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Title of Thesis

Nusrat Jabeen
Institute/University/Department Details
Department of Microbiology University of Karachi
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
bacteriocins, phytopathogenic bacteria, biological control, biocontrol agents, anitimicrobial peptides, corynebacterium, erwinia, pseudomonas, xanthomonas, agrobacterium, antimicrobial substances, erwinia, erwiniocin na5, niab

Interest in biological control has increased recently fuelled by public concerns over the use of chemicals in the environment in general, and the need to find alternatives to the use of chemicals for disease control. Use of biocontrol agents against plant diseases has been advocated to get the high yields of crops and safe breeding of livestock. Bacteriocins have been described as extracelluar macromolecular protein/peptide antibiotics produced by certain bacteria which exert their lethal effects on bacteria of the same or the related groups. Bacteriocins and anitimicrobial peptides have attracted attention as potential substitutes or as additions to currently used antimicrobial compounds. Probably 99% of all bacteria generate at least one bacteriocin, while both gram-positive and gram-negative bacteria are capable of producing the bacteriocins. Many phytopathogenic bacteria including members of the Corynebacterium, Erwinia, PseudomoNA5, XanthomoNA5 and Agrobacterium produce bioactive bacteriocins. These bacteriocins are highly specific, cost effective and are even eco- friendly.

Eight strains namely XanthomoNA5 oryzae NA1, XanthomoNA5 oryzae NA2, XanthomoNA5 citri NA3 PseudomoNA5 andropogonis NA4 Erwinia carotovora NA5 Agrobacterium radiobacter. NA6 Agrobacterium radiobacter NA 7 and Erwinia arotovora NA8 were isolated from diseased fruits, vegetables and soil. All the isolates were screened for bioactivity against phytopathogenic bacteria whereby only three i.e. Erwinia carotovora NA5, Agrobacterium radiobacter NA6 and Agrobacterium radiobacter NA 7 were found to produce bacteriocin. Their bacteriocins are designated as Erwiniocin NA5, Agrocin NA6 and Agrocin NA 7 respectively. Agrocin NA6 and NA 7 were found active against Agrobacterium tumefaciens B6, however, Erwiniocin NA5 proved to be a mega bacteriocin. The inhibitory activity could not be related to bacteriophages and hydrogen peroxide. Erwiniocin NA5 has a broad-spectrum activity against many gram-positive and gram-negative bacteria (including phytopathogens). It showed stability against a wide range of temperatures (60°C, 80°C, 100°C and pressured 121°C) for varied time periods and remained stable at wide (2-14) pH range. The substance (Erwiniocin NA5) was resistant to treatment with several metal ions and organic solvents, lipase, lysozyme, and catalase but sensitive to proteiNA5e K and protease (suggesting its proteinaceous nature). The antibacterial titre of Erwiniocin NA5 was found to be 160 AU/mL. Its production starts in early logarithmic phase and continues till late stationary phase. Erwiniocin NA5 revealed bactericidal effect on XanthomoNA5 oryzae NA 1 as well as on Ewrinia carotovora NA8. Erwiniocin NA5 is dialyzable through dialysis membrane; its molecular weight was estimated to be less than 12 kDa, (which was confirmed to be 11 kDa by SDS-P AGE). Bacteriocin was precipitated (up to 80% saturation) by ammonium sulphate. The precipitate was found to contain 290 mg total protein. When partially purified bacteriocin was subjected to gel filtration (using sephadex G75 column) a major active peak of protein (containing 11 mg of total protein as estimated by biuret method) with 2.18 fold purification was detected. The amino acid composition (processed by NCBI genomics server) data revealed that Erwiniocin NA5 contains about 12.44% acidic and 13.57% basic and 73.9<)0-10 neutral amino acid components (with 49.8% polar and 50.16% non-polar amino acid components). The electron micrograph of native negatively stained Erwiniocin NA5 revealed spherical particles, which may be presumed to be membrane vesicle from lysed cells or defective bacteriophage particles (i.e. empty head) like structure. The production of the Erwiniocin NA5 is regulated by extra chromosomal genetic factor(s). The in vivo effect of Erwiniocin NA5 was monitored against XanthomoNA5 oryzae subsp. Olyzae the causative agent of bacterial leaf blight (BLB) of rice. Accordingly, the bacteriocin was found effective (in controlled conditions as well as in field trials). Bacteriocins (like Erwiniocin NA5) could be preferred over antibiotics and synthetic chemicals. The production of Erwiniocin NA5 may be studied by shake flask, Lab and pilot scale fermenter technologies. (before the question of mass scale production can be addressed).

