungicide at one half dose of the

recommended dose rate repress the disease of rosemary even more than the single

application of biocontrol agent and fungicide.

Foliar application of chemical fungicide consistently provided significant disease

suppression caused by F. moniliforme. Present finding was consistent with previous

findings for foliar application of chemical fungicide was effective in the control of

Fusarium stalk rot in maize under field conditions (Wilson et al., 2002; Byrne et al.,

2005). Carbendazim reduced the disease severity index and per cent disease incidence

in maize with an increase in plant height, fresh and dry weights (Rani et al., 2013).

We have used six PGPR strains P. aeruginosa 4nm, P. aeruginosa JYR,

Pseudomonas sp., B. endophyticus, B. pumilus, B. firmus which have performed well

in controlled condition of greenhouse by decreasing the disease severity and

improving the plant growth of maize plants. In the field experiment only three

Pseudomonas strains and one Bacillus strain was found to be effective in controlling

the Fusarium stalk in maize plant by eliciting ISR which may verify these

antagonistic PGPR as potent biocontrol agents. Furthermore, the combined

application of these antagonistic PGPR with half dose of fungicide yielded much

better results in controlling the fungal pathogens in maize plants as compared to

single application.


171

It might be inferred that, the combined application of PGPR and chemical

fungicide not only is an economical approach but also an environment friendly

alternative for minimizing the adverse effects of chemical fungicide on plant, soil and

human being.

Molecular detection of antibiotics biosynthetic genes in antagonistic PGPR Chapter 5

172

5.1 Introduction

Biological control of fungal diseases by PGPR (plant growth promoting

rhizobacteria) is a well-known and recognized phenomenon and antibiotics has a

promising role in suppression of numerous phytopathogens (Handelsman and Stabb,

1996). In fact, Antibiosis is among the most potent, effective, powerful and well-

studied biocontrol mechanisms for the suppression of plant pathogens (as reviewed by

following references (Dowling and Gara, 1994; Cook et al., 1995: Loper et al., 1997;

Haas et al., 2000; Haas and Keel, 2003; Brodhagen et al., 2004). Antibiotics contain

an extensive array of low molecular weight organic compounds produced by a large

number of rhizobacteria (Raaijmakers et al., 2002; George, 2002).

The antibiotics produced by biological control strains isolated from the

rhizosphere of various plants, growing in agro-climatic different locations, propose an

evolutionary advantage and efficient antagonistic activity (Hassan, 2010). The

rhizobacteria producing antibiotics are potent candidates for the biological control of

numerous soil borne fungal pathogens by reinforcement of the plants natural defence

system (Thomashow and Weller, 1996; Weller, 1988; Raaijmakers et al., 2002; Haas

and Defago, 2005). In rhizosphere, these antibiotics inhibit the development of

pathogen by interfering with germination of fungal spores and initiation of root

infection procedure (Hass and defago, 2005). Antibiotics may have an inhibitory

effect on a variety of pathogenic microbes (Leclere et al., 2005). Each antibiotic use a

different mode of action e.g. inhibition and disruption of the appropriate formation of

cell wall, protein synthesis and membrane integrity, membrane function, DNA

synthesis and synthesis of small essential molecules (Walker et al., 2001).

Antibiotics are also involved in the improvement of the ecological fitness of

rhizobacteria in the competitive niche, which is an additional and long term advantage

to the biological control efficiency (Mazzola et al., 1992). This makes antibiotic

producing rhizobacteria more appealing, effective and potent biocontrol agents

including the Streptomyces, Bacillus and Pseudomonas species (Hass and defago,

2005).

The PGPR produce various antibiotics e.g. phenazine-1-carboxylic acid,

zwittermycin A, butyrolactones, pyoluteorin, kanasamine, pyrrolnitrin, oligomycin A,

2,4-diacetylphloroglucinol (2,4- DAPG), oomycin A, viscosinamide and xanthobaccin

(Whipps, 2001). In many cases, the biocontrol capability of PGPR is directly


173

associated with the production of antibiotics (Blumer et al., 2000; Raaijmakers et al.,

2002; Haas and Keel, 2003).

Antibiotic producing Bacillus and Pseudomonas have been widely studied to

be used as biocontrol agents for the better growth and yield of plants in agriculture. In

various plant pathogen interactions, fluorescent pseudomonads use antibiotics (such

as phenazine-1-carboxylate, 2, 4-diacetylphloroglucinol (PHL), pyrrolnitrin and

pyoluteorin), as primary mechanism to inhibit disease (Thomashow, and Weller,

1996). B. cereus strain was also reported to produce kanosamine and zwittermicin for

biocontrol of fungal pathogen (Glandorf et al., 2001). Furthermore, P. fluorescens

suppress the Pythium sp. by the production of 2,4-diacetylphloroglucinol and B.

subtilis use iturin A against the biocontrol of R. solani and Botrytis cinerea (Kloepper

et al., 2004). The production of mycosubtilin has also been reported against Pythium

aphanidermatum by B. subtilis (Leclere et al., 2005). B. amyloliquefaciens inhibit F.

oxysporum by producing the bacillomycin and fengycin (Koumoutsi et al., 2004).

Phenazine is produced by P. fluorescens strains against Gaeumannomyces (Wilhite et

al., 2001).

The capabilities to produce multiple antibiotics (use different mode of action

to inhibit the pathogens) enhance the efficacy of biocontrol agents. The genetically

engineered P. putida strains with the ability to produce two antibiotics, phenazine and

DAPG, has the improved the biocontrol activity (Glandorf et al., 2001).

The genetic loci for the antibiotics synthesis in rhizobacteria are well studied

and their sequences are accessible (Mavrodi et al., 1998; Nowak-Tompson et al.,

1999; Bangera and Thomashow 1999). Hence it is possible to detect and characterize

the biological control agents on the basis of antibiotic synthesis genes present in their

genome. These sequences can be used to design primers on the basis of conserved

regions for PCR (polymerase chain reaction), to detect antibiotic gene in antagonistic

PGPR (Gardener et al., 2001; De Souza and Raaijmakers, 2003).

Previously, six antagonistic rhizobacteria were screened against F.

moniliforme in vivo and in vitro. They significantly controlled the fungal mycelial

growth and reduced the incidence and severity of stalk on maize (Zea mays L.). The

inhibition of fungal growth in test tubes by the application of cell free supernatant by

all six strains suggested that antibiosis is possibly a promising mechanism for disease

suppression by antifungal activity. Detection of the antibiotic genes enables us to

understand the molecular mechanism involved in biological control approaches.


174

Aims and Objective

Therefore the present study was aimed to evaluate the genes involved in inhibiting the

fungal pathogens and thereby disease suppression so as to develop the selected strains

into an efficient biocontrol agent.


175


5.2.1 Antibiotic production by agar well diffusion method

The selected rhizobacteria which have shown the inhibition against F.

moniliforme were streaked on LB agar plates and incubated at 28oC for 24 h. A

loopful of rhizobacterial inoculum was transferred in to 100 mL minimal defined

medium to initiate the production of antibiotic substances (Sadfi et al., 2001). The

inoculation media was incubated for 72 h at 28oC at 180 rpm. Afterwards, the

bacterial suspension was centrifuged at 12, 000 x g at 4oC for 10 min. The cell free

substrate was used to detect the antibiotic production by agar well diffusion method.

The agar well diffusion method was used following the method of Sen at al.,

(1995). The seven day old culture of F. moniliforme was swabbed on the surface of

SDA agar plates by using sterilized cotton swab and let it dry for 15 minutes.

Sterilized cork borer (8 mm) was used to make the wells and 100 μl of cell free

culture supernatants was shifted into each well. The agar plates were then incubated at

28oC for 48 h and development of inhibition zones around each well was considered

as positive for antibiotic production (Appendix 22).

5.2.2 Detection of genes related to antibiotic biosynthesis by polymerase chain

reaction

For the detection of the genes that encodes biosynthesis of antibiotics was

accomplished by polymerase chain reaction using gene-specific primers.

5.2.2.1 Design of oligonucleotide primers for molecular detection of antibiotic

gene fragments

For the construction of primers, nucleotide sequences of different genes of

antibiotics involved in their synthesis were retrieved from the National Centre for

Biotechnology Information (NCBI) and aligned using the program BioEdit 7.1.11.

Primers (Table 5.1) were generated by using Primer 3 (Ebersbach, Germany). To

ensure for the primer specificity, the sequences of all primers were put to blast once

again with NCBI Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) which showed 100%

similarity with the antibiotic biosynthetic genes.

