GM Maize Crops and Plant Growth-Promoting Characterization of the Isolated Strains

Analysis of Bacterial Diversity in the Rhizosphere of GM and Non- GM Maize Crops and Plant Growth-Promoting Characterization of the Isolated Strains

By NASEER AHMAD

Department of Plant Sciences Faculty of Biological Sciences Quaid-i-Azam University Islamabad-Pakistan 2013

Analysis of Bacterial Diversity in the Rhizosphere of GM and Non-GM Maize Crops and Plant Growth-Promoting Characterization of the Isolated Strains

A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy By NASEER AHMAD

Department of Plant Sciences Faculty of Biological Sciences Quaid-i-Azam University Islamabad-Pakistan 2013

DECLARATION OF ORIGINALITY

I hereby declare that the work accomplished in this thesis is the result of my own research carried out in the Molecular Systematics and Applied Ethnobotany Lab, Department of Biotechnology, Quaid-i-Azam University Islamabad. This thesis has not been published previously nor does it contain any material from the published resources that can be considered as the violation of international copyright law.

Furthermore I also declare that I am aware of the terms ‘copyright’ and ‘plagiarism’ and if any copyright violation was found out in this work I will be responsible of the consequences of any such violation.

Signature: ______

Name: Naseer Ahmad

Date: / /2013

CERTIFICATE

This thesis submitted by Mr. Naseer Ahmad, 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 requirements for the degree of Doctor of Philosophy (Plant Sciences).

Supervisor Prof. Dr. Zabta Khan Shinwari Chairman Department of Biotechnology

External examiner

Chairperson Prof. Dr. Asghari Bano Chairperson Department of Plant Sciences

Date: / /

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This thesis is dedicated with love and gratitude To

My Parents

Who taught me the first word to speak, the first alphabet to write, the first step to take and have raised me to be the person I am today.

AUTHOR DECLARATION

I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners and supervisor. Any errors in the thesis are only mine.

Naseer Ahmad

LIST OF CONTENTS

DECLARATION OF ORIGINALITY ...... ii

CERTIFICATE ...... iii

LIST OF TABLES ...... xvi

LIST OF FIGURES ...... xviii

LIST OF ABBREVIATIONS ...... xx

ACKNOWLEDGEMENTS ...... xxiii

ABSTRACT ...... xxvi

1. INTRODUCTION ...... 1

1.1. Brief summary of the genetically modified (GM) plants ...... 1

1.1.1. Biosafety concerns of GM plants ...... 2 1.1.2. The impact of GM crops on soil microorganisms ...... 2 1.1.3. Impacts of GM plants on environment ...... 4 1.1.4. Impact of GM maize on animals ...... 6 1.1.5. Effects of GM crops on fungi ...... 6 1.1.6. Effects GM crops on non-target insects ...... 7 1.1.7. Cross pollination and gene flow in GM maize crops ...... 8

1.2. Rhizospheric bacterial community ...... 9

1.2.1. Soil as a reservoir for microbial diversity ...... 9 1.2.2. Why soil serves as a habitat for such enormous microbial diversity ...... 9 1.2.3. Rhizosphere bacterial diversity analysis based on cultured based approaches ...... 10 1.2.4. Rhizosphrere bacterial diversity on direct metagenomic based approaches 11

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1.2.5. Next-generation sequencing (NGS) ...... 11 1.2.6. Pyrosequencing based approaches for rhizosphere microbial community analysis ...... 11

1.3. Plant microbe interactions ...... 15

1.3.1. The way plant and microbe interacts with each other ...... 16 1.3.2. Culturing uncultured microbes as a source of secondary metabolites ...... 17 1.3.3. Plant growth promotion characterization ...... 22 1.3.4. Plant growth promoting rhizobacteria ...... 22 1.3.5. Mechanisms of plant growth promotion ...... 22 1.3.6. PGPR as biofertilizers ...... 23 1.3.7. Phosphate solubilization ...... 23 1.3.8. Nitrogen fixation ...... 23 1.3.9. PGPR as phytostimulators ...... 24 1.3.10. Indole acetic acid (IAA) ...... 24

1.4. Functional characterization ...... 25

1.4.1. PGPR as biocontrol agents ...... 25 1.4.2. Biocontrol mechanisms of PGPR ...... 25 1.4.3. Competition for nutrients ...... 25 1.4.4. Antibiotics production ...... 25 1.4.5. Production of lytic enzymes ...... 26 1.4.6. Niche exclusion ...... 26

1.5. Hypothesis ...... 27

1.6. Research objectives ...... 27

2. REVIEW OF LITERATURE ...... 28

2.1. Biosafety issues related to transgenic crops ...... 28

2.2. Transgenics plants and their impacts on soil microbes ...... 30

2.3. Rhizospheric bacterial diversity ...... 31

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2.4. Culture based approaches ...... 33

2.5. Culture independent approaches ...... 33

2.6. Plant microbes interaction ...... 36

2.7. Functional characterization ...... 38

3. MATERIALS AND METHODS ...... 42

3.1. Field experimental design ...... 42

3.2. Rhizospheric soil sampling and processing ...... 42

3.3. Isolation and characterization of rhizospheric bacterial community ...... 43

3.3.1. Isolation and identification of culturable bacterial strains...... 43

3.4. Culture based approach to evaluate bacterial diversity ...... 43

3.4.1. Bacterial culturing medias ...... 43 3.4.2. Tryptic soy broth agar (TSB+A) ...... 44 3.4.3. Low-nutrient R2A medium ...... 44 3.4.4. Mixed media ...... 44 3.4.5. Cultured analysis ...... 45 3.4.6. Colony forming unit (CFU) calculation ...... 45 3.4.7. Sub-culturing and isolation of pure strains ...... 45 3.4.8. Bacterial glycerol stock ...... 46 3.4.9. Bacterial colony counting and identification ...... 46

3.5. PCR of the culture strains for 16S rRNA gene analysis ...... 46

3.5.1. Bacterial strain identification ...... 46 3.5.2. DNA isolation by using direct cell lysis approach ...... 47 3.5.3. Recipe for lysis buffer ...... 47 3.5.4. Bacterial genomic DNA isolation by commercially available kit ...... 47 3.5.5. Selection of PCR primers ...... 48 3.5.6. PCR amplification of bacterial 16S rRNA gene fragments ...... 48

3.5.7. Gel electrophoresis ...... 48 3.5.8. PCR product purification ...... 49 3.5.9. DNA sequencing ...... 49 3.5.10. Sequences analysis ...... 49 3.5.11. Phylogenetic analysis ...... 49

3.6. Metagenomics approach to evaluate bacterial diversity ...... 50

3.6.1. Metagenomic DNA extraction ...... 50 3.6.2. DNA extraction and PCR conformation ...... 50 3.6.3. Pyrosequencing ...... 50

3.7. Characterization of plant growth promotion traits assay ...... 51

3.7.1. IAA production assay ...... 51 3.7.2. Phosphate solubilization ...... 51 3.7.3. Siderophores detection ...... 52 3.7.4. Nitrate reduction ...... 52

3.8. Plant growth promotion traits and in vitro plant growth promotion assay ...... 53

3.8.1. Inoculation procedure and plant growth ...... 53

3.9. Enzymatic characterization ...... 53

3.9.1. Protease activity ...... 54 3.9.2. Cellulase activity ...... 54 3.9.3. Lipase activity ...... 54 3.9.4. Chitinase activity ...... 55 3.9.5. Pectinase activity ...... 55

3.10. Functional characterization of the cultured isolates ...... 56

3.10.1. Antimicrobial characterization...... 56 3.10.2. Antibacterial activity ...... 56 3.10.3. Antifungal activity ...... 56

3.11. Statistical analyses ...... 57

4. RESULTS ...... 58

4.1. Bacterial diversity ...... 58

4.1.1. Analysis of pre sowing rhizospheric bacteria on the basis of 16S rRNA gene sequence ...... 58 4.1.2. Phylogenetic analysis of pre sowing rhizospheric bacteria on the basis of 16S rRNA gene sequence ...... 62 4.1.3. Phylogenetic analysis of vegetative stage collected rhizospheric bacteria on the basis of 16S rRNA gene sequence...... 64 4.1.4. Phylogenetic analysis of vegetative stage rhizospheric bacteria on the basis of 16S rRNA gene sequence ...... 67 4.1.5. Phylogenetic analysis of GM and Non-GM maize harvesting stage rhizospheric bacteria on the basis of 16S rRNA gene sequence ...... 69 4.1.6. Phylogenetic analysis of the isolates at harvesting stage ...... 72

4.2. Metagenomics analysis ...... 74

4.2.1. Pyrosequencing based GM and Non-GM maize rhizosphere bacterial diversity ...... 74 a. Bacterial diversity based on phylum level distribution of field soil sample ...... 74 b. Bacterial diversity based on family level distribution of field soil sample ...... 74 c. Bacterial diversity based on genus level distribution of field soil sample ...... 75 4.2.2. Pyrosequencing based GM and Non-GM maize rhizosphere bacterial diversity during vegetative stage ...... 79 a. Bacterial diversity based on phylum level distribution of GM and Non-GM maize rhizosphere at vegetative stage...... 79 b. Bacterial diversity based on family level distribution of GM and Non-GM maize rhizosphere at vegetative stage...... 80 c. Bacterial diversity based on genus level distribution of GM and Non-GM maize rhizosphere at vegetative stage...... 81 4.2.3. Pyrosequencing based GM and Non-GM maize rhizosphere bacterial diversity during harvesting stage ...... 84

a. Bacterial diversity based on phylum level distribution of GM and Non-GM maize rhizosphere at harvesting stage...... 84 b. Bacterial diversity based on family level distribution of GM and Non-GM maize rhizosphere at harvesting stage...... 84 c. Bacterial diversity based on genus level distribution of GM and Non-GM maize rhizosphere at harvesting stage ...... 85

4.3. Isolates characterization plant growth promoting activity ...... 88

4.3.1. In vitro screening of GM and Non-GM maize rhizosphere pre sowing isolates for plant growth promoting potential ...... 88 a. Characterization of pre sowing isolates on maize shoot and root fresh weigh .... 88 b. Characterization of first stage isolates on maize seedling root fresh weight ...... 88 c. Characterization of first stage isolates on maize seedling shoot dry weight ...... 89 d. Characterization of first stage isolates on maize seedling root dry weight ...... 89 e. Characterization of first stage isolates on maize seedling shoot length ...... 91 f. Characterization of first stage isolates on maize seedling root length ...... 91 g. Characterization of first stage isolates on number of lateral roots of maize seedling ...... 91 4.3.2. Plant growth promotion traits of the pre sowing collected rhizosphere isolates ...... 93 a. Siderophores production ...... 93 b. Nitrate reduction ...... 93 c. Phosphate solubilization...... 94 d. Indole acetic assay (IAA) ...... 94 4.3.3. In vitro screening of isolates from GM and Non-GM maize Rhizosphere during vegetative stage isolates for plant growth promoting potential ...... 96 a. Characterization of vegetative stage isolates on maize seedling shoot fresh weight ...... 96 b. Characterization of vegetative stage isolates on maize seedling root fresh weigh ...... 97 c. Characterization of vegetative stage isolates on maize seedling shoot dry weight ...... 97 d. Characterization of vegetative stage isolates on maize seedling root dry weight 98 xii

e. Characterization of vegetative stage isolates on maize seedling shoot length ... 101 f. Characterization of vegetative stage isolates on maize seedling root length ..... 101 g. Characterization of vegetative stage isolates on number of lateral roots of maize seedling ...... 102 4.3.4. Plant growth promotion traits of the vegetative stage rhizosphere isolated 105 a. Siderophores production ...... 105 b. Phosphate solubilization...... 105 c. Nitrate reduction ...... 106 d. Indole acetic assay (IAA)...... 106 4.3.5. In vitro screening of the isolates from GM and Non-GM maize Rhizosphere during harvesting stage for plant growth promoting potential 109 a. Characterization of harvesting stage isolates on maize seedling shoot fresh weight ...... 109 b. Characterization of harvesting stage isolates on maize seedling root fresh weight ...... 110 c. Characterization of harvesting stage isolates on maize seedling shoot dry weight ...... 110 d. Characterization of harvesting stage isolates on maize seedling root dry weight ...... 111 e. Characterization of harvesting stage isolates on maize seedling shoot length ... 114 f. Characterization of harvesting stage isolates on maize seedling root length ..... 114 g. Characterization of harvesting stage isolates on number of lateral roots of maize seedling ...... 115 4.3.6. Plant growth promotion traits of the harvesting stage rhizosphere isolated 117 a. Phosphate solubilization ...... 117 b. Siderophores production ...... 117 c. Nitrate reduction ...... 118 d. Indole acetic assay (IAA) ...... 118

4.4. Production of cell wall hydrolyzing enzymes by pre sowing GM and Non-GM rhizosphere isolates ...... 121 xiii

4.4.1. Cellulase activities of the isolated bacterial strains ...... 121 4.4.2. Chitinase activities of the isolated bacterial strains ...... 121 4.4.3. Protease activities of the isolated bacterial strains ...... 122 4.4.4. Pectinase activities of the isolated bacterial strains ...... 122 4.4.5. Lipase activities of the isolated bacterial strains ...... 123

4.5. Functional characterization of isolated bacterial strains...... 125

4.5.1. Antifungal activities of pre sowing isolated bacterial strains ...... 125 4.5.2. Antibacterial activities of pre sowing isolated bacterial strains...... 125

4.6. Production of cell wall hydrolyzing enzymes by isolates collected at vegetative stage from GM and Non-GM maize rhizosphere ...... 127

4.6.1. Cellulase activities of the vegetative stage isolated bacterial strains ...... 127 4.6.2. Chitinase activities of the vegetative stage isolated bacterial strains ...... 127 4.6.3. Protease activities of the vegetative stage isolated bacterial strains ...... 128 4.6.4. Pectinase activities of the vegetative stage isolated bacterial strains ...... 129 4.6.5. Lipase activities of the vegetative stage isolated bacterial strains ...... 129

4.7. Functional characterization of isolated bacterial strains...... 132

4.7.1. Antifungal activities of vegetative stage isolated bacterial strains ...... 132 4.7.2. Antibacterial activities of vegetative stage isolated bacterial strains ...... 132

4.8. Production of cell wall hydrolyzing enzymes by isolates collected at harvesting stage from GM and Non-GM maize rhizosphere ...... 134

4.8.1. Cellulase activities of the harvesting stage isolated bacterial strains ...... 134 4.8.2. Chitinase activities of the harvesting stage isolated bacterial strains ...... 134 4.8.3. Protease activities of the harvesting stage isolated bacterial strains ...... 135 4.8.4. Pectinase activities of the harvesting stage isolated bacterial strains ...... 136 4.8.5. Lipase activities of the harvesting stage isolated bacterial strains ...... 136

4.9. Functional characterization of isolated bacterial strains...... 139

4.9.1. Antifungal activities of harvesting stage isolated bacterial strains ...... 139

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4.9.2. Antibacterial activities of harvesting stage isolated bacterial strains ...... 139

5. DISCUSSION ...... 141

CONCLUSION...... 152

6. REFERENCES ...... 153

Appendix 1 Pyrosequencing based bacterial diversity of GM and Non GM maize rhizosphere ...... 185

Appendix: 2A-1 Eztaxon classification of isolated bacterial strains pre sowing stage ...... 213

Appendix: 2A-2 RDP base classification of the pre sowing isolates ...... 214

Appendix: 2B-2 RDP base classification of the isolated from GM and non GM maize Rhizosphere ...... 216

Appendix 3 The GenBank (NCBI) accession number allotted for 16S rRNA sequence of the isolates ...... 219

LIST OF TABLES

Table 1.1:Potential impacts of GM crops on the environment...... 5

Table 1.2: Brief summary of various bacterial groups found in the rhizosphere of a wide variety of plants...... 14

Table 1.3: The list of commercial companies dealing in a variety of products, isolated directly or indirectly from microorganisms...... 19

Table 4.1. Identification of bacterial strains from pre sowing soil samples using analysis of partial 16S rRNA gene sequences...... 60

Table 4. 2: Identification of isolates from GM and Non-GM maize Rhizosphere Bacterial strains using analysis of partial 16S rRNA gene sequences...... 65

Table 4. 3: Identification of isolates from GM and Non-GM maize Rhizosphere during harvesting stage using analysis of partial 16S rRNA gene sequences...... 70

Table 4. 4: Growth promotion traits and in vitro plant growth promotion assay of rhizospheric strains isolated from pre sowing of GM and Non-GM plants...... 95

Table 4. 5: In vitro screening of isolates from GM and Non-GM maize rhizosphere during vegetative stage for plant growth characterization...... 108

Table 4. 6: In vitro screening of the isolates from GM and Non-GM maize rhizosphere during harvesting stage for Plant Growth Promoting activities...... 120

Table 4. 7: In vitro screening of pre sowing isolates for cell wall degrading enzymes production...... 124

Table 4. 8: Antifungal and antibacterial activities of the pre sowing isolates...... 126

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Table 4. 9: In vitro screening of GM and Non-GM maize rhizosphere isolates collected during vegetative stage for cell wall degrading enzymes...... 131

Table 4. 10: Antifungal and antibacterial activities of the isolates from GM and Non-GM maize rhizosphere during vegetative stage isolates...... 133

Table 4. 11: In vitro screening of GM and Non-GM maize rhizosphere isolates collected during harvesting stage for cell wall degrading enzymes...... 138

Table 4. 12: . Antifungal and antibacterial activities of the isolates from GM and Non- GM maize rhizosphere during harvesting stage...... 140

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LIST OF FIGURES

Figure 4-1: Percentage of various bacterial strains isolated from pre sowing soil samples ...... 61

Figure 4-2: The phylogenetic tree of pre sowing isolates was constructed by using the neighbor-joining method...... 63

Figure 4-3: Percentage of various genera isolated from GM and Non-GM maize rhizosphere samples...... 66

Figure 4-4: The phylogenetic tree of isolates from GM and Non-GM maize rhizosphere during vegetative stage...... 68

Figure 4-5: Percentage of various genera isolates from GM and Non-GM maize Rhizosphere during harvesting stage...... 71

Figure 4-6: The phylogenetic tree of the isolates from GM and Non-GM maize rhizosphere during harvesting stage...... 73

Figure 4-7: Bacterial diversity based on family level distribution of (pre sowing) field soil sample...... 77

Figure 4-8: Bacterial diversity based on family level distribution of field soil sample collected after one year interval from GM and Non-GM maize field trial...... 78

Figure 4-9: GM and Non-GM maize vegetative stage rhizosphere bacterial diversity based on phyla's...... 83

Figure 4-10: GM and Non-GM maize harvesting stage rhizosphere bacterial diversity based on phyla's...... 87

Figure 4-11: Effect of pre sowing isolates on maize shoot (SFW) and root (RFW) fresh weight...... 90

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Figure 4-12: Effect of pre sowing isolates on maize shoot (SDW) and root (RDW) dry weight...... 90

Figure 4-13: Effect of pre sowing isolates on maize shoot (SL), root (RL) length and No of roots (RN) per plant ...... 92

Figure 4-14: Effect of isolates from GM and Non-GM maize Rhizosphere during vegetative stage ...... 99

Figure 4-15: Effect of isolates from GM and Non-GM maize Rhizosphere during vegetative stage ...... 100

Figure 4-16: Effect of isolates from GM and Non-GM maize Rhizosphere during vegetative stage ...... 104

Figure 4-17: Effect of the isolates from GM and Non-GM maize Rhizosphere during harvesting stage...... 112

Figure 4-18: Effect of the isolates from GM and Non-GM maize Rhizosphere during harvesting stage...... 113

Figure 4-19: Effect of the isolates from GM and Non-GM maize Rhizosphere during harvesting stage...... 116

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LIST OF ABBREVIATIONS

Abbreviations Full Name

 A Sample collected before cultivation (Pre sowing)  A Soil sample from same field after one year

 AgNO3 Silver nitrate  BAC Bacterial artificial chromosome  oC Centigrade  CAS Chrome azurol S  CCA Colloidal chitin agar  CFU Colony forming unite  cm Centimeter  CMC Carboxy methyl cellulose  CMV Cucumber mosaic virus  EDTA Ethylene diamine tetra acetate  Fe Iron  GM Genetically modified  GMB Vegetative stage GM-maize rhizosphere sample  GMC Harvesting stage GM-maize rhizosphere sample  GMOs Genetically modified organisms  GMPs Genetically modiﬁed plants  HCl Hydrochloric acid  IAA Indole acetic acid  IG Islamabad gold (Maize variety)  IGB Vegetative stage Non GM-maize variety (IG) rhizosphere sample  IGC Harvesting stage Non GM-maize variety (IG) rhizosphere sample  ISR Induced systemic resistance  IW Islamabad white (Maize variety)  IWB Vegetative stage Non GM-maize variety (IW) rhizosphere sample xx

 IWC Harvesting stage Non GM-maize variety (IW) rhizosphere sample  K Potassium  LSD Least significant difference  M Molar  Mg Magnesium  Mg Milligram(s)  MHC Moisture holding capacity  Min Minute(s)  ml Milliliters  mM Millimolar  NaCl Sodium chloride  NCBI National Center for Biotechnology Information  Non-GM Non transgenic  OD Optical density  P Phosphorous  PCR Polymerase chain reaction  PGPB Plant growth promoting bacteria  ppm Part per million  PRAPS Partial ribosomal amplification and pyrosequencing  PVK Pikovskaya medium  RCBD Randomized complete block design  RDP Ribosomal database classifier  RISA Ribosomal intergenic spacer analysis  rpm Revolution per minute  RS-A Pre sowing collected soil sample  RS-BGM Vegetative stage GM rhizosphere soil sample  RS-BIG Vegetative stage Islamabad gold variety rhizosphere soil sample  RS-BIW Vegetative stage Islamabad white variety rhizosphere soil sample  RS-CGM Harvesting stage GM rhizosphere soil sample

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 RS-CIG Harvesting stage Islamabad gold variety rhizosphere soil sample  RS-CIW Harvesting stage Islamabad white variety rhizosphere soil sample  WHO World Health Organization  % Percentage  µ moles/g Micro moles per gram  µg ml-L Microgram per millilitre  µl Microliter

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ACKNOWLEDGEMENTS

I have no words to express my deepest sense of gratitude and numerous thanks to Almighty Allah, who enabled me to complete this study and with innumerable blessing for the Holy prophet peace be Upon Him who is forever a source of guidance and knowledge for the whole humanity.

I express very special thanks to my supervisor, Prof. Dr. Zabta Khan Shinwari Chairman Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad, for his affection, consistent encouragement, sincere attitude and incentive guidance throughout my research work whose guidance, concrete suggestion and incisive criticism made this work possible. I sincerely thanks for his understanding and patience. Besides other, his valuable suggestions for thesis write-up will serve as beacon of light through the course of my life.

I extend my sincere gratitude towards Dr. Muhammad Yasir, for his valuable suggestion and guidance's during my Ph.D research work. I am really very grateful to him for concrete suggestion and consistent help during my entire Ph.D research work. I am impressed from his scientific thinking and politeness.

I also pay my thanks and regards to my ex-supervisor Prof. Dr. Asghari Bano, Dean Faculty of Biological Sciences, Quaid-i-Azam University Islamabad for her affectionate efforts, sympathetic attitude, valuable guidance, support and encouragement.

I would like to record my sincere thanks and grateful appreciation to Dr. Zhanyuan Zhang Director Plant Transformation Core Facility Division of Plant Sciences University of Missouri Columbia- USA for providing research facilities in his lab for six months under IRSIP program sponsored by Higher Education Commission of Pakistan (HEC).

I am thankful to Dr. Gul for his critical comments and valuable suggestion during my dissertation write up.

I am also grateful to Higher Education Commission Pakistan (HEC) for providing me entire financial support to complete this doctoral research work.

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I hereby once again acknowledge to all my teachers who even taught me a single word during my student life and research work.

I want to express my cordial thanks to my lab fellows M. Zada, Imran Afzal, Nadia Rizvi. Nisar, Javed, Hummaira Zafar, Fazal Akbar and Farees Mufti. I will always remember you all for your help, company and cooperation during critical moments. I hereby acknowledge all the members of our research team Samina Bashir, Raees Khan, Riaz Ullah, Muhammad Ibrahim, Rabia Saba and Saira Karimi for their consistent support and encouragement during the critical stage of my career.

I want to express my cordial thanks to my friends and colleagues Dr. Faizan Ullah, Ishtiaq Hussain, Muhammad Adil, Zahid Ullah, Aftab Alam, Sami Ullah, Huusain Badshah, Dr. Samiullah, Syed Jalal Shah, Nasrullah, Adnan Shah, Hammad Pasha, Rahman Ali, Imran Khan, Ilyas Ahmad, and Khurram Shahzad, I will always remember for their help, company and cooperation during critical moments during my carrier. I believe their sincere encouragements, which were a constant source of strength and inspiration to me that despite many constraints, I was able to complete my Ph. D dissertation.

Heartiest thanks to my other Research fellows for their cooperation during critical moments and quick response.

The chain of gratitude would definitely be incomplete if I would not express a deep sense of appreciation for my late Father and ever loving Mother who formed part of my dreams and taught me the good things that really matter in practical life. Father, I am sure you still watch over me and happy memories of you provide me with the strength to carry on. Mother, you know you are the greatest mother ever and I love you so much. Thanks for your constant prayers and strength. I would like to express my deepest thanks to my family, and especially to my brothers Prof. Gulzar Ahmad, Mr. Nisar Ahmad, Mr. Bashir Ahmad and Mr. Shabir Ahmad for collectively nurtured me with tireless love and support before, during, and since I wrote this dissertation. I wish to pay my sincere gratitude to my loving sister Miss. Razia And Miss Ruqaia for their good wishing and

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continue prayers. I would also like to thanks my nephews Waqar Ahmad Irshad Ahmad and Shakeel Ahmad; nieces Saeeda and Saima.

I am very grateful to my fiancee for her moral support and encouragement which is a source of inspiration for me.

Finally as customary the errors that remain are mined alone.

Naseer Ahmad

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ABSTRACT

The rhizosphere is a critical interface supporting the exchange of resources between plants and their associated soil environment. Rhizosphere microbial diversity is inﬂuenced by the physical and chemical properties of the rhizosphere, some of which are determined by the genetics of the host plant. However, within a plant species, the impact of genetic variation on the composition of the bacterial biota of GM and Non-GM maize rhizosphere is poorly understood. Here, we studied the bacterial diversity and population dynamics in the rhizosphere of one GM and two Non-GM maize varieties (IG and IW) grown under field conditions, by traditional cultivation techniques and 16S rRNA gene- based molecular analysis of DNA directly extracted from pre cultivated soil and rhizosphere samples. Rhizosphere and pre cultivated soil samples were taken at three different plant growth stages. The isolated bacterial strains were further screened for different functional characterization.

Using pyrosequencing of bacterial 16S rRNA genes, around 160,000 sequences were obtained (20,000 reads per sample) representing 21 phyla's, 184 families, 469 genera and a small amount of unclassified bacteria. The predominant bacterial groups in the rhizosphere of GM and Non-GM maize as well as in bulk soil were Proteobacteria (39.455%), Actinobacteria (24.453%), Bacteroidetes (11.990%), Firmicutes (7.532%) and Planctomycetes (4.478%). Other groups that were consistently found, although at lower abundance were Fibrobacteres, Thermi, Euryarchaeota, Tenericutes, Synergistetes and Thermotogae. There was no indication of consistent bacterial variation in the rhizosphere of GM and Non-GM maize as well as in bulk soil sample. We observed no significant variation in bacterial richness, diversity, and relative abundances of taxa between different growth stages of GM and Non-GM maize rhizosphere.

Bacterial diversity in the rhizosphere of GM and Non-GM maize as well as bulk soil sample was explored using a culture based approach at different growth stages. A total of 52 bacterial strains were isolated from the rhizosphere as well as from bulk soil samples (different growth stages) and subjected to further analysis. Based on the analysis of the

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16S rRNA gene sequences, all the bacterial isolates were classified into four major phyla's of the domain bacteria. The culturable component of the bacterial community revealed that the predominant groups were Firmicutes and Proteobacteria at Pre sowing stage). Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria (vegetative stage) and Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes (harvesting stage) were the pre dominant groups at different growth stages of GM and Non-GM maize rhizosphere.

Zea mays, one of the most important cereals crop worldwide. The use of plant growth promoting (PGP) rhizobacteria may constitute a biological alternative to increase crop yield and plant resistance to diseases. In search for PGP rhizobacteria strains, all the cultured isolated bacterial strains (52) were in vitro screened for their PGP characteristics and biocontrol against plant pathogenic strains. Some of the bacterial isolates (different stages) were shown to produce indole acetic acid, phosphate solubilization, nitrate reduction and siderophore when tested in vitro for their plant growth promoting abilities. Their further application in a greenhouse experiment using Zea mays indicated that plant traits such as root and shoot elongation and biomass production, were influenced by the inoculation. Plant growth promoting traits of these strains indicated beneficial relationship between rhizobacteria and Zea mays plant.

To understand the antagonistic potential, an in vitro antagonistic assay was performed to characterize and identify strains that were antagonistic to the plant pathogens Fusarium oxysporum, Aspergillus niger and Pseudomonas syringae, Xanthomonas axonopodis. Some of the strains (from different stages) exhibited in vitro inhibitory activity against the target plant pathogenic but none of the isolates were found to have antibacterial activity. Total 46 strains were screened (stages like pre sowing-14, vegetative-19 and harvesting-13) for enzymatic activities. Most of the isolates produced cell wall degrading enzymes. About isolates exhibited cellulase (43%), chitinase (43%), protease (54%), pectinase (63%) and lipase (70%). There was no significant variation in functional characterization of the isolates of GM maize rhizosphere as compared to their Non-GM counterpart.

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INTRODUCTION CHAPTER 1

1. INTRODUCTION

1.1. Brief summary of the genetically modified (GM) plants

Plants are the primary source of life on earth. They provide not only necessary nutrients and protection but also determine the micro climate for soil organisms in the upmost soil layers. Now a days, the GM crops play a very important role in an agriculture sector all around the world. The extraordinary trend toward transgenic crops started during 1996. In 2012, the GM crops were cultivated in 28 different countries covering a total of 420 million acres of land around the globe (ISSA, 2012). There is a change in tradition from having transgenic crops contained only one gene of interest or one introduced novel trait (single trait GM crops towards the combination or stacking different traits or genes in over the last few years (Garbeva, 2004; Leimanis et al., 2008). However, multi traits transgenic plants are observed in different counties by national authorities before the release into environment (Leimanis et al., 2008). Although GM crops have a great impact and potential for commercial use but still there are some concerns regarding to their safety impact on environment (Brusetti et al., 2004a). With the development of agricultural biotechnology and the release of GM crops into the environment help the agriculture sector but on the other hand it also have some drastic effect on the ecosystem some of these are direct or indirect effect on natural flora and fauna. The rapid improvement of agricultural biotechnology and transgenic crops release into the environment have provided many agricultural benefits. However they also raised a multitude of concerns including transgene, direct or non-direct effects on non-target flora and finally the development of resistance in target pests, fauna, microorganisms and ecosystems (Chelius and Triplett, 2001a, b; Elvira-Recuenco and van Vuurde, 2000; Pereira et al., 2011; Rijavec et al., 2007).

Although the above ground effects of GM crops have been studied in detail, underground impacts have also been recognized to some extent as a result of recent methodological advances in soil microbial ecology (Zhang et al., 2005). But due to inherent technical difficulties, the impact of GM crops on soil-associated microfauna, including soil 1

INTRODUCTION CHAPTER 1 microbial communities still signifies one of the least understood areas. Perhaps due to the inherent technical difficulties involved in the study of soil-borne living organisms (Filion. M, 2008).

1.1.1. Biosafety concerns of GM plants

Since the release of first genetically modified plant, there is a strong debate about their influence on microbial population density and diversity primarily due to the introduction of GM products and secondly due to different composition of root exudates (Oger et al., 1997). Other concerns includes that the insertion of tansgene may unintentionally change GM plant properties others then the required one. The composition and growth of soil microorganisms present in rhizosphere are changed, and in turn they may have some environmental effects (Donegan et al., 1995). These changes in composition and growth induce a different community of microorganisms associated the roots (Rengel et al., 1998). Different studies were carried out this aspect to check whether these changes in microbial communities of GM and Non-GM root associate bacteria can catalyze changes in the environment. These kind of studies have great impact to understand the ecology of rhizosoheric micro-organisms and their effect on the environment and also helpful in the management to reduce the potential negative impact of the GM crops (Fang et al., 2007).

1.1.2. The impact of GM crops on soil microorganisms

Releasing GM crops to the environment is a strong debate since very beginning as many concerns over their potential risks on human health and environment have been identified. A variety of studies both cultured based and non-cultured direct metagenomic based approaches have been utilized so far to assess the effects of GM crops on environment. Most of the studies performed so far correlates to Bt crops, as these plants release Cry proteins through roots to their surroundings (Saxena et al., 1999a). These studies also found that the toxin released by these microorganisim bind to clays and humic acids and its insecticidal activity remain for at least 6 months, thus both target and non-target soil organisms are at potential risk (Saxena and Stoztky, 2001). However, some studies have not found any visible evidence about deleterious effects of root

INTRODUCTION CHAPTER 1 released cry proteins on earthworms, nematodes, soil bacteria and saprophytic fungi (Koshella and Stotzky, 2002). Same was the case with the microflora of rice, cotton, soyabean and potato expressing cry proteins (Ferreira et al., 2003; Wu et al., 2004). Furthermore, PLFA, CLPP or CLCP, T-RFLP DGGE and SSCP data obtained on the basis of uncultured method did not showed any effects of Bt crops on the soil microflora (Blackwood and Buyer 2004). In an another study focusing on automated ribosomal intergenic spacer analysis of rhizospheric bacterial communities of Bt and Non-Bt corn plants it was determined that fingerprints of communities exposed to Bt and Non-Bt plant exudates clustered separately after principal component analysis, which indicates that different root exudates have a selective pressure (Brusetti et al., 2004b). DGGE analysis have also showed that differences exist between GM and Non-GM rhizosphere community profile with special emphasis on metabolically active fractions. Microbial activity in terms of carbon released as CO2 have been also shown to be affected by GM crops (Castaldini et al., 2005). Indirect effect of the GM plants includes growth reduction of bacteria (Escher et al., 2000).

Studies on potato lines producing concanavalin A and Galanthus nivalis agglutinin showed a great reducing impact on microbial activity and other physiological profiles significantly. (Griffiths et al., 2000). Moreover, same results were obatained with potato plants expressing the cysteine proteinase inhibitors, developed to control potato-cyst nematode (Cowgill et al., 2002). GM crops having foreign chitinases and glucanases genes have also been found to significantly decrease the number of intraradical fungi, which in turn is accompanied by a tenfold increase in the number of root associated bacteria (Liu et al., 2004).

The impacts of GM plants modified regarding the chemical improvements have been studied very less so far. A study reported that GM potato with modified composition of starch influence Pseudomonas population exhibit in rhizosphere (Milling et al., 2004). However, Medicago Sativa convey a lignin peroxidase encoding gene demonstrated noteworthy distinctions, as for Non-GM plants, in rhizospheric bacterial communities composition and their activity (Di Giovanni et al., 1999). GM alfalfa plants with over- expressing malate dehydrogenase genes were found to pose qualitative changes in 3

INTRODUCTION CHAPTER 1 rhizospheric bacterial communities (Tesfaye et al., 2003). Glyphosate and glufosinate- resistant Brassica napus and corn were not found to induce permanent changes in the composition and diversity of rhizospheric microbial communities (Dunfield and Germida, 2003).

From the above discussion it is clear that the assessment of the impact of genetically modified plants on agricultural and natural ecosystems should be assessed. Monitoring particular impacts of single transformation events on the support of distinctive studies using different target and non-target organisms. Besides, multimodal trial methodologies including the evaluation of biochemical, physiological and molecular parameters ought to be done.

1.1.3. Impacts of GM plants on environment

Since the first commercial release of GM plants into the environment, one of the major debates concerning the potential threats of GM plants is that how they affect the environment. A variety of questions that arise in this case includes, might they be able to influence non-target insects? Their outcross ability to produce uncontrollable weeds? or have adverse effects on wildlife?. How can they benefit the environment by reducing certain chemical contributions into agriculture?.

The essential inquiries regarding natural effect are the same regardless of the strategy for plant breeding used: what is the way of the genetic change being made, how is the genetic structure and physical appearance of the plant in question altered, and what is/are the natural results of that change. The environmental impacts of the GM plants can be divided into two categories, namely direct and indirect (Philip et al., 2002). However a brief summary of both direct and indirect effects of GM plants on environment has been summarized in

Table 1.1.

INTRODUCTION CHAPTER 1

Table 1.1:Potential impacts of GM crops on the environment.

Class Example Direct impact of novel traits on the environment Chemical interaction with living Non-target effects of insect resistance things Fate and consequences of insecticidal toxins in soil Changes in persistence or Persistence in agriculture habitat (weediness) invasiveness of the crop Invasiveness in natural habitats Gene flow by pollination to weeds Transfer of herbicide tolerance to weeds and feral plants Transfer to biotic and abiotic stress tolerance to weeds or feral species

Indirect impact of changing agricultural practices on the environment Reduce the efficiency of pest, disease Development of weeds tolerant to herbicides by and weed control evolution and selection from within the weed genepool Development of resistance to Bt toxins in pests Effect on wildlife biodiversity Effect on broad spectrum herbicides Effect on soil and water Change in herbicide use Change in sol level cultivation patterns *Impacts can be detrimental of beneficial. Risk assessment focuses primarily on the analysis of potential negative effects on the environment. Source of table: (Dale et al., 2002)

INTRODUCTION CHAPTER 1

1.1.4. Impact of GM maize on animals

The first commercial scale planting of insect resistant corn hybrids commonly known as Bt corn arises in 1996. The genetic modification made to these plants was scientifically termed as “Event 176” which is related to Cry1Ab expression in plants that enables them to be insect resistant. In order to determine the adverse effects that may be posed by the GM maize, a variety of experiments were performed.Brake and Vlachos (1998) take chickens as the feeding hosts, but no significant toxic effects were observed. Subchronic animal feeding studies were performed in female BN rats and B10A mice by Teshima et al. (2002) in order to examine the effect of genetically modified corn CBH351 on the immune system, which contains the Cry9C protein derived from Bacillus thuringiensis subspecies tolworthi. From the results it was concluded that no immunotoxic activity was detected in the GM-corn-fed rats and mice in this subchronic dietary study. Hammond et al. (2004) also reported that GM crops are as safe as Non-GM crops after assessing the dietary effects on rats. It was found by Mendoza et al. (1998) that utilization of hereditarily adjusted, low-phytic acid strains of maize, may enhance iron retention in human population that expend maize-based diets, incorporating those that are reliant fundamentally on plant-derived diets.

1.1.5. Effects of GM crops on fungi

The effects of GM plants on symbiotic fungi have been studied recently. Especially Arbuscular mycorrhizal fungi which establishes mutualistic symbiosis with a variety of plants are considered as one of the important non target organisms in this case (Giovannetti and Avio, 2002). An experimental model framework developed utilizing this class of plant symbionts was approved utilizing transgenic corn. Different stages of AM fungi life cycle were focused in this case namely host recognition responses, spore germination and pre-symbiotic development, tainting structures separation and host root colonization (Turrini et al., 2004). On the other hand aubergine plants transformed to constitutively express the defensin Dm-AMP1 were assessed for their effects on saprophytic fungi. The defensins released by these plants roots were found to stop the

INTRODUCTION CHAPTER 1 development of phytopathogenic fungi by lessening hyphal stretching and by binding to particular sphingolipid locales of hyphal membrans (Thevissen et al., 2004).

In an another study Bt11 and 176 lines lessened the measure of viable tainting hyphae accompanied by the preparation of functional intraradical mycelium, by 72% and 67%, respectively as contrasted with Non-GM plants (Turrini et al., 2004). In an additional study two Nicotiana sp. genetically modified for PR-2 proteins and chitinases activity were studied separately in order to determine whether these plants effects symbiotic fungi or not. Results showed that tobacco plants modified for the expression of PR-2 proteins demonstrated a delayed root colonization by AM fungi regarding non-GM plants (Medina et al., 2003), whereas no significant effects were observed in case of the plants expressing chitinase gene.

1.1.6. Effects GM crops on non-target insects

One of the most significant studies carried out on the assessment of the potential hazard of the GM crops on non-target species includes the detrimental effects of Bt insecticidal proteins on the monarch butterfly. In 1999 a laboratory study reported that "Bt maize" was a potentially danger to monarch butterfly larvae consuming milkweed leaves (Asclepias sp.) with pollen from corn holding a Bt gene sprinkled on the milkweed leaf surface (Losey et al., 1999). However, on the basis of this initial study overall ecological consequences cannot be predicted. Thus, more non target species should be emphasized in this regard.

Other studies in this field are based on determining the effects of Bt toxin on other pests and insect predators. Research by Hilbeck et al. (1998) reported that first, lacewings reared on Ostrinia nubilalis (corn borer, ECB) or Spodoptera littoralis that had ingested BtCry1Ab toxin expressing corn leaves, showed delayed development accompanied by increased mortality and second, high concentrations of Cry1Ab in an synthetic diet fed directly to lacewing larvae were harmful. However these laboratory studies also highlight the difficulties of conducting environmentally relevant laboratory experiments therefore, caution must be used while illustrating conclusions from results of small sets of laboratory experiments that usually test non-target species under unnatural controlled 7

INTRODUCTION CHAPTER 1 conditions or focus on hazard singly, without bearing in mind the level of exposure that occurs under natural environmental conditions (Philip et al., 2002).

1.1.7. Cross pollination and gene flow in GM maize crops

Another concern related to GM plants is that of cross pollination, as they may cross hybridize with compatible species and thus by producing hybrids and their progeny. They may affect the environment. However the four basic elements in this area that should be kept in mind include the distance travelled by the pollen, synchrony of flower between two plants, sexual compatibility and environmental condition of the recipient. The plausible outcomes of hybridization between oilseed rape and related species have been broadly evaluated, it has been verified that oilseed rape can hybridize with hoary mustard, with wild radish and with other wild brassicas species (Scheffler et al., 1995). Maize is potentially the world's most paramount food crop, with worldwide production of ∼700 million metric tons in 2005, which is 10% more than wheat and rice production (Faostat, 2006). Maize (Zea mays) is a monoecious allogamous plant which is progressively upgraded by the expression of technological traits, namely resistance to pests, vaccines production, enzymes and pharmaceuticals (Bates et al., 2005). Under such conditions, establishing robust measures to guarantee appropriate confinement of GM crops is of utmost importance (Ervin et al., 2003). In fact, pollen dispersal can lead to the transmission of foreign genes to conventional genotypes which are in vicinity to GM fields (Eastman and Sweet, 2002).

Gene flow in maize could be followed by measuring the rate of cross fertalization rates from the source plant to the recipient plant. Pollen intervened gene flow to non-transgenic plants of the same species is especially important in maize on account of its biology and pollen attributes. A single maize inflorescence handles from 2-5 million pollen grains (Goss, 1968) and pollen shedding may last for 5–6 days (Westgate et al., 2003) and is normally not synchronous with silking, conditions that normally favors a high level of cross-fertilization. A study on the impact of gene flow on the expression of Bt toxin was performed by utilising cross pollination between MON810 a GM maize variety and non- GM white maize variety. Results showed that the F1 plants from Non-GM variety after

INTRODUCTION CHAPTER 1 cross pollination were found to express even a significantly lower level of Cry1AB compared to the parental variety but the trend in Bt production was more likely to that of the Bt parent plants. Moreover an increasing level of Bt was determined throughout the developmental stages of the plant. Thus reduced levels of this Bt toxin may lead to a sub- lethal dose of Cry1Ab. This may contribute to target insect resistance development (Bates et al., 2005).

1.2. Rhizospheric bacterial community

1.2.1. Soil as a reservoir for microbial diversity

In order to define microbial diversity, several criteria are used for example phylogeny, metabolism, physiology, and genomics. However diversity is usually defined as the sum of all different species in a given environment. Phylogenetic surveys have revealed that the number of prokaryotic species present in a single sample exceeds that of all known cultured prokaryotes to date (Daniel, 2005). The total number of prokaryotic cells on earth are expected to be about 4–6 ×1030 (Whitman et al., 1998) which are considered to comprise between 106-108 distinct genospecies (Amman et al., 1995). One gram of thick forest soil contains approximately 4×10 7 prokaryotic cells (Markewitz and Richter, 1998) and the same amount of soil in addition contains about 2000-18000 distinct prokaryotic genomes (Torsvik and Ovreas, 2006). This diversity suggests a huge (and largely untapped) genetic and biological pool that can be explored for the recovery of novel genes, and their products namely; bioactive metabolites, enzymes in special, and entire metabolic pathways controlling their production (Cowan, 2000).

1.2.2. Why soil serves as a habitat for such enormous microbial diversity

Soil is composed of mineral particles of different sizes, shapes and chemical compositions, along with the soil biota (the organisms that occupy the ecosystem) and finally innumerable organic compounds in various stages of compositions. Dominant structural characteristics of the soil matrix includes the formation of clay-organic matter complexes, stabilization of clay, sand and silt particles through the formation of

INTRODUCTION CHAPTER 1 aggregates, ranging from about ≥ 2 mm to fractions of a micrometer for bacteria and colloidal particles. Prokaryotes are the most abundant organisms on earth surface and are able to form the largest component of soil biomass (Daniel, 2005).

In Soil microorganisms often strongly adhere or adsorb onto the surface of various soil particles including sand grains or clay organic matter complexes. Microhabitats for soil microorganisms include either the surfaces of soil aggregates or they may reside inside the aggregates, also they occupy the complex pore spaces in between (Daniel, 2005). Owed to size limitations some of the pore spaces are unapproachable for microorganisms. Primarily the metabolism and the survival of soil microorganisms in addition are strongly influenced by the water and nutrients availability. Compared to aquatic habitats, surfaces of soil environments undergoes intensive cyclic changes in water content, ranging from water clogging to extreme drought. During each drying and wetting cycle, a fraction of the microbial community is perished. Consequently the composition of soil microbial communities are affected, depending on changes either in the water content, or other environmental factors namely pH, oxygen availability or temperature, however the effects of these factors has not been studied intensively so for (Daniel, 2005).

To archive this immense microbial diversity and the relating gene pools, there must be substantial scale of soil studies. The flexibility of these unexplored soil microorganisms is additionally vital for biotechnological industry, as soil microbes have been the primary source of natural products, incorporating antibiotics, enzymes and other bioactive components (Daniel, 2005).

1.2.3. Rhizosphere bacterial diversity analysis based on cultured based approaches

Culture based approaches revealed that bacteria in rhizosphere are likely to be present one or two fold more than bulk soil. These bacteria mainly include gram negative symbionts and r strategists (Curl and Truelove, 1986). Nutrient factor is responsible for the presence of a wide variety of physiologically different bacterial species in the rhizosphere. Various cultured based studies have isolated a variety of gram positive and gram negative bacteria both r strategists (copiotrophs) and k strategists (oligotrophs) (Semenov et al., 1999 ). Isolation and cultured based approaches applied in case of grass 10

INTRODUCTION CHAPTER 1 rhizosphere community analysis using diluted TSB medium and soil extract have revealed that mostly Proteobacteria, Actinobacteria and Firmicutes resides in these plants rhizosphere (Nijhuis et al., 1993). These isolates were tested for their colonization ability and were found potent to be colonizing on grass roots.

1.2.4. Rhizosphrere bacterial diversity on direct metagenomic based approaches

16S rRNA gene analysis is one of the most to date, efficient, applied, time and cost effective method for studying total rhizosphere community. In a study of Medicago sativa rhizosphere soil and Chenopodium album based on 16S rRNA gene sequences from isolates and library clones derived from rhizosphere DNA extracts has revealed that the most abundant isolates in this case were Variovorax sp., strain WFF052 (β proteobacteria), Acinetobacter calcoaceticus (γ proteobacteria) and Arthrobacter ramosus (high GC Gram-positives). Howevere many other studies have found out that the Proteobacteria and the Actinobacteria form are the most common of the dominant populations in the rhizosphere of many plant species (Singh et al., 2007). In case of Pinus contorta nineteen percent of a 16S rRNA gene-based clone library was found to originate from Acidobacteria (Chow et al., 2002). In an another study Acidobacterial sequences were found relatively higher in the rhizosphere of Thlaspi caerulescens, Lolium perenne and Castanea crenatam (Gremion et al., 2003). Verrucomicrobial sequences were found to be present in the rhizosphere of Pinus contorta (Chow et al., 2002), T. caerulescens (Gremion et al., 2003), L. truncatulum and L. xanthoconus (Stafford et al., 2005).

1.2.5. Next-generation sequencing (NGS)

1.2.6. Pyrosequencing based approaches for rhizosphere microbial community analysis

Most recent advancements in NGS technologies have made conceivable that various strategies could be utilized for sequencing, despite the fact that with variable expenses and proficiencies. The ﬁrst instrument dependent upon the 454 pyrosequencing technology called GS20, was proficient to sequence up to 25 million bases in a 4 h run

INTRODUCTION CHAPTER 1 time, with normal read lengths of 110 bp accompanied with 96% crude read accuracy (Margulies et al., 2005). A more recent model 454 GS-FLX sequencer utilizing Titanium chemistry that read lengths of up to 500 bp, with anticipated enhancements in read length expected. By comparison, the Illumina Solexa platform dependent upon ﬂuorescently named sequencing by synthesis generates 35-76 bp on average.

Recent developments in non-culture analytical tools have greatly expanded bacterial quantification and identification. Those molecular based methods, pyrosequencing has proven a novel non-culturable technology that could be used to not only measure the biodiversity of microorganisms, but also to characterize exposure to these microorganisms in occupational settings (Nonnenmann et al., 2010). The bacterial tag- encoded flexible (FLX) amplicon pyrosequencing (bTEFAP) approach utilizes the ribosomal DNA 16s gene for phylogenetic analysis. These highly conserved structures can be used to identify individual genera or species of bacteria from varied and diverse samples. In addition to being an exceptionally sensitive method, pyrosequencing is relatively low in cost and allows for a relatively quick turnaround in the identification, distribution and concentration of bioaerosols (Nonnenmann et al., 2010). However, reliable sampling methods in use with pyrosequencing have yet to be investigated. A standardized sampling protocol for use with bTEFAP would be highly valuable in providing occupational scientists a novel and effective tool in the quest to characterize bioaerosol exposure and its subsequent relationship to worker health.

The more latest version from Applied Biosystems of the short read sequencer, called the SOLiD4, generates 100 Gb per run accompanied by read length of 50 base pairs. Though the specaility of NGS technologies is that these provide good overall coverage for single genomes, but on other side the short read lengths can be a serious limitation for efﬁcient assembly of metagenomic sequences.

Teixeira et al. (2010a), used 454 Pyrosequencing approach in order to study the rhizosphere bacterial communities of Colobanthus quitensis (Kunth). From results they concluded that Firmicutes was the most abundant phylum. Besides some phyla that are usually in greater part in generally mild and tropical soils, for example Acidobacteria,

INTRODUCTION CHAPTER 1 were barely introduce in the analyzed examples. Analyzing all the sample libraries together, the leading genera discovered were Arcobacter (phylum Proteobacteria), Bifidobacterium (phylum Actinobacteria) and Faecalibacterium (phylum Firmicutes).

Using high-throughput next generation 454 pyrosequencing Uroz et al. (2010) reported that the generally plentiful bacterial groups in oak rhizosphere were Acidobacteria. Alternately, more unclassiﬁed microbes were documented in the bulk soil as contrasted with the rhizosphere, speaking to that the soil remains a challenging reservoir of complexity.

INTRODUCTION CHAPTER 1

Table 1.2: Brief summary of various bacterial groups found in the rhizosphere of a wide variety of plants.

Taxon Abundance (% of Quantification References total) methods Proteobacteria 27-66 Clone libraries Chow et al. (2002): Filcon et al. (2004): McCaig et al. (1999) Α 1-24 Clone libraries Di Cello et al. (1999): Ramette et al. (2005) Β 2-9 Clone libraries Burkholderia Up to 93b Clone libraries, isolates, colony hybridization Γ 0-9 Clone libraries Pseudomonas 0-19 Flourscent insitu Gochnaeur et al. hybridization, isolates (1989): Watt et al. (2003, 2006) Δ 2-3 Clone libraries Acidobacteria 0-65 Clone libraries Lee et al. (2008): Singh et al. (2009) Actinobacteria 1-3 Clone libraries Chelius and triplett. (2001): Singh et al. (2007): Stafford et al. (2005) Cytophage- 0-3 Clone libraries Chow et al. (2002): flavobactrium- Marilley and Aragano Bacteroridetes (1999) Verrucomicrobia 0-3 Clone libraries Chow et al. (2002): Lee et al. (2008) Unclassified 15-30 Clone libraries Source of table: (Buee et al., 2009)

INTRODUCTION CHAPTER 1

1.3. Plant microbe interactions

Microorganisms are essentially important parts of the soil habitat where they assume enter parts in environment by regulating supplement cycling responses irreplaceable for supporting soil fertility, and they additionally add to the establishment and upkeep of soil structure (Kirk et al., 2004). Especially in arable soils microbial community play a key role in biologically mediated processes namely; degradation of organic residues, transformation of organic matter, mineralization of nutrients, formation of soil aggregates and soil nutrient balance. Other well-known functions of soil microorganisms important for soil fertility includes catalysis of nutrient transformations, formation and stabilization of soil structure, storage of nutrients and finally control of plant pathogens (Gomoryova et al., 2009).

Soil microbial communities have great potential for temporal and spatial change (Garland, 1997). Also, microbes interact with plants throughout their whole life cycles and thusly can go about as exact and quick indicator of ecological progressions.

Plants have a huge impact on the size and composition of the microbial community and their distribution in ecosystem. Bacteria are the dominant soil organisms accounting for more than 80% of the overall biomass of most soil environments. They largely determine the functioning of terrestrial ecosystems, and they have direct interactions with plants, thereby creating a strong feedback between plants and microorganisms. There is a global hypothesis and common scientific saying that the upper ground biodiversity controls the underneath ground diversity. So the genetic make-up of plants seems to have an impact on the community structure of soil microorganisms. In this regard the study of the effects of genetically modified plants (GM) on the diversity of microbes in soil will be worthy to predict the possible risk associated with the field cultivation of GM crops (Saha et al., 2007).

Plants depend on upon the capability of roots to communicate with microorganisms through a micro-zone at the root-soil interface and alternately numerous bacteria and fungi are subject to companionships with plants that are regularly managed by exudates of root (Bais et al., 2004). Rhizosphere which is a complex habitat composed of the 15

INTRODUCTION CHAPTER 1 fraction of soil adhering to plant roots and altered by root activity, hosts a complex network of microorganisms which interacts very closely with plant roots (Brusetti et al., 2004b). It is well known fact that rhizosphere exerts a selective pressure on the associated microbial communities which in turn have a great influence on root biology.

Also they impact plant development, nutrition and development and are essential for enduring supportability of agricultural soils (Mantelin and Touraine, 2004).

Various studies have cleared that the rhizosphere has much higher population densities of bacteria and fungi contrasted and bulk soil (Semenov et al., 1999 ).

1.3.1. The way plant and microbe interacts with each other

Plants modify their rhizosphere environment by releasing root exudates (Secondary Metabolites) (Uren, 2001). Ingredients of these exudates mainly includes amino acids, fatty acids, carbohydrates and nucleotides, other compounds may include steroids, growth hormones, organic acids, flavones and enzymes, whose effects still needs to be studied in detail (Zhang et al., 2005). This complex mixture of organic compounds acts a source of decreased carbon, nitrogen, and other different nutrients for soil occupying microorganisms (Buyer et al., 2002). The composition of compounds which are discharged from the roots and the way they interact with in the rhizospheric bacteria differ relying upon the plant species, cultivar, and physiological status of the plants (Saxena et al., 1999a). Root exudates might increase both positive and negative interactions in the rhizosphere. The positive connections incorporate upgrade of bacterial development rates (Hartwig et al., 1991) acting as chemo-attractants (Dharmatilake and Bauer, 1992), and finally as transcriptional signals in the communication between soil bacteria and host plants during nodule formation and biological nitrogen fixation by plants (Redmond et al., 1986). Plant-microbe interactions often involve the release of bactericidal substances and that may include toxic components like benzofurans, terpenoids, butyrolactones, and other phytoalexins, leading to bacterial growth suppression or death (Bowen and Rovira, 1991). The quality and quantity of exudates (mainly carbon compounds) in the rhizosphare governed the microbial communities (Maloney et al., 1997). Root exudation may make a 16

INTRODUCTION CHAPTER 1 particular force that impacts which microorganisms colonize the rhizosphere, in this manner bringing about the alteration in the creation and differences of rhizomicrobial communities in more probable plant-specific way (Grayston et al., 1998).

As little contrasts that exist between different cultivars of the same plant species may cause changed organization of root exudates, which can at last bring about the event of distinctive microbial neighborhoods in the rhizosphere (Rengel et al., 1998).

1.3.2. Culturing uncultured microbes as a source of secondary metabolites

About 50,000 known secondary metabolites have been characterized till now and mostly these are produced by microorganisms, especially bacteria (Berdy, 1995). Structurally and functionally diverse bioactive compounds have also been isolated from a variety of prokaryotic organisms, including members of the cyanobacteria (i.e., Nostoc) and myxobacteria (i.e., Sorangium), including antibiotics with antiviral, antimicrobial and antitumor activities (Reichenbach, 2001).

Enzymes produced by microorganisms, either through large-scale fermentation or recombinant DNA technology are involved in the production of more than 500 products. These products are involved in 50 different industrial applications (Cherry and Fidantsef, 2003). Today, more than 90% of industrial enzymes are produced by microorganisms in order to boost production economy and product purity (Cherry and Fidantsef, 2003). Large numbers of new enzymes are discovered by Short and co-workers through cloning genes directly from the environment, thereby retrieving the remarkable microbial diversity of yet-to-cultured microorganisms.

NovoBiotic (Cambridge, MA, USA), a biotechnological company is using the diffusion chamber technology, to grow uncultured actinomycetes as a source of new antibiotics. Thus soil cultivation of microbes has been proved beneficial. Soil serves as a source of various biotechnologically potent microorganisms capable of producing a large variety of important enzymes and antimicrobials.

Moreover “Recombinant Bio catalysis limited” and “TerraGen Discovery Inc.” are dominant in the field in question; especially the former one is the well-recognized leader, 17

INTRODUCTION CHAPTER 1 involved in the production of a variety of biotechnologically important enzymes (Table 1.3). Many other smaller biotechnology companies (Table 1.3) seems to be competing in the same market sector (Cowan et al., 2005b).

INTRODUCTION CHAPTER 1

Table 1.3: The list of commercial companies dealing in a variety of products, isolated directly or indirectly from microorganisms.

Company Target products class Product and Commercial market intrest BASF Enzymes Amylase, Acidophilic Food industry. www.corporate.basf.com Hydrase glucoamylase Aiding with digestion of starch Bioresearch Italia, SpA, Anti infectives N.D Dalbavancin Development of Italy human genes targeting novel and therapeutic anti infective B.R.A.I.N Bioactive N.D Nitrile Degussa AG www.brain.biotech.de peptides and hydratases. partnership for enzymes for Cellulases the industrial pharmaceuticals processes and agrochemicals Cubist pharmaceuticals Anti-infectives N.D N.D Various www.cubist.com commercial relationships, variety of products in stage 2 and stage 3 trails Diversa Enzymes Nitrase, Discovery of Drug lowering www.diversa.com greater than cholesterol levels, 100 nitriliases Broad spectrum glycosidase, Production of β-mannanase and lipitior β-glucunase phytase Pyrloase 160, added to animal

INTRODUCTION CHAPTER 1

Phyzyme feed to breakdown in digestable pyhtate in grains and oil seeds to release digestables Biometabolites Flourescent Green FP and Novel green and proteins Cyan FP Cyan flourscent proteins for the potential use in drug discovery Diversa and Invitrogen Enzymes DNA ThermalAce Research and www.invitrogen.com polymerse and Replicase diagnostics DNA for research diagnostics EMetagen Enzymes, Polyketides Gene and Food and www.emetagen.com antibiotics, pathway banks, agricultural small active lalrge clone research and molecules DNA libraries other commercial encoding applications biosynthetic pathways for 5000-20,000 secondary metabolites Kosan technology Antibiotics Polyketides Adriamycin, Therapeutic drugs www.kosan.com erythromycin, mevacor, rapamycin Genecore Enzymes Lipases, Washing Cleaning industry www.genecore.com proteases powder and alkaline

INTRODUCTION CHAPTER 1

tolerant portease Libragen Antibiotics and N.D Antiinfectives Medicine and www.libragen.com biocatalysis for and antibiotic synthesis of pharmaceuticals discovery pharmaceuticals Prokaria www,proteus.fr Enzymes, Not Research and Anti antibiotics, specified Diagnostic pyhtopathogenic antigens products for fungal agent, agricultural development of and novel environment, biomolecules food, medical and chemical industries Xanagen Libraries Gene Unspecified Services in www.xanagen.com products library construction, screening and annotation Source:(Cowan et al., 2005a)

INTRODUCTION CHAPTER 1

1.3.3. Plant growth promotion characterization

1.3.4. Plant growth promoting rhizobacteria

The term plant growth-promoting rhizobacteria (PGPR) was first used by Kloepper and Schroth in 1978. They defined it as a group of rhizobacteria that readily colonize roots and enhance plant growth. According to the mode of action, PGPR have been divided into two groups: PGPR that directly affect plant growth, seed emergence, or improve crop yields and biocontrol PGPR that indirectly benefit the plant growth (Glick et al., 1999). In direct methods PGPR directly contributes in enhancing plant growth by secreting phytohormones or increasing the availability of nutrients while in indirect method PGPR helps plant by suppressing phytopathogens or inducing plants to express defence response against phytopathogen. Based on their mechanism of action, PGPR can be categorized into three general groups namely biofertilizing PGPR (microorganisms with the ability to fix nitrogen, solubilize phosphorous complexes or solubilize some other essential mineral), phytostimulating PGPR (microorganism with the ability to produce phytohormones) and biopesticide (microorganisms that promote plant growth by suppressing phytopathogenic growth) (Bhattacharyya and Jha, 2012).

1.3.5. Mechanisms of plant growth promotion

PGPR exert beneficial effects on plant growth by using one or more mechanisms of actions. These mechanisms may have a direct or indirect effect on plant growth. Direct mechanisms include facilitating nutrient availability like phosphorus for plant uptake, nitrogen fixation, iron sequestration (i.e. acting as biofertilizer) or secreting phytohormones (i.e. phytostimulation) (Antoun and Kloepper, 2001). While in indirect mechanisms PGPR suppress or prevent the deleterious effects of phytopathogens through the production of antibiotics, siderophores, cell wall hydrolyzing enzymes, cyanide and eliciting induced local or systemic resistance (Whipps, 2001a). A particular PGPR may enhance plant growth using one or more of these mechanisms simultaneously or at different stages of the life cycle.

INTRODUCTION CHAPTER 1

1.3.6. PGPR as biofertilizers

Biofertilizer is a contraction of term Biological Fertilizer. It has been defined as any microorganism aiding in the supply and availability of primary nutrients to the host plant upon its application to soil, seed or plant surface by colonizing the interior of the plant or its rhizosphere (Vessey, 2003b).

1.3.7. Phosphate solubilization

Phosphorous (P) is second in mineral nutrients that limit the growth of terrestrial plants. Although soils are usually rich in total P but only a small amount is available for plants as large amounts of soil P is in insoluble (Glass, 1989). Even soluble inorganic phosphorous applied as chemical fertilizer is immobilized soon after its application (Glick, 2012). Rhizosphere is usually rich in phosphate solubilizing bacteria which secret organic acids and phosphatases facilitating the conversion of insoluble forms of P to plant-available forms (Kim et al., 1998). Typically, the solubilization of inorganic phosphorus occurs due to the action of organic acids secreted by various soil bacteria and mineralization of organic phosphorus occurs through the production of a number of phosphatases, catalysing the hydrolysis of phosphoric esters (Rodriguez and Fraga, 1999). Agricultural microbiologists are taking huge interest in utilizing the P-solubilizing strains of rhizobacteria for enhancing P uptake of crops (Stefan et al., 2013).

1.3.8. Nitrogen fixation

Nitrogen is an essential component in plant development and is usually considered as one of the key nutrient limiting plant growth (Santi et al., 2013). Although molecular nitrogen is abundantly available in (about 80% of total gases) atmosphere but is inaccessible to plant as it cannot directly utilize molecular nitrogen (N2). Plants utilize nitrogen in the form of ammonium and nitrates by absorbing it through the roots from soil (Drogue et al., 2012).

Microorganisms improve nitrogen availability to plants by a process known as biological nitrogen fixation. It is defined as the process of conversion of atmospheric nitrogen to a

INTRODUCTION CHAPTER 1 form that can be easily utilized by plants. In nature some prokaryotes are responsible for carrying out this process and hence are known as biological nitrogen fixers (Lam et al., 1996). Such microorganisms are known as diazotrophs. They have the ability to colonize rhizosphere and interact with the host plant.

1.3.9. PGPR as phytostimulators

Plants respond to their environment by secreting phytohormones which play an important role in their growth and development (Davis et al., 2004). They face a number of biotic and abiotic stresses during their lifetime in order to decrease the negative effects of these stresses they try to adjust their endogenous phytohormones levels (Glick et al., 2007). Research have shown that some rhizospheric microorganisms produce phytohormones under in vitro conditions. Many PGPR have been reported to possess the ability to produce phytohormones hence they alter the phytohormone levels of plants affecting plant’s hormonal balance and its response to stress. This phenomenon has been termed as phytostimulation and PGPR with this activity are known as phytostimulators (Bloemberg and Lugtenberg, 2001).

1.3.10. Indole acetic acid (IAA)

Indole acetic acid (IAA) affects the physiology of plants in a number of ways. It controls processes of vegetative growth; affects plant cell division, extension and differentiation; increases the root development and initiates lateral and adventitious root formation; it increases the rate of xylem, mediates responses to light, gravity and florescence; it also affects the biosynthesis of various metabolites, pigment formation, photosynthesis and resistance to stressful conditions (Spaepen and Vanderleyden, 2011). Furthermore plant response to IAA is tissue dependent (Pilet & Saugy, 1987). IAA secreted by PGPR has the ability to change the plant’s endogenous pool of IAA. The endogenous levels of IAA in plant roots may be optimal or suboptimal for growth hence additional IAA acquired from PGPR may stimulate or suppress the plant growth resulting in plant growth promotion or inhibition, respectively (Pilet and Saugy, 1987). Generally PGPR secreted IAA increases plant’s access to soil nutrients by increasing root surface area and length.

INTRODUCTION CHAPTER 1

It increases root exudation by loosening plant cell walls which helps in providing nutrients to rhizospheric bacteria (Mathesius et al., 1998).

1.4. Functional characterization

1.4.1. PGPR as biocontrol agents

Plant diseases cause 10-12% loss in production, using rhizobacteria as biocontrol agents means that they can reduce or prevent plant diseases that are caused by fungi or bacteria (Bloemberg and Lugtenberg, 2001). Therefore these PGPR indirectly enhance plant growth by suppressing diseases. Number of mechanisms have been used by PGPR to suppress the growth of pathogenic organisms which include niche competence, production of lytic enzymes, siderophores, antimicrobials, cynide or stimulation of host plant’s defenses called induced systemic resistance (ISR).Well known biocontrol PGPR are members of Pseudomonas, Bacillus and Streptomyces (Antoun et al., 1998).

1.4.2. Biocontrol mechanisms of PGPR

Mechanisms used by biocontrol agents to suppress growth of phytopathogens are discussed in following section.

1.4.3. Competition for nutrients

Under iron limiting conditions some PGPR produce siderophores which are iron chelating compounds. These siderophores sequesters available iron by forming siderophore and iron complex and hence limit the growth of phytopathogens (Osullivan and Ogara, 1992). Such PGPR are thought to be effective as they metabolize nutrients more rapidly and out compete fungal pathogens for available iron (Glick, 2012).

1.4.4. Antibiotics production

Synthesis of antibiotics to prevent phytopathogenic growth is one of the most effective mechanisms that PGPR employ. Some biocontrol strains with this property have been commercialized. However increased use of such strains may pose a problem of antibiotic 25

INTRODUCTION CHAPTER 1 resistance among phytopathogens. To prevent this from happening researchers have designed an alternative approach of utilizing biocontrol bacteria producing hydrogen cyanide along with production of one or more antibiotics. The synergistic effect of both of these secretary products made it an effective approach (Glick, 2012).

1.4.5. Production of lytic enzymes

Rhizobacteria produce range of defence enzymes like chitinases, β 1,3- glucanases, cellulases, proteases and lipases (Bashan et al., 2005). These enzymes have the ability to lyse a portion of the cell walls of many phytopthogenes. PGPR that synthesize one or more of these enzymes have been found to have biocontrol activity against a range of pathogenic fungi. Some such bacteria can destroy oospores of phytopathogenic fungi (El- Tarabily, 2006) and also affect the spore germination and germ-tube elongation of phytopathogenic fungi (Frankowski et al., 2001). In addition, enzyme producing bacteria were successfully used in combination with other biocontrol agents, leading to a synergistic inhibitory effect against pathogen (Dunne et al., 1998).

1.4.6. Niche exclusion

Well known PGPR have been observed to be rhizosphere competent. It means that they have the ability to colonize root surface more effectively as compared to other microbes. Some indirect evidences showed that this competition between PGPR and other microorganisms for colonizing root surface helps in decreasing incidences of pathogenic attacks and also reduces disease severity. It may be justified by the fact that PGPR have the ability to rapidly colonize root surface and hence increase their population, this increased population may rapidly consume all the available nutrients exuded from roots. Such conditions render pathogens difficult to grow. For example, Innerebner et al. (2011) reported that infection in tomato by bacterial pathogen Pseudomonas syringae was prevented by applying species of Sphingomonas, a leaf bacterium.

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1.5. Hypothesis

Releasing GM crops to the environment is a strong debate since very beginning as many concerns over their potential risks on soil bacterial community. Rhizosphere is the important niche for bacterial community which directly or indirectly effect plant growth. The hypothesis of the present research work was to evaluate the impact of GM and Non- GM maize crops on rhizosphere bacterial community. Isolation of GM and Non-GM maize rhizosphere bacterial stains and their potential for plant growth promotion characterization and biocontrol. In order to evaluate the bacterial diversity of GM and Non-GM maize rhizosphere and their potential as a PGPR and biocontrol the present study was conducted to addressed these concern.

Specific objectives of the proposed research work were as follows.

1.6. Research objectives

 Isolation, Identification and phylogenetic characterization of the GM and Non- GM maize rhizosphere bacteria at different developmental stages.  Bacterial diversity of GM and Non-GM maize rhizosphere at different developmental stages based on culture and uncultured approach.  Plant growth promoting characterization of the isolated bacterial strains.  Functional characterization of the isolated strains for the production of hydrolytic enzymes and antimicrobial activity.

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2. REVIEW OF LITERATURE

2.1. Biosafety issues related to transgenic crops

A large number of transgenic crops emerge into the surroundings due to new transgenic technology and is being entered into industries (Barber, 1999; Fernadez- Cornejo and McBride, 2000; Huang et al., 2002). Recent world food security and advancement in biological sciences are due to the advent of biotechnology and transgenic crops. However, there are many concerns of biosafety regarding to the release and utilization of transgenic products (Crawley et al., 2001; Ellstrand, 2001; Prakash, 2001; Snow, 2002). The possible environmental risks associated with transgene escape through gene flow (cross-pollination) are foremost among these concerns (Amand et al., 2000; Ellstrand et al., 1999; Halfhill et al., 2001; Lavigne et al., 2002; Lefol et al., 1996). Improvement of agricultural production and the future versatility are dependent on the rational utilization of technologies.

In this new era we have advance innovations, for example recombinant DNA technology, informative content innovation and high throughput genomics are, to upgrade our understanding of the structure and function of the genomes and to apply this informative data for plant and animal improvement. New opportunities are being made available by modern biotechnology which results into new things such as genetically modified (GM) or transgenic crops to achieve sustainable productivity. Insertion of genes of tolerance to biotic and abiotic stresses in crops plants lead into increase in crop production and the crop plants are environmentally friendly (Ortiz, 1998), At the same time other believed that such crops as being just as un suit to environment (Rifkin, 1998). GE of crops for lessened fertilizer necessity through in planta nitrogen fixation could be gainful through lessening the negative effect on the soil and the resulting impacts of run-off into rivers and drainage into ground water. Several reasons relating to the balance of associated risks and benefits leads to objections development and deployment of transgenic crops arise. A variety of crops have been intended to overcome herbicide application, and this achieves much talks over, provided that this will lead to more or less herbicide being used for crops, which sorts of herbicide will be applied, the persistence and effects of herbicide residues, the

REVIEW OF LITERATURE CHAPTER 2 possibility of herbicide resistance developing in target species and their genes being in turn passed onto non-target relatives to make nosy herbicide safe weeds (Kling, 1996). There is adequate confirmation that transgenic crops and their genes, through poleen dispersal, can spread even between species that are for the most part inbreeders (Cavan et al., 1998). Regarding transgene and transplastomic regulation, there are no standard situations yet it has been established that there is a low chance of chloroplast development from oilseed rape into wild species (Scott and Wilkinson, 1999). The impacts of transgene break on the environment are dubious, yet latest technology could reduce such "hereditary pollution" through, in a few cases, building sterility into the transgenics to guarantee immeasurably diminished gene flow into the cultivating and natural environment. WHO have conducted an assessment of the genetically modified (GM) products that are currently on the international market,and concluded that there is no risk to human health. Evidence is absent that GM plants and food are toxicologically safe (Domingo, 2007).

Plants have modified with foreign gene are abled for guarding against natural stresses (biotic and abiotic) with improved survival, persistence and competitive ability, producing biofuels, antibodies and vaccines and more better quality products also novel compounds of commercial worth and enhancing the wholesome quality of nourishment goods. To date, a number of foreign genes have been effectively inserted into the nuclear genomes of various plant species. Major crops where transgenics are commercially available include soybean, potato, maize, cotton, canola, squash, cassava, papaya, oilseeds, rice, groundnut and fruits and various vegetables (Hutchison et al., 2010). European Corn Borer (ECB, Ostrinia nubilalis) cause huge damage which consequence is reduced maize yield and quality (Munkvold et al., 1999). Cry1A toxins provide by transgenic cultivars of the natural can potentially solve this problem in an suitable way to environment ((Baldwin, 2000).

Dangerous influences on non-target organisms rarely occur in the environment suggested by recent extensive field studies. Besides, it is demonstrated by biosafety parts that there is contrast in hybridisation between GMPs or non-GMPs and associated wild species. Henceforth hazard is because of both of exposure and risk, biosafety examination may as well obviously target gene flow introduction as well as specifically focus on wanted threats rising up from successful transgene flow to wild relatives of GMPs (Bartsch and Schuphan, 2002). 29

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To guarantee the biosafety of transgenic products, there is a need for an extensive investigation of transgenic plants and their descendants, and their complex communication that can inﬂuence their execution. It is basic to fabricate scientiﬁc aptitude and resources for assessment of conceivable dangers before commercialization of GEOs are pondered. The Biosafety Laws build safety measures and enforcment mechanisms on the development, growth, handling, taking care of, transportation, exchange, import, send out, space, exploration, promoting, utilization, release into nature's release into the environment and disposal of transgenics and their derivatives for protection of human and animal health and the environment. The accompanying areas give a diagram of the concerns of nourishment and environment security and the methodologies to investigate them, which have been put forth in a type of artwork.

2.2. Transgenics plants and their impacts on soil microbes

Field trials were used to evaluate the possible non-target inflences of transgenic chili pepper on the on microbial population of bacteria in rhizosphere.

These plants having a viral cover protein (CP) gene that shows resistance to cucumber mosaic infection (CMV). For correlation, a non-transgenic parental cultivar (wild sort) and an alternate non-transgenic commercial varieties were subjected to examination.

Soil samples were taken at ﬂowering stage (June) and twice throughout the fruit maturing stage (August and September). It is inferred that the effect of CMV-resistant transgenic pepper on soil microbial arrays is feeble and minor, compared with the dominating natural variations associated with plant growth stages.

The rhizosphere hosts a broad spectrum of microbiota that signiﬁcantly inﬂuence plant growth and health (Lynch and Whipps, 1990). Biological and chemical changes in the composition of root exudations may select a different community of rhizosphere microorganisms, further affecting soil functions (Hawes, 1990; Maloney et al., 1997; Sorensen, 1997). Viral coat protein is communicated constitutively in all parts of a CMV-resistant plant, rendering the probability that plant deposits and root exudates could hold an excessive amount of that protein. In this manner, the handling

REVIEW OF LITERATURE CHAPTER 2 of substantial amounts of transgenic protein raises worries about the potential for non- target consequences for rhizosphere soil microorganisms, e.g., updates in the organization or capacity of microbial groups.

Here, it is estimated that preparation of viral CP may directly or indirectly inﬂuence soil microbial assembles. Specifically, such proteins radiated from the roots may influence the development of a few microorganisms when digested, and additionally cause alteration in the sythesis of soil composition when disintegrated. The presence of overproduced metabolites can then specifically regulate the improvement of certain microbial community that use those metabolites. What's more, backhanded impacts may show up when transgenic plants develop bigger than original plants, change the physical structure of a substrate and microenvironment, or disturb interactions at the trophic level.

2.3. Rhizospheric bacterial diversity

The rhizosphere, which is the narrow zone of soil that is influenced by root secretions, can contain up to 1011 microbial cells per gram root (Dilfuza, 2010) and more than 30,000 prokaryotic species (Mendes et al., 2011). The collective genome of this microbial community is much bigger than that of the plant and is additionally alluded to as the plant's second genome. The bacterial population in the rhizosphere is essential for the execution of the plant, as bacterial species can have useful, independent or harmful associations with the root (Atkinson and Watson, 2000; Buchenauer, 1998; Sylvia and Chellemi, 2001). Root exudates and rhizo-deposits from the substrates for rhizosphere microorganisms, and it has been distinguished that the organization of these substrates can contrast between plant species, even between cultivars, and throughout plant growth (Rengel, 2002; Whipps, 2001b). These contrasts in root-determined substrates are accepted to demonstrate the plant particular rhizosphere bacterial communities that have been watched for distinctive plant species become under comparative conditions (Kowalchuk et al., 2002; Marschner et al., 2001b; Smalla et al., 2001).

Yet, the root exudate composition is additionally influenced by the plant's development conditions in the soil e.g. pH, nutrition confinement, dampness stress and the presence of pathogens (Hertenberger et al., 2002; Yang and Crowley, 2000).

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In addition, diverse soil type are known to own altogether different bacterial groups, which give off an impression of being free of plant species organization to a huge degree (Kowalchuk et al., 2000). It is not yet known to what degree plants can select a consistent rhizosphere community from remarkably differentiating supplies of mass soil population. Hence, while plant species or genotype might assume a vital part in directing bacterial groups of the rhizosphere, soil type and the composition of the rhizo-competent populaces present in the mass soil might additionally be critical (Buyer et al., 2002; Marschner et al., 2001b).

Rhizospheric soil is one the largest reservoirs of microbial genetic diversity. Before conducting a large-scale metagenomic analysis of an environment, such as a rhizospheric soil, it is necessary to perform a pre-screening of the resident genetic diversity. A Study analyzed the bacterial diversity associated with the rhizosphere of wheat plants by PCR amplification, construction of a library and sequencing of 16S rDNA genes. Our results suggest that the rhizosphere of wheat plants is highly dense with bacteria (Velazquez-Sepulveda et al., 2012).

Recent confirmation infers that differences between plant genotypes in a solitary gene can have a huge effect on the rhizosphere microbiome. The production of a solitary exogenous glucosinolate essentially changed the microbial community on the root of transgenic Arabidopsis (Bressan et al., 2009). Alphaproteobacteria and fungal groups were fundamentally affected, as demonstrated by denaturing gel gradient electrophoresis of particular amplified fragments of 16s and 18s rRNA genes. In addition, it has been accounted for that an ABC transporter mutant of Arabidopsis, abcg30, had root exudates with expanded phenolic compounds and reduced sugars, which additionally brought about a different root microbiome (Badri et al., 2009).

An investigation of bacterial rhizosphere communities in two distinctive soils on three cultivars of potato utilizing the Phylo-Chip engineering reported 2432 operational taxonomic units in the potato rhizosphere, of which less than half had a site-specific wealth (Weinert et al., 2011). Just less than one tenth of the operational taxonomic units had a cultivar-dependent abundance in one or the other soil, though the abundance of one twentieth of the operational taxonomic units was cultivar dependent in both soils. These effects underline not just the significance of the soil in verifying rhizosphere communities, additionally that a few microorganisms have a specific

REVIEW OF LITERATURE CHAPTER 2 liking for certain plant genotypes. Interestingly, distinctions in the abundance on the three cultivars were predominantly watched for microorganisms fitting in with the Pseudomonales, Streptomycetaceae and Micromonosporaceae, which are an order and two groups of bacteria, individually, that have been widely concentrated on for their capability to control plant pathogens. These studies demonstrate that the plant genotype can influence the amassing of microorganisms that help the plant to safeguard itself against pathogen attack. Surely, distinctions have been watched in the capability of wheat cultivars to aggregate naturally occurring DAPG-producing Pseudomonas sp., bringing about contrasts in the disease suppressiveness (Gu and Mazzola, 2003 ; Meyer et al., 2010). Moreover, the amount of antibiotics produced by specific biocontrol strains on roots has been found to differ between wheat cultivars (Okubara and Bonsall, 2008). Furthermore, particular wheat cultivars were accounted for to underpin particular biological control microbes differentially, which further secures that there is a level of specificity in the interaction between plant genotype and the arrangement of their microbial community (Meyer et al., 2010).

2.4. Culture based approaches

Cultivation-based techniques address just the culturable microorganisms, which are thought to just a modest extent (0.1-10%) of the total bacteria available in soil and in the rhizosphere (Amann et al., 1995).

In this study, we analyzed the bacterial diversity associated with the rhizosphere of wheat plants by PCR amplification, construction of a library and sequencing of 16S rDNA genes. Thirty operational taxonomic units (OTUs) were detected, including the different classes like Alfaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobateria, Actinobacteria, Bacilli, Clostridia and Uncultivable bacteria. Within the Gammaproteobacteria class, the genera Pseudomonas, Stenotrophomonas and Bacillus were the most abundant, since they corresponded to 40% of the whole ribosomal library.

2.5. Culture independent approaches

Here the application of the bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) diversity method is presented, which will promote studies in soil

REVIEW OF LITERATURE CHAPTER 2 microbiomes. Dominating phyla in this soil were Actinobacteria, Bacteriodetes and Fermicutes. Bacteriodetes were more overwhelming in soil under farming system (Ct–W–Cr and Ct–Ct) contrasted with the same soil under non-disturbed grass system (P and CRP). The inverse pattern was discovered for the Actinobacteria, which were more dominating under nondisturbed grass system (P and CRP). The bTEFAP procedure provided to be a compelling technique to sort out the bacterial diversity of the soil examinations under the diverse management and land use in in terms not only on the presence or absence, as well as regarding distribution (Acosta-Martıineza et al., 2008).

Incomplete ribosomal amplification and pyrosequencing (PRAPS) is a later and influential strategy which could be utilized to turn in profundity toward microbial diversity and type of specimen (Dowd et al., 2008a) using 454 Genome Sequencer FLX System (Roche, Nutley, New Jersey). The bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) method (Dowd et al., 2008b) enhances this PRAPS methodology expanding the cost-benefit of diversity pyrosequencing through the utilization of sample-specific sequence tags fused onto secondary amplification primers. An E. coli metagenomic library was constructed based on bacterial artificial chromosome (BAC) clones with large genomic inserts from metagenomic DNA from the rhizosphere of wheat plants. The average of the DNA cloned segments were varied from 5 to 80 kb, with an average size of 38 kb. The metagenomic sequences belonged mostly to Proteobacteria, Firmicutes, Archaea, Actinobacteria, Fungi, Virus, Unclassified Bacteria and Unknown taxa. It is expected that the metagenomic library from rhizospheric soil of wheat plants keeps growing. At the same time, functional analyses are conducted in the search for genes of agrobiotech interest (Hernandez-Leon et al., 2012).

The DNA of the microbial community connected with the rhizospheres of Deschampsia antarctica Desv (Poaceae) and Colobanthus quitensis (Kunth) Barti (Caryophyllaceae), the main two local vascular plants that are discovered in Antarctic ecosystem, was assessed utilizing a 16S rRNA multiplex 454 pyrosequencing approach. This examination uncovered comparative examples of bacterial diversity between the two plant species from distinctive areas, contending against the speculation that there might be contrasts between the rhizosphere communities of diverse plants. Moreover, the phylum distribution introduced an impossible to miss design, with a bacterial group structure not quite the same as those reported of

REVIEW OF LITERATURE CHAPTER 2 numerous different soils. Firmicutes was the most plentiful phylum in just about all the analyzed specimens, and there were large amounts of anaerobic reprisentatives. Likewise, some phyla that are predominant in generally mild and tropical soils, for example Acidobacteria, were seldom reported in the investigated specimens. Breaking down all the specimen libraries together, the transcendent genera discovered were Bifidobacterium (phylum Actinobacteria), Arcobacter (phylum Proteobacteria) and Faecalibacterium (phylum Firmicutes). To the best of our information, this is the first major bacterial sequencing exertion of this sort of soil, and it uncovered more than needed differences inside these rhizospheres of both oceanic Antarctica vascular plants in Admiralty Bay, King George Island, which is part of the South Shetlands archipelago (Teixeira et al., 2010b).

Recently, high-throughput sequencing utilizing sequencing by synthesis engineering (454 pyrosequencing) was presented as another approach fit for better revealing the taxonomic assorted qualities inside surviving microbial communities (Acosta- Martinez et al., 2008; Roesch et al., 2007a; Sogin et al., 2006) Acosta-Martinez et al., 2008). (Roesch et al., 2007b). Before pyrosequencing Partial ribosomal amplification was used to detect the actual microorganisms communities in environmental specimens (Acosta-Martinez et al., 2008; Binladen et al., 2007).

Regardless of the innate predisposition in PCR and all molecular techniques (Dowd et al., 2008a), which are the same for all examined specimens, it is fascinating to join the selectivity of primer based PCR with high-throughput sequencing method. Additionally, the utilization of the bacterial tag-encoded FLX amplicon pyrosequencing technique takes into consideration for mixed specimens to be run on the same sequencing run and later binned (Acosta-Martıineza et al., 2008; Binladen et al., 2007; Dowd et al., 2008a).

Due to metagenomic techniques we can bitterly access to the diverse microbial community because soil microbes have prokaryotic diversity on highest degree.

Most studies done on the soil biodiversity and function shows that the extracted DNA from microbial community in the soil, but it is little clear by the DNA extracted from the soil. Unfortunately, these methods do not give a complete and conform subsample of metagenomic DNA, and as a result we unable to determine the precise distribution

REVIEW OF LITERATURE CHAPTER 2 of species. Furthermore any inaccurate reports may exclude some groups/class of these soil microbes from study and its identification.

To enhance metagenomic methodologies, examine DNA extraction biases, and provide methodes for evaluating the relative plenitudes of distinctive assemblies, we investigated the biodiversity of the accessible group DNA by fractioning the metagenomic DNA as a capacity of (i) vertical soil specimens, (ii) thickness angles (cell suspension), (iii) cell lysis stringency, and (iv) DNA fragment estimate dispersion. Every fraction had an extraordinary genetic alteration, with distinctive overwhelming and rare species (dependent upon ribosomal intergenic spacer investigation (RISA) fingerprinting and phylochips). All fractions take part to the number of bacterial communities uncovered in the metagenome, hence expanding the DNA pool for further requisitions. Surely, we were equipped to gain entrance to an all the more genetically altered extent of the metagenome (an addition of more than 80% contrasted with the best single extraction methodology), limit the predominance of a couple of genomes, and boost the species richness for every sequencing. This work stresses the distinction between extracted DNA pools and the at present blocked off complete soil metagenome (Delmont et al., 2011).

In comparative metagenomic latest developments has occured due which pyrosequencing engineering seems, by all accounts, to be a greatly significant tool for the characterization of complex microbial communities. It has tried to present this approach for the examination of microbial diversity and capacity in the rhizosphere soil. In any case, on account of rhizosphere soil, as just restricted measure of soil DNA might be acquired and the crude extract holds impressive measure of plant inferred DNA, along these lines it has tried to make the strategy to extract microbial DNA from the rhizosphere soil for the metagenome examination by utilizing pyrosequencing engineering is inferred that these devices will probably give a significant tools for the investigation of rhizosphere microbial dynamics.

2.6. Plant microbes interaction

The rhizosphere (the soil in the vicinity of roots) having high microbial density; the stimulation of microbial development by roots is generally reputed to be the rhizosphere impact. The advantageous part of rhizosphere microorganisms could be

REVIEW OF LITERATURE CHAPTER 2 help the plant development, for example quickening plant nourishment uptake, breaking down natural compounds, transforming plant hormones-like compounds, and interaction with plant pathogens, and so on. In a study by Germida and Siciliano (2001), the evolutionary relationship in plant-microorganism communications was uncovered: old wheat cultivars were colonized by phylogenetically altered rhizobacteria, inasmuch as the rhizosphere of present day cultivars was overwhelmed by quickly developing Proteobacteria. Cultivar specificity was additionally reported for oilseed rape (Graner et al., 2003). A plant species-dependent composition of bacterial and fungal rhizosphere isolates with in vitro antagonistic activity towards the plant-pathogenic fungus Verticillium dahlia was shown by (Berg et al., 2005a; Berg et al., 2006); Berg et al. (2002); (Berg et al., 2005b) Generally, interactions between plants and microorganisms can be classified as pathogenic, saprophytic, and beneficial (Lynch, 1990). Beneficial interactions involve plant growth promoting rhizobacteria (PGPR), generally refers to a group of soil and rhizosphere free-living bacteria colonizing roots in a competitive environment and exerting a beneficial effect on plant growth (Bakker et al., 2007). However, numerous researchers tend to enlarge this restrictive definition of rhizobacteria as any root-colonizing bacteria and consider endophytic bacteria in symbiotic association: Rhizobia with legumes and the actinomycete Frankia associated with some phanerogams as PGPR genera. Among PGPRs are representatives of the following genera: Acinetobacter, Agrobacterium, Arthrobacter, Azoarcus, Azospirillum, Azotobacter, Bacillus, Burkholderia, Enterobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, and Thiobacillus. Some of these genera such as Azoarcus sp., Herbaspirillum, and Burkholderia include endophytic species. However, Pseudomonas and Bacillus species constitute, together with Streptomyces species, the most bacteria often found in the rhizosphere of many crop plants. In recent years, interest in the use of PGPR to promote plant growth has increased. Beneficial effect of PGPR on plant growth involves abilities to act as phytostimulators; biofertilizers.

PGPR could enhance crop yield through nutrient uptake and plant growth regulators. PGPR could also act as biocontrol agents by production of antibiotics, triggering induced local or systemic resistance, or preventing the deleterious effects of xenobiotics by degradation (rhizoremediators) by acting as rhizoremediators (Aseri et al., 2008). Their application as crop inoculants for biofertilization would be an

REVIEW OF LITERATURE CHAPTER 2 attractive option to reduce the use of chemical fertilizers (Vessey, 2003a). In addition, PGPR have great adaptation to harsh environments including drought stress (Arshad et al., 2008; Arzanesh et al., 2011), salt stress (Mayak et al., 2004), high temperatures, dryness or heavy rain falls in tropical countries (Da Mota et al., 2008), and contaminated environments (Dell’Amico et al., 2008), indicating that they could contribute to ameliorate plant crops in areas with poor agricultural potential.

Biocontrol by rhizobacteria could involve PGPR and non-PGPR bacteria in the way that suppression of plant diseases could result in no enhancement of plant growth but only in protection against plant pathogens. Action of bacteria in the rhizosphere is also restricted by their ability to colonize the rhizosphere. Indeed, in practice, we cannot conclude that a bacterium is a PGPR only after its isolation from rhizobacteria and its reintroduction by plant inoculation followed by assessment of its ability to colonize rhizosphere and beneficial effect. This corresponds to only 2–5% of rhizobacteria (Kloepper and Schroth, 1978).

2.7. Functional characterization

A 44-kda 1,3-b-glucanase was filtered from the culture medium of a Paenibacillus strain with a 28-fold expand in particular action with 31% recuperation. The cleansed enzyme specially catalyzes the hydrolysis of glucans with 1,3-b-linkage and has an endolytic mode of action. The enzymes additionally indicated tying action to different insoluble polysaccharides incorporating unhydrolyzable substrates, for example xylan and cellulose. The antifungal action of this Paenibacillus enzyme and an previously purified 1,3-b-glucanase from Streptomyces sioyaensis were analyzed in this study. Both compounds could harm the cell wall of the developing mycelia of phytopathogenic fungi Pythium aphanidermatum and rhizoctonic solani AG-4.

Nonetheless, the Paenibacillus enzyme had a much stronger impact to stop the growth of fungi tested. A solid medium containing ashed, acid-washed cellulose and a dye, Congo red, has been developed for enumeration of cellulose-utilizing bacteria in soil. Bacteria able to use the cellulose in this medium produced distinct zones of clearing around their colonies. A vivid contrast between the uniform red color of the medium and these halos made this method of differentiation of these organisms superior to other methods (Hendricks et al., 1995).

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To test the effectiveness of the new medium, the growth of known cellulose-utilizing bacteria (Streptomyces lividans TK23 (Crawford et al., 1993), non-cellulose-utilizing bacteria (Escherichia coli, Proteus mirabilis, and Pseudomonas putida (Cruden and Markovetz, 1979), and celluloseutilizing fungi (Acremonium sp. and Alternaria sp. (Pfender and Wootke, 1988) was examined. Cultures of Acremonium sp. and Alternaria sp. Were maintained on potato dextrose agar; S. lividans TK23 was maintained on yeast-malt extract agar (Dekleva et al., 1985); and E. coli, P. mirabilis, and P. putida were maintained on Luria-Bertani agar (Difco). Bacterial cultures (positive and negative controls) were grown in liquid culture for 18 h at 30 6 28oC (mean 6 range), serially diluted by decades, spread plated on the cellulose-Congo red agar, and incubated for 7 days at 25 6 28oC. Following incubation, the colonies exhibiting zones of clearing were counted. Replicate plates of the bacterial cultures were flooded with a 1% solution of HAB (J. T. Baker) to examine the ability of HAB to enhance visualization of the zones of clearing (Cruden and Markovetz, 1979).

Two chitinolytic bacterial strains, Paenibacillus sp. 300 and Streptomyces sp. 385, suppressed Fusarium wilt of cucumber (Cucumis sativus) caused by Fusarium oxysporum f. sp. cucumerinum in nonsterile, soilless potting medium. A mixture of the two strains in a proportion of 1:1 or 4:1 gave altogether (P < 0.05) preferred control of the disease over each of the strains utilized separately or than mixtures in different degrees (Singh et al., 1999b).

Those bacterial endophytes that additionally give some benefit to plants may be measured to be plant growth promoting microscopic bacteria (PGPB) and can expedite plant development by various diverse mechanisms. In the work that is accounted for here, soil spacimens from a few regions far and wide were utilized as a beginning point for the separation of new endophytes. In this manner, those recently isolated endophytes that were fit to use the plant compounds 1-aminocyclopropane-1- carboxylate (ACC) as a sole source of nitrogen, as an outcome of holding the enzyme ACC deaminase, were chosen for extra characterization. All the more particularly, ACC deaminase-communicating strains were tried for IAA amalgamation, siderophore processing, phosphate solubilization action, optimal development temperature, salt tolerance, and anti-microbial sensitvity. Likewise, the partial DNA sequencing of the16S rRNA genes of the analyzed strains were resolved so the taxonomic position of every strain could be surveyed, and the capability of some of 39

REVIEW OF LITERATURE CHAPTER 2 these strains to expedite the development of canola plant roots under regulated gnotobiotic conditions was determined (Rashid et al., 2012). A novel outlined microbiological growth medium, National Botanical Research Institute's (NBRIP) phosphate growth medium, which is more proficient than Pikovskaya medium (PVK), was created for screening phosphate solubilizing microorganisms. In plate examine the efficiency of NBRIP was comparable to PVK; in any case, in stock test NBRIP reliably showed about of 3-fold higher effectiveness contrasted with PVK. The effects showed that the model for isolation of phosphate solubilizers dependent upon the formation of noticeable halo/zone on agar plates is not a solid procedure, as numerous isolates which did not indicate any reasonable zone on agar plates solubilized insoluble inorganic phosphates in fluid medium. It may be presumed that soil organisms ought to be screened in NBRIP broth examine for the distinguishing proof of the most productive phosphate solubilizers.

Six strains of Bacillus cereus exhibiting antifungal activity were isolated from local environment and optimized for growth conditions. The isolates appeared neutrophiles, grew best at aeration and were represented by thermophiles and mesophiles. Growth inhibition zones of varying diameters were observed when the isolates were tested against Saccharomyces cerevisiae on nutrient agar. Further work on these bacterial isolates is likely to define their antimycotic mode of actions and limitations regarding therapeutic applications of their antifungal exoproducts.

This study evaluated the antifungal action of biomolecules produced from the secondary metabolism of bacterial strains found in the rhizosphere of semi-arid plants against human pathogenic Candida albicans. Crude extracts were obtained using ethyl acetate as an organic solvent and the bioactivity was assessed with a bioautography technique. These results suggested that microorganisms found in the environment could have bioprospecting potential. Untapped source of microbial diversity is represented by microbes associated with plants. These microorganisms produce antimicrobial agents and seem to have unique genetic and biological systems that may have applications outside the host plants, in which they normally reside. Bioprospection of such organisms and their products represents a promising source in the discovery of new antifungal drugs, which could be applied in the pharmaceutical industry (Colwell, 1997; Demain, 2000; Strobel and Daisy, 2003). This study was aimed to determine the antifungal activity against the human pathogen, C. albicans 40

REVIEW OF LITERATURE CHAPTER 2

(CCMB 286), found in biomolecules obtained from bacterial strains of the rhizosphere of Stachytarpheta crassifolia, collected in Chapada Diamantina, Bahia, Brazil.

Thirty seven bacterial cultures isolated from soil samples obtained from different locations were tested for their antagonistic activity against some fungal pathogens, viz., Sclerotium rolfsii, Fusarium oxysporum and Rhizoctonia solani, causal agents of collar rot of sunfl ower, wilts and root rots, respectively. These strains showed their extensive inhibition effect particularly against gram-positive test bacteria (Staphylococcus aureus and Bacillus subtilis) and the test fungal strain (Candida albicans). On the other hand, B. brevis M1 66 and B. brevis T1 22 strains had an inhibitory effect against gram positive and gram-negative test bacteria (Escherichia coli and Proteus vulgaris) as well as the test fungal strain. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the K- Proteobacteria. The sponge isolates show antimicrobial activities against Gram- positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential (Hentschel et al., 2001).

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3. MATERIALS AND METHODS

3.1. Field experimental design

A field experiment was conducted in a randomized complete block design (RCBD) with three replicates at National Agriculture Research Council (NARC), Islamabad-Pakistan (Latitude 33° 43' N; longitude 73° 04' E). Annual average rainfall in this region ranges from 500- 900 mm. The climate has a typical version of humid subtropical region, with hot summers accompanied by a monsoon season followed by fairly cold winters. The weather ranges from a minimum of 3.9 °C (39.0 °F) in January to a maximum of 46.1 °C (115.0 °F) in June. The experiment was carried out in 2 m × 8 m plots in 2011-2012. Seeds of genetically modified (insect resistant) and non-modified maize (Zea mays L.) cv Islamabad gold and Islamabad white were obtained from Plant Genetic Resources Institute (PGRI) gene bank, NARC Islamabad-Pakistan. The field was net covered to minimize the environmental impact of horizontal gene transfer from transgenic maize to other crop species.

3.2. Rhizospheric soil sampling and processing

Rhizospheric soil sampling was carried out at three developmental stages of one transgenic and tow non transgenic maize varieties, prior to cultivation (Stages A), vegetative (Stage B), and senescent (Stage C) as described (Hack H, 1993) during 2011- 2012. Five plants per plot were carefully removed from the soil. After the plants were shaken, the roots with adhering soil were combined, cut into pieces, and treated as a composite sample. Three composite samples of each cultivar and transgenic line were processed at each stage. For isolation of the root-associated bacterial community, samples (10 g) was transferred into a sterile zipper bag and homogenized with 30 ml Milli-Q water for 60 sec at high speed. This homogenization step was repeated three times and the combined suspensions were collected in two 50 ml tubes. The first tube was centrifuged for 15 minutes at 4 °C and 10,000 rpm, the supernatant was discarded and the tube was filled with the contents of the second tube prior to another centrifugation. The

MATERIALS AND METHODS CHAPTER 3 resulting pellets containing the root-associated bacteria were stored at -80 °C until DNA was extracted.

3.3. Isolation and characterization of rhizospheric bacterial community

3.3.1. Isolation and identification of culturable bacterial strains.

Sample (10 g) was mixed with autoclaved distilled water (100 ml) in an sterilized Pyrex flask (250 ml). The flask was placed on a shaker at 150 rpm for 2 hours. Then serial dilutions were made from 101-106. About 100 µl aliquots of each sample from various dilutions ranging from 101 to 106 were spread onto petri plates containing different media composition. Different medias used were TSB+A (high nutrient concentration tryptic soy broth agar (Oxid LTD Basingstoke, Hampshire England), lower nutrient (R2A) medium (Sccharlab Barcelona,Spain) and nutrient broth with agar added medium. The plates were incubated at 25± °C for 3-6 days. The number of colonies on each plate of corresponding medium was counted after third and six day inoculation. The same plates were also counted on day six and these plates were used for sub-culturing and isolation of pure strains. From each sample morphologically different colonies were picked at random and subcultured. Once the purity of each of these cultures had been verified, they were stored at 4 °C as described previously (Ellis et al., 1999) and were used for all further work.

3.4. Culture based approach to evaluate bacterial diversity

3.4.1. Bacterial culturing medias

Three different type of media were used to isolate bacterial strains from transgenic and non transgenic maize rhizosphere. In order to study bacterial diversity of transgenic and non transgenic maize rhizosphere in more detail we slightly changed the concentration and composition of the culturing medias.

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3.4.2. Tryptic soy broth agar (TSB+A)

Tryptic soya agar media from Oxoid LTD Basingstoke Hampshire England (Lot No 996291) was modified and reduced from the full strength (30 gL-1) manufactured recommended concentration to 1\20 strength (1.5 gL-1). The manufactured recommended concentration was modified in order to isolate the slow growing bacterial strains. Originally this medium comprise (g/L) of pancreatic digest of casein (17 g), Enzymatic digest of soya bean (3 g), Glucose (2.5 g), sodium chloride (5 g) and Di-potassium hydrogen phosphate (2.5 g). TSB (0.75 g) and agar (7.5 g) was dissolved in 500 ml of sterilized distilled water. The final pH of this medium was 7.3 at 25 ºC and autoclaved at 121 ºC for 20 minutes.

3.4.3. Low-nutrient R2A medium

The second culture media used was a conventional R2A agar medium from Sccharlab (S. L. Barcelona, Spain European Union). We also modified the media concentration from full strength (18 gL-1) to three-fold (6 gL-1) reduced strength. Originally This medium comprised (gm/L) of yeast extract (0.5 gm/L), Starch (0.5 gm/L), Proteose peptone (0.5 gm/L), Glucose (0.5 gm/L), Casein hydrolysate (0.5 gm/L), Dipotassium phosphate (0.30 gm/L), Sodium pyruvate (0.30 gm/L), Magnesium sulphate anhydrous (0.024 gm/L), and agar (15 gm/L). Normally 4 gm/L of R2A is dissolved in one liter of autoclaved distilled water with 4 g agar addition. Final pH of the media was adjusted to 7-7.5 at 25 ºC with NaOH/HCL and autoclaved at 121 ºC for 20 minutes.

3.4.4. Mixed media

In this strategy we mixed tryptic soy broth (TSB), lower-nutrient (R2A) and nutrient broth (Difco) and reduced the actual manufactured recommended concentration to our own desired concentration. We dissolved TSB (1 g), R2A (4.5 g) and nutrient broth (LB) (1g) along with agar (4 g) to sterilized distilled water (500 ml), the pH was adjusted to 7- 7.5 at 25 ºC with NaOH/HCL and autoclaved at 121 ºC for 20 minutes.

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For sterilization all the above mentioned media were autoclaved at 121 ºC for 15 minutes after cooling the media to 50 ºC. Amphotericin B (fungicide) was added to prevent any fungal contamination. If needed the medium was stored at 4 °C. Each of the above prepared medias were poured into disposable plastic petri plates for bacterial culturing and further processing.

3.4.5. Cultured analysis

Rhizosphere sample (100 µl) from each dilution were spread on selected solid media plates. Each of the above steps were carefully carried out in laminar flow under sterile conditions to avoid any kind of contamination. The spread plates were incubated at 25± °C in incubator (Memmert-German) for 3-6 days. The number of colonies forming on each medium was counted after three and six days of inoculation. Plates with different dilution carried diverse colonies were counted after three days interval and subcultured on the same media for purification. Those colonies which showed slow growth their plates were kept up to six days, and subculturing was repeated as mentioned. While doing subculturing from each sample morphologically different colonies were considered, picked at random and streaked onto fresh plates of the same media. Once the purity of each of these cultures was confirmed they were stored at 4 °C as described previously (Ellis et al., 1999) and were used for all further work.

3.4.6. Colony forming unit (CFU) calculation

After 3- 6 days incubation, several morphologically different colonies appeared on all the above three types of media. All the incubated plates were carefully observed and the numbers of colonies were calculated for each plate/media and finally colony numbers were converted to colony forming unites (CFU).

3.4.7. Sub-culturing and isolation of pure strains

Different bacterial colonies were selected on the basis of morphological characters like color, shape, type of growth and spreading nature and subcultured onto a separate plate.

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We focused on small and morphologically different colonies in order to find novelty and continues this process till reached to pure single bacterial strains.

3.4.8. Bacterial glycerol stock

Glycerol stocks are important for long-term storage of bacterial strains. We prepared LB broth according to our need and autoclaved for 20 minutes at 121oC. Then we transferred LB broth (70ul) to each sterilized eppendorf tubes and with the help of sterilized inoculation loop we transmit full loop of pure bacterial strains into the broth and incubate overnight at 37 oC on a shaker. Next day we prepared glycerol stock solution comprised of 100% glycerol (65 ml), 1M MgSO4 (10 ml), 1M tris buffer (2.5 ml) and sterilized distilled water (22.5 ml). The stock solution were autoclaved at 121 oC for 20 minutes. We added 70 ul of the glycerol stock to the overnight cultured, vortex gently so the cultured and glycerol solution properly mixed, labeled and then freeze rapidly in liquid nitrogen and stored at -80 oC for further work.

3.4.9. Bacterial colony counting and identification

Dilution plates that carried bacterial colonies were counted after three days of incubation. The same plates were also counted on day 6 and these plates were used for sub-culturing and isolation of pure strains. Different colonies were selected on the basis of color, shape, type of growth and nature of spreading of colonies. Morphologically different colonies were picked at random from each plates and subcultured onto fresh plates. Once the purity of each of these cultures had been verified, they were stored at 4 °C for further work.

3.5. PCR of the culture strains for 16S rRNA gene analysis

3.5.1. Bacterial strain identification

The bacterial colonies were investigated for the correct identification of different strains. We used 16S rRNA gene for transgenic and non transgenic rhizospheric culture bacterial strains identification. DNA isolation/extinction was pre requisite to analyze 16S rRNA

MATERIALS AND METHODS CHAPTER 3 gene by polymerase chain reaction (PCR). For this purposes we used two approaches (i) Direct cell lysis approach (ii) Genomic DNA isolation approach using commercially available genomic DNA extraction kit. The detailed procedure of the above mention approaches are below.

3.5.2. DNA isolation by using direct cell lysis approach

Purified bacterial colonies were selected and processed for DNA isolation. Each colony was carefully transferred to a PCR tube (0.2 ml) with the help of sterilized loop and 100 µl of lysis buffer was added to each tube and the contents were thoroughly mixed. PCR tubes were heated at 95 oC for 10 minute in thermo-cycler (PCR machine) followed by centrifugation at 14000 rpm for 10 minutes to pellet-out the debris. The supernatant was collected and properly labeled for further processing.

3.5.3. Recipe for lysis buffer

We freshly prepared lysis buffer according to our requirement. For 2 ml lysis buffer, 1.75 ml nuclease free water, 200 µl 10% Triton X-100, 40 µl 1M Tris HCl (pH 8.0) and 8 µL 0.5 M of EDTA (pH 8.0) was added in a tube. For each reaction/cell lysis we used 100 µl of this buffer.

3.5.4. Bacterial genomic DNA isolation by commercially available kit

The bacterial strains were cultured in TSB medium. Sterilized the medium in 250 ml flask by autoclaving and distributed 1.5 ml medium into each sterilized eppendorf tube. Each of the bacterial colonies from the purified bacterial plates were picked with a sterile inoculation loop, inoculated into eppendorf and labeled. These cultures were then placed in a shaker incubator at 25 oC and 250 rpm for 24-48 hours. Bacterial cell suspension was obtained and were centrifuged at 8000 rpm for 12 minutes at 27 °C. Supernatant was discarded and the pellet was used for bacterial genomic DNA extraction. The bacterial DNA was isolated using the Wizard® Genomic DNA purification Kit by Promega (Lot No: 323556) according to the manufacturer’s instructions.

MATERIALS AND METHODS CHAPTER 3

3.5.5. Selection of PCR primers

In order to correctly identify the bacterial strains, the universal set of primers for the bacterial identification was used. The primers described below were used for the polymerase chain reaction (PCR) amplification of the 16S rRNA gene fragment. 27F (AGAGTTTGATCMTGGCTCAG) 1492 (GGTTACCTTGTTACGACTT) Most of the isolates were recognized on the basis of 16S RNA sequencing.

3.5.6. PCR amplification of bacterial 16S rRNA gene fragments

The isolated strains from pre sowing as well as from GM and Non-GM maize rhizospheric were selected for 16S RNA gene analysis. The genomic DNA of the selected strains was extracted using DNA extraction kit and were amplified using 16S rRNA gene primers. For 16S rRNA gene amplification we used forward 27F (AGAGTTTGATCMTGGCTCAG) and reverse1492 (GGTTACCTTGTTACGACTT) primers. For PCR, 16S rRNA gene fragments were amplified in 25 μl reaction mixtures containing 12.5 ul master mix (Promega), 1ul of each of the forward and reverse primers, 1 μl DNA template and the final volume of the reaction was raised to 25ul by nuclease free water. For all PCR reactions DNase/RNase-free distilled water (Promega) was used. The thermal cycling programmes were performed with an initial denaturing at 94 °C for 5 minutes followed by 35 cycles denaturation at 94 °C for 30 sec. annealing at 55oC for 30 sec and extension 72 oC for 90 sec. while final extension at 72 oC for 7 minutes.

3.5.7. Gel electrophoresis

PCR products were checked by gel electrophoresis, 3 μl PCR products load to 1% agarose gel (1g agarose was dissolved in 10 ml of 10X TBE buffer and its volume was raised to 100 ml) and ethidium bromide for staining was also added. The mixture was boiled in microwave for 40 sec. to dissolved agarose and get a clear liquid and was cooled in water bath and added 3 ul (0.5 ug/ml) of ethidium bromide. The melted agarose was poured into gel tray (mini and midi), comb was fixed and allowed to solidify. samples (3-5 ul) were loaded in the wells along with 100 bp DNA ladder (Fermentas) and

MATERIALS AND METHODS CHAPTER 3 run in 1X TBE buffer at 100 volts for 25-30 minutes. Amplify fragments were visualized under UV illumination and photographed using Dolphin gel documentation system ( Wheeltech,USA).

3.5.8. PCR product purification

Positive PCR product were purified, using two type of kits (i) Wizard® SV Gel and PCR Clean-Up System of Promega having catalogue number A9285 and (ii) Axygen® AxyPrep™ PCR Clean-Up Kit (Kit No: AP-PCR-250) according to the manufactures instruction.

3.5.9. DNA sequencing

The Purified PCR products were commercially sequenced using 16S rRNA gene partial sequencing through Sanger sequencing technology through Macrogen, South Korea.

3.5.10. Sequences analysis

All the bacterial isolates were classified by BLAST analysis of their respective 16S rRNA gene partial sequences. Various databases including eztaxon server version 2.1, NCBI and Ribosomal Database Classifier (RDP) were used in order to determine the exact taxonomical classification of individual organisms. For the determination of closest type strains eztaxon server version 2.1 was used. The partial 16S rRNA Gene sequences of the closest type strains were obtained from the NCBI database for phylogenetic tree construction. The entire hierarchy of all the organisms was determined by using RDP Naive Bayesian rRNA Classifier Version 2.5.

3.5.11. Phylogenetic analysis

The evolutionary linkages among the isolated strains and with related type strains were identified through phylogenetic analysis. The related type strains were identified from gene bank. Multiple alignment was performed with ClustalW (Larkin et al., 2007). The phylogenetic tree was constructed by distance method (Neighbor-joining) using MEGA4 software (Tamura et al., 2007).

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3.6. Metagenomics approach to evaluate bacterial diversity

3.6.1. Metagenomic DNA extraction

The field soil sample (before conducting the GM and Non-GM field trial) were collected in two different interval. One sample soil was collected before sowing of GM and Non- GM maize crops (represented by code A) while the second sample was collected after one year interval of GM and Non-GM maize crops from the same field (represented by code A). Whereas rhizosphere samples were also collected from GM and Non-GM maize (varieties IG and IW) at different developmental stages (vegetative and harvesting). Genomic DNA were extracted from rhizosphere samples (0.25 g) using Power Soil DNA Isolation Kit (Mo Bio USA) following the manufacturers instruction. The extracted DNA was confirmed on 1% agarose gel and was amplified for 16S RNA gene. The soil was rich in PCR inhibitors e.g. humic acid that's why different dilutions were used for 16S rRNA gene amplification.

3.6.2. DNA extraction and PCR conformation

Rhizosphere sample (0.25 g) was treated with Power Soil DNA Isolation Kit (Mo Bio USA) exactly following the protocol of the DNA extraction kit. The extracted DNA was confirmed on 1% agarose gel. The extracted metagenomic DNA was amplified for 16S RNA gene. A single-step 35 cycle PCR were used under the following conditions: 94 oC for 5 minutes followed by 28 cycles of 94 oC for 30 seconds, 55 oC for 40 seconds and 72 oC for 1 minute; after which a final elongation step at 72 oC for 5 minutes was performed.

3.6.3. Pyrosequencing

The DNA samples which were PCR positive sent for pyrosequencing to Molecular Research DNA (MR DNA) Laboratory of US. Bacterial diversity was assessed through pyrosequencing technology using 515F primer. Minimum 200 bp region of the 16S rRNA was sequenced and nominal 20,000 reads per sample were given.

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3.7. Characterization of plant growth promotion traits assay

3.7.1. IAA production assay

In order to measure the indole-3-acetic acid (IAA) production of the selected bacterial strains Patten and Glick (2002) method was followed with slight modification. All the strains were processed in triplicates and strict measures were taken to avoid any kind of contamination. About 20 µl aliquots of an overnight grown bacterial culture were used to inoculate 5 ml TSB without and with tryptophan (500 µg ml-1) and incubated at 30 oC for 24 hours. After 24 hours incubation the cultures were centrifuged for 30 minutes and 1 ml supernatant was mixed with 4 ml Salkowski’s reagent (Gordon and Weber, 1951). The mixture was incubated for 20 minutes at room temperature before the absorbance was measured at 535 nm. The concentration of each sample was calculated from a standard plot which was prepared from different dilution of pure IAA (Sigma-Aldrich) ranging from 0.01 to 0.4 µg ml-1. IAA value was calculated by the formula (x= ay+b) and was expressed in µgml-1

3.7.2. Phosphate solubilization

Phosphate solubilizing activity of rhizobacteria was determined qualitatively by using Nautiyal (1999) method. Bacterial strains were assessed for their ability to solubilize inorganic phosphate. Tri-calcium orthophosphate was used in agar medium as insoluble inorganic form of phosphate and was used as a source of indication for phosphate sulubilization property of the bacterial strains. Medium for PB was prepared according to Nautiyal (1999) with slight modifications. The medium was comprised of agar (15 g), glucose (10 g), NH4Cl (5 g), NaCl (1 g), MgSO4.7H2O (1 g), Ca3(HPO4) (0.8 g) and yeast extract (0.5 g) per liter while pH of the medium was adjusted to 7.2. On each plate three bacterial strains were checked with triplicate and the plates were incubated at 30±1 °C for 4 days where as uninoculated medium with tricalcium phosphate source served as control. A clear halo formed around some of the colonies after 4 days indicated that these isolates were positive for phosphate solubilization.

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3.7.3. Siderophores detection

In order to determine siderophores detection assay for selected bacterial strains isolated from transgenic and non transgenic rhizosphere method as described by Perez-Miranda et al. (2007) was used. In this assay CAS medium was used which was prepared according to (Schwyn and Neilands, 1987) procedure with some modifications in the absence of nutrients. The CAS medium (1L) contained Chrome azurol S (CAS) 60.5 mg, hexadecyltrimetyl ammonium bromide (HDTMA) 72.9 mg, Piperazine-1,4-bis (2- ethanesulfonic acid) (PIPES) 30.24 g, and 1 mM FeCl3·6H2O in 10 mM HCl 10 mL. Agarose (0.9%, w/v) was used as gelling agent. Siderophores detection was achieved after 10 ml (standard, 80 mm diameter Petri dishes) overlays of this medium were applied over those agar plates containing cultivated microorganisms to be tested for siderophores production. After a maximum period of 80 minutes, a change in color was observed in the overlaid medium exclusively surrounding producer microorganisms, from blue to purple (as described in the traditional CAS assay for siderophores of the catechol type) or from blue to orange (as reported for microorganisms that produce hydroxamates). Assay specificity was evaluated by repeating the above mentioned protocol with non-deferrated media (thus, lacking induction of siderophores production). All these experiments were performed at least three times each in three replicates.

3.7.4. Nitrate reduction

The ability of bacterial isolates to reduce nitrates to nitrites or beyond the nitrate stage was determined using method described by Cappuccino and Sherman (1996). Bacterial isolates were grown on TSB supplemented with 0.1% potassium nitrate and 0.1% agar (to make the media semisolid). Semisolidity resists the diffusion of oxygen into medium and hence favors anaerobic environment necessary for nitrate reduction. Cultures were incubated at 28±2 ºC for 48 hours. Following incubation two reagents were added to cultures to determine the ability to reduce nitrates. Reagents used were solution A (sulfanilic acid) followed by solution B (alphanaphthylmine). In case of reduction addition of above mentioned reagents produced immediate cherry red colour. Zinc powder was added to cultures not showing colour change to confirm whether nitrates were not reduced or isolate rapidly reduced nitrates beyond nitrites to ammonia or 52

MATERIALS AND METHODS CHAPTER 3 nitrogen gas. Reduction of nitrates beyond nitrites was inferred in cases where addition of Zinc did not produced any colour change.

3.8. Plant growth promotion traits and in vitro plant growth promotion assay

Green house experiment was conducted in the Department of Biotechnology Quaid-i- Azam University Islamabad for inoculation of seed with isolated strains.

3.8.1. Inoculation procedure and plant growth

The study of the effect of bacterial strains on growth of maize was carried out in pots using sterilized loamy sand (sieved and autoclave) in the greenhouse under control condition with 16/8 photoperiod and 24-28 oC temperature. The seeds of maize (Islamabad gold variety) were surface sterilized with 70% ethanol for 5 minutes and 0.1% mercuric chloride (HgCl2) for 15 minutes. The sterilized seeds were rinsed three times with sterilized distilled water . Bacterial strains were grown in TSB medium and agitated on a rotary shaker (120 rpm; 37 oC) for two days and before harvesting centrifuged and resuspended. Cell suspensions was adjusted to about 108 colony forming units (CFU/ml). The seeds were soaked for 30 minutes in bacterial suspension where for control seeds were just inoculated in broth without any bacterial strains and were grown in 10 cm diameter pots contained sterilized loamy sand in greenhouse under control conditions with an average temperature of 24–28 oC. The inoculation treatments were set-up in a randomized design with three replicates. Six seeds of maize were sown per pot and after germination plants were thinned to four plants per pot. The loamy sand was moistened with autoclaved water and maintained at 60% of moisture holding capacity (MHC). Six weeks after germination, the plants were uprooted and washed to remove the particles and data was recorded for different parameters.

3.9. Enzymatic characterization

Different enzymatic activities of the isolated bacterial strains from transgenic and non transgenic maize rhizosphere were conducted and are discussed as under. 53

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3.9.1. Protease activity

In order to determine the protease activity of the selected bacterial isolates from transgenic and non transgenic maize rhizosphere at different developmental stages. The protease activity of selected bacterial strains were determined by Smibert and Krieg (1994) using skim milk agar medium. All the strains were processed in triplicates and strict measures were taken to avoide any kind of contamination. About 500 ml of modified TSB medium was prepared, while in another flask 1.5% (W/V) of skimmed milk was dissolved in distilled water (100 ml). Both of these flasks were properly plugged, labeled and autoclaved at 121 ºC for 20 minutes. From each of the strains to be tested for protease activity, a colony was picked with a sterile inoculation loop and was spotted on the media plate containing skim milk. On each plate five morphologically different strains were spotted. All the plates were then placed in an incubator at 30 oC and were regularly checked after 24, 48 and 72 hours to find out if there were any protease activity.

3.9.2. Cellulase activity

In order to determine the cellulase activity of the selected bacterial strains, all the strains were processed in triplicates and strict measures were taken to avoid any kind of contamination. Cellulase activity of the bacterial strains were analyzed by Cattelan et al. (1999) method. Carboxy Methyl Cellulose (CMC) medium (0.2%) was prepared (1g CMC and 3 g of TSB dissolved in 500 ml of distilled water). The CMC medium was properly plugged, labeled and autoclaved at 121 ºC for 20 minutes. After autoclaving the media was cooled and poured into plates, five different strains were inoculated onto a single plate. All the plates were then placed in an incubator at 37 oC for 48 hours. After 48 hours incubation all the plates were flooded with 0.1% of congo red dye solution (0.1 g CRD in 100 ml of distilled water), the plates were shaken carefully in shaker for about 20-30 minutes. After shaking the plates were washed with 1 M NaCl solution. Data was recorded by examining yellow halos against red background.

3.9.3. Lipase activity

In order to determine the Lipase activity of the selected bacterial strains, all the strains

MATERIALS AND METHODS CHAPTER 3 were processed in triplicates and strict measures were taken to avoid any kind of contamination. For lipase activity 1% of tween 20 was added to TSB medium and was properly plugged, labeled and autoclaved at 121 ºC for 20 minutes. The media was cooled and poured into plates; five different strains were spotted onto a single plate with sterile inoculation loop. All the plates were then placed in an incubator at 25 oC and were regularly checked after 24 and 48 hours to find out if there was any protease activity. Data was recorded by examined white type precipitation surrounding their colony.

3.9.4. Chitinase activity

Chitinase activity was determined by using Renwick et al. (1991) method, in which carbon was the sole source in a defined medium having colloidal chitin. All the strains were processed in triplicates and strict measures were taken to avoid any kind of contamination. TSA medium (0.5g of MgSO4-7H2O, 0.7g of K2HPO4, 0.3 g of KH2PO4,

0.01 g of FeSO4 · H2O, 0.001 g of ZnSO4, 0.001 g of MnCl2) with 0.6% (w/v) colloidal chitin was used. For colloidal chitin two grams of chitin from crab shell (UniChem) was dissolved in concentrated HCl (200 ml), by shaking the mixture overnight at 4 oC in shaker. To decrease the viscosity of mixture it was incubated in water bath at 37 oC. These isolates were screened to determine chitinase production. Each isolate was inoculated on colloidal chitin agar (CCA) and incubated at 28 ºC in the dark until (after 7 days incubation) zones of chitin clearing were seen around colonies. Clear zone diameters are measured (mm) and used to indicate the chitinase activity of each isolate.

3.9.5. Pectinase activity

The pectinase activity was determined by Raju and Divakar (2013) method. After 48 hours incubation at 28 °C, the plates were flooded with iodine solution (50 mM) and incubated for 15 minutes at 37 oC. Strains surrounded by clear halos around colonies were considered positive for pectinase activity. Composition of media used for pectinolytic activity was pectin (0.2%), KH2PO4 (0.3%), MgSO4.7H2O (0.01%), NaCl

(0.5%), NH4Cl (0.2%), Na2HPO4 (0.6%). Bacterial isolates were spot inoculated on plates and incubated at 28± 2 °C for three days. The medium was properly plugged, labeled and autoclaved at 121 ºC for 20 minutes. From each of the strains to be tested for

MATERIALS AND METHODS CHAPTER 3 pectinase activity, single colony was picked with a sterile inoculation loop and was spotted on the media plates. After incubation, plates were observed for pectinase activity by flooding plates with iodine solution. Isolates possessing pectinolytic activity formed clear zones around colonies

3.10. Functional characterization of the cultured isolates

3.10.1. Antimicrobial characterization

Different functional assays of the isolated bacterial strains from transgenic and non transgenic maize rhizosphere were conducted which are discussed as under.

3.10.2. Antibacterial activity

In order to determine the antibacterial activities by disk diffusion method of the selected bacterial isolates from transgenic and non transgenic maize rhizosphere at different developmental stages was conducted. All the strains were processed in triplicates and strict measures were taken to avoid any kind of contamination. Antibacterial activity of test strains were tested against Pseudomonas syringae (FCBP 009) and Xanthomonas axonopodis (FCBP 001). The bacterial strains were first grown in TSB broth overnight in a shaking incubator at 25 oC. First these selected target strains were overnight grown in TSB broth. About 50 ul of these selected target strains were spread on solidified TSB+Agar plates with a sterile glass spreader. Then 5 sterile filter disks were placed on top of solidified media plates, the control disk was placed in the center while the other four disks were placed at equal distance from the center. From each test strains 10 µl was poured onto separate filter disks, while onto the center filter disk 10 µl antibiotic solution (kanamycin) was applied at 20 µg/ml and was considered as positive control. These plates were then incubated at 37 oC and were observed clear zones after 24-72 hours.

3.10.3. Antifungal activity

In order to determine the antifungal activities of the selected bacterial isolates from transgenic and non transgenic maize rhizosphere at different developmental stages all the

MATERIALS AND METHODS CHAPTER 3 strains were processed in triplicates and strict measures were taken to avoid any kind of contamination. Antifungal activity of bacterial isolates was tested against Aspergilus niger (FCBP 1109) and Fusrium oxysporum (FCBP 1114). Medium used for antifungal activity contained 1:1 PDA and TSA. Fungal disc was placed in the middle of the plate and bacterial isolates were inoculated 2 cm away from fungal discs. Bacterial isolates were streaked on three sides around the fungal discs while the fourth side was left uninoculated to serve as negative control. Plates were incubated at 28± 2 °C for seven days. Isolates possessing antifungal activity restricted fungal growth while it grows extensively at uninoculated portion of plate.

3.11. Statistical analyses

Statistical analysis was performed using one way analysis of variance (ANOVA) followed by least significant difference (LSD) using the statistix software version 8.1 (USA).

RESULTS CHAPTER 4

4. RESULTS

4.1. Bacterial diversity

4.1.1. Analysis of pre sowing rhizospheric bacteria on the basis of 16S rRNA gene sequence

From the pre sowing soil sample, different bacterial strains were isolated through cultured method. Eighteen morphologically different bacterial strains were selected for sequence analysis. On the basis of RDP Naive Bayesian rRNA Classifier Hierarchy analysis, all the pre sowing isolated strains were grouped to two major phyla’s namely Firmicutes (61%) and Proteobacteria (39%) three genera, 56% Bacillus, 33% Pseudomonas and 11% Enterobacter (Appendix 2A-2).

On the basis of partial 16S rRNA analysis 10 strains were identified as Bacillus species. Four strains, RS-A-1, RS-A-6, RS-A-8 and RS-A-17 were belong to Bacillus anthracis, while three strains RS-A-7, RS-A-9 and RS-A-17 were identified as Bacillus cereus two strains RS-A-3 and RS-A-9 were identified as Bacillus tequilensis and one strains RS-A- 2 was identified as Bacillus aerophilus. Five of the strains were found to be Pseudomonas species i.e., RS-A-10, RS-A-11, RS-A-12, RS-A-14, and RS-A-16 were identified as Pseudomonas taiwanensis. While the remaining three strains RS-A-15, RS- A-18 and RS-A-4 were identified as Enterobacter cloacae, Enterobacter cancerogenus and Pseudomonas plecoglossicida respectively. On the basis of genus criteria, 5 major genera were identified as Bacillus, Pseudomonas, Enterobacter, Klebsiell and Salirhabdus (Appendix 2A-2). Bacillus was the major genera representing about ten of all the isolated strains. The isolates RS-A-1, RS-A-2, RS-A-3, RS-A-5, RS-A-6, RS-A-7, RS-A-8, RS-A-9, RS-A-11 and RS-A-17 belong to genus Bacillus. Pseudomonas was the second major genera representing fifth of all the isolated strains. The isolates RS-A-4, RS-A-10, RS-A-12, RS-A-14 and RS-A-16 belong to genus Pseudomonas. Whereas Enterobacter, Klebsiell and Salirhabdus were the minor genera. The isolates RS-A-18, RS-A-15 and RS-A-13 belong to genera Enterobacter, Klebsiell and Salirhabdus respectively. About 55.5% of the strains were found to belong to genus Bacillus, whereas

RESULTS CHAPTER 4

27.7% of the strains belong to genus Pseudomonas. While individually each genera of Enterobacter, Klebsella and Salirhabdus constitute for 5.6% of all the strains. On the basis of 16S rRNA gene sequences blast, 28% isolates were identification Bacillus anthracis, 22% Pseudomonas taiwanensis, 17% Bacillus cereus, 11% Bacillus tequilensis, whereas Bacillus aerophilus and Enterobacter cancerogenus constitute 6% each, while Enterobacter cloacae subsp. and Pseudomonas plecoglossicida constitute 5% each of the total pre sowing stage isolated strains (Appendix 2A-1).

RESULTS CHAPTER 4

Table 4.1. Identification of bacterial strains from pre sowing soil samples using analysis of partial 16S rRNA gene sequences.

Isolate Organism Accession Closely related type strain % 16S identified numbers rRNA identity RS-A-1 Bacillus sp. KC430942 Bacillus anthracis ATCC 14578T 100 RS-A-2 Bacillus sp. KC430943 Bacillus aerophilus 28K T 100 RS-A-3 Bacillus sp. KC430944 Bacillus tequilensis 10b T 99 RS-A-4 Pseudomonas sp. KC430945 Pseudomonas plecoglossicida FPC951 T 96 RS-A-5 Bacillus sp. KC430946 Bacillus cereus ATCC 14579 T 100 RS-A-6 Bacillus sp. KC430947 Bacillus anthracis ATCC 14578 T 100 RS-A-7 Bacillus sp. KC430948 Bacillus cereus ATCC 14579 T 100 RS-A-8 Bacillus sp. KC430949 Bacillus anthracis ATCC 14578 T 98 RS-A-9 Bacillus sp. KC430950 Bacillus tequilensis 10b T 99 RS-A-10 Pseudomonas sp. KC430951 Pseudomonas taiwanensis BCRC 17751 T 99 RS-A-11 Pseudomonas sp. KC430952 Pseudomonas taiwanensis BCRC 17751 T 100 RS-A-12 Pseudomonas sp. KC430953 Pseudomonas taiwanensis BCRC 17751 T 99 RS-A-13 Bacillus sp. KC430954 Bacillus anthracis ATCC 14578 T 94 RS-A-14 Pseudomonas sp. KC430955 Pseudomonas taiwanensis BCRC 17751 T 99 RS-A-15 Enterobacter sp. KC430956 Enterobacter cloacae subsp. dissolvens LMG 2683 T 96 RS-A-16 Pseudomonas sp. KC430957 Pseudomonas taiwanensis BCRC 17751 T 100 RS-A-17 Bacillus sp. KC430958 Bacillus cereus ATCC 14579 T 99 RS-A-18 Enterobacter sp. KC430959 Enterobacter cancerogenus LMG 2693 T 99

RESULTS CHAPTER 4

Stage A* Bacterial Strains Bacillus cereus

Pseudomonas taiwanensis 6% 6% 17% Enterobacter cloacae subsp. 5% Bacillus anthracis 11% Bacillus tequilensis 22% Pseudomonas plecoglossicida

Bacillus aerophilus 28% 5% Enterobacter cancerogenus

Figure 4-1: Percentage of various bacterial strains isolated from pre sowing soil samples

RESULTS CHAPTER 4

4.1.2. Phylogenetic analysis of pre sowing rhizospheric bacteria on the basis of 16S rRNA gene sequence

The 18 pre sowing rhizospheric bacteria that were used for PGPR and enzymatic characterization were identified by partial 16S rRNA gene sequence analysis (

Table 1.1). Five different genera were identified and assigned to two major classes: Firmicutes (n=10; 55.6%), Proteobacteria (n=8; 44.4%) respectively. A phylogenetic tree of pre sowing rhizosphere isolates was generated from the pre sowing stage sequence data using the neighbor-joining method with the Jukes and Cantor model in a MEGA4 program (Table 1.2). For the 16S rRNA gene data, branching patterns remained consistent, depending on which sequences of other related type strains were included in the data set. The correspondingly high bootstrap values resulted in significant branching points in the 16S rRNA gene sequence based phylogenetic tree. Sequence identity within 94%–100% and their phylogenetic relationships to representative type strains of genera are shown in Figure 4.2. Strains representative of phylum Firmicutes were placed in the cluster recovered with bootstrap values 99%. Strains RS-A-8, RS-A-13 and RS-A-17 were almost related to each other. In addition, RS-A-6, RS-A-5, RS-A-1, RS-A-11 and RS-A-7 were identical to each other with high bootstrap value (86%). While strains RS- A-2, RS-A-3 and RS-A-9 were identical to each other with bootstrap value (99%) These isolates mainly belonged to the genus Bacillus and were placed in two clusters of different species, where three isolates were identical to type strains Bacillus anthracis, five isolates to Bacillus cereus, two isolates to Bacillus aerophilus and one isolate was closely related to Bacillus tequilensis. Strains representative of phylum Proteobacteria were placed in two clusters. Strains RS-A-18 was almost identical to Enterobacter cancerogenus, whereas strains RS-A-15 was closely related to type strain Enterobacter cloacae. Strains RS-A-16, RS-A-12 and RS-A-10 were identical to each other and closely related to type strain Pseudomonas taiwanensis. While isolate RS-A-4 was closely related to type strains Pseudomonas plecoglossicida.

RESULTS CHAPTER 4

RS-A-8 (KC430949) RS-A-13 (KC430954) RS-A-17 (KC430958) Bacillus anthracis (KF150341) 86 RS-A-11 (KC430952) RS-A-7 (KC430948) Bacillus cereus (AE016877) RS-A-1 (KC430942) 99 RS-A-5 (KC430946) RS-A-6 (KC430947)

99 RS-A-2 (KC430943) Bacillus aerophilus (NR 042336)

95 RS-A-9 (KC430950) RS-A-3 (KC430944) 99 Bacillus tequilensis (HQ223107)

98 RS-A-18 (KC430959) 71 Enterobacter cancerogenus(NR 044977) 99 Enterobacter cloacae (NR 044978) RS-A-15 (KC430956)

16 RS-A-16 (KC430957) Pseudomonas taiwanensis (EU103629) RS-A-10 (KC430951) 99 RS-A-12 (KC430953) Pseudomonas plecoglossicida (AY032725) 37 RS-A-4 (KC430945) RS-A-14 (KC430955)

0.20 0.15 0.10 0.05 0.00

Figure 4-2: The phylogenetic tree of pre sowing isolates was constructed by using the neighbor-joining method (compute linearized) data obtained from aligned nucleotides. Bootstrap values (expressed as percentage of 1000 replications) greater than 50 % are shown a at the branch points. Bar, 0.01 changes per nucleotide position. GenBank accession numbers for each sequence are shown in parentheses.

RESULTS CHAPTER 4

4.1.3. Phylogenetic analysis of vegetative stage collected rhizospheric bacteria on the basis of 16S rRNA gene sequence.

Analysis of the vegetative rhizosphere bacterial isolates

On the basis of RDP Naive Bayesian rRNA Classifier Hierarchy analysis (Table 4.2), all the vegetative stage isolated strains were grouped into four major phyla’s namely Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. On the basis of other ranks of classification there were 11 genera namely Pseudomonas, Acinetobacter, Enterobacter, Stenotrophomonas, Sphingomonas, Chryseobacterium, Sphingobacterium, Bacillus, Solibacillus, Aerococcus and Microbacterium (appendix 2A-2).

On the basis of 16S rRNA analysis it was explored that 5 strains belong to Pseudomonas. RS-BGM-23, RS-BIW-26, RS-BIW-32 and RS-BIG-38 have greater similarities with Pseudomonas, Pseudomonas linimoorei, Pseudomonas lini and Pseudomonas cremoricolorata respectively. Five strains belong to Chryseobacterium (RS-BGM-19, RS-BIW-25, RS-BIW-28, RS-BIG-36, RS-BGM-24). They were most closely related to Chryseobacterium indologenes, Chryseobacterium indologenes, Chryseobacterium indologenes, and Chryseobacterium hagamense respectively. Three isolates RS-BGM-21, RS-BIW-31, and RS-BIG-37 were belong to Enterobacter genus. They were considered Enterobacter cloacae, Enterobacter cloacae and Enterobacter ludwigii respectively on the basis of partial 16S rRNA sequence. Two strains RS-BGM-20, RS-BIW-33 were related to Microbacterium genus and were resembled to Micrococcus antarcticus and Microbacterium oleivorans respectively. During vegetative stage 33% bacterial strains were isolated from GM maize rhizosphere. While remaining 67% bacterial strains were isolated from two Non-GM maize rhizosphere (BIW-48%; BIG 19%). In the GM maize rhizosphere Micrococcus antarcticus and Sphingobacterium multivorum were present but were absent in the other two Non-GM maize rhizosphere samples.

RESULTS CHAPTER 4

Table 4. 2: Identification of isolates from GM and Non-GM maize Rhizosphere Bacterial strains using analysis of partial 16S rRNA gene sequences.

% 16S Accession Isolate Organism identified Closest type strain in eztaxon database rRNA Variety numbers identity RS-BGM-19 Chryseobacterium sp. KC430960 Chryseobacterium indologenes LMG 8337 T 98 RS-BGM-20 GM Micrococcus sp. KC430961 Micrococcus antarcticus T2 T 99 RS-BGM-21 Enterobacter sp. KC430962 Enterobacter cloacae subsp. dissolvens LMG 2683 T 99 RS-BGM-22 Bacillus sp. KC430963 Bacillus cereus ATCC 14579 T 100 RS-BGM-23 Pseudomonas sp. KC430964 Pseudomonas moorei RW10 T 99 RS-BGM-24 Sphingobacterium sp. KC430965 Sphingobacterium multivorum IAM14316 T 98 RS-BGM-24A Pseudomonas sp. KC430965 Pseudomonas hibiscicola ATCC 19867 T 94 RS-BIW-25 Non- Chryseobacterium sp. KC430966 Chryseobacterium indologenes LMG 8337 T 98 RS-BIW-26 GM Pseudomonas sp. KC430967 Pseudomonas lini CFBP 5737 T 99 Ism. T RS-BIW-27 white Bacillus sp. KC430968 Bacillus isronensis B3W22 98 RS-BIW-28 variety Chryseobacterium sp. KC430969 Chryseobacterium indologenes LMG 8337 T 98 RS-BIW-29 Acinetobacter sp. KC430970 Acinetobacter pittii LMG 1035 T 99 RS-BIW-30 Sphingomonas sp. KC430971 Sphingomonas koreensis JSS26 T 99 RS-BIW-31 Enterobacter sp. KC430972 Enterobacter cloacae subsp. dissolvens LMG 2683 T 100 RS-BIW-32 Pseudomonas sp. KC430973 Pseudomonas lini CFBP 5737 T 99 RS-BIW-33 Microbacterium sp. KC430974 Microbacterium oleivorans DSM 16091 T 99 RS-BIW-34 Sphingomonas sp. KC430975 Sphingomonas koreensis JSS26 T 99 RS-BIG-35 Non-GM Aerococcus sp. KC430976 Aerococcus urinaeequi IFO 12173 T 97 RS-BIG-36 Ism. gold Chryseobacterium sp. KC430977 Chryseobacterium hagamense RHA29 T 97 variety RS-BIG-37 Enterobacter sp. KC430978 Enterobacter ludwigii DSM 16688 T 99 RS-BIG-38 Pseudomonas sp. KC430979 Pseudomonas cremoricolorata NRIC 0181 T 100

RESULTS CHAPTER 4

Stage B* Isolates Chryseobacterium Micrococcus 5% 5% 19% Enterobacter 9% Bacillus

5% 5% Pseudomonas

5% Sphingobacterium 14% Acinetobacter Sphingomonas 24% 9% Microbacterium Aerococcus

Figure 4-3: Percentage of various genera isolated from GM and Non-GM maize rhizosphere samples.

Where B* is representing the vegetative stage rhizosphere isolates

RESULTS CHAPTER 4

4.1.4. Phylogenetic analysis of vegetative stage rhizospheric bacteria on the basis of 16S rRNA gene sequence

All the bacterial isolates were classified by BLAST analysis using their 16S RNA partial sequences. Various data bases like NCBI, RDP and Eztaxon were used for the determination of exact taxonomic position of each isolate. RDP Naive Bayesian rRNA Classifier version 2.5 were used for the delineation of whole hierarchy of the isolates. The partial sequences of 16S rRNA closet strains were obtained from NCBI for the phylogenetic tree construction.

All the sequences were aligned with reference sequences from NCBI with ClustalW and phylogenetic tree (Figure 4.4) was constructed by neighbor-joining method using MEGA 4 software. There were isolated 17 strains from GM and Non-GM maize rhizosphere belonging to 19 genera. Pseudomonas was the dominant genus having 4 isolates and constitute 24% of the total isolates.

Chryseobacterium was considered the second major genus contained 4 isolates and comprising 19% of the total strains. Third major genus was Enterobacter constituting 14% of the total isolated bacterial strains. Bacillus and Sphingobacterium genera were comprised 9% each of the total isolates. The remaining 5 genera Micrococcus, Sphingobacterium, Acinetobacter, Microbacterium, and Aerococcus were contained 1 isolate each on comprising 25% collectively of the total bacterial strains (appendix 2B-1).

RESULTS CHAPTER 4

93 RS-BIW-32 (KC430973) Pseudomonas putida (AB680667) 36 RS-BIW-26 (KC430967) 10 Pseudomonas sp.(KC012939) 0 RS-BGM-23 (KC430964) 66 RS-BIG-38 (KC430979) RS-BIW-31 (KC430972) Enterobacter ludwigii(KC139450) 77 100 RS-BIG-37 (KC430978) RS-BGM-21 (KC430962) 66 Enterobacter cloacae(JX885522) 100 RS-BIW-29 (KC430970) 100 Acinetobacter sp.(KC853193) 62 RS-BGM-24A 100 Stenotrophomonas maltophilia(AY748889) RS-BIW-30 (KC430971) RS-BIW-34 (KC430975) 100 Sphingomonas sp.(GU184189) 100 RS-BGM-20 (KC430961) 100 Micrococcus antarcticus (KC494324) RS-BIW-33 (KC430974) 100 Microbacterium oleivorans(KC764962) 81 10 RS-BIG-35 (KC430976) 0 Aerococcus sp.(HE979850) 100 RS-BIW-27 (KC430968) 58 RS-BGM-22 (KC430963) 100 Bacillus sp.(KC466270) 100 RS-BGM-24 (KC430965) Sphingobacterium sp.(JF703657) 100 RS-BIG-36 (KC430977) RS-BIW-25 (KC430966) 100 RS-BIW-28 (KC430969) 99 RS-BGM-19 (KC430960) 91 Chryseobacterium sp.(AY468461)

0.15 0.10 0.05 0.00

Figure 4-4: The phylogenetic tree of isolates from GM and Non-GM maize rhizosphere during vegetative stage was constructed by using the neighbor-joining method (compute linearized) data obtained from aligned nucleotides. Bootstrap values (expressed as percentage of 1000 replications. GenBank accession numbers for each sequence are shown in parentheses.

RESULTS CHAPTER 4

Part C

4.1.5. Phylogenetic analysis of GM and Non-GM maize harvesting stage rhizospheric bacteria on the basis of 16S rRNA gene sequence

Analysis of harvesting stage bacterial isolates

From GM and Non-GM maize field trial rhizosphere bacterial were isolated during harvesting stage. On the basis of morphology differences total 16 bacterial strains, 5 from GM while 11 from two Non-GM maize varieties rhizosphere were sequenced. All the isolates were grouped into 4 major phyla on the basis RDP Naïve Bayesian rRNA Classifier hierarchy analysis. The major identified phyla's were Firmicutes (50%), Proteobacteria (25%), Actinobacteria (19%) and Bacteroidetes (6%). On the basis of genera classification rank there were 6 major genera, Bacillus (50%), Enterobacter (12%), Pseudomonas (12%), Bordetella (12%), Microbacterium (19%), Flavobacterium (6%) (Appendix: 2C-2).

On the basis of 16S rRNA analysis it was revealed that 5 strains namely, RS-CGM-39, RS-CGM-40, RS-CIG-48, RS-CIG-49 and RS-CIG-52 were closely related to type strain Bacillus cereus. Whereas strains RS-CGM-43, RS-CIW-46 and RS-CIG-53 were closely related to Bacillus aryabhattai, Bacillus idriensis and Bacillus anthracis respectively. Two isolate of phylum Proteobacteria RS-CGM-41 and RS-CGM-42 were identical to Enterobacter cloacae while similarly RS-CGM-43A and RS-CIW-44 were closely related to type strains Microbacterium hominis. While isolates RS-CIG-50, RS-CIW-47, RS-CIW-45 and RS-CIG-51 were identical to Pseudomonas stutzeri, Bordetella petrii, Microbacterium trichothecenolyticum and Flavobacterium sasangense respectively. (Appendix: 2C-2).

RESULTS CHAPTER 4

Table 4. 3: Identification of isolates from GM and Non-GM maize Rhizosphere during harvesting stage using analysis of partial 16S rRNA gene sequences.

% 16S Organism Accession Isolate Varieties Closest type strain in eztaxon database rRNA identified numbers rhizosphere identity RS-CGM-39 Bacillus sp. KC430980 Bacillus cereus ATCC 14579 T 100 RS-CGM-40 GM Bacillus sp. KC430981 Bacillus cereus ATCC 14579 T 100 RS-CGM-41 Enterobacter sp. KC430982 Enterobacter cloacae subsp. dissolvens LMG 2683 T 98 RS-CGM-42 Enterobacter sp. KC430983 Enterobacter cloacae subsp. dissolvens LMG 2683 T 98 RS-CGM-43 Bacillus sp. KC430984 Bacillus aryabhattai B8W22 T 93 RS-CIW-44 Microbacterium sp. KC430985 Microbacterium hominis IFO 15708 T 97 RS-CGM-43A Non- Microbacterium sp. AB004727 Microbacterium hominis IFO 15708 T 97 RS-CIW-44 GM Microbacterium sp. KC430985 Microbacterium hominis IFO 15708 T 97 Ism. white RS-CIW-45 Microbacterium sp. KC430986 Microbacterium trichothecenolyticum IFO 15077 T 98

RS-CIW-46 Bacillus sp. KC430987 Bacillus idriensis SMC 4352-2 T 99 RS-CIW-47 Bordetella sp. KC430988 Bordetella petrii DSM 12804 T 97 RS-CIG-48 Non- Bacillus sp. KC430989 Bacillus cereus ATCC 14579 T 99 RS-CIG-49 GM Bacillus sp. KC430990 Bacillus cereus ATCC 14579 T 100 RS-CIG-50 Ism. gold Pseudomonas sp. KC430991 Pseudomonas stutzeri ATCC 17588(T) 99

RS-CIG-51 Flavobacterium sp. KC430992 Flavobacterium sasangense YC6274(T) 97 RS-CIG-52 Bacillus sp. KC430993 Bacillus cereus ATCC 14579(T) 100

RESULTS CHAPTER 4

Stage C* Isolates

Bacillus 6% 6% Enterobacter 6% Microbacterium 44%

Bordetella

25% Pseudomonas

Flavobacterium 13%

Figure 4-5: Percentage of various genera isolates from GM and Non-GM maize Rhizosphere during harvesting stage.

Where C* represent the harvesting stage bacterial isolates.

RESULTS CHAPTER 4

4.1.6. Phylogenetic analysis of the isolates at harvesting stage

All the bacterial isolates were classified by BLAST analysis with respect to their 16S RNA partial sequences (Table 4.3). Various data bases like NCBI, RDP and Eztaxon were used for the determination of exact taxonomic position of each isolate. RDP Naive Bayesian rRNA Classifier version 2.5 were used for the delineation of whole hierarchy of the isolates. The partial sequences of 16S rRNA closet strains were obtained from NCBI for the phylogenetic tree construction. All the sequences were aligned with reference sequences from NCBI with ClustalW and phylogenetic tree was constructed by neighbor- joining method using MEGA 4 software .

Total 6 genera were isolated from the GM Non-GM rhizosphere at the harvesting stage. In these genera bacillus genus comprised the dominant one containing 7 bacterial isolated strains and was the 44% of all the isolates genera. The second major genus was Microbacterium containing 4 isolates and constituting 25% of the total isolates genera. The third abundant was genus is Enterobacter comprising 13% of the all genera. Brodetella, Pseudomonas and Flavobacterium were the minor genera collectively contributing 18%. During harvesting stage 38% bacterial strains were isolated from GM maize rhizosphere while 62% were isolated from two Non-GM maize rhizosphere samples (IGC-31%; IWC-31%). Bacillus cereus, Enterobacter cloacae, Bacillus aryabhattai and Microbacterium hominis were predominant species in the GM maize rhizosphere whereas Microbacterium hominis, Microbacterium trichothecenolyticum, Bacillus idriensis and Bordetella petrii were identified in Non-GM maize (IW-Variety) rhizosphere.

RESULTS CHAPTER 4

RS-CIG-48 (KC430989) RS-CIG-53 (KC430994) RS-CGM-39 (KC430980) 100 Bacillus cereus(KC876035) RS-CIG-49 (KC430990) 59 RS-CIG-52 (KC430993) RS-CGM-40 (KC430981) 100 RS-CIW-46 (KC430987) 100 Bacillus idriensis(KC429594)

79 RS-CGM-43 (KC430984) RS-CGM-43A Microbacterium sp.(JQ977248) RS-CIW-45 (KC430986) 100 RS-CIW-44 (KC430985) Microbacterium sp.(HM629344)

100 RS-CIW-47 (KC430988) Betaproteobacterium (AB470444)

100 RS-CIG-50 (KC430991) 100 Pseudomonas stutzeri(JQ782508)

61 RS-CGM-42 (KC430983)

100 RS-CGM-41 (KC430982) 67 Enterobacter cloacae (JX885522) RS-CIG-51 (KC430992) 100 Flavobacterium sp.(KC157036)

0.20 0.15 0.10 0.05 0.00

Figure 4-6: The phylogenetic tree of the isolates from GM and Non-GM maize rhizosphere during harvesting stage was constructed by using the neighbor-joining method (compute linearized) data obtained from aligned nucleotides. Bootstrap values (expressed as percentage of 1000 replications) GenBank accession numbers for each sequence are shown in parentheses.

RESULTS CHAPTER 4

4.2. Metagenomics analysis

4.2.1. Pyrosequencing based GM and Non-GM maize rhizosphere bacterial diversity a. Bacterial diversity based on phylum level distribution of field soil sample

Rhizosphere samples were collected from GM and Non-GM maize during vegetative stage (NARC Islamabad- Pakistan). Genomic DNA was extracted from rhizosphere samples (0.25 g) using Power Soil DNA Isolation Kit (Mo Bio USA), followed the manufactured instruction. The extracted DNA was confirmed on 1% agarose gel and amplified with 16S RNA gene. The DNA samples were sent for pyrosequencing to Molecular Research DNA (MR DNA) Laboratory of US. Bacterial diversity was assessed through pyrosequencing technology using 515F primer. Minimum 200 bp region of the 16S rRNA was sequenced and nominal 20,000 reads per sample were obtained. From pre sowing soil sample 19 phyla's (Fibrobacteres, Actinobacteria, Crenarchaeota, Planctomycetes, Euryarchaeota, Tenericutes, Proteobacteria, Synergistetes, Nitrospirae, Firmicutes, Bacteroidetes, Cyanobacteria, Dictyoglomi, Acidobacteria, Aquificae, Spirochaetes, Chloroflexi, Thermotogae, Verrucomicrobia) were identified through pyrosequencing analysis (Figure 4.7). Among these phyla 47% were Proteobacteria, 21% Bacteroidetes, 10% Actinobacteria, 7% Firmicutes, 4% each Planctomycetes and Verrucomicrobia, 2% Crenarchaeota. While phyla Fibrobacteres, Euryarchaeota, Tenericutes, Synergistetes, Nitrospirae, Cyanobacteria, Dictyoglomi, Aquificae, Spirochaetes and Chloroflexi contribute lowest percentage of total bacterial population in the pre sowing soil sample (Appendix 1-A). b. Bacterial diversity based on family level distribution of field soil sample

The field soil sample (before conducting the GM and Non-GM field trial) were collected in two different interval. One soil sample was collected before sowing of GM and Non- GM maize crops (represented by code A) while the second sample was collected after one year of harvesting GM and Non-GM maize crops from the same field (represented by

RESULTS CHAPTER 4

code A). The bacterial diversity of the samples was assessed through pyrosequencing technology using 515F primer. Total 185 families were obtained from pyrosequencing analysis while some families were unclassified and placed in others group. Total 26 major groups were found in higher percentage and contributed the maximum population. Among the major phyla's average 6.281% Flexibacteraceae (A-8.222%; A4.34%), 6.022% Ectothiorhodospiraceae (A-5.785%; A-6.258%), 4.596% Hyphomicrobiaceae (A-4.468%; A-4.723%) 4.590% Nitrososphaeraceae (A-1.891%; A-7.288%), 3.546% Rhodospirillaceae (A-3.159%; A-3.933%), 3.351% Rubrobacteraceae (A-2.518%; A- 4.183%), 2.885% Nitrosomonadaceae (A-3.493%; A-2.276%), 2.534% Bacillaceae (A- 1.918%; A-3.149%), 2.079% Polyangiaceae, 1.824% Pirellulaceae, 1.563% Streptomycetaceae, 1.967% Micromonosporaceae , 1.879% Moraxellaceae, 1.687% Sphingomonadaceae, 1.563% Streptomycetaceae, 1.390% Acidimicrobiaceae, 1.346% Nocardioidaceae, 1.270% Planococcaceae and 1.175% Desulfobacteraceae, contribute maximum population. While the reaming 167 families were found in lower percentage in both the samples (Appendix 1-B). c. Bacterial diversity based on genus level distribution of field soil sample

The field soil samples (before conducting the GM and Non-GM field trial) were collected in two different interval. One sample soil was collected before sowing of GM and Non- GM maize crops (represented by code A) while the second sample was collected after one year interval of GM and Non-GM maize crops from the same field (represented by code A). The bacterial diversity of the samples was assessed through pyrosequencing technology using 515F primer. On the basis of genera level distribution of bacterial flora, total 446 different genera were obtained from pyrosequencing analysis while some genera were unclassified and placed in others group (specified with name of others) (Appendix 1-C). Total 24 genera were found in higher percentage and contributed the maximum population. Among the major genera average 4.590% Nitrososphaera (A- 1.891%; A-7.288%), 4.196% Chitinophaga (A-5.934%; A-2.457%), 4.673% Ectothiorhodospira (A-4.77%; A-4.576%), 2.075% Rhodoplanes (A-2.193%; A- 1.957%), 2.080% Bacillus (A-1.516%; A-2.643%) and 2.014% Nitrosovibrio (A-

RESULTS CHAPTER 4

2.356%; A-1.672%) contributed the significant portion of total bacterial flora of the pre sowing as well as after one year interval analyzed soil samples. Whereas some genera like average 1.521% Streptomyces (A-1.241%; A-1.8%),1.076% Blastopirellula (A- 1.038%; A-1.113%), 1.076% Blastopirellula (A-1.038%; A-1.113%), 1.085% Opitutus (A-1.39%; A-0.78%), 1.660% Hyphomicrobium (A-1.593%; A-1.726%), 0.127% Halomonas (A-0.19%; A-0.064%), 0.028% Hydrogenophaga, 1.220% Thiothrix, 1.349% Lewinella, 1.852% Azospirillum, 1.973% Flexibacter, 1.253% Desulfovibrio, 1.039% Skermanella, 1.390% Acidimicrobium, 1.185% Clostridium, 1.917% Chondromyces and 1.095% Moraxella were the higher genera and contributed some portion of bacterial flora in both samples. While all the remaining genera were in minor population (Appendix 1-C).

RESULTS CHAPTER 4

Phya based bacterial diversity of pre sowing soil sample Euryarchaeota Acidobacteria Chloroflexi Verrucomicrobia 0% 1% Fibrobacteres Aquificae 1% 4% Others Crenarchaeota Thermotogae 0% 0% 4% 2% Dictyoglomi Spirochaetes 0% Actinobacteria Planctomycetes 0% 0% 10% 4% Cyanobacteria 0% Tenericutes 0% Bacteroidetes 21%

Proteobacteria 47% Firmicutes 7% Nitrospirae 0%

Synergistetes 0%

Figure 4-7: Bacterial diversity based on family level distribution of (pre sowing) field soil sample.

RESULTS CHAPTER 4

Phya based bacterial diversity of soil sample collected after one year interval

Deferribacteres Aquificae Chloroflexi Others 0% 0% 1% Verrucomicrobia 3% Fibrobacteres 2% 0% Acidobacteria Spirochaetes 1% 0%Thermotogae Thermi 0% Cyanobacteria Dictyoglomi 0% Crenarchaeota 1% 0% 7% Bacteroidetes Actinobacteria 8% 18% Planctomycetes Nitrospirae 4% 0% Firmicutes 10%

Synergistetes 0% Proteobacteria Euryarchaeota 44% 1%

Tenericutes 0%

Figure 4-8: Bacterial diversity based on family level distribution of field soil sample collected after one year interval from GM and Non-GM maize field trial.

RESULTS CHAPTER 4

4.2.2. Pyrosequencing based GM and Non-GM maize rhizosphere bacterial diversity during vegetative stage a. Bacterial diversity based on phylum level distribution of GM and Non-GM maize rhizosphere at vegetative stage.

From one GM and two Non-GM maize crops rhizosphere (during vegetative stage) total 19 phyla's (Fibrobacteres, Thermi, Actinobacteria, Crenarchaeota, Planctomycetes, Euryarchaeota, Tenericutes, Proteobacteria, Synergistetes, Nitrospirae, Firmicutes, Bacteroidetes, Cyanobacteria, Dictyoglomi, Acidobacteria, Deferribacteres, Aquificae, Spirochaetes, Chloroflexi, Thermotogae and Verrucomicrobia) were identified through pyrosequencing analysis. While some bacterial were new and placed in candidate division (represented with code: others). Total eight major groups were found in higher percentage and contributed the maximum population during vegetative stage of GM and Non-GM maize rhizosphere. Among the major phyla's average 127% Proteobacteria, 58% Actinobacteria, 41% Bacteroidetes, 21% Firmicutes, 14% Planctomycetes, 10% Verrucomicrobia, 9% and 3% Chloroflexi were found in vegetative stage rhizosphere soil of GM and Non-GM maize. Whereas 12 phyla's Fibrobacteres, Thermi, Euryarchaeota, Tenericutes, Synergistetes, Nitrospirae, Cyanobacteria, Dictyoglomi, Deferribacteres, Aquificae, Spirochaetes and Thermotogae were contribute average 1.5% of the total population (Appendix 1-A). While average 14% of the isolates were placed in new group (candidate division). In GM maize rhizosphere the major phylum were found 44.087% Proteobacteria, whereas the population of this phylum is lower in Non-GM maize rhizosphere (43.204 % IGB; 40.434% IWB). Similarly in GM maize rhizosphere the population of Bacteroidetes (16.862%) and were more than Non-GM maize varieties rhizosphere (13.305% IGB; 11.466% IWB ). While the population of other major phyla's like Actinobacteria (GMB-15.769%; IGB-21.266%; IWB-21.222%), Crenarchaeota (GMB-2.57%; IWB-4.698%), Planctomycetes (GMB-4.318%; IWB-5.35%), and Chloroflexi (GMB-0.885%; IWB-1.021%), were found more in Non-GM is compared to GM maize rhizosphere (Figure 4.9)

RESULTS CHAPTER 4

b. Bacterial diversity based on family level distribution of GM and Non-GM maize rhizosphere at vegetative stage.

Rhizosphere samples were collected from GM and Non-GM maize field during vegetative stage. Total three rhizosphere samples were selected, one from GM maize rhizosphere and two from Non-GM maize varieties (IG- Islamabad gold; IW- Islamabad white) and used for culturing as well as unculturing bacterial studies. The bacterial diversity of the samples were assessed through pyrosequencing technology using 515F primer. Total 185 families were obtained from pyrosequencing analysis while some families were unclassified and placed in others group. Total 28 major groups were found in higher percentage and contributed the maximum population. Total eight major groups were found in higher percentage and contributed the maximum population during vegetative stage of GM and Non-GM maize rhizosphere. Among the major phyla's average 5.771% Hyphomicrobiaceae, 5.541% Flexibacteraceae, 3.775% Ectothiorhodospiraceae, 2.364% Nocardiaceae, 2.098% Pirellulaceae, 2.670% Rhodospirillaceae, 2.618% Sphingomonadaceae, 2.384% Bacillaceae, 2.614% Nocardioidaceae, 1.242% Bradyrhizobiaceae, 1.301% Desulfobacteraceae, 1.708% Moraxellaceae, 1.769% Micromonosporaceae, 1.649% Acidimicrobiaceae, 1.215% Saprospiraceae, 1.055% Piscirickettsiaceae, 1.532% Planococcaceae, 1.350% Opitutaceae, 1.391% Polyangiaceae, 1.232% Mycobacteriaceae, 1.499% Thiotrichaceae, 1.116% Desulfovibrionaceae and 1.963% Streptomycetaceae contributed the major population of GM and Non-GM maize rhizosphere during vegetative stage. In GM maize rhizosphere the major families, average 5.541% Flexibacteraceae (GM- 6.694%; IG-5.203%, IW-4.726%), 2.618% Sphingomonadaceae (GM-4.21%; IG- 1.832%; IW-1.812%), 3.775% Ectothiorhodospiraceae (GM-4.102%; IG-3.79%, IW- 3.433%), 2.073% Opitutaceae (GM-2.073%; IG-0.997%, IW-0.98%), 1.215% Saprospiraceae (GM-1.875%; IG-1.039%, IW-0.73%), 1.301% Desulfobacteraceae (GM-1.586%; IG-1.323%, IW-0.994%) were pre dominate as compared to Non-GM maize rhizosphere. While the population of some families like Nitrososphaeraceae (GM-2.566%; IG-2.514%, IW-4.698%), Rubrobacteraceae (GM-3.383%; IG-4.211%, IW-5.77%), Pirellulaceae (GM-1.811%; IG-2.101%, IW-2.383%), Hyphomicrobiaceae

RESULTS CHAPTER 4

(GM-5.104%; IG-5.95%, IW-6.26%) and Nocardioidaceae (GM-1.798%; IG-3.891%, IW-2.154%) were pre dominant in Non-GM maize rhizosphere (Appendix 1-B). c. Bacterial diversity based on genus level distribution of GM and Non-GM maize rhizosphere at vegetative stage.

On the basis of genera level distribution of bacterial flora, total 470 different genera were obtained from pyrosequencing analyzed data, while some genera were unclassified and placed in others group (specified with name of others) which contributed average 3.984% of the total bacterial flora (Appendix 1-C). The major genera which contributed maximum portion of the GM and Non-GM maize rhizospheric bacterial community during crop vegetative stage were identified. Among the major genera average 4.455% Rubrobacter (GM-3.383%; IG-4.211%, IW-5.77%), 4.538% Chitinophaga (GM-5.371%; IG-4.545%, IW-3.697%), 3.259% Nitrososphaera (GM-2.566%; IG-2.514%, IW- 4.698%), 2.262% Rhodococcus (GM-1.319%;IG-3.697%,IW-1.769%), 2.534% Hyphomicrobium (GM-2.141%; IG-2.773%, IW-2.689%), 2.379% Rhodoplanes (GM- 2.231%; IG-2.197%, IW-2.710%) and 2.825% Ectothiorhodospira (GM-3.139%; IG- 2.781%, IW-2.555%) were the dominant genera in both GM and Non-GM maize rhizosphere during vegetative. While the population of other genera like, average 1.921% Streptomyces, 1.589% Blastopirellula, 1.589% Blastopirellula, 1.232% Mycobacterium, 1.350% Opitutus, 1.114% Thiothrix, 1.110% Chthoniobacter, 1.175% Lewinella, 1.259% Azospirillum, 1.454% Flexibacter, 1.947% Bacillus, 1.116% Desulfovibrio, 1.012% Segetibacter, 1.108% Bradyrhizobium, 1.649% Acidimicrobium, 1.530% Kaistobacter, 1.367% Nocardioides, 1.224% Chondromyces, 1.612% Nitrosovibrio and 1.037% Moraxella also contributed significant portion of GM and Non-GM maize rhizospheric flora. While the remaining 440 genera were found in lowest population level in both GM and Non-GM maize varieties rhizospheres during vegetative stage. Some bacterial genera were found pre dominant in GM maize rhizosphere like Chitinophaga (GM-5.371%; IG- 4.545%, IW-3.697%), Ectothiorhodospira (GM-3.139%; IG-2.781%, IW-2.555%), Opitutus (GM-2.073%; IG-0.997%, IW-0.98%), Lewinella (GM-1.829%; IG-1.011%, IW-0.684%), Azospirillum (GM-1.423%; IG-1.149%, IW-1.205%), Desulfovibrio (GM-

RESULTS CHAPTER 4

1.328%; IG-1.039%, IW-0.980%) and Kaistobacter (GM-2.322%; IG-1.124%, IW- 1.145%). But overall the bacterial diversity of GM and Non-GM maize rhizosphere during vegetative stage were not significantly differ from each other (Appendix 1-C).

RESULTS CHAPTER 4

Phya based bacterial diversity of GM and Non GM maize during Veg. Stage

45 40

35 30 GMB 25 20 15 IGB Percentage 10 5

0 IWB

Others

Thermi

Aquificae

Firmicutes

Nitrospirae

Chloroflexi

Tenericutes

Dictyoglomi

Spirochaetes

Synergistetes

Thermotogae

Fibrobacteres Bacteroidetes

Acidobacteria

Euryarchaeota

Cyanobacteria

Crenarchaeota

Proteobacteria

Actinobacteria

Deferribacteres

Planctomycetes Verrucomicrobia Phyla

Figure 4-9: GM and Non-GM maize vegetative stage rhizosphere bacterial diversity based on phyla's.

Where codes represent GMB-Vegetative stage GM maize rhizosphere sample. IGB-Vegetative stage Non-GM maize (Islamabad Gold variety) rhizosphere sample. IWB-Vegetative stage Non-GM maize (Islamabad White variety) rhizosphere sample.

RESULTS CHAPTER 4

4.2.3. Pyrosequencing based GM and Non-GM maize rhizosphere bacterial diversity during harvesting stage a. Bacterial diversity based on phylum level distribution of GM and Non-GM maize rhizosphere at harvesting stage

From one GM and two Non-GM maize crops rhizosphere (during vegetative stage) total 19 phyla's (Fibrobacteres, Thermi, Actinobacteria, Crenarchaeota, Planctomycetes, Euryarchaeota, Tenericutes, Proteobacteria, Synergistetes, Nitrospirae, Firmicutes, Bacteroidetes, Cyanobacteria, Dictyoglomi, Acidobacteria, Deferribacteres, Aquificae, Spirochaetes, Chloroflexi, Thermotogae and Verrucomicrobia) were identified through pyrosequencing analysis. Total seven major groups were found in higher percentage and contributed the maximum population during harvesting stage of GM and Non-GM maize rhizosphere. Among the major phyla's average 36% Actinobacteria, 32% Proteobacteria, 8% Bacteroidetes, 7% Firmicutes, 4% each Crenarchaeota, Planctomycetes and 1% Verrucomicrobia were found in harvesting stage rhizosphere soil of GM and Non-GM maize. Whereas 14 phyla's Fibrobacteres, Thermi, Euryarchaeota, Tenericutes, Synergistetes, Nitrospirae, Cyanobacteria, Dictyoglomi, Acidobacteria, Deferribacteres, Aquificae, Spirochaetes, Chloroflexi and Thermotogae were the minor phyla's which contribute less the 1% (0.1623%) of the total population. In GM maize rhizosphere the 4 major phyla's were found like Proteobacteria (35.96%), Firmicutes (8.629%) Planctomycetes (4.921%) and Verrucomicrobia (2.037%) as compared to Non-GM maize rhizosphere (Figure 19). whereas the population of these phyla's were comparative lower in Non-GM maize rhizosphere. While the population of other 3 major phyla's Actinobacteria, Crenarchaeota and Bacteroidetes were higher in Non-GM maize rhizosphere as compared to GM maize rhizosphere (Appendix 1-A). b. Bacterial diversity based on family level distribution of GM and Non-GM maize rhizosphere at harvesting stage.

Total 185 families were obtained from pyrosequencing analysis while some families were unclassified and placed in others group. Total 25 major groups were found in higher

RESULTS CHAPTER 4

percentage and represent the maximum population of the rhizosphere bacterial community based on family level. Among the major groups the pre dominant families were, average 7.467% Micrococcaceae, 6.460% Nocardioidaceae, 5.316% Hyphomicrobiaceae, 5.857% Rubrobacteraceae, 5.013% Streptomycetaceae, 4.521% Flexibacteraceae, 4.415% Nitrososphaeraceae, 3.155% Rhodospirillaceae, 2.543% Bacillaceae and 2.054% Pirellulaceae contributed the major population of GM and Non- GM maize rhizosphere during harvesting stage (Appendix 1-B). In GM maize rhizosphere the major families, average 5.316% Hyphomicrobiaceae (GM-6.694%; IG- 5.673%, IW-4.216%), 2.543% Bacillaceae (GM-2.817%; IG-2.62%, IW-2.192%), 3.155% Rhodospirillaceae (GM-3.871%; IG-3.143%, IW-2.453%), 2.073% Opitutaceae (GM-2.073%; IG-0.997%, IW-0.98%),1.215% Saprospiraceae (GM-1.875%; IG- 1.039%, IW-0.73%), 1.301% Desulfobacteraceae (GM-1.586%; IG-1.323%, IW- 0.994%) were pre dominate as compared to Non-GM maize rhizosphere. While the population of some families like Nitrososphaeraceae (GM-2.566%; IG-2.514%, IW- 4.698%), 1.360% Mycobacteriaceae (GM-1.986%; IG-1.339%, IW-0.757%), 1.809% Sphingomonadaceae (GM-2.192%; IG-1.622%, IW-1.614%), 1.078% Polyangiaceae (GM-1.385%; IG-1.211%, IW-0.639%), 1.671% Acidimicrobiaceae (GM-2.111%; IG- 1.644%, IW-1.26%) and 1.791% Promicromonosporaceae (GM-2.158%; IG-1.553%, IW-1.664%) were per dominant in harvesting stage GM rhizosphere. While Other families like average 7.467 % Micrococcaceae (GM-2.003%; IG-2.236%, IW-18.161%), 4.415% Nitrososphaeraceae (GM-3.33%; IG-5.753%, IW-4.163%) and 5.013% Streptomycetaceae (GM-3.908%; IG-3.522%, IW-7.611%) were pre dominantly found in Non-GM maize rhizosphere (Appendix 1-B). c. Bacterial diversity based on genus level distribution of GM and Non-GM maize rhizosphere at harvesting stage

RESULTS CHAPTER 4

maximum portion of the GM and Non-GM maize rhizospheric bacterial community during crop harvesting stage were identified. Among the major genera average 5.857% Rubrobacter, 4.898% Streptomyces, 4.415% Nitrososphaera, 3.465% Nocardioides, 2.213% Rhodoplanes, 2.262% Hyphomicrobium, 2.1405% Bacillus and 2.347% Chitinophaga were found the major genera in GM and Non-GM maize rhizosphere during harvesting stage. While some other genera like average 1.765% Promicromonospora, 1.275% Blastopirellula, 1.275% Blastopirellula, 1.527% Ectothiorhodospira, 1.361 Mycobacterium, 1.475 Kribbella, 1.287% Azospirillum, 1.451% Flexibacter, 1.102% Methylobacterium, 1.369% Bradyrhizobium, 1.294% Skermanella, 1.672% Acidimicrobium, 1.113% Kaistobacter, 1.275% Arthrobacter and 1.070 Nitrosovibrio also contributed higher portion of the bacterial population in GM and Non-GM maize rhizosphere. While the remaining 447 genera were found in lowest population in both GM and Non-GM maize rhizospheres. There was no significant variation in the population of bacteria (based on genera level distribution) in GM maize rhizosphere as compared to their counterpart Non-GM maize varieties rhizospheric soil (Appendix 1-C).

RESULTS CHAPTER 4

Phya based bacterial diversity of GM and Non GM maize during hervesting Stage 50 45 40 35 30 25 20

Percentage 15 10 GMC 5

0 IGC

IWC

Others

Thermi

Aquificae

Firmicutes

Nitrospirae

Chloroflexi

Tenericutes

Dictyoglomi

Spirochaetes

Synergistetes

Thermotogae

Fibrobacteres Bacteroidetes

Acidobacteria

Euryarchaeota Cyanobacteria

Crenarchaeota

Proteobacteria

Actinobacteria

Deferribacteres

Planctomycetes Verrucomicrobia

Phyla

Figure 4-10: GM and Non-GM maize harvesting stage rhizosphere bacterial diversity based on phyla's.

Where codes represent GMC-Harvesting stage GM maize rhizosphere sample. IGC- Harvesting stage Non-GM maize (Islamabad Gold variety) rhizosphere sample. IWC- Harvesting stage Non-GM maize (Islamabad White variety) rhizosphere sample.

RESULTS CHAPTER 4

4.3. Isolates characterization plant growth promoting activity

4.3.1. In vitro screening of GM and Non-GM maize rhizosphere pre sowing isolates for plant growth promoting potential a. Characterization of pre sowing isolates on maize shoot and root fresh weigh

Isolates that were collected prier to sowing of GM and Non-GM maize plants and were evaluated for plant growth promotion activities in vitro assay. The results of pot experiment showed that inoculation of maize seeds with bacterial strains significantly enhanced seedling vigor. The shoot fresh weight of maize seedling was significantly increased by all the isolates as compared to control (p<0.05). The maximum increased in shoot fresh weight were recorded in maize seedling inoculated with isolates RS-A-10, RS-A-11 and RS-A-3 respectively (Figure 4.11). Those isolates (RS-A-10,RS-A-11 and RS-A-3) were identified as species of the two genera Pseudomonas and Bacillus respectively. While majority of the isolates significantly increased maize seedling shoot fresh weight as compared to control but the variation was not significantly differ among the isolates. b. Characterization of first stage isolates on maize seedling root fresh weight

In vitro experiment was carried out to evaluate the impact of bacterial isolates collected from field prior to conduct GM and Non-GM maize field trial. Inoculations of bacterial isolates were done with maize seeds and were grown in sterilized pot under control condition. The results of pot experiment shown that some isolates like RS-A-2, RS-A-4, RS-A-5, RS-A-6, RS-A-10 and RS-A-11 significantly increased maize seedling root fresh weight as compared to control (p<0.05) but there were no significant variation among the isolates (Figure 4.11). Those isolates (RS-A-2, RS-A-5, RS-A-6 and RS-A-4, RS-A-10, RS-A-11) were identified as species of the genera Bacillus and Pseudomonas respectively. While all the other isolates did not increased maize seedling root fresh weight considerably as compared to control. The strains RS-A-16 and RS-A-18 (belong to genera Bacillus and Enterobacter respectively) also significantly increased maize

RESULTS CHAPTER 4

seedling root fresh weight as compared to control. The Isolates RS-A-10 and RS-A-11 consistently significantly increased maize seedling shoot and root fresh weight as compared to control (Figure 4.11) c. Characterization of first stage isolates on maize seedling shoot dry weight

In vitro experiment was carried out to evaluate the impact of bacterial isolates collected from field prior to conduct GM and Non-GM maize field trial. The results (Figure 4.12) showned that all the isolates (n=14; 100%) significantly increased maize seedling shoot dry weight as compared to control. The highest increased in shoot dry weight was observed in seedling inoculated with strains RS-A-8 and RS-A-3 which were identified, belong to Bacillus genus. While all the other isolates also increased maize seedling shoot dry weight but those were not significantly differ with each other as compared to control. The maximum significant increased (p<0.05) in maize seedling shoot dry weight was recorded in isolate RS-A-8 as compared to control but effect was not significantly differ with other isolates. d. Characterization of first stage isolates on maize seedling root dry weight

The collected bacterial isolates were in vitro evaluated for maize seedling root dry weight under control condition. The results shown in Figure 4.12 indicated that maximum number (n=13; 92.9%) of isolates significantly (p<0.05) increased maize seedling root dry weight as compared to control. The highest significant increased was recorded in isolate RS-A-8 which belong to Bacillus genus. While the other isolates like RS-A-1,RS- A-4, RS-A-5, Rs-A-10, RS-A-11 and Rs-A-18 also maximum increased in maize seedling root dry weight as compared to control but the impact of these isolates were not significantly differ with each other. Whereas isolates RS-A-1 and RS-A-5 belong to genus Bacillus, RS-A-4, Rs-A-10 and RS-A-11 belong to genus Pseudomonas, Rs-A-18 belong to genus Enterobacter respectively. The isolate RS-A-7 have no effect on maize seedling root dry weight as compared to control.

RESULTS CHAPTER 4

SFW (g) RFW (g)

3.5 a ab ab 3 abc abc abc abc 2.5 bc bc c c c c c a a a a 2 a a ab abcde abc abcd 1.5 bcde d e de e cde 1

Root & & FreshShootRoot Weight 0.5

Isolates

Figure 4-11: Effect of pre sowing isolates on maize shoot (SFW) and root (RFW) fresh weight.

SDW (g) RDW (g)

0.4 a ab 0.35 bc abc abc bc abc 0.3 a bcd cde de ab abc de abc 0.25 abc e abc abc abc de abc bc bc c bc bc 0.2 f f 0.15 d 0.1

0.05 Root & & Dry ShootRoot Weight 0

Isolates

Figure 4-12: Effect of pre sowing isolates on maize shoot (SDW) and root (RDW) dry weight. Means in each bar followed by the same letter indicated not significantly different at the P value (p<0.05).

RESULTS CHAPTER 4

e. Characterization of first stage isolates on maize seedling shoot length

Isolates of first stage were in vitro evaluated under control condition for maize seedling shoot length. The isolates RS-A-5, RS-A-8, RS-A-10, RS-A-11 and RS-A-15 significantly increased (p<0.05) maize seedling shoot length as compared to control. The maximum significant increased in shoot length was recorded in maize seedling inoculated with RS-A-10, RS-A-11 (belong to genus Pseudomonas) and RS-A-15 (genus Enterobacter) as compared to control but there were no significant variation among these isolates. While all the other isolates have no significant effect on the maize seedling shoot length as compared to control (Figure 4. 13). f. Characterization of first stage isolates on maize seedling root length

The results (Figure 4. 13) indicated that all the first stage isolates have increased the maize seedling root length. Maximum increased in root length was recorded in maize seedling inoculated with RS-A-10, RS-A-6 and RS-A-18 which belong to genera Pseudomonas, Bacillus and Enterobacter respectively. While all the other isolates also significantly increased (p<0.05) maize seedling root length as compared to control. The highest significant increased were noted in maize seedling inoculated with isolates like RS-A-10, RS-A-6 and RS-A-18 as compared to control but the variation was not differ among the these isolates. g. Characterization of first stage isolates on number of lateral roots of maize seedling

Maize seedling were uprooted from the pot experiment and were evaluated for number of lateral roots. The results indicated the maximum isolates have no significant effect on number of lateral roots of the inoculated maize seedling (Figure 4.13). The isolates RS- A-10 and RS-A-12 significantly increased (p<0.05) number of lateral roots of the inoculated maize seedling as compared to control. Both the effective isolates belong to genus Pseudomonas.

RESULTS CHAPTER 4

SL (cm) RL (cm) RN

a ab ab 25 abcde abcde abcd abc abcd abc abcde a bcde bcde 20 abc cde ab abc ab abc abcd de bcde bcde bcd e bcde cde 15 de de

f 10 a a ab ab ab ab ab ab ab ab ab ab ab ab

5 b Shoot/ RootShoot/Length & RootsofNo per Plant

0 Control RS-A-1 RS -A-2 RS -A-3 RS -A-4 RS -A-5 RS -A-6 RS -A-7 RS -A-8 RS -A-10 RS -A-11 RS -A-12 RS -A-15 RS -A-16 RS -A-18 Isolates

Figure 4-13: Effect of pre sowing isolates on maize shoot (SL), root (RL) length and No of roots (RN) per plant

RESULTS CHAPTER 4

4.3.2. Plant growth promotion traits of the pre sowing collected rhizosphere isolates

Bacterial strains were collected from GM and Non-GM maize field before crop sowing. A large number of bacteria were isolated through culturing method and morphologically different strains were selected for PGP traits. The effective strains which showed positive results were identified through 16S rRNA gene sequencing. Total fourteen bacterial strains were selected for in vitro screening for PGP traits like siderophores, nitrate reduction, phosphate solubilization and indole acetic production. The detail of each activity is given below. a. Siderophores production

Siderophores activity of the selected bacterial strains was assessed by the method of Schwyn and Neilands on Chrome azurol S agar (CAS) medium. Eight isolates RS-A-1, RS-A-4, RS-A-6, RS-A-8, RS-A-10, RS-A-11, RS-A-12 and RS-A-18 changed the colour of CAS medium from blue to orange by producing siderophores. While seven isolates RS-A-2, RS-A-3, RS-A-5, RS-A-7, RS-A-15 and RS-A-16 showed growth but not changed the colour of CAS medium, Hence these isolates were consider negative for siderophores production (Table 4.4). The maximum isolates which showed siderophores activity, belong to genus Pseudomonas. b. Nitrate reduction

The ability of bacterial isolates to reduce nitrates to nitrites or beyond the nitrate stage was determined. Bacterial isolates were grown on TSB supplemented with 0.1% potassium nitrate. Cultures were incubated at 28±2 ºC for 48 hours. Zinc powder was added to cultures not showing colour change to confirm whether nitrates were not reduced or isolate rapidly reduced nitrates beyond nitrites to ammonia or nitrogen gas. Reduction of nitrates beyond nitrites was inferred in cases where addition of Zinc did not produced any colour change. Total 10 isolates RS-A-1, RS-A-2, RS-A-4, RS-A-5, RS-A- 6, RS-A-8, RS-A-10, RS-A-15, RS-A-16 and RS-A-18 showed nitrate reduction activity

RESULTS CHAPTER 4

when Zinc was not added to the culture. While 4 isolates RS-A-3, RS-A-7, RS-A-11 and RS-A-12 showed activity when culture supplemented with Zinc. c. Phosphate solubilization.

Phosphate solubilizing activity of rhizobacteria was determined qualitatively on media containing tricalcium phosphate (TCP) as substrate. Total seven bacterial strains RS-A-1, RS-A-3, RS-A-5, RS-A-10, RS-A-12, RS-A-16 and RS-A-18 displayed the development of a clear zone around colonies as an indication of phosphate solubilization activity. Five bacterial strains RS-A-1, RS-A-3, RS-A-10, RS-A-12 and RS-A-16 showed cooperatively high phosphate solubilization activity with zone diameter (4-5mm). Whereas two isolates RS-A-5 and RS-A-18 showed lower phosphate solubilization activity with zone diameter (2-3 mm). While seven isolates did not show any phosphate solubilization activity. Three isolates of Pseudomonas and two Bacillus sp. showed maximum activity (Table 4.4). d. Indole acetic assay (IAA)

Indole acetic production assay of rhizobacteria was determined quantitatively on TSB media. Overnight grown bacterial culture was used to inoculate 5 ml TSB without and with tryptophan (500 µgmL−1) and incubated at 30 oC in rotary shaker for 24 houre. the indole acetic acid concentration was spectrometrically detected. Maximum bacterial strains showed IAA production. The culture without added tryptophan relative showed lower IAA activity. The highest IAA production (4.7282 and 3.2302 mgL-1) was recorded in isolate RS-A-10 and RS-A-3 while the lowest (0.5615 mgL-1) was recorded in RS-A-15. Whereas the culture with tryptophan (500 µg mL−1) showed relatively high IAA production. The bacterial strains RS-A-10, RS-A-12 and RS-A-3 showed highest (12.655 , 12.397 and 10.383 mgL-1 respectively ) IAA production (Table 4.4).

RESULTS CHAPTER 4

Table 4. 4: Growth promotion traits and in vitro plant growth promotion assay of rhizospheric strains isolated from pre sowing of GM and Non-GM plants.

Nitrate reduction Indole acetic assay Strains Closely related type strain % Siderophores Phosphate production Code Identity Without With solubilization Without With Zinc Zinc tryptophan tryptophan 5.4028 BCD RS-A-1 Bacillus anthracis ATCC 14578 T 100 + + - ++ 1.5734 CD

4.5694 BCD RS-A-2 Bacillus aerophilus 28K T 100 - + - - 2.9425 B

10.383 AB RS-A-3 Bacillus tequilensis 10b T 99 - - + ++ 3.2302 B

0.4722 D RS-A-4 Pseudomonas plecoglossicida FPC951 T 96 + + - - 0.8194 DE

4.8968 BCD RS-A-5 Bacillus cereus ATCC 14579 T 100 - + - + 2.2480 BC

0.3333 D RS-A-6 Bacillus anthracis ATCC 14578 T 100 + + - - 0.7897 DE

0.6508 D RS-A-7 Bacillus cereus ATCC 14579 T 100 - - + - 0.6508 DE

0.8393 D RS-A-8 Bacillus anthracis ATCC 14578 T 98 + + - - 0.8393 DE

RS-A-10 Pseudomonas taiwanensis BCRC 17751 T 99 + + - ++ 4.7282 A 12.655 A RS-A-11 Pseudomonas taiwanensis BCRC 17751 T 100 + - + - 0.6210 DE 0.7004 D RS-A-12 Pseudomonas taiwanensis BCRC 17751 T 99 + - + ++ 1.5933 CD 12.397 A RS-A-15 Enterobacter cloacae subsp. dissolvens 96 - + - - 0.5615 E 3.2302 CD LMG 2683 T RS-A-16 Pseudomonas taiwanensis BCRC 17751 T 100 - + - ++ 1.3849 CDE 7.9325 ABC RS-A-18 Enterobacter cancerogenus LMG 2693 T 99 + + - + 1.0377 DE 7.5556 ABC

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4.3.3. In vitro screening of isolates from GM and Non-GM maize Rhizosphere during vegetative stage isolates for plant growth promoting potential a. Characterization of vegetative stage isolates on maize seedling shoot fresh weight

The isolates were collected at vegetative stage from GM and Non-GM maize field trial at NARC Islamabad. All the isolates were in vitro evaluated in a pot experiment under control condition for plant growth promotion activities. For evaluating the effect of the isolated bacterial strains (isolated from GM and Non-GM maize rhizosphere), pot experiment was conducted in autoclaved sterilized sand under control condition where maize were used as indication crop. The results have shown that inoculation of maize seeds with bacterial strains significantly enhanced seedling vigor. All the vegetative stage isolates increased maize seedling shoot fresh weight as compared to control. All the seven isolates of GM maize rhizosphere like RS-BGM-19, RS-BGM-20, RS-BGM-21, RS-BGM-22, RS-BGM-22A, RS-BGM-23 and RS-BGM-24 significantly increased (p<0.05) maize seedling shoot fresh weight as compared to control. Among the GM maize rhizospheric bacterial isolates, maximum significant increased was recorded in maize shoot fresh weight, inoculated with isolates RS-BGM-20 and RS-BGM-22 respectively as compared to control. Where these isolates RS-BGM-20 and RS-BGM-22 belonged to genera Micrococcus and Bacillus respectively. The isolates of Non-GM maize verities (Islamabad white and gold) were also evaluated for maize seedling fresh weight. The results shown in Figure 4.14 indicated that eight isolates of Non-GM maize (Islamabad white variety) were in vitro screened for maize growth. Seven out of eight isolates significantly increased (p<0.05) maize seedling shoot fresh weight as compare to control. The maximum significant shoot fresh weight were recorded in maize seedling inoculated with the isolate RS-BIW-27, RS-BIW-31 and RS-BIW-33 respectively as compared to control. Where the isolates RS-BIW-27, RS-BIW-31 and RS-BIW-33 were identified and belong to genera Bacillus, Enterobacter and Microbacterium respectively. While all the four isolates (RS-BIG-35, RS-BIG-36, RS-BIG-37 and RS-BIG-38) of Non-GM

RESULTS CHAPTER 4

maize variety (Islamabad gold) have also significantly increased (p<0.05) maize seedling shoot fresh weight as compared to control (Figure 4.14). b. Characterization of vegetative stage isolates on maize seedling root fresh weigh

All the nineteen isolates of vegetative stage were in vitro evaluated in a pot experiment under control condition for plant growth promotion activities. Some of the isolates increased maize seedling root fresh weight while maximum have no significant impact on the root fresh weight of inoculated maize seedling. Out of seven only tow GM maize rhizosphere significantly increased (p<0.05) maize seedling shoot fresh weight as compared to control. The tow isolates RS-BGM-21 and RS-BGM-22A, which significantly increased maize seedling shoot fresh weight belong to genera Enterobacter and Pseudomonas. While all the other isolates of GM maize rhizosphere have no significant effect on maize seedling root fresh weight as compared to control. The Non- GM maize variety (Islamabad white) rhizosphere isolates have no effect on the maize seedling root fresh weight as compared to control. All the eight isolates of Non-GM maize Islamabad white variety have no effect on root fresh weight of maize seedling. While two out of four Non-GM maize gold variety rhizosphere isolates significantly increased (p<0.05) maize seedling root fresh weight as compared to control. The isolate two isolates RS-BIG-37 and RS-BIG-38 which belong to genera Enterobacter and Pseudomonas significantly increased maize root fresh weight respectively (Figure 4.14). c. Characterization of vegetative stage isolates on maize seedling shoot dry weight

The GM and Non-GM maize rhizosphere isolate of vegetative stage were in vitro evaluated for maize seedling shoot and root dry weight. All the vegetative stage isolates increased maize seedling shoot dry weight as compared to control. All the eight isolates of GM maize rhizosphere significantly increased (p<0.05) maize seedling shoot dry weight as compared to control. The maximum increased in shoot dry weight was recorded in maize seedling inoculated with isolates RS-BGM-19, RS-BGM-20 and RS- BGM-22.The isolate RS-BGM-19, RS-BGM-20 and RS-BGM-22 of GM maize rhizosphere belong to genera Chryseobacterium, Micrococcus and Bacillus significantly

RESULTS CHAPTER 4

increased maize seedling shoot dry weight as compared to control. While the isolates of Non-GM maize Islamabad white variety have also increased maize seedling shoot dry weight. Seven out of eight Islamabad white variety rhizosphere isolates significantly increased (p<0.05) maize seedling shoot dry weight as compare to control (Figure 4. 15). The isolates RS-BIW-33, RS-BIW-31, RS-BIW-29, RS-BIW-27 and RS-BIW-26 which belong to genera Microbacterium, Enterobacter, Acinetobacter, Bacillus and Pseudomonas respectively increased maize seedling shoot dry weight as compared to control. The rhizospheric isolates of Islamabad gold variety have also effected the maize seedling shoot dry weight. All the four isolates RS-BIG-35, RS-BIG-37, RS-BIG-36 and RS-BIG- 38 which belong to genera Aerococcus, Chryseobacterium, Enterobacter and Pseudomonas significant increased maize seedling shoot dry weight as compared to control (Figure 4.15). d. Characterization of vegetative stage isolates on maize seedling root dry weight

The GM and Non-GM maize rhizosphere isolate of vegetative stage were in vitro evaluated for maize seedling root dry weight. Some isolates of vegetative stage increased maize seedling root dry weight as compared to control. All the eight isolates of GM maize rhizosphere increased maize seedling root dry weight. The isolates RS-BGM-23 and RS-BGM-22 which belong to genera Pseudomonas and Bacillus significantly increased (p<0.05) maize seedling root dry weight as compared to control (Figure 4.15). Whereas one (RS-BIW-33) out of eight Islamabad white rhizosphere isolates significant increased maize seedling root dry weight as compared to control. While the other isolates of Islamabad. RS-BIW-33 belong to genus Microbacterium which maximum increased the root dry weight of maize seedling as compared to control. While the other Isolates of Islamabad white did not affect the root dry weight. Whereas the Islamabad gold maize variety rhizosphere isolates also increased maize seedling root dry weight. The isolates RS-BIG-36, RS-BIG-37 and RS-BIG-35 which belong to genera Chryseobacterium, Enterobacter and Aerococcus significantly increase (p<0.05) maize seedling root dry weight as compared to control (Figure 4.15).

RESULTS CHAPTER 4

SFW (g) RFW (g)

a 4.5 ab abc 4 abcd bcdef 3.5 cdef bcdef defg cdef cdef defg 3 fg fg fg efg defg fg 2.5 fg a a ab gh ab 2 abcd ab abcd abc abc bcdefbcde bcde 1.5 h efghi cdefgh bcdefg cdefgh ghi fghi 1 hi i

Root & Shoot & Root Freshweight 0.5 0

Isolates

Figure 4-14: Effect of isolates from GM and Non-GM maize Rhizosphere during vegetative stage on maize shoot (SFW) and root (RFW) fresh weight. Means in each bar followed by the same letter indicated not significantly different at the P value (p<0.05).

RESULTS CHAPTER 4

SDW (g) RDW(g)

0.45

0.4 a a ab abc 0.35 abcd ab abc abc ab bcde bc bcdef bcd cdefg bcdefg bcdef 0.3 bcd cdefgh bcde cdefg efghi defghi 0.25 defghghi fghi fghi efghi efghi hi fghi ghi efghi ij fghi 0.2 hi i hi hi i 0.15 j 0.1

Shoot & RootDry & Shoot Weight 0.05 0

Isolates

Figure 4-15: Effect of isolates from GM and Non-GM maize Rhizosphere during vegetative stage on maize shoot (SDW) and root (RDW) dry weight. Means in each bar followed by the same letter indicated not significantly different at the P value (p<0.05).

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e. Characterization of vegetative stage isolates on maize seedling shoot length

The GM and Non-GM maize vegetative stage rhizospheric isolates were in vitro evaluated in a pot experiment under control condition. Maize seeds were inoculated with 19 isolates and grown in pot under control condition, where the control plants were just applied broth without any inoculation. The plants were grown in pots and were applied Hoagland solution after two days interval while the control plants received autoclave sterilized water. The results shown in figure 4.16 indicated that some of the isolates increased maize seedling shoot length. Two isolates RS-BGM-22 and RS-BGM-20 which belong to genera Bacillus and Micrococcus significantly increased (p<0.05) maize seedling shoot length as compared to control (Figure 4.16) . While the five isolates of GM maize rhizosphere have no significant effect on the maize seedling shoot length as compared to control. Whereas seven out of eight isolate of Non-GM Islamabad white variety have significantly increased maize seedling shoot length as compared to control (Figure 4.16). The maximum increased in shoot length were recorded in seedling inoculated with isolates RS-BIW-27, RS-BIW-29 and RS-BIW-32 which belong to genera Bacillus, Acinetobacter and Pseudomonas respectively. The four isolates of Non- GM maize Islamabad gold variety also increased maize seedling shoot length but the effect was not significant as compared to control. f. Characterization of vegetative stage isolates on maize seedling root length

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recorded in seedling inoculated with isolates RS-BGM-22 and RS-BGM-20 which belong to genera Bacillus and Micrococcus respectively. Whereas five out of eight Non-GM maize rhizospheric isolates (Variety; Islamabad white) also significantly increased (p<0.05) maize seedling root length as compared to control. The maximum increased in root length were recorded in seedling inoculated with isolates RS-BIW-26, RS-BIW-27 and RS-BIW-31 which belong to genera Pseudomonas, Bacillus and Enterobacter respectively (Figure 4.16). While the Isolates of other Non-GM maize rhizosphere (Islamabad gold) also increased maize seedling root weight. Three out of four isolates (Variety; Islamabad gold) significantly increased maize seedling root length as compare to control. The maximum increased in root length were recorded in seedling inoculated with isolates RS-BIG-35 and RS-BIG-37 which belong to genera Aerococcus and Enterobacter respectively. g. Characterization of vegetative stage isolates on number of lateral roots of maize seedling

The GM and Non-GM maize vegetative stage rhizospheric isolates were in vitro evaluated for their effect on maize seedling lateral roots growth. Maize seeds were inoculated with 19 isolates and grown in pot under control condition. After reach to certain level the plants were uprooted and were washed with tap water and the number of lateral roots were counted. The results indicated in Figure 4.16 shown that some of the isolates increased the number of lateral roots of the inoculated maize seedling. Four out of seven GM maize rhizosphere isolates significantly increased number of lateral roots of maize seedling as compared to control. Whereas all the eight Non-GM maize rhizospheric isolates (variety; Islamabad white) also significantly increased (p<0.05) the number of lateral roots of the inoculated maize seedling as compared to control. The maximum increased in number of lateral roots were recorded in seedling inoculated with isolates RS-BIW-30 and RS-BIW-32 which belong to genera Sphingomonas and Pseudomonas respectively. While the Isolates of other Non-GM maize rhizosphere (variety; Islamabad gold) also increased number of lateral roots of maize seedling. All the four

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isolates (variety; Islamabad gold) significantly increased number of lateral roots of maize seedling as compare to control (Figure 4.16).

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SL (cm) RL (cm) RN

a 35 ab abc abc abcd abcd 30 abcde abcdef abcdef bcdefg 25 cdefg defg bcdefg bcdefg a abcd a defg efg efg abc abcd ab a 20 g abcde fg abcdef bcdefg g abcdef cdefg defg cdefg 15 efgh fgh efgh ghi hi ab ab ab abc 10 abcd i a abc abc abcd bcd cde bcd bcd def bcd cde def g efg fg 5

Shoot/ RootShoot/Length & RootsofNo per plant 0

Isolates

Figure 4-16: Effect of isolates from GM and Non-GM maize Rhizosphere during vegetative stage on maize shoot (SL), root (RL) length and No of roots (RN) per plant. Means in each bar followed by the same letter indicated not significantly different at the P value (p<<0.05).

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4.3.4. Plant growth promotion traits of the vegetative stage rhizosphere isolated

Bacterial strains were collected from GM and Non-GM maize field during vegetative stage. A large number of bacteria were isolated through culturing method and morphologically different strains were selected for PGP traits. The effective strains which showed positive results were identified through 16s rRNA gene sequencing. Total nineteen (6 from GM and 13 from Non-GM maize rhizosphere) bacterial strains were selected for in vitro screening for PGP traits like siderophores, nitrate reduction, phosphate solubilization and indole acetic production. The detail of each activity is given below. a. Siderophores production

Siderophores activity of the selected bacterial strains isolated from GM and Non-GM maize rhizosphere was assessed. Total nine isolates RS-BIG-38, RS-BIW-25, RS-BIW- 26, RS-BIW-27, RS-BIW-31, RS-BIW-32, RS-BGM-21, RS-BGM-22 and RS-BGM-23 changed the colour of CAS medium from blue to orange by producing siderophores. Three bacterial strains isolated from GM maize rhizosphere were positive for siderophores production. Whereas six bacterial strains isolated from Non-GM maize rhizosphere (n=5 from Islamabad white; n=1 from Islamabad gold) were positive for siderophores production. While ten isolates showed growth up to some extent but not changed the colour of CAS medium, Hence these isolates were negative for siderophores production (Table 4.5). The maximum isolates which show siderophores activity were belong to genera Bacillus and Pseudomonas. b. Phosphate solubilization.

Phosphate solubilizing activity of the selected bacterial strains isolated from GM and Non-GM maize rhizosphere were screened on media containing Tricalcium phosphate (TCP) as substrate. Total eleven bacterial strains RS-BIG-38, RS-BIW-25, RS-BIW-26, RS-BIW-27, RS-BIW-29, RS-BIW-31, RS-BIW-33, RS-BGM-19, RS-BGM-21, RS- BGM-23 and RS-BGM-24 displayed the development of a clear zone around colonies as

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an indication of phosphate solubilization activity. Nine bacterial strains RS-BIG-38, RS- BIW-25, RS-BIW-26, RS-BIW-29, RS-BIW-31, RS-BIW-33, RS-BGM-19, RS-BGM- 21, and RS-BGM-24 showed cooperatively high phosphate solubilization activity with zone diameter (4-5 mm). Whereas two isolates RS-BIW-27 and RS-BGM-23 showed comparatively lower phosphate solubilization activity with zone diameter (2-3 mm). While eight isolates did not show any phosphate solubilization activity. Three bacterial strains isolated from GM maize rhizosphere were highly positive ( zone diameter 4-5 mm) for phosphate solubilization (Table 4.5). Whereas six bacterial strains isolated from Non-GM maize rhizosphere (n=5 from Islamabad white; n=1 from Islamabad gold) were positive for phosphate solubilization. c. Nitrate reduction

The ability of bacterial isolates to reduce nitrates to nitrites or beyond the nitrate stage was determined. Bacterial isolates were grown on TSB supplemented with 0.1% potassium nitrate. Cultures were incubated at 28±2 ºC for 48 hours. Zinc powder was added to cultures not showing colour change to confirm whether nitrates were not reduced or isolate rapidly reduced nitrates beyond nitrites to ammonia or nitrogen gas. Reduction of nitrates beyond nitrites was inferred in cases where addition of Zinc did not produc any colour change. Total 9 isolates RS-BIG-37, RS-BIW-25, RS-BIW-26, RS- BIW-27, RS-BIW-31, RS-BIW-32, RS-BGM-21, RS-BGM-22 and RS-BGM-23 showed nitrate reduction activity when Zinc was not added to the culture. While 13 isolates showed activity when culture was supplemented with Zinc (Table 4.5). d. Indole acetic assay (IAA).

Indole acetic production of the selected bacterial strains isolated from GM and Non-GM maize rhizosphere were screened on TSB media. Overnight grown bacterial culture was used to inoculate 5 ml TSB without and with tryptophan (500 µg mL−1) and incubated at 30◦C in rotary shaker for 24 houre. the indole acetic acid concentration was spectrometrically detected. Maximum bacterial strains showed IAA production. The culture without added tryptophan relative showed lower IAA activity. Among cultured

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with tryptophan (500 µg mL−1) showed relative lower IAA production. The significant increased (P<0.05) in IAA production (4.609, 2.694, 1.295 and 1.0972 mgL-1) with tryptophan was recorded in isolates RS-BGM-23, RS-BIG-36, RS-BIG-37 and RS-BGM- 21 respectively. When tryptophan was added to the culture some isolates namely; RS- BIG-36, RS-BGM-21, RS-BGM-19 and RS-BIG-37 increased IAA production (10.214, 6.980 and 3.974 and 3.002 mgL-1) as compare to other isolates (Table 4.5).

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Table 4. 5: In vitro screening of isolates from GM and Non-GM maize rhizosphere during vegetative stage for plant growth characterization.

Indole acetic assay Nitrate reduction Closely related type strain production Strains ID Variety % P. sol Sideroph Without Without With Identity ores With Zinc Zinc tryptophan tryptophan RS-BIG-38 Ism. Pseudomonas parafulva AJ 2129 T 98 - + - + 0.9385 0.3036 GH gold DEFG T RS-BIG-36 variety Chryseobacterium hagamense RHA2-9 97 ++ - + + 2.6944 B 10.214 A

RS-BIG-37 Enterobacter ludwigii DSM 16688 T 99 - - - + 1.2956 C 3.0020 CDE RS-BIW-25 Chryseobacterium indologenes LMG 8337 98 ++ + + + 0.4425 FGH 0.9881 FGH T RS-BIW-26 Non Pseudomonas lini CFBP 5737 T 99 ++ + + - 0.1250 H 0.6409 FGH RS-BIW-27 GM Bacillus isronensis B3W22 T 98 - + + - 0.1746 H 0.3135 GH RS-BIW-28 Ism. Chryseobacterium indologenes LMG 8337 98 - - - + NA NA white T RS-BIW-29 variety Acinetobacter pittii LMG 1035 T 99 ++ - - + 0.2937 H 0.8690 FGH RS-BIW-30 Sphingomonas koreensis JSS26 T 99 - - - + 0.3135 GH 1.444 EFGH RS-BIW-31 Enterobacter cloacae subsp. dissolvens 100 ++ + + - 1.1270 0.4573 FGH LMG 2683 T CDE RS-BIW-32 Pseudomonas lini CFBP 5737 T 99 - + + - 1.1895 CD 1.84 DEFG

RS-BIW-33 Microbacterium oleivorans DSM 16091 T 99 ++ + - + 0.998 DEF 0.531 FGH

RS-BIW-34 Sphingomonas koreensis JSS26 T 99 - - - + NA NA RS-BGM-19 Chryseobacterium indologenes LMG 8337 98 ++ - - + 1.0873 CDE 3.9742 C GM T RS-BGM-20 Micrococcus antarcticus T2 T 99 - - - + 0.0536 H 0.0536 H RS-BGM-21 Enterobacter cloacae subsp. dissolvens 99 ++ + + - 1.0972 6.9802 B LMG 2683 T CDE RS-BGM-22 Bacillus cereus ATCC 14579 T 100 - + + + 0.2242 H 0.7996 FGH

RS-BGM-23 Pseudomonas moorei RW10 T 99 + - + - 4.6091 A 2.0397 DEF RS-BGM-24 Sphingobacterium multivorum IAM14316 T 98 ++ - - + 0.5119 0.3433 GH EFGH

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Part C

4.3.5. In vitro screening of the isolates from GM and Non-GM maize Rhizosphere during harvesting stage for plant growth promoting potential a. Characterization of harvesting stage isolates on maize seedling shoot fresh weight

GM and Non-GM maize (verities; Islamabad white and gold) rhizospheric bacteria were isolated at crop harvesting stage and in vitro evaluated for maize seedling shoot and root fresh weight. An experiment was conducted in pot with sterilized sand under control condition and maize seeds were inoculated with isolates in grown. Maize seeds were inoculated with total 16 isolates and grown in pot under control condition, where the control plants were just applied broth without any inoculation. The inoculated plants were applied Hoagland solution after two days interval while the control plants received autoclaved sterilized water. When the plants reached to certain level, then uprooted and carefully separated to different portion. The results indicated in Figure 4.17 shown that maximum isolates increase shoot fresh weight of maize seedling as compared to control. All the five isolates of GM maize rhizosphere significantly increase shoot fresh weight of maize seedling as compared to control. Maximum increased in shoot fresh weight was recorded in seedling inoculated with isolates RS-CGM-42 and RS-CGM-44 which belong to genera Enterobacter and Microbacterium respectively (Figure 4.17). Whereas all the four Non-GM maize rhizospheric isolates (variety; Islamabad white) also significantly increased (p<0.05) shoot fresh weight of maize seedling as compared to control. The maximum increased in shoot fresh weight was recorded in isolates RS-CIW-46 and RS- CIW-47 which belong to genera Bacillus and Bordetella respectively. While six out of seven rhizosphere isolates (variety; Islamabad gold) significantly increased shoot fresh weight of maize seedling as compared to control. Maximum increased in shoot fresh weight was recorded in maize seedling inoculated with isolate RS-CIG-52, which belong to genus Bacillus.

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b. Characterization of harvesting stage isolates on maize seedling root fresh weight

The harvesting stage isolate of GM and Non-GM maize (varieties; Islamabad white and gold) rhizosphere were in vitro evaluated for root fresh weight of the inoculated maize seedling under control condition. Maize seeds were inoculated with total 16 isolates and were grown in pot under control condition. The plants were uprooted after reached to certain level (5 weeks) and were carefully separated to different portion. The results (Figure 4.17) indicated that none of the isolates increased root fresh weight of maize seedling as compared to control. The isolates of GM maize rhizosphere had no effect on the root fresh weight as compared to control. Whereas the Non-GM maize rhizosphere, four of Islamabad white and seven isolates Islamabad gold variety have no effect on the root fresh weight of maize seedling as compared to control. The root fresh weight of inoculated maize seedling were not significantly affected by the harvesting stages rhizospheric isolates either from GM or Non-GM maize. c. Characterization of harvesting stage isolates on maize seedling shoot dry weight

The harvesting stages rhizospheric isolates of GM and Non-GM maize were in vitro screened for plant growth promotion activity. Maximum isolates increased shoot dry weight of the inoculated maize seedling as compared to control. All the five isolates of GM maize rhizosphere were significantly increased shoot dry weight of maize seedling as compared to control. Maximum increased in shoot dry weight were recorded in seedlings inoculated with isolates RS-CGM-42, RS-CGM-43 and RS-CGM-44 which were belong to genera Enterobacter, Bacillus and Microbacterium respectively. Whereas the four isolates of Non-GM maize rhizosphere (variety; Islamabad white) were also significantly increased (p<0.05) shoot dry weight of maize seedling as compared to control (Figure 4.18). While some of the isolates of Non-GM maize rhizosphere (Islamabad gold) increased shoot dry weight of maize seedling. Five out of seven isolates significantly increased shoot dry weight of maize seedling as compared to control. The maximum increased in shoot dry weight was recorded in seedling inoculated with isolates RS-CIG- 49, RS-CIG-52 which belong to genus Bacillus.

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d. Characterization of harvesting stage isolates on maize seedling root dry weight

The harvesting stages rhizospheric isolates of GM and Non-GM maize were in vitro screened for plant growth promotion activity. Few isolates increased root dry weight of the inoculated maize seedling as compared to control. One out of five GM maize rhizosphere isolates significantly increased root dry weight of inoculated maize seedling as compared to control. While none of the harvesting stage isolates of Non-GM maize either to Islamabad white or gold have significant effect of the root dry weight of maize seedling as compared to control (Figure 4.18).

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SFW (g) RFW (g)

3.5 ab cde cde 3 def abc a def abc defg defg 2.5 bcd efg cd cde fg 2 gh a ab ab a 1.5 abc abc abc h abc abc abc abc abc abc c bc bc 1

0.5 d Root & Shoot & Root FreshWeight 0

-0.5

Isolates

Figure 4-17: Effect of the isolates from GM and Non-GM maize Rhizosphere during harvesting stage on maize shoot (SFW) and root (RFW) fresh weight. Means in each bar followed by the same letter indicated not significantly different at the P value (p<0.05).

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SDW (g) RDW (g)

0.4 a 0.35 a ab abc abcd 0.3 bcde bcde cde cde cde 0.25 b e de de de e bc bc bc 0.2 bcd bc bcd bc f bcd cd bcd cd f cd cd 0.15 f cd d 0.1

0.05 Root & Shoot & Root Dry Weight 0

Isolates

Figure 4-18: Effect of the isolates from GM and Non-GM maize Rhizosphere during harvesting stage on maize shoot (SDW) and root (RDW) dry weight. Means in each bar followed by the same letter indicated not significantly different at the P value (p<0.05).

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e. Characterization of harvesting stage isolates on maize seedling shoot length

The harvesting stages rhizospheric isolates of GM and Non-GM maize were in vitro screened for plant growth promotion activity. The maize seeds were inoculated with the harvesting stage GM and Non-GM maize rhizosphere isolates. The inoculated seedling were received Hoagland solution after two days interval, while the control plants were applied autoclaved sterilized water. The impact of the GM and Non-GM maize rhizosphere were evaluated on the root and shoot length as well as on the number of lateral roots. All the harvesting stage five isolates (GM maize) were effective in increasing the shoot length of inoculated seedling. Maximum increased in shoot length were recorded in seedling inoculated with isolates RS-CGM-39, RS-CGM-42, and RS- CGM-44 which were belong to genera Bacillus, Enterobacter and Microbacterium respectively. Whereas three out of five Non-GM rhizosphere isolates (variety; Islamabad white) significantly increased shoot length of maize seedling as compare to control. The maximum increased were recorded in seedling inoculated with isolates RS-CIW-46, RS- CIW-47 and RS-CIW-48A as compared to other isolates as well as with control. While three out of seven Non-GM maize rhizosphere isolates (variety; Islamabad gold) were significantly increased shoot length of inoculated maize seedlings as compared to control. The maximum increased in shoot length were recorded in seedling inoculated with isolate RS-CIG-49, which belong to genus Bacillus. Comparative the harvesting stage isolates of GM and Non-GM maize (variety; Islamabad white) were more effective as compared to the rhizosphere isolates of Islamabad gold variety. f. Characterization of harvesting stage isolates on maize seedling root length

The harvesting stage rhizosphere isolates of one GM and two Non-GM maize varieties were in vitro screened for root length of the inoculated maize seedling. The maize seedling were inoculated with 16 isolates and were grown in pot under control condition. The results indicated in Figure 4.19 showed that three out of five GM maize rhizosphere isolates significantly increased root length of maize seedling as compared to control. Two isolates RS-CGM-39, RS-CGM-43 belong to genus Bacillus and one RS-CGM-41

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belong to Enterobacter significantly increased root length of maize seedling as compared to control. Whereas three out of four harvesting stage isolates (RS-CIW-45, RS-CIW-46 and RS-CIW-47) of Non-GM maize (variety; Islamabad white) significantly increased root length of inoculated maize seedling as compared to control. While three out of seven harvesting stage rhizosphere isolates of Islamabad gold variety were effective an increasing the root length of inoculated maize seedlings as compared to control. The isolates RS-CIG-52, RS-CIG-53 and RS-CIG-54 significantly increased the root length of inoculated seedlings as compare to control (Figure 4.19). g. Characterization of harvesting stage isolates on number of lateral roots of maize seedling

The GM and Non-GM maize harvesting stage isolates were in vitro evaluated for number of lateral roots of inculcated maize seedling. Total 16 isolate were screened, after certain time the plants were uprooted and washed with tap water and the lateral roots were counted. Four out of five GM maize rhizosphere isolates increased number of lateral roots of inoculated maize seedling. Whereas maximum rhizosphere isolates of Islamabad white variety increased number of lateral roots of the inoculated maize seedling. While all the rhizosphere isolates of Islamabad gold variety also increased the lateral root of the inoculated seedling as compared to control (Figure 4.19).

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SL (cm) RL (cm) RN

40 a a a 35 a a a ab ab 30 abc abcd abcde abcde abcde 25 a a bcde ab abc ab cde ab 20 abc abc de

abcd e bcde bcde abcde 15 cde de a de de e ab abcd abc 10 abcde abcde bcdef bcdef bcdef f def ef ef cdef def ef g 5

0 Shoot/ RootShoot/Length & RootsofNo per plant

Isolates

Figure 4-19: Effect of the isolates from GM and Non-GM maize Rhizosphere during harvesting stage on maize shoot (SL), root (RL) length and No of roots (RN) per plant. Means in each bar followed by the same letter indicated not significantly different at the P valu e (p<0.05).

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4.3.6. Plant growth promotion traits of the harvesting stage rhizosphere isolated

Bacterial strains were collected from GM and Non-GM maize field during harvesting stage. A large number of bacteria were isolated through culturing method and morphologically different strains were selected for PGP traits. The effective strains which showed positive results were identified through 16s rRNA gene sequencing. Total thirteen (4 from GM and 9 from Non-GM maize rhizosphere) bacterial strains were selected for in vitro screening of PGP traits like siderophores, nitrate reduction, phosphate solubilization and indole acetic production. The detail of each activity is given below. a. Phosphate solubilization

Phosphate solubilizing activity of the selected bacterial strains isolated from GM and Non-GM maize rhizosphere during harvesting stage were screened on media containing Tricalcium phosphate (TCP) as substrate. Total nine bacterial strains RS-CIG-48, RS- CIG-49, RS-CIG-50, RS-CIG-52, RS-CIG-53, RS-CGM-41, RS-CGM-42, RS-CGM-43 and RS-CIW-46 displayed the development of a clear zone around colonies as an indication of phosphate solubilization activity. Three bacterial strains RS-CIG-50, RS- CGM-42 and RS-CGM-43 showed comparatively high phosphate solubilization activity with zone diameter (4-5 mm). While five isolate showed phosphate solubilizing activity but relatively lower than other with zone diameter (2-3 mm). Three isolates of GM maize rhizosphere (n=3) were positive for phosphate solubilization, whereas 6 isolates of Non- GM maize rhizosphere (n=5 from Islamabad white; n=1 from Islamabad gold) were positive for phosphate solubilization. While four isolate did not show phosphate solubilizing activity (Table 4.6). b. Siderophores production

Siderophores activity of the selected bacterial strains isolated from GM and Non-GM maize rhizosphere during harvesting stage were assessed on Chrome azurol S agar (CAS) medium. Total eight isolates RS-CIG-48, RS-CIG-49, RS-CIG-50, RS-CIG-52, RS-

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CGM-39, RS-CGM-43, RS-CIW-45 and RS-CIW-46 changed the colour of CAS medium from blue to orange by producing siderophores. Tow bacterial strains isolated from GM maize rhizosphere were positive for siderophores production. Whereas six bacterial strains isolated from Non-GM maize rhizosphere (n=2 from Islamabad white; n=4 from Islamabad gold) were positive for siderophores production. While five isolates showed growth up to some extent but not changed the colour of CAS medium, Hence these isolates were negative for siderophores production (Table 4.6). c. Nitrate reduction

The ability of bacterial isolates to reduce nitrates to nitrites or beyond the nitrate stage was determined. Bacterial isolates were grown on TSB supplemented with 0.1% potassium nitrate. Cultures were incubated at 28±2 ºC for 48 hours. Zinc powder was added to cultures not showing colour change to confirm whether nitrates were not reduced or isolate rapidly reduced nitrates beyond nitrites to ammonia or nitrogen gas. Reduction of nitrates beyond nitrites was inferred in cases where addition of Zinc did not produc any colour change. Total 3 isolates RS-CGM-39, RS-CGM-41 and RS-CIW-46 showed nitrate reduction activity when Zinc was not added to the culture. While 10 isolates (n=2 from GM; n=8 from Non-GM maize rhizosphere) showed nitrate reduction activity when culture was supplemented with Zinc. d. Indole acetic assay (IAA)

Indole acetic production of the selected bacterial strains isolated from GM and Non-GM maize rhizosphere during harvesting stage were screened. Overnight grown bacterial culture was used to inoculate 5 ml TSB without and with tryptophan (500 µg mL−1) and incubated at 30 ◦C in rotary shaker for 24 houre. the indole acetic acid concentration was spectrometrically detected. majority of the bacterial strains showed IAA production. The culture with tryptophan showed relatively higher IAA production as compared to the culture without tryptophan. The culture with no tryptophan, isolate RS-CIG-50 produced maximum IAA (1.523 µg mL−1) as compared to other isolates. Whereas the culture with

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tryptophan, isolate RS-CIG-53 produced maximum IAA (11.216 µg mL−1) as compared to other isolates (Table 4.6).

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Table 4. 6: In vitro screening of the isolates from GM and Non-GM maize rhizosphere during harvesting stage for Plant Growth Promoting activities.

Indole acetic assay % Phosphate Nitrate reduction Variety production Strains ID Closely related type strain Identity Siderophores solubilization Without With Without With

Zinc Zinc tryptophan tryptophan RS-CIG-48 Ism. Bacillus cereus ATCC 14579T 99 - + - + 0.551 D 2.129 FG RS-CIG-49 gold Bacillus cereus ATCC 14579 T 100 - + - + 0.065 EF 0.3929 HI RS-CIG-50 variety Pseudomonas stutzeri ATCC 99 + + - + 1.5238 A 7.6548 C 17588 T RS-CIG-51 Flavobacterium sasangense 97 - - - + 1.017 BC 9.8869 B YC6274 T RS-CIG-52 Bacillus cereus ATCC 14579 T 100 - + - + 0.0952 EF 2.2183 F RS-CIG-53 Bacillus cereus ATCC 14579 T 100 - - - + 0.9782 BC 11.216 A RS-CGM- Bacillus cereus ATCC 14579 T 100 - + + - 0.0536 F 0.0536 I 39 GM RS-CGM- Enterobacter cloacae subsp. 98 + - + - 1.0972 B 7.4167 C 41 dissolvens LMG 2683 T RS-CGM- Enterobacter cloacae subsp. 98 ++ - - + 0.0536 F 0.0536 HI 42 dissolvens LMG 2683 T RS-CGM- Bacillus aryabhattai B8W22 T 93 ++ - - + 0.6508 CD 0.2440 HI 43 RS-CIW-45 Ism. Microbacterium 98 - + - + 0.5218 D 3.5476 white trichothecenolyticum IFO E variety 15077 T RS-CIW-46 Bacillus idriensis SMC 4352-2 99 + + + - 0.7698 CD 5.0556 D T RS-CIW-47 Bordetella petrii DSM 12804 T 97 - - - + 0.6905 1.355 FGH BCD

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4.4. Production of cell wall hydrolyzing enzymes by pre sowing GM and Non-GM rhizosphere isolates

All the 14 pre sowing rhizospheric bacterial isolates were in vitro characterized for production of fungal cell wall hydrolyzing enzymes such as cellulase, chitinase, protease and pectinase (Table 4.7). The detail of each individual enzyme is given below.

4.4.1. Cellulase activities of the isolated bacterial strains

All the 14 bacterial strains were screened for cellulase enzyme production. After two days of incubation and treatments of the individual plates with Congo red dye and NaCl solution, cellulase activity of the strains were estimated by measuring the yellowish brown halo around individual colonies. The pre sowing isolated bacterial strains were screened, 29% showed cellulase positive activity while 71% did not show any activity. From the diameter of the zone it was concluded that 4 strains RS-A-6, RS-A-7, RS-A-8 and RS-A-11 showed positive cellulase activities (Table 4.7). While 10 bacterial strains did not showed cellulase activity. The effective bacterial strains, RS-A-6, RS-A-7, RS-A- 8 and RS-A-11 which showed positive cellulase activity with zone diameter (< 3 mm) were belong to Bacillus anthracis, Bacillus cereus, Bacillus anthracis and Pseudomonas taiwanensis respectively (Table 4.7).

4.4.2. Chitinase activities of the isolated bacterial strains

Chitinase activity was screened for all the 14 pre sowing rhizosphere collected bacterial strains on TSA medium, in which carbon was the sole source in a defined medium having colloidal chitin. These isolates were screened to determine chitinase production. Each isolate was inoculated on colloidal chitin agar (CCA) and incubated at 28 ºC in the dark until (7 days incubation) zones of chitin clearing were observed around colonies. Colony zone diameters were measured (mm) and used to indicate the chitinase activity of each isolate. Maximum isolates showed chitinase activity while few strains did not showed any activity. Total eight isolates out of fourteen were positive for chitinase production. Results showed that 14 out of 24 selected strains were positive for protease activity.

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Among these isolates RS-A-2, RS-A-10 which belong to genera Bacillus and Pseudomonas respectively, showed comparatively high chitinase activity with zone diameter (4-5 mm) as compared to other isolates. Whereas the isolates RS-A-4, RS-A-5, RS-A-6, RS-A-7, RS-A-8 and RS-A-11 showed comparatively low chitinase activity with zone diameter (< 3 mm). While the remaining six isolates RS-A-1, RS-A-3, RS-A-12, RS-A-15, RS-A-16 and RS-A-18 were found negative and did not showed any chitinase activity.

4.4.3. Protease activities of the isolated bacterial strains

Protease activities was determine of the 14 pre sowing rhizosphere isolated bacterial strains bacterial strains on their respective modified medium with added skimmed milk. All the bacterial strains after three days inoculation on medium were screened for protease activities. Strains producing a clear zone around colony were assumed to have positive for protease production. Results showed that 6 out of 14 selected strains RS-A-2, RS-A-4, RS-A-6, RS-A-8, RS-A-11 and RS-A-15 were proved to possess the protease activity. Among these one RS-A-6 and tow RS-A-4, RS-A-11 isolates which belong to genera Bacillus and Pseudomonas respectively, showed comparatively highest protease activity with zone diameter (6-7 mm) as compared to other isolates. Whereas the strains RS-A-2 and RS-A-15 showed comparatively high protease activity with zone diameter (4-5mm). While the lowest protease activities was recorded in isolate RS-A-8 with zone diameter (< 3 mm). While other 8 Strains namely; RS-A-1, RS-A-3, RS-A-5, RS-A-7, RS-A-10, RS-A-12, RS-A-16 and RS-A-18 did not showed any protease activities (Table 4.7).

4.4.4. Pectinase activities of the isolated bacterial strains

Pectinase activities was determine of the 14 pre sowing rhizosphere isolated bacterial strains on pectinase screening agar medium (PSAM). Results showed that 9 out of 14 selected strains namely; RS-A-4, RS-A-5, RS-A-6, RS-A-7, RS-A-8, RS-A-10, RS-A-11, RS-A-12 and RS-A-15 were proved to possess the pectinase activities. Among these two Bacillus RS-A-6 and RS-A-7 and one Pseudomonas strains RS-A-11 showed

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comparatively highest pectinase activities with zone diameter (6-7 mm) as compared to other isolates. Whereas the two strains RS-A-4 and RS-A-10 showed comparatively high pectinase activities with zone diameter (4-5 mm). While the lowest pectinase activities was recorded in isolates RS-A-5, RS-A-8, RS-A-12 and RS-A-15 with zone diameter (< 3 mm). While other 5 strains namely; RS-A-1, RS-A-2, RS-A-3, RS-A-16 and RS-A-18 did not showed any pectinase activities (Table 4.7).

4.4.5. Lipase activities of the isolated bacterial strains

Lipase activities was determine of the 14 pre sowing rhizosphere isolated bacterial strains on TSB medium with 1% tween 20. After 48 hours incubation at 28°C. Strains surrounded by white precipitation were considered positive for lipase activities. The results indicated that 6 out of 14 selected strains namely; RS-A-2, RS-A-4, RS-A-6, RS- A-7, RS-A-8, RS-A-11 were proved to possess the lipase activities with zone diameter (< 3 mm). Most of the strains which showed lipase activities were belong to genus Bacillus. While the remaining 8 bacterial strains did not showed any lipase activities (Table 4.7).

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Table 4. 7: In vitro screening of pre sowing isolates for cell wall degrading enzymes production.

Cell Wall Degrading Enzymes Strains Closely related type strain % Code Identity Cellulase Chