bipartite interaction of acc deaminase producing fluorescent pseudomonas with rice for alleviating

301 Pages·2015·13.1 MB·English

“BIPARTITE INTERACTION OF ACC DEAMINASE
PRODUCING FLUORESCENT PSEUDOMONAS WITH
RICE FOR ALLEVIATING ABIOTIC STRESS”

Ph. D. Thesis

by

Renu Kushwah

DEPARTMENT OF PLANT MOLECULAR BIOLOGY AND
BIOTECHNOLOGY
COLLEGE OF AGRICULTURE
INDIRA GANDHI KRISHI VISHWAVIDYALAYA
RAIPUR (Chhattisgarh)
2015
“BIPARTITE INTERACTION OF ACC DEAMINASE
PRODUCING FLUORESCENT PSEUDOMONAS WITH
RICE FOR ALLEVIATING ABIOTIC STRESS”

Thesis

Submitted to the

Indira Gandhi Krishi Vishwavidyalaya, Raipur
by
Renu Kushwah

IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE DEGREE OF

Doctor of Philosophy
in
Plant Molecular Biology and Biotechnology

Roll No. 15437 ID No. 130111007

JANUARY, 2015

CONTENTS
CHAPTERS PARTICULARS PAGE
I INTRODUCTION 1
II REVIEW OF LITERATURE 8
2.1 Fluorescent Pseudomonas 8
2.2 Biochemical and Molecular characterization of Fluorescent 9
Pseudomonas
2.3 Fluorescent Pseudomonas as Plant growth promoting rhizobacteria 13
(PGPR)
2.3.1 Production of Auxin - Indole 3- acetic acid 13
2.3.2 Solubilization of phosphates by fluorescent Pseudomonads 15
2.3.3 Nitrogen fixation by Pseudomonas 16
2.3.4 Siderophore production 16
2.3.5 HCN production 18
2.3.6 Production of Antibiotics 18
2.4 Impact of water stress and flooding in Rice 19
2.5 Ethylene in Plant physiology 22