5.2.2.2 Amplification and detection of antibiotic biosynthesis genes

The amplification reaction (25 μl) contained 1µL of each primer, 50 ng of

genomic DNA, 12.5 µL Econo taq master mix, 9.5 µL DNA/RNAase free water.

http://blast.ncbi.nlm.nih.gov/Blast.cgi


176

Amplification was performed in thermo cycler by using the primers set for the

screening of genes encoding different antibiotics. The PCR conditions are listed in

Table 5.2. To check the results of amplified product, 5 µL of PCR product

electrophoresed on 1% agarose gel in 1× TAE buffer for 45 min at 50 V, stained with

gel red dye and the PCR products were visualized with a Gel Doc System UV trans-

illuminator. Purification of PCR product was done by using the CR purification

columns (Promega, Madison, USA). For Sequencing by ABI 3730 Capillary

Sequencer at UNSW, Sydney the PCR product was purified by making a reaction

mixture of 20 µL containing 1 µL BigDye terminator V 3.1, 20-50 ng PCR product,

3.2 pmol primer, 3.5 µL 5x buffer, nuclease free H2O up to 20 µL and amplified for

25 cycles of 96oC for 10 sec, 50

oC for 5 sec and 60

oC for 4 min. After this PCR

product was again purified by using the purification protocol to remove the

unincorporated dye-labelled terminators and the purified PCR product was sent for

Sequencing. The comparison of the antibiotic gene fragments was done in the NCBI

nucleotide database using the Basic Local Alignment Search Tool (blastx).


177

Table 5.1: Primers of antibiotics biosynthetic genes used in this study

Primer Genus Sequence Function Ref.

phzFf

phzFr

Pseudomonas

5‘-CGCCCCTTAAGTTTCAAGC-3‘

5‘-GCTCACTGGTTTGGAGAAGC-3‘

PCR,RT-

PCR,

sequencing

of phzF gene

this study

phzFf

phzFr

5‘-AGATGAACCTGTCGGAGAGC- 3‘

5‘-ACACTTCCACCGTCGAGACC-3‘

PCR,RT-

PCR

sequencing

of phzF gene

this study

phzDf

phzDr

5‘-GGCATTCCCGAAATCACC-3‘

5‘-TGGATGTCGTTGGAGTAGGC-3‘

PCR,RT-

PCR

,sequencing

of phzD gene

this study

PhzFf

phzFr

Bacillus

5‘-TGACGCATTTACGAATAAACC-3‘

5‘-TTCACATACACCGCTGTTCC-3‘

PCR,RT-

PCR,

sequencing

of phzF gene

this study

phzFf

phzFr

5‘-GTGGTCATGGGACAGTAGGG -3‘

5‘CTTTCACATATACCGCTGTTCC-3‘

PCR,RT-

PCR,

sequencing

of phz C and

F gene

this study

PhzDf

PhzDr

5‘-ATTGGTGTCAGCAGTGATCG-3‘

5‘-ATCTTCGGGTATCCCAATCC-3‘

PCR,RT-

PCR,

sequencing

of phzD gene

this study

prnDf

prnDr

Pseudomonas

5‘-GTGGAACGCACCTTGAACC-3‘

5‘-AGGTGAGCGTGAGTAGATCG-3‘

PCR,RT-

PCR,

sequencing

of

pyrrolnitrin

D gene

this study

prnCf

prnCr

5‘-GGATCCTGGCCAAACAACAGTTTC-

3‘

5‘-ACTAGTTGCGTCCAGTACATCAGC -

3‘

PCR,

sequencing

of

pyrrolnitrin

D gene

Upadhyay

and

Srivastava

,2010

PhlCf

PhlCr

5‘-AGGAAATGATCGTCGAGTCC- 3‘

5‘-GTAACCGCCAAGGTTCTGC-3‘

PCR,

sequencing

of PhlC gene

this study

PhlDf

PhlDr

5‘-ACCCACCGCAGCATCGTTTATGAG

-3‘

5‘-

CCGCCGGTATGGAAGATGAAAAAGTC

3‘

PCR,

sequencing

of PhlD gene

Raaijmakers

and

Thomashow,

1997

ZmaR f

ZmaR r

Bacillus

5‘-ATGTGCACTTGTATGGGCAG

-3‘

5‘-TAAAGCTCGTCCCTCTTCAG -3‘

PCR,

sequencing

of

Zwitterimicin

A gene

Milner et al.,

1996


178

Table 5.2: Conditions of PCR for amplification of antibiotic biosynthetic genes

Conditions Amplification of antibiotic biosynthetic genes


Denaturation 94°C 30 sec

Annealing 51-59 (for different antibiotic

genes)

30 sec

Extension 72°C 45 sec

Final extension 72°C 10 min


179

Fig 5.1: Schematic presentation of steps followed for the detection for

biosynthesis genes of antibiotic by using gene specific primers


180

5.2.3 Quantification of antibiotics (Phenazine) by HPLC

Antibiotic substances were extracted following the modified method of

Ayyadurai et al., (2007). Phenazine was quantitatively determined by analytical

HPLC.

5.2.3.1 Culture Extracts of phz Positive Strains

The 24 h old culture was inoculated in 100 mL minimal medium broth and

incubated for 3 d in a rotary shaker. The cultures were transferred into 50 mL falcon

tubes for centrifugation at 5,000×g for 15 min to eliminate any residual bacteria. The

resulting supernatant was filtered through 22 μm syringe filter and transferred to new

falcon tubes. Acidification (pH 2±0.2) of supernatant was done by using 1 N HCl and

equal volume of ethyl acetate was used for extraction. The ethyl acetate extracts were

dried in vacuum at 35°C and were dissolved in 1.5 mL of 90% methanol. The organic

phase containing phenazine derivatives was evaporated to dryness and suspended in 3

mL of acetonitrile (ACN) and further purified by solid phase extraction method. For

this, sample was passed through the column (Chromafix C18 cartridge (Machery-

Nagel, Duren, Germany) and the analytes are adsorbed on the stationary phase. The

bounded metabolites were eluted with acetonitrile (ACN), and concentrated it in

Labcone concentrator 745500 and re-suspended in 100 µL ACN.

5.2.3.2 HPLC Analyses

Reversed phase HPLC anlayses were conducted by using C18 column

(Phenomenex Onyx Monolithic C18, 100 × 3.00 mm) and applying an gradient of

H2O (A)/ MeCN (B) with 0.1% formic acid mixed in both solvents (gradient 0 min

15% B, 3 min 15 % B, 10 min 60% B, 11 min 60% B, 11.50 min 100% B, 13 min

100% B, 13.01 15% B, 16 min 15% B, flow 2 mL/min) on a Waters 2695 XE

Separations Module system coupled to an Waters photodiode array detector (Waters

2695). HPLC gradient profiles were monitored at the spectral peak maxima (247.6

and 368.2 nm) that are characteristic of phenazine-1-caboxylic acid.

5.3.4 Well plate assay

Antagonistic activity of fractionated extracts of Pseudomonas strains was

determined in a well plate assay using PDA agar plates inoculated with the suspension

of F. moniliforme (107

cfu/mL in PDA broth) and allowed to dry at room temperature.


181

Fig 5.2: Schematic illustration for the quantitative analysis of

antibiotics through HPLC


182

Test samples of crude phenazine extract from Pseudomonas strains and added

to a 4 mm well, and the plates were incubated at 37°C for 24 h. The antagonistic

activity was observed by the development of halo zone around the wells. The

antifungal activity is measured by subtracting the diameter of the well from the zone

of growth inhibition (Kerr et al., 1999).

5.2.5 RNA Isolation for RT-PCR

Pseudomonas and Bacillus strains were grown at 28oC in minimal medium for

18 h. The cells were harvested by centrifugation at 4000 x g for 10 min and Qiagen

RNeasy Plus Mini Kit (cat. no. 74134 and 74136) was used for RNA extraction

according to the recommendation of manufacturer. RNA quality was determined by

the Bioanalyser (Agilent 2100). Furthermore, for the removal of genomic DNA the

DNase I (Ambion Life Technologies) was used on column digestion. The resultant

RNA was run on agarose gel. RNA was reverse-transcribed by using QuantiTect

Reverse Transcription Kit (cat. no. 205310).

5.2.6 Quantitative RT-PCR

The RT-PCR was performed by using a QIAGEN 2-step RT-PCR kit.

Aliquots (1 mL) of cDNA (2 ng per reaction) were used as template for RT-PCRs

along with Fast PCR Master Mix and primers (500 nM final concentration). RT-PCR

amplifications were carried out in BioRad CX96 thermocycler with following

conditions 50oC for 2 min and 95

oC for 10 min, followed by 40 cycles of 95

oC for 15

s and 60oC for 1 min and a final dissociation curve analysis step from 65

oC to 95

o C.

Technical replicate experiments were performed for each biological triplicate sample

and for negative controls same volume of water was used. Amplification specificity

for each reaction was confirmed by the dissociation curve analysis. The 16S rDNA

genes were used as the reference genes to normalize samples and similar results were

obtained. A relative quantification value was calculated for each gene (Wang et al.,

2011 and 2012).

Accession numbers of nucleotide sequences

The antibiotic gene fragment sequence data presented in the present

investigation were submitted in the gene bank nucleotide sequence database under

their accession numbers of Phz D genes were KF281757, KF281758, KF281759.


183

Fig 5.3: Schematic illustration of steps followed for gene expression studies using

Real time PCR


184

5.3 RESULTS

In the present study evidence related to the biosynthesis of various antibiotics

by the selected antagonist PGPR strains was provided by PCR amplification with

gene specific primers and by detecting their expression through RT-PCR. Antibiotic

production was further confirmed by the quantification through HPLC and formation

of inhibition zone by the antibiotic extracted.

5.3.1 Antibiotic production assay by agar well diffusion method

All the rhizobacteria exhibited the production of antibiotic by the development

of halo zone around the wells. . The inhibition zone diameter for the antibiotic

production by antagonistic PGPR was in the range of 1.13 to 3.93 cm (Fig 5.4).

Maximum inhibition (3.93 cm) was exhibited by P. aeruginosa JYR followed by B

.endophyticus Y5, P. aeruginosa 4nm and Pseudomonas sp. B. firmus PTWz and B.

pumilus Yio produced the least inhibition (1.13 and 1.46 cm).

5.3.2 Detection of DAPG, PRN gene by Polymerase chain reaction (PCR)

When genomic DNA was used as template three Pseudomonas strains

including P. aeruginosa 4nm, P. aeruginosa JYR and Pseudomonas sp. NDY showed

the amplification of pyrrolnitrin (PRN) (500bp) genes with primer prnD and prnC

(Fig 5.5).

PCR amplification for 2,4diacetylphlourogluionol (DAPG) biosynthesis genes

did not provide any amplicon by using the gene-specific primers although all these

strains showed antifungal activity against phytopathogenic fungi.

5.3.3 Detection of zwittermicin A self-resistance gene by Polymerase chain

reaction (PCR)

A 950 bp PCR product was amplified with B. endophyticus Y5 using

zwittermicin A gene-specific primers zmaR (Fig 5.6). No PCR product was amplified

from B. pumilus Yio by using zmaR primer. In this study, B. endophyticus and

B.firmus was found to have zmaR gene. This is an important report for the detection

of zmaR gene in bacillus species other than B. cereus and B. thuringiensis.


185

Fig 5.4: Development of inhibition zone by antagonistic PGPR PA1: P. aeruginosa 4nm, BE: B. endophyticus, PA2: P. aeruginosa JYR, Ps: Pseudomonas sp., BF: B.

firmu PTWz, BP: B. pumilus. Results are expressed as means of three replicate. All means sharing the

common letter differ non-significantly at P< 0.05 LSD: 0.155.

Fig 5.5: Agarose gel electrophoresis of the PCR products of Pseudomonas

strains with PrnC primer M is Marker 1Kb ladder and Lane 1 – 6 are PCR products with genus specific primers of pyrrolnitrin.

Lane 1-2 (P. aeruginosa 4nm), Lane 3-4 (P. aeruginosa JYR), Lane 5-6(Pseudomonas sp. NDY)

0

1

2

3

4

PA1 PA2 Ps BF BP BE

c

a

d

e e

b In

hib

itio

n z

on

e d

iam

ete

r (c

m)

Antagonistic PGPR

Inhibition zone diameter


186

Fig 5.6: Agarose gel electrophoresis of the PCR products of Bacillus strains

with zamR primer. M is Marker 1Kb ladder and Lane 1 – 6 are PCR products with genus specific primers of Zwittermicin

A. Lane 1-2 (B.endophyticys Y5), Lane 3-4 (B.firmus PTWz), Lane 5-6(B.pumulis Yio.)

Fig 5.7: Agarose gel electrophoresis of PCR products of Pseudomonas strains

with PhzD/PhzF primers M is Marker 1Kb ladder and Lane 1 – 6 are PCR products with genus specific primers of phenazine

genes (Phz D and F). Lane 1(P. aeruginosa 4nm), Lane 2 (P. aeruginosa JYR) with Phz D primers,

Lane 3 (P.aeruginosa 4nm), Lane 4 (P .aeruginosa JYR), Lane 5 (Pseudomonas sp. NDY) with Phz

F primers.


187

Fig 5.8: Agarose gel electrophoresis of the PCR products from genomic DNA of

selected Pseudomonas strains with Phz D primers M is Marker 1Kb ladder and Lane 1 – 6 are PCR products with genus specific primers of Phenazine

genes (Phz F). Lane 1-2 (P. aeruginosa 4nm), Lane 3-4(P.aeruginosa JYR) Lane 5-6

(Pseudomonas sp. NDY).

Fig 5.9: Agarose gel electrophoresis of the PCR products from genomic DNA of

selected Pseudomonas strains with Phz F primers.

M is Marker 1Kb ladder and Lane 1 – 6 are PCR products with genus specific primers of phenazine

genes (Phz F). Lane 1-2 (P. aeruginosa 4nm), Lane 3-4(P. aeruginosa JYR) Lane 5-6

(Pseudomonas sp. NDY).


188

5.3.4 Detection of phenazine genes by polymerase chain reaction (PCR)

When total DNA of the bacterial antagonists was tested by PCR using gene-

specific primers, all the Pseudomonas strain amplified the DNA fragment of 700 bp

for phzD (Fig 5.7 and 5.8). For phzF (all Pseudomonas strains) yielded the specific

500 bp amplification product (Fig 5.9). The products when sequenced and searched

with the blast nucleotide yielded very high similarity with the phzD and phzF genes of

P. aeruginosa M 18 and P. aeruginosa PAb1. All the three Bacillus strains showed

good antagonistic activity against phytopathogens but did not indicate the presence of

phenazine genes when assayed in PCR by using the phenazine genes specific primers

phzF and phzD.

5.3.5 Quantitative determination of antibiotic substances (Phenazine)

Biosynthesis of phenazine was demonstrated by extracting them from the

broth cultures and resolving them on HPLC. Phenazine was detected at 275 nm with a

retention time 8.77 min. P. aeruginosa JYR produced maximum amount of phenazine

(0.47 mg/mL) and the strains P. aeruginosa 4nm and Pseudomonas sp. NDY produced

minimum amounts of phenazines (0.216 and 0.277 mg/mL, respectively indicating

that phzD and phz F are involved in the biosynthesis of phenazines (Fig 5.10).

5.3.6 Well plate assay

In well plate assay inhibition of F.moniliforme growth occurred for phenazine

extracted from all the strains of Pseudomonas as indicated in fig 5.11 and 5.12. The

inhibition zone diameter for the antibiotic extracted from antagonistic PGPR was in

the range of 1.3 to 1.66 cm (Fig 5.11). Maximum inhibition (1.6 cm) was exhibited by

treatments PA2 (P. aeruginosa JYR) and P. aeruginosa 4nm and Pseudomonas sp.

produced the least inhibition (1.3 cm).

5.3.7 Gene expression of phenazine

The result presented in Fig 5.14 is consistent with quantity of phenazine

produced and the inhibition zone shown by the extracted phenazine from the

antagonistic PGPR strains. The Phz F and Phz D expression was significantly higher

in the P. aeruginosa 4nm. The expression of phz D was significantly lower in the

Pseudomonas sp. as compare to P. aeruginosa strains. These results indicate that

phzF and phzD are involved in the expression of the phenazine biosynthetic genes.


189

HPLC Chromatograms for crude phenazine extracts from Pseudomonas Strains

Fig 5.10: Phenazine production by antagonistic PGPR

PA1: P. aeruginosa 4nm, BE: B. endophyticus, PA2: P. aeruginosa JYR, Ps: Pseudomonas sp. NDY,

BF: B.firmus PTWz, BP: B.pumilus Yio. Results are expressed as means of three replicate. All means

sharing the common letter differ non-significantly at P< 0.05, LSD: 0.0584.

0

0.1

0.2

0.3

0.4

0.5

PA1 BE PA2 Ps BF BP

b

d

a

c

d d

Ph

en

azin

e (m

g/m

L)

Antagonistic PGPR

Phenazine


190

Fig 5.11: Inhibition of fungal growth crude phenazine extract of

antagonistic PGPR

PA1:P. aeruginosa 4nm, PA2: P. aeruginosa JYR, Ps: Pseudomonas sp. NDY, Control: Standard.

Results are expressed as means of three replicate. All means sharing the common letter differ non-

significantly at P< 0.05, LSD: 0.061.