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
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2 1 Introduction / Literature Review 1
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  1.1 What Are Bacteriocins? 1
  1.2 Historical Perspective Of Bacteriocins 5
  1.3 How Do They Work? 7
  1.4 Genetical Aspects Of Bacteriocins 8
  1.5 Bacteriocins Vs Other Antimicrobial Substances L0
  1.6 Application Of Bacteriocins 12
  1.7 Control Of Phytopathogenic Bacteria By Bacteriocins 17
  1.8 Genitcs Of Bacteriocin From Erwinia 34
  1.9 Bacterial Leaf Blight (BLB) In Pakistan 36
3 2 Materials And Methods 38
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  2.1 Materials 38
  2.2 Methods 40
4 3 Results 67
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5 4 Tables 71
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  4.1 Morphological And Cultural Characteristics Of Isolated Strains/Bacteria 71
  4.2 Biochemical Characteristics Of The Isolates 72
  4.3 Plants, Diseases And The .Isolates 73
  4.4 Cross Bacteriocinogenic Activity Of Isolated Phytopathogenic Bacteria (By Cross- Streak Method ) 74
  4.5 Cross Bacteriocinogenic Activity Of Isolated Phytopathogenic Bacteria (By Stab-Overlay And Agar-Well Diffusion Method ) 75
  4.6 Titration Of Erw-Iniocin NA5 76
  4.7 Activity Of Erwiniocinna5 At Extended pH Range 76
  4.8 Stability Of Erwiniocin NA5 At Extended pH Range 77
  4.9 Effect Of Temperature Range On The Bioactivity Of Erwiniocin NA5 78
  4.10 Effect Of Different Enzymes On The Bioactivity Of Erwiniocin NA5 78
  4.11 Effect Of Organic Solvents On The Bioactivity Of Erwiniocin NA5 79
  4.12 Effect Of Metal Ions On The Bioactivity Of Erwiniocin NA5 79
  4.13 Effect Of Agar Concentration On The Production Of Erw-Iniocin NA5 80
  4.14 Effect Of Different Media On The Production Of Bacteriocins 80
  4.15 Differential Purification Prof "De Of Erwiniocin NA5 81
  4.16 Amino Acid Composition Of Erwiniocin NA5 By Computational Analysis 82
  4.17 Bacteriocinogenic Potential Of Erwiniocin NA5 On Diverse (Miscellaneous) Bacteria 3
  4.18 Location Of Genetic Determinants By Curing Technique 84
  4.19 Counter Selective Antibiotic Markers For Bacterial Conjugation 84
  4.20 Recombination Via Conjugation (For Invivo Bac + Marker Transfer ) 85
  4.21 A Efficacy Of Erwiniocin NA5 To Control BLB Disease Of Rice In Controlled Conditions (Average Disease Scores Of Three Replicates) Rice Varities From Faislabad ( Niab ) 85
  4.21 B. Efficacy Of Erwiniocin NA5 To Control BLB Disease Of Rice In Controlled Conditions (Average Disease Scores Of Three Replicates) Rice Varieties From Sheikhupura 86
  4.22 A. Effect Of Erwiniocin NA5 To Control BLB Disease Of Rice (In Field Conditions ) 86
  4.22 B. Efficacy Of Erwiniocin NA5 To Control BLB Disease Of Rice In Field Conditions (Average Disease Scores Of Three Replicates ) 87
  4.23 Bacteriocinogenic Potential Of Miscellaneous Bacteria Against Isolated Phytopathogenic Bacteria 87
6 5 Graphs 88
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  5.1 Production Of Erwiniocin NA5 During Growth Cycle Of Erwinia Carotovora NA5 88
  5.2 Bactericidal Effect Of Erwinioicin NA5 On Stationary Phase Cells And Growing Phase Cells 89
  5.3 Column Chromatography Showing Single Active Peak Of Er-W-Iniocin NA5 90
7 6 Figures 91
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  6.1 Collection Sites 91
  6.2 Infected Fruits, Leaves And Vegetable 92
  6.3 Soft Rot Of Potato 93
  6.4 A. Api 20 E Strips Showing The Biochemical Reactions (For Erwinia Carotovora NA5) 94
  6.4 B. Api 20 Ne Strip Showing The Biochemical Reactions (For Agrobacterium Radiobacter Na6) 94
  6.5 Cross-Streak Method, Demonstrating Bacteriocinogenesis By Erwinia Carotovora NA5 Against Different Indicator Strains 95
  6.6 Stab-Overlay Approach Producer Erwinia Carotovora NA5 Against Different Indicator Strain .96
  6.7 Agar- Well Diffusion Method 97
  6.8 Titration Of Erwiniocin NA5 98
  6.9 Residual Activity Of Erwiniocin NA5 At Extended Ph Range 99
  6.10 Stability Of Erwiniocin NA5 At Extended Ph Range Treatment Followed By Neutralization 100
  6.11 Effect Of Different Temperatures On The Bioactivity Of Erwinia Carotovora NA5 Against Agrobacterium Radiobacter Na6 1 01
  6.12 Effect Of Different Enzymes On Erwiniocin NA5 102
  6.13 Effect Of Organic Solvent (5%), On Thebioactivity Of Erwiniocin NA5, Against Erwinia Carotovora NA8 103
  6.14 Approximated Molecular Mass Estimation Of Erwiniocin NA5 (Using Dialysis Membrane Of 12 KDA Cut Out Size ) 104
  6.1.5 Bio-Activity Of Crude And Ammonium Sulphate Precipitated (80%) Erwinioccin NA5 Against Erwinia Carotovora NA8 105
  6.16 Fractions After Size Exclusion Column Chromatography 106
  6.17 SDS Page Of ERW- Iniocin NA5 107
  6.18 Tem Of Crude ERW- Iniocin NA5 108
  6.19 Plasmid Curing Strategies 109
  6.20 Characterization Of Cured And Uncured Strains (Plasmid Isolation And Agarose Gel Electrophoresis ) 110
  6.21 Molecular Characterization Of Mini Prep Plasmid Dna (Molecular Weight Estimation ) 111
  6.22 Field Trials (Under Controlled Conditions) Showing Effect Of Erwiniocin NA5 Against BLD Infected Rice Varieties 112
  6.23 Bioactive Potential Of Bacteriocin ( Erwiniocin NA5), On BLD Affected Rice Varieties 113
  6.24 Rice Fields (General View At Niab ( Faislabad, Pakistan ) 114
  6.25 Open Field Trials Showing The Efficacy Of Erwiniocin NA5 Against BLB Infected Rice (Bas-370) 115
  6.26 Cut And Paste Pictures Of The BLB Infected Rice Leaves From Different Rice Varieties 116
8 7 Discussion 117
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  7.1 Isolation And Identification Of Phytopathogenic Bacteria 117
  7.2 Bacteriocins Lbiocontrol Agents: The Emerging Threat For Phytopaths 117
  7.3 Screening For Bacteriocinogenic Strains 119
  7.4 Excluding The Possibility Of The Inhibitory Effect Of Lytic Phage Or 8202 122
  7.5 Titration Of Erwiniocin NA5 122
  7.6 Physicochemical Characterization Of Erwiniocin NA5 123
  7.7 Growth Kinetics 125
  7.8 Mode Of Action Of Erwiniocin NA5 126
  7 .9 Immunity 126
  7.10 Lacuna Counts 127
  7.11 Molecular Weight Estimation Of Erwiniocin NA5 (By Using Dialysis Membrane ) 128
  7.12 Activity Directed Purification Of Erwiniocin NA5 128
  7.13 Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SOS Page) Analysis 131
  7.14 Transmission Electron Microscopy 132
  7.15 Computational Analysis For Amino Acid Composition 133
  7.16 Inhibitory Spectrum Of Erwiniocin 134
  7.17 Determination Of BAC + Marker! Genetic Determinants Of Bacteriocin Production 135
  7.18 Field Trials. 137
9 8 Conclusion 146
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10 9 Expected Benefits 149
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11 10 Bibliography 150
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12 11 Appendices 186
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