2.5.1 Historical perspective of ethylene in plant physiology 22
2.5.2 Enzymes involved in regulation of ethylene level in plants 23
2.6 The enzyme ACC Deaminase 24
2.6.1 ACC deaminase genes in microorganisms and plants 25
2.6.2 Regulation of ACC Deaminase genes 26
2.6.3 Inheritance of Acc Deaminase genes 28
2.6.4 Expression levels 29
CHAPTERS PARTICULARS PAGE
2.6.5 Mode of action of bacterial ACC deaminase 30
Role of Fluorescent Pseudomonas ACC deaminase in stress
2.7 31
agriculture
2.7.1 Salinity stress 32
2.7.2 Drought stress 33
2.7.3 Flooding stress 34
2.7.4 Temperature stress 35
2.7.5 Pathogenicity stress 36
2.8 Recent advances at molecular level 37
III MATERIALS AND METHODS
3.1 Material 38
3.1.1 Fluorescent Pseudomonas Isolates 38
3.1.2 Plant materials 38
3.2 Methods 39
3.2.1 Isolation of Fluorescent Pseudomonas and culture conditions 39
Biochemical characterization of Fluorescent Pseudomonas
3.2.2 39
isolates
3.2.2.1 Gelatin liquefaction test 40
3.2.2.2 Nitrate reduction test 40
3.2.2.3 Lipolytic activity with Tributyrin agar 41
3.2.2.4 Casein hydrolysis 41
3.2.2.5 Carbohydrate utilization 42
3.2.2.6 Antibiotic sensitivity test 42
CHAPTERS PARTICULARS PAGE
3.3 Screening of 1-Aminocyclopropane-1-carboxylic acid 43
deaminase (ACC Deaminase) containing Fluorescent
Pseudomonas isolates
3.3.1 Qualitative estimation of ACC Deaminase activity 44
3.3.2 Quantitative Estimation of ACC Deaminase activity 44
3.3.3 Determination of Protein concentrations 46
3.4 Characterization for Plant growth promoting potentials 47
3.4.1 Screening and characterization of Siderophore 47
3.4.2 49
Determination of Indole Acetic acid (IAA) production
3.4.3 Screening isolates of Fluorescent Pseudomonas spp. for in vitro 49
Phosphate solubilisation
3.4.4 HCN production 50
3.4.5 50
In vitro Screening for antagonistic activity against soil borne
phytopathogens
Evaluation of Fluorescent Pseudomonas on plant growth
3.5 51
promoting activity in Rice under drought conditions
Evaluation of Fluorescent Pseudomonas on plant growth
3.6 52
promoting activity in Rice under flooding conditions
3.7 Estimation of PGPR activity of Fluorescent Pseudomonas in 52
field conditions
3.8 Study of Root characteristics 53
3.9 54
Molecular Characterization of Fluorescent Pseudomonas isolates
3.9.1 Procedure for genomic DNA isolation of Fluorescent 54
Pseudomonas
3.9.2 Quantification of genomic DNA
CHAPTERS PARTICULARS PAGE
3.9.3 Characterisation of Fluorescent Pseudomonas using genus and 56
species-specific loci
3.9.4 Characterization of acdS gene of Fluorescent Pseudomonas 57
Screening of Fluorescent Pseudomonas for HCN production
3.9.5 57
using HCN primers
3.10 Visualization of PCR products 62
3.11 Sequencing of PCR product 63
3.11.1 Elution of bands 64
Enrichment of eluted product
3.11.2 67
3.11.3 Purification of eluted PCR product 67
3.11.4 Sequencing of purified products 69
3.12 In silico analysis of sequences 69
3.13 Statistical analysis 69
IV RESULTS
Isolation and Phenotypic characterization of fluorescent
4.1 81
Pseudomonas isolates
4.1.2 Differential utilization of carbon sources by isolates of 82
fluorescent Pseudomonas by Hi-carbohydrateTM kit test
Differentiation of fluorescent Pseudomonas into P. aeruginosa,
4.1.3 82
P.putida and
P.fluorescens
Screening of 1-Aminocyclopropane-1-carboxylic acid
4.2 85
deaminase (ACC Deaminase) containing Fluorescent
Pseudomonas isolates
4.2.1 Qualitative estimation of ACC Deaminase activity 85
CHAPTERS PARTICULARS PAGE
4.2.2 Quantitative estimation of ACC Deaminase 85
4.2.2.1 Classification of fluorescent Pseudomonas based on ACC 86
deaminase producing ability
4.3 Characterization for Plant growth promoting potentials 95
4.3.1 Screening and characterization of Siderophore 95
4.3.2 Determination of Indole Acetic acid (IAA) production 97
Screening isolates of Fluorescent Pseudomonas for in vitro
4.3.3 98
Phosphate solubilisation
4.3.4 Screening of different isolates of Fluorescent Pseudomonas for 99
hydrogen cyanide (a volatile inhibitory compound) production
4.4 In vitro antagonistic activity of Fluorescent Pseudomonas 100
isolates against R. solani and S. rolfsii
Evaluation of Fluorescent Pseudomonas on plant growth
4.5 103
promoting activity in Rice under drought conditions
4.6 Evaluation of Fluorescent Pseudomonas on plant growth 108
promoting activity in Rice under flooding conditions
Estimation of PGPR activity of Fluorescent Pseudomonas in
4.7 112
field conditions
4.8 Analyzing root system morphology 116
4.8.1 Water stress experiments 116
4.8.2 Flooding experiments 120
4.9 Molecular Characterization of Fluorescent Pseudomonas isolates 123
4.9.1 123
Characterisation of Fluorescent Pseudomonas using genus and
species- specific loci
4.9.2 Characterization of acdS gene of Fluorescent Pseudomonas 128