Fig 5.12: Development of inhibition zone by crude phenazine antibiotic extracted

from antagonistic PGPR (Pseudomonas strains)

a: P. aeruginosa 4nm b: P. aeruginosa JYR c: Pseudomonas sp. NDY T: Terbinafine synthetic

antifungal compound

0

0.5

1

1.5

2

2.5

3

3.5

PA1 PA2 Ps Control

b b

c

a

zon

e d

iam

ete

r (c

m)

Antagonistic PGPR (Pseudomonas strains)

Zone diameter


191

Fig 5.13: Agarose gel electrophoresis of the RNA isolated from the Phz D PCR

product of selected Pseudomonas strains. M is Marker 1Kb ladder and Lane 1 – 4 are RNA isolated from PCR product (Phz D), Lane 1-2

(P.aeruginosa 4nm), Lane 3(P.aeruginosa JYR) Lane 4 (Pseudomonas sp. NDY).

Fig 5.14: Phenazine gene expression in antagonistic PGPR of

Pseudomonas strains

PA1: P. aeruginosa 4nm, PA2: P. aeruginosa JYR, Ps= Pseudomonas sp. NDY. Relative expression of

phz F and phz D in Pseudomonas strains was compared by RT-PCR. Vertical bars represent means of

three replicates and according to statistical analysis confirmed that for each gene tested, non-

overlapping error bars indicate significant differences between strains in gene expression. These

experiments were repeated at least three times and similar results were obtained.

0

1

2

3

4

5

6

7

8

PA2 PA1 Ps

Re

lati

ve t

ran

scri

pt

leve

l

antagonistic PGPR (Pseudomonas strains)

Phz F Phz D


192

5.4 DISCUSSION

Antibiotic production by bacterial antagonists is considered as an essential

element in the biological control of fungal phytopathogens as reviewed by

Raaijmakers et al., 2002; Weller et al., 2002; Haas and Keel, 2003 and Mavrodi et al.,

2006. The better understanding of the mechanism behind this phenomenon will assist

to enhance the performance of the biocontrol strains. A strong correlation was

observed between the production of secondary metabolites and percentage inhibition

of the fungal pathogens. Antibiotics biosynthesis is the mechanism positively related

with the capability of a PGPR to inhibit the development and growth of

phytopathogens (Mazurier et al., 2009). These antibiotics target the electron transport

chain through phenazines and pyrrolnitrin production, copper containing cytochrome

oxidases (hydrogen cyanide), plasma membrane (2,4-diacetylphloroglucinol) (Haas

and Defago 2005). Although the exact mecahnism of action used by the antibiotics

involved in the inhibition of phytopahtogen growth is still unknown.

The antagonistic rhizobacteria producing antibiotics are the potential

candidates to be applied as biological control agents by reinforcement of the plant

natural defence against several plant diseases. These rhizobacteria are well known for

their capability to control various soil borne pathogens (Raaijmakers et al., 2002;

Haas and Defago, 2005). In the present study we detected the antibiotic biosynthetic

genes in the rhizobacterial strains (P. aeruginosa 4nm, P. aeruginosa JYR,

Pseudomonas sp. NDY and B. endophyticus Y5) which have exhibited the strong

antifungal activity not only in plate assay but also reduce the disease severity in the

greenhouse and field experiment. Antibiosis might be an extensive mechanism

involved in inhibition of fungal growth as reported by Weller et al., 2002. Antifungal

antibiotics produced by rhizobacteria have the ability to antagonize the pathogens and

at the same time they are naturally biodegradable hence, limiting the extensive use of

chemical fungicides (Yamaguchi, 1996).

Antibiotics are extensively implicated for the biocontrol of a wide range of

plant pathogens. In the last few decades, several antibiotics have been extracted from

numerous biocontrol strains belonging to different genera. Pseudomonas strains are

among the important group of antagonistic PGPR which suppress the pathogens by

the production of antibiotics. Whipps, (1997) has emphasized on various reasons of

extensive studies on antibiotics production by various Pseudomonas sp. As they are


193

the most abundant inhabitant of rhizosphere, can be easily isolated from their native

environments, easy to culture and manipulate genetically. Although, there are other

several reports for antibiotics production by other bacterial biocontrol agents like

Bacillus. The Biocontrol strain producing the multiple antibiotics is considered more

efficient in suppressing the pathogen e.g. B. cereus strain UW85 and P. fluorescens

strains (CHA0 and Pf5) by producing the multiple antibiotics with strong antagonistic

activity against fungal pathogens (Keel et al., 1996; Bender et al., 1999)

For this study we designed genus specific sets of primer for phenazine (phz F,

phz D, and Phz C) DAPG (Phl C and Phl D), Pyrrolnitrin (Prn C) and Zwittermicin A

(ZamR) (Table 5.2). All of them have dissimilar sequences but all target the

respective gene. These oligonucleotide primers were utilized to screen the selected

antagonistic bacteria which have shown significantly high antagonistic activity.

In the present investigation, all the Pseudomonas species showed the

production of phenazine synthesis as confirmed by the PCR analysis for the specific

primers designed for phenazine. These results are further confirmed by evaluating the

phenazine production quantitatively by high performance liquid chromatography.

Fernande and Pizarro, (1997) have demonstrated that phenazine derivatives can be

extracted from the cell free supernatant for the quantitative analysis by using HPLC.

Our results of The HPLC UV visible spectral analyses presented the high probability

for the production of phenazine antibiotic by Pseudomonas strains. These results are

in accordance with the finding of Mavrodi et al., (1998) that phz gene in

Pseudomonas strains were directly involve in the condensation reactions for the

production of the phenazines and its derivatives. Phenazines are considered as one of

the efficient secondary metabolites having the broad spectrum antibiotic activity. The

most commonly identified antibiotics produced by rhizobacteria are the derivatives of

phenazine including phenazine-1- Carboxylic acid, pyocyanin, and different

hydroxyphenazines (Ramamoorthy et al., 2001). Lee et al., (2003) showed the

complete inhibition of mycelial growth of B. cinerea and C. cucumerinum by the

production of phenazine-1- carboxylic acid. It is suggested by Mavrodi et al., (2010)

that most phenazine producers are soil inhabiting bacteria or plant associated species.

The synthesis of phenazine-1- carboxylic acid and its derivatives biosynthesis has

been investigated extensively in Pseudomonas strains (Mavrodi et al., 2006).

Six antagonistic PGPR, two P. aeruginosa strains and one strain of

Pseudomonas sp. was tested positive for the presence of phenazine and pyrrolnitrin


194

biosynthetic genes. The P. chlororaphis and P. aurantiaca strains were considered as

potent biocontrol agents by the production of phenazine and pyrrolnitrin antibiotics

(Ramarathnam and Fernando, 2006). Pyrrolnitrin have a major role in suppression of

fungal growth (Fernando et al., 2005). It is supposed that the pyrrolnitrin production is

a key factor in the inhibition of rhizospheric fungal pathogens which make them an

effective candidate for the biological control of phytopathogens (Fernando et al.,

2005).

Absence of 2,4- DAPG antibiotic is also in accordance with earlier finding

which revealed that the biosynthetic genes of DAPG has the limited distribution and

their biosynthetic pathways co-occurrence is found only in a very specific group of

antibiotic producing biocontrol agents (Mavrodi et al., 2001).

Zwittermicin A is a new class of antibiotic (He et al., 1994) that has been

identified for its importance in biocontrol of phytopathogens by using B. cereus (Silo-

Suh et al., 1994). It is well known for its broad spectrum biocontrol activity by

inhibiting various gram-negative and gram-positive eukaryotic microorganisms (Silo-

Suh et al., 1998). This antibiotic has also been detected in this study in two Bacillus

strains (B. endophyticus and B. firmus). All Bacillus strains were evaluated negative

for most of the antibiotics screened except for zmaR self-resistance gene. However;

they have indicated significant antifungal activity in in vitro assays and greenhouse

experiment, signifying their potent role by the use of some other antifungal

mechanisms or the involvement of some other novel or unique antibiotics.

These results were further confirmed by the quantification through HPLC. The

HPLC analyses revealed phenazine in the rhizobacteria extract appearing at the

identical retention time as standard. UV visible spectrum revealed identical spectrum

and wavelength for Phenazine and bacterial (Pseudomonas strains) extract. Agar well

diffusion assay also demonstrated the higher antifungal activity of Pseudomonas

strains against Fusrium sp. Finally the gene expression experiment through RT-PCR

clearly indicates the up-regulation of phenazine gene by the Pseudomonas strains.

The present investigation demonstrated the multifunctional potential of

rhizobacteria as potent antagonist against fungal pathogens and as efficient plant

growth promoter. It is also found that the phenazine and pyrrolnitrin antibiotics have

active role in biological control of fungal pathogens in indigenous Pseudomonas

antagonists and antibiosis was one of the key mechanisms for disease suppression.

Production of these antibiotics in these strains provides them a competitive advantage


195

in the rhizosphere. Identification of genes for antibiosis mechanism will explore the

opportunities for the further manipulation of these antagonistic strains. We have

indicated in this study the presence of phenazine and pyrrolnitrin biosynthetic genes

in Pseudomonas strains and Zwittermicin A in Bacillus strains. Knowledge related to

genes involved in specific biocontrol traits would help to enlighten the efficiency of

biological control agents.

Concluding chapter Chapter 6

196

6.1 CONCLUDING CHAPTER The hazards related to health and environment due to the extensive use of chemical

pesticides for the disease control of important cereal corps urged for the implementation of

biological control by using indigenous rhizobacteria (Gliessman, 2001). The use of plant

growth promoting rhizobacteria is a multipurpose approach to protect plants from various soil

borne pathogens. The present work was focussed on

i. The isolation and characterization of the antagonistic PGPR from rhizosphere of

maize infected with and without stalk rot.

ii. Screening of their potential in vitro and in vivo to control stalk rot disease, detection

of the mechanisms involved in antagonism and the genes involved in antibiotic

biosynthesis by selected rhizobacteria.

iii. Evaluation of their performance under field conditions with the aim to develop

efficient bio-fungicide/bio-pesticide.

During the present investigation, 117 rhizobacteria have been isolated from three

different agro-climatic regions (irrigated, arid, semi-arid) and divided in 2 groups on the basis

of their habitat (i) rhizospheric soil of non-infected maize and (ii) rhizospheric soil of maize

infected with stalk rot disease. In vitro screening of rhizobacteria showed efficient

antagonistic activity of isolates against F. moniliforme, H. sativum and A. flavus with a

variable range of percentage inhibition. All types of agricultural soils have some suppressive

effect over several soil borne plant pathogens owing to the antagonistic activities of the

rhizobacteria inhabiting in soil (Weller et al., 2002).

The selected rhizobacteria were further evaluated under axenic conditions in pot

experiment to evaluate their effect on plant growth promotion. Rhizobacteria including B.

endophyticus Y5 and B. pumilus YiO from semi-arid region, P. aeruginosa JYR and B.

firmus PTWz from arid region, P. aeruginosa 4nm and Pseudomonas sp. NDY from irrigated

region were good colonizer of maize roots as evidenced by high cfu count. The rhizobacteria

isolated from the rhizosphere soil of non-infected maize fields also have shown more survival

efficiency as compared to the rhizobacteria from the maize infected with stalk rot. The ability

of rhizobacteria to promote plant growth and suppress the plant pathogen is related to their

potential to colonize the roots. The plants, inoculated with P. aeruginosa 4nm, P. aeruginosa

JYR, B. endophyticus Y5 of group 1 and Pseudomonas sp. NDY, B. firmus PTWz, B. pumilus

Yio of group 2, showed significant increase in fresh and dry weight, length and leaf are of

maize plants as compared to un-inoculated control.


197

Fig 6.1: Summary of induction of induced systemic resistance by the application of

antagonistic PGPR under axenic conditions of greenhouse


198

Fig 6.2: Summary of the experiments conducted for the evaluation of isolated PGPR as biocontrol agents under field conditions


199

It is inferred from results that rhizobacteria with in vitro antagonistic activity and

exhibit the potential to promote plant growth in vivo as reported by Rani et al., (2005).

The antagonistic rhizobacteria acting as PGPR have dual role in the rhizosphere

acting as biocontrol agent against fungal pathogens as well as plant growth regulators.

The molecular identification of these effective antagonistic isolates have shown that

they belong to genus Pseudomonas (4nm, JYR, JYG, NDY) and Bacillus (Y5, PTWz,

Yio) on the basis of 16S rRNA gene sequencing and successive molecular phylogeny

analyses. The defense related potential of these two genera are well documented in the

literature (Schisler et al., 2004).

Furthermore these selected strains were applied to plants grown in greenhouse

experiment in order to evaluate the mechanism of action by the selected rhizobacteria.

The single application of B. endophyticus Y5 was as effective as fungicide the effect

of which being mediated via increase in antioxidants enzymes activities PPO, POD,

SOD, ascorbate peroxidase, total soluble phenol, PR related proteins and decreased

the MDA content, as observed in experiments conducted under axenic conditions. The

fungicide (0.1%) augmented the effect of B. endophyticus Y5 isolated from semi-arid

region and its effect was even higher ten the full dose of chemical fungicide in

increasing the activities of antioxidant enzymes and defense-related enzymes.

Whereas, the single application of P. aeruginosa JYR isolated from arid region and P.

aeruginosa 4nm isolated from irrigated region also increased the reduction in stalk rot

disease by stimulating some of the defense related enzyme activities and the

endogenous level of secondary metabolites. But, the magnitude of fungal growth was

lesser than that of B. endophyticus Y5. The combined application of P. aeruginosa

JYR and P. aeruginosa 4nm with fungicide significantly improved the photosynthetic

pigments, PPO, protease and chitinase activities than its single application. It was

further revealed from the present investigation that the endogenous level of secondary

metabolites determine the extent of defense related processes. Most importantly the

combined application of B. firmus PTWz and Pseudomonas sp. NDY with fungicide

induced resistance in maize against Fusarium stalk rot by improving all the

physiological and biochemical attributes by induction of defense related proteins and

production of antibiotics as shown in Figure 5.1. While the application of B. pumilus

Yio alone and with fungicide also provided defense by enhancing some of the

activities of defense-related enzymes, indicating that the mode of action of

antagonistic PGPR is by the induction of systemic resistance (ISR).


200

Performance of these antagonistic PGPR was also observed under natural

condition of field and it was found that the single application of P.aeruginosa JYR

and Bacillus endophyticus Y5 significantly reduced the stalk rot disease incidence and

improved the total soluble phenol, antioxidant activities and yield attributes. The

application of P. aeruginosa JYR and B. endophyticus with fungicide supplemented

the effect of fungicide and exhibited much higher increase in antioxidant enzyme and

defense-related enzymes, endogenous phytohormones content and yield. The single

and combined applications of P .aeruginosa 4nm and Pseudomonas sp. NDY have

also contributed to induce defense in maize plants against stalk rot pathogen (F.

moniliorme), which eventually resulted in higher yield. It has also been observed that

Pseudomonas sp. predominate in group 1(isolated from the rhizosphere of non-

infected maize fields from arid region) whereas Bacillus sp. predominates in the group

2 rhizobacteria (isolated from the rhizosphere of non-infected maize fields from arid

region). Combined application of B. endophyticus Y5 + half dose of fungicide (0.1%)

was at par with full dose of fungicide (0.2%) in increasing yield hence, 50% cost can

be minimized by using the low concentration of fungicide.

The single application of Bacillus firmus PTWz and Bacillus Pumilus Yio

along with fungicide was non-responsive for physiological and biochemical

parameters while combined application of Bacillus firmus PTWz and Bacillus Pumilus

Yio with fungicide at 0.1% showed very little increase in antioxidant enzymes (SOD,

POD), defense-related enzymes (PPO, chitinase, proteases), endogenous level of

phytohormone (IAA) and exhibited 14 and 13% increase in yield as compared to

infected control, respectively.

Results obtained from both the greenhouse and field experiments revealed that

the combined application of antagonistic PGPR were more effective in controlling

disease and inducing defense against the pathogen attack than their single

applications. The effect of antagonistic PGPR was more pronounced in greenhouse

experiment under axenic conditions than that of the field experiment under natural

conditions, indicating that the affectivity of antagonistic PGPR is possibly affected by

the competitive effect of pathogenic and other indigenous microbes present in soil

under natural conditions. Whereas, the sole effects of these rhizobacteria were

demonstrated under axenic condition in greenhouse plants.

Biosynthetic genes of antibiotic phenazine and pyrrolnitrin were detected in

the antagonistic Pseudomonas strains (P. aeruginosa 4nm, P. aeruginosa JYR,


201

Pseudomonas sp. NDY) while, the genes for zwittermicin A has been detected in

Bacillus strain (Bacillus endophyticus Y5) by amplifying the specific genes involved

in the synthesis of these antibiotics by using the genus specific primers. The

antibiotics producing biocontrol strains isolated from the rhizosphere of host plant

growing in different agro-climatic locations suggest an efficient antagonistic activity

and evolutionary advantage of these rhizobacteria to others. The gene expression

experiment through RT-PCR indicated the up-regulation of phenazine gene in the

Pseudomonas strains. It was found that the phenazine and pyrrolnitrin antibiotics have

their active role in biological control of fungal pathogens by indigenous Pseudomonas

antagonists. Antibiosis appears one of the key mechanisms for disease suppression.

Antibiotic producing rhizobacteria are promising candidates to be used as biocontrol

agents of various diseases. They act by strengthening the natural plant defense. These

bacteria are well known as manager of several soil borne diseases (Raaijmakers et al.,

2002).

The production of hydrolytic enzymes, secondary metabolites, siderophore,

HCN and IAA by the antagonistic rhizobacteria may be responsible for inhibition of

pathogen by inducing defense related proteins and improving the physical and

chemical barriers of host plants to resist pathogen attack. Furthermore, antibiotic were

also involved in controlling the stalk rot.

Two stains of Bacillus didn’t exhibit the significant disease protection in

maize plants under field conditions which may be due to poor competence of the

rhizobacteria with the indigenous rhizobacteria of the maize rhizosphere as evidenced

by cfu of these PGPR (Kumar et al., 2011). These rhizobacteria did not show any

increase in the antioxidant activities, PR related proteins, endogenous level of IAA

and yield as compared to infected maize plants. Their inability to reduce the disease

intensity may also be related to absence of antibiotic related genes in them.

The inhibition of fungal growth by the fungicide was higher than single

application of antagonistic PGPR while the combined application of P. aeruginosa

JYR and B. endophyticus Y5 was at par with the chemical fungicide against stalk rot

infection. It was also found that the rhizobacteria P. aeruginosa JYR, P. aeruginosa

4nm, and B. endophyticus Y5 were isolated from the rhizosphere of non-infected

maize. It supports the concept of that the rhizobacteria in the non-infected soils have

the better ability to supress the phytopathogens and keeps the plants healthy. It was

also observed from the present investigation that the rhizobacteria like P. aeruginosa


202

JYR and B. endophyticus Y5 were isolated from arid and semi-arid regions have

greater potential to survive under stress conditions prevailing in these agro-climatic

regions. The present result also suggested that the combined application of

antagonistic PGPR can replace 50% requirement of the chemical fungicide to combat

fungal diseases.

It can be inferred that combinations of antagonistic rhizobacteria with

fungicide could provide promising integrated alternatives in the suppression of fungal

diseases of maize, in addition it also help to improve the plant growth. The

antagonistic PGPR P. aeruginosa JYR and B. endophyticus Y5 were the potent

biocontrol agent to reduce the use of chemical fungicide. The antagonistic PGPR was

also capable of suppressing the oxidative stress by inducing the ISR and enhancing

the plant growth and yield.

1. The antagonistic PGPR (P.aeruginosa JYR, B.endophyticus Y5, P.aeruginosa

4nm and Pseudomonas sp. NDY) can be used with low concentration of

chemical fungicide (0.1%) to provide resistance to host plant against fungal

pathogens. it can be inferred from present investigation that

2. The antagonistic PGPR can be used to reduce the health hazards and protect

the ecosystem and food as they directly affect the crop plant, further these

PGPR rhizobacteria are not assumed to have adverse residual effects on the

indigenous soil micro flora.

3. Antagonistic PGPR in combination with low concentration of fungicide are

economical than commercial fungicide and can be applied with the seed

soaking treatment for seed-borne fungal pathogens.

4. Antibiosis is the most prominent mechanism involved in F.moniliforme

suppression. Production of these antibiotics provides them a competitive

advantage with other antagonistic PGPR.

6.2 Recommendations and Future perspectives On the basis of above discussion to improve the plant yield by reducing the disease

incidence being eco-friendly and sustainable bio-fungicide further investigations are

required;


203

1. The antibiotics in the antagonistic PGPR involved in the suppression of the

fungal growth need further characterization through NMR spectroscopy and

LC-MS analyses.

2. Further investigation related to time of application, carrier material, and

inoculation methodology is required.

3. The residual effect of bio-fungicide with the commercial fungicide on pests

and rhizosphere micro-flora need to be ascertained.

4. The persistence and shelf life of the bio-fungicide in storage need further

investigation.

5. Caution need to be exercised referring biosafety of these strains as the strains

belonging to the genus P.aeruginosa are opportunistic pathogens to human

(Betazmann and plesiat, 2011)

References Chapter 7

205

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APPENDICES

Appendix 1

Soil Analysis reagents

i. Determination of P contents

Total extractable P was determined by the method described by Soltanpour (1985).

Preparation of mixed reagent

Mixed reagent was prepared by mixing 1 L of 5 NH2SO4 with 1 L distilled water

containing potassium tartarate (0.2908).

Working colour reagent

For the preparation of working colour reagent, 0.74 of ascorbic acid were dissolved in

140 mL of mixed reagent.

Preparation of standard solutions

KH2PO4 solution (100 ppm) was prepared by dilution stock (100 ppm) solution. From

this (100 ppm) solution, 0, 0.5, 1, 1.5, 2, 2.5, and 3.0 ppm solution were prepared.

Samples preparation

Samples along with standard were prepared as follows: one mL of sample (or

standards solution ), 9.0 mL of distilled water and 2.5 mL of working colour reagent (

color reagent + ascorbic acid ) were mixed and analyzed after 15 to 20 minutes on

Spectronic 21 at 880 nm.

ii. Determination of K+, Ca

++, Mg

++ IONS

REAGENTS

1. Lanthanum diluting solution

Lanthanum oxide (La2O3; 5, 9 g) was dissolved in 20 mL distilled H2O in a 500 mL

flash and placed in a cold water bath. Concentrated HCL (10.5 mL) and HNO3, (14

mL) were added to 100 mL flask containing lanthanum oxide. The final volume was

diluted with 200 mL-distilled H2O.

II. High stock solutions

i. K+ = (2000pmm): 3.815g KCL diluted to volume (1 L) with distilled

water.

ii. Ca++

= (10.000pmm): 24.97 g CaCO3 dissolved in 1 L- distilled water.

iii. Mg++

= (1000PMM): 1.0 G MG ribbon dissolved In 1 L distilled water.

253

III. Low stock solutions

Employing following alts low stock solutions were made;

i. K+=(1000ppm): 0.1907 g of KCI dissolved in 1 L distilled water

ii. Ca++

=(500ppm): 1.25G CaCO3 dissolved in 1 L distilled water

iii. Mg++

=(500ppm): 0.829 G MgO in 10 mL of HNO3 and final volume was

made in L

Low stock solutions were added in 100 ml flask and final volume was made with

appropriate extracting solutions extracting solutions. Extraction of K+, Mg

++, Ca++,

from the soil samples were dome according to Mehlich 1953 and 1984.

Procedure

Aliquot (1.5 mL) of each working standard and all soil extracts were diluted with

CaCO3 working solution to final volume of 1.5 mL. K+,Ca

++ and Mg

++ were measured

by atomic Absorption Spectrophotometer (Shimazu, AA-670) at the wavelength of

766.5,422.7 and 285.2 nm respectively.

Appendix 2

Gram Staining

Preparation of solutions

a. Crystal Violet (Hucker’ s)

Solution A

Crystal violet (90% dye content) 2 g

Ethyl alcohol 20 mL

Solution B

Ammonium oxalate 0.8 g

Distilled water 80 mL

Mix solution A and B

b. Gram, s Iodine

Iodine 1 g

Potassium Iodide 2 g


c. Ethyl alcohol (95%)

Ethyl alcohol (100%) 95 mL


254

d. Safranin

Safranin O (2.5% solution

in 95% ethyl alcohol) 10 mL


Appendix 3

LB (Lubria-Bertani) Medium, (Miller 1972)

Tryptone 10 g

Yeast Extract 5 g

NaCl 10 g

Agar 18 g

H2O 1000 mL

pH (final) 7.0

Appendix 4

Chitin agar medium

i. Colloidal chitin Preparation

Colloidal chitin was prepared from commercial chitin by the method of Roberts and

Selitrennikoff, (1988) with a few modifications described herein. In the first step acid

hydrolysis of commercial chitin was done by suspending 5.0 g of chitin in 60 mL

Conc. HCl by constant stirring using a magnetic stirrer at 4oC (refrigerator) overnight.

Second step was the extraction of colloidal chitin by ethanol neutralization. To the

resulting slurry (as obtained in step one), 2000 mL of ice-cold 95% ethanol was added

and kept at 26oC for overnight. It was then centrifuge at 3000 rpm for 20 min at 4

oC.

The pellet was washed with sterile distilled water by centrifugation at 3000 rpm for 5

min at 4oC. The washing of the pellets was done till the smell of alcohol vanished.

Colloidal chitin thus obtained was stored at 4oC until further use.

ii. Chitin agar medium (g/L)

Colloidal chitin (crab shell) 10 g

Yeast Extract 5 g

MgSO4 0.5 g

255

Sodium nitrate 2 g

KC1 0.5 g

FeSO4 pinch

K2HPO4 1 g

Agar 20 g

Final pH (using 1N NaOH) 6.0±0.2

Appendix 5

Pikovskaya’s agar (g/L)

Yeast extract 0.50 g

Dextrose 10.00 g

Calcium phosphate 5.00 g

Ammonium sulfate 0.50 g

Potassium chloride 0.20 g

Magnesium sulphate 0.10 g

Manganese sulphate 0.0001 g

Ferrous sulphate 0.0001 g

Agar 15.00 g

Appendix 6

King's medium B (g /L)

Proteose peptone 20 g

Dipotassium hydrogen phosphate 1.5 g

Magnesium sulphate, heptahydrate 1.5 g

Agar 20 g

Final pH (at 25°C) 7.2±0.2

Appendix 7

CAS agar

The following is a detailed, step-by-step procedure for preparing the CAS agar Clean

all glassware with 6M HCl to remove any trace elements, then rinse with ddH2O.

A. Blue Dye:

256

a. Solution 1:

i. Dissolve 0.06 g of CAS (Fluka Chemicals) in 50 mL of ddH2O.

b. Solution 2:

i. Dissolve 0.0027 g of FeCl3-6 H2O in 10 mL of 10 mM HCl.

c. Solution 3:

i. Dissolve 0.073 g of HDTMA in 40 mL of ddH2O.

d. Mix Solution 1 with 9 mL of Solution 2. Then mix with Solution 3.

Solution should now be a blue color. Autoclave and store in a plastic

container/bottle.

B. Mixture solution:

a. Minimal Media 9 (MM9) Salt Solution Stock

i. Dissolve 15 g KH2PO4, 25 g NaCl, and 50 g NH4Cl in 500 mL

of ddH2O.

b. 20% Glucose Stock

i. Dissolve 20 g glucose in 100 mL of ddH2O.

c. NaOH Stock

i. Dissolve 25 g of NaOH in 150 mL ddH2O; pH should be ~12.

d. Casamino Acid Solution

i. Dissolve 3 g of Casamino acid in 27 mL of ddH2O.

ii. Extract with 3% 8-hydroxyquinoline in chloroform to remove

any trace iron.

iii. Filter sterilizes.

C. CAS agar Preparation:

a. Add 100 mL of MM9 salt solution to 750 mL of ddH2O.

b. Dissolve 32.24 g piperazine-N,N′-bis(2- ethanesulfonic acid) PIPES.

i. PIPES will not dissolve below pH of 5. Bring pH up to 6 and

slowly add PIPES while stirring. The pH will drop as PIPES

dissolves. While stirring, slowly bring the pH up to 6.8. Do not

exceed 6.8 as this will turn the solution green.

c. Add 15 g Bacto agar.

d. Autoclave and cool to 50 °C.

e. Add 30 mL of sterile Casamino acid solution and 10 mL of sterile 20%

glucose solution to MM9/ PIPES mixture.

257

f. Slowly add 100 mL of Blue Dye solution along the glass wall with

enough agitation to mix thoroughly.

g. Aseptically pour plates.

Appendix 8

Peptone water

Peptone 10 g

Sodium Chloride 5 g

Final pH (25°C) 7.2 ± 0.2

Appendix 9

Skim Milk Agar (g/L)

Skim milk powder 28 g

Casein enzymatic hydrolysate 5 g

Yeast Extract 2.5 g

Dextrose 1 g

Agar 15 g

Final pH (25°C) 7.0±0.2

Appendix 10

Minimal Agar media (g/L)

Glucose 20 g

Glutamic acid 5 g

MgSO4 0.5 g

K2HPO4 1 g

KCL 0.5 g

Trace elements 1 mL

MnSO4. H2O 0.5 g

Trace elements CuSO4. 5H2O 0.16 g in 100 mL

FeSO4.7H2O 0.015 g

Final pH (at 25°C) 6.2±0.2

258

Appendix 11

Protein Analysis Reagents

Following reagents were prepared for the determination of protein content

determination.

Phosphate Buffer (Stock solution)

i. Monobasic sodium phosphate

27.6 was dissolved in distilled water (1000 mL)

ii. Dibasic sodium phosphate

53.6 g was dissolved in 1000 mL distilled water.

Monobasic sodium phosphate (16 mL) and dibasic sodium phosphate (84

mL) was mixed together to obtain the desired pH (7.5).

i. Reagent A

2.0 g sodium carbonate (Na2CO3)

0.4 g NaOH (0.1 N) and 1 g Na-K tart rate was dissolved in 100 mL of distilled water.

ii. Reagent B The Cu SO4.5H2O (0.5) dissolved in 100 of distilled water.

iii. Reagent C

Solution A (50) and solution B (1 mL) were mixed together.

iv. Reagent D

Folin phenol reagent was diluted with distilled water in the ratio 1:1.

259

Appendix 12

Morphological characteristics of 3 day old colonies of selected rhizobacteria

isolated from the maize rhizosphere

Appendix 13

Morphological characteristics of 3 day old colonies of rhizobacteria isolated from

rhizosphere of non-infected maize fields from irrigated region

Rhizoba

cteria

Shape Size

(mm)

Odour Colour Elevation Surface Margins Cell

shape

Arrangem

ent

Grams

Test

4nm Irregular 1.5 pungent Yellowish

green

Raised Rough Undulate Rod Paired -

JYR

Irregular 2 Odorless green Raised Rough Undulate Rod Scattered -

Y5

Round 1.5 Odorless Pinkish

white

Raised Smooth Entire Rod Paired +

PTWz Round 1.5 Odorless Yellow Raised Smooth

Shiny

Entire Rod Scattered +

NDY

Round 1.2 Odorless Yellow Raised Smooth Entire Paired -

Yio

Round 1 Odorless Orange Raised Smooth Entire Rod Scattered +

Rhizob

acteria Shape

Size

(mm) odor Colour Elevation Surface

Margin

s

Cell

shape Arrangement

Grams

Test

1nm Round 4 Odorless orange Raised Smooth

shiny

Entire Rod Scattered -

3nm Round 1 Odorless Brown Raised Smooth

shiny

Entire Rod Paired -

6nm Round 3.5 Odorless pale Raised Smooth

shiny

Entire Rod Paired +

7nm Round 1.6 Odorless White Raised Rough Entire Round Scattered +

11nm Round 1 Odorless White Raised Smooth

shiny

Entire Rod Paired -

13nm Round Punctifo

rm

Odorless Golden

brown

Raised Smooth

shiny

Entire Round Paired -

14nm Irregular 4 Odorless Yellow Flat Smooth

shiny

Undulat

e

Rod Scattered -

15nm Round 1.2 Odorless Creamy

white

Raised Smooth Entire Rod Scattered +

16nm Irregular 3.8 Odorless silver convex Smooth

shiny

undulate Rod Paired +

19nm Round 3.5 Odorless cream flat Rough Entire Round Paired -

20nm Round puntifor

m

Odorless yellowish Raised Smooth

shiny


2nm Round 1.2 Odorless Creamy

white


5nm Irregular 3.8 Odorless silver convex Smooth

shiny

undulate Rod Paired -

8nm Round 3.5 Odorless cream flat Rough Entire Round Paired -

9nm Round 1.3 Odorless yellow Raised Smooth

shiny


12nm Round 4 Odorless white Raised Rough Undulat

e

Round Paired -

17nm Round 1 Odorless Brown Raised Smooth

shiny

Entire Rod Paired

18nm Round 3.5 Odorless pale Raised Smooth

shiny

Entire Rod Paired

260

Appendix 14


rhizosphere of infected maize fields from irrigated region

Bacterial

isolates Shape Size (mm) odor Color Elevation Surface Margins

Cell

shape Arrangement

Grams

Test

NLY irregular 1.5 Odorless Yellowish Raised Smooth

shiny

Undulate Round Scattered -

Nws Round 1 Odorless white Raised Smooth

shiny


Nst Round puntiform Odorless white Raised Smooth Entire Rod Scattered -

N2C Round 1 Odorless cream Raised Smooth

shiny

Entire Rod Paired +

NSP Round 2 Odorless cream Raised Smooth

shiny


NY Round 1 Odorless yellowish Raised Smooth

shiny


NpY Round 1.5 Odorless yellow Raised Smooth

shiny


NLW Irregular 2.8 Odorless white Flat Smooth

shiny


NWR1 irregular 1.5 Odorless white Raised Rough Undulate Round Scattered -

Nwcir Round 1 Odorless white Creteriform Rough Entire Round Paired +

Nwp2 Round 1 Odorless white Raised Rough Entire Rod Scattered -

Np Round 4 Odorless orange Raised Smooth

shiny


NWsm Round 1 Odorless Brown Raised Smooth

shiny

Entire Rod Paired -

Nwp Round 3.5 Odorless pale Raised Smooth

shiny

Entire Rod Paired +

N91 Round 1.6 Odorless White Raised Rough Entire Round Scattered +

NFY Round 1 Odorless White Raised Smooth

shiny

Entire Rod Paired -

Nwce Round Punctiform Odorless Golden

brown

Raised Smooth

shiny


Nwce2 Irregular 4 Odorless Yellow Flat Smooth

shiny

Undulate Rod Scattered -

261

Appendix 15


rhizosphere of non-infected maize fields from arid region

Rhizob

acteria

Shape Size (mm) odor Color Elevation Surface Margins Cell

shape

Arrangement Grams

Test

JP Round puntiform Odorless Cream Raised Smooth

shiny

Entire Rod Paired -

JY Round 3.5 Odorless yellowish Raised Smooth

shiny


JWIR1 irregular 3.2 Odorless white Raised Smooth

shiny

undulate Rod Scattered +

JDW Round 1 Odorless white Raised Smooth

shiny


JFW irregular 2 Odorless Creamy

white

Raised Smooth

shiny

undulate Round Scattered +

JYD irregular 1.5 Odorless yellowish Raised Smooth

shiny

undulate Round Scattered -

Jpe Round 1 Odorless orange Raised Smooth

shiny


JTz Round 1.2 Odorless Creamy

white


Jshi Irregular 3.8 Odorless silver convex Smooth

shiny

undulate Rod Paired +

JMT Round 3.5 Odorless cream flat Rough Entire Round Paired -

JYT Round puntiform Odorless yellowish Raised Smooth

shiny


JY1 Round 4 Odorless orange Raised Smooth

shiny


JWIR Round 1 Odorless Brown Raised Smooth

shiny

Entire Rod Paired -

JWIR2 Round 3.5 Odorless pale Raised Smooth

shiny

Entire Rod Paired +

JSIR Round 1.6 Odorless White Raised Rough Entire Round Scattered +

JSIR2 Round 1 Odorless White Raised Smooth

shiny

Entire Rod Paired -

JWCH1 Round Punctiform Odorless Golden

brown

Raised Smooth

shiny


JLPO Irregular 4 Odorless Yellow Flat Smooth

shiny


JYG Irregular 2.5 Odorless Green Raised Rough Undulate Rod Paired -

262

Appendix 16


rhizosphere of infected maize fields from arid region

Rhizoba

cteria Shape Size (mm) odor Color Elevation Surface Margins

Cell

shape Arrangement

Grams

Test

PW Round 1 Odorless white Raised Smooth

shiny


Pyz irregular 2 Odorless Creamy

white

Raised Smooth

shiny


PYT2b irregular 1.5 Odorless yellowish Raised Smooth

shiny


PFW Round 1 Odorless white Raised Rough Entire Rod Scattered -

PCIR Round puntiform Odorless Cream Raised Smooth

shiny

Entire Rod Paired -

PYD Round 3.5 Odorless yellowish Raised Smooth

shiny


Ppe Round 3.5 Odorless cream flat Rough Entire Round Paired -

PTz Round 1.3 Odorless yellow Raised Smooth

shiny


Pshi Round 4 Odorless white Raised Rough Undulate Round Paired -

Pwa Round 3.5 Odorless cream flat Rough Entire Round Paired -

PO Round 1.3 Odorless yellow Raised Smooth

shiny


PY1a Round 4 Odorless white Raised Rough Undulate Round Paired -

PYT2a Round 1.7 Odorless Sea green Raised Smooth

shiny


PCP Round 1.5 Odorless white Raised Smooth

shiny

Entire Round Paired +

PTW irregular 2 Odorless white Raised Rough Undulate Round Scattered -

PCWIR Round 1.5 Odorless Yellowish Raised Smooth

shiny


PTW1 Round punctiform Odorless Yellowish Raised Smooth

shiny

Entire Round Scattered +

263

Appendix 17


rhizosphere of non-infected maize fields from semiarid region

Rhizobacteria Shape Size (mm) odor Color Elevat-

ion

Surface Margins Cell

shape

Arrangeme-

nt

Grams

Test

YLY Round puntiform Odorless yellowish Raised Smooth

shiny


YCC Round 2.1 Odorless Skin Raised Smooth

shiny


YCH Round 1.3 Odorless Yellowish Raised Smooth

shiny


YLB Round 1 Odorless Golden Raised Smooth

shiny


Y1 Round 4 Odorless Cream Raised Smooth Entire Rod Scattered -

Y1a Round 1.5 Odorless Cream Raised Smooth

shiny

Entire Rod Paired +

Y2 Round 3.5 Odorless Green Raised Smooth

shiny


Y8 Round 1.7 Odorless Sea green Raised Smooth

shiny


Yw Round 1.5 Odorless white Raised Smooth

shiny


YWD

irregular 2 Odorless white Raised Rough Undulate Round Scattered -

YDY Round 1.5 Odorless Yellowish Raised Smooth

shiny


YDYs Round punctiform Odorless Yellowish Raised Smooth

shiny

Entire Round Scattered +

YCC1 Round 1.5 Odorless White Raised Smooth

shiny


YCH1 Round 1 Odorless Pink Raised Smooth

shiny


Y4 Irregular 4 Odorless Yellow Raised Smooth Undulate Rod Scattered -

Yys Round 1 Odorless Yellow Raised Smooth

shiny

Entire Rod Paired -

Y3 Round 3.5 Odorless White Raised Smooth Entire Round Paired -

264

Appendix 18


rhizosphere of non-infected maize fields from semiarid region

Rhizobac

teria

Shape Size (mm) odor Color Elevation Surface Margins Cell

shape

Arrangement Grams

Test

Yiy Irregular 2.8 Odorless white Flat Smooth

shiny


yiys irregular 1.5 Odorless white Raised Rough Undulate Round Scattered -

YicL Round 1 Odorless white Creterifor

m

Rough Entire Round Paired +

Yiw Round 1 Odorless white Raised Rough Entire Rod Scattered -

Yips Round puntiform Odorless Cream Raised Smooth

shiny

Entire Rod Paired -

Yiws Round 3.5 Odorless yellowish Raised Smooth

shiny


Yiwp irregular 3.2 Odorless white Raised Smooth

shiny

undulate Rod Scattered +

Yicst Round 1 Odorless white Raised Smooth

shiny


Yipy irregular 2 Odorless Creamy

white

Raised Smooth

shiny


YiC1 irregular 1.5 Odorless yellowish Raised Smooth

shiny


Yi1a Round 1 Odorless orange Raised Smooth

shiny


YiPe Round 4 Odorless orange Raised Smooth

shiny


YiBa Round 1 Odorless Brown Raised Smooth

shiny

Entire Rod Paired -

YiLy Round 3.5 Odorless pale Raised Smooth

shiny

Entire Rod Paired +

YiC Round 1.6 Odorless White Raised Rough Entire Round Scattered +

YiH Round 1 Odorless White Raised Smooth

shiny

Entire Rod Paired -

YiBs Round Punctiform Odorless Golden

brown

Raised Smooth

shiny


Yi16 Irregular 4 Odorless Yellow Flat Smooth

shiny


265

Appendix 19: Development of halo zone by rhizobacteria for the production of (a and

b) siderophore, (c and d}) protease, (e and f) solubilization of phosphate

Appendix 20: Change in colour from yellow to orange indicating HCN production

266

Appendix 21: Quick Test system (QTS) kits for the determination of

carbon/nitrogen utilization pattern

Appendix 22: Development of halo zone by antagonistic PGPR indicating

the production of antibiotics 1 and 2: PA2 (P. aeruginosa JYR), 3 and 4: BF (B. firmus PTWz), 5 and 6: BP (B. pumilus Yio), x:

Terbinafine synthetic antifungal compound

267

Appendix 23: Effect of rhizobacteria of different regions on antifungal activity

against F.moniliforme

268

Appendix 24: Effect of rhizobacteria on plant growth

Appendix 25: Effect of antagonistic PGPR on maize plant infected with

F.moniliforme

Appendix 26: Symptoms of Fusarium stalk rot

204

List of Publications

1. Uzma Farooq, Asghari Bano. 2007. Effect of Abscisic acid and Chlorocholine chloride

on nodulation and biochemical content of Vigna radiata L. under Water stress.

Pakistan Journal of Botany. 38(5):1511-1518.

2. Asia Nosheen, Asghari Bano, Faizan Ullah, Uzma Farooq, Humaira Yasmin and Ishtiaq

Hussain. 2011. Effect of plant growth promoting rhizobacteria on root morphology of

Safflower (Carthamus tinctorius L.) African Journal of Biotechnology. 10(59):

12669-12679.

3. Rabia Naz, Asghari Bano, Humaira Yasmin, Samiullah and Uzma Farooq. 2011.

Antimicrobial potential of the selected plant species against some infectious microbes

used. Journal of Medicinal Plants Research.5 (21): 5247-5253.

4. Humaira yasmin, Asghari Bano, Samiullah, Rabia Naz, Uzma Farooq, Asia Nosheen,

Shah Fahad. 2011. Growth promotion by P-solubilizing, siderophore and bacteriocin

production rhizobacteria in Zea mays L. Journal of Medicinal Plants Research.

Journal of Medicinal Plants. 6(3): 553-559.

5. Uzma Farooq and Asghari Bano. 2013. Screening of indigenous bacteria from

rhizosphere of maize (Zea mays L.) For their plant growth promotion ability and

antagonism against fungal and bacterial pathogens. The Journal of Animal & Plant

Sciences. 23(6): 1642-1652.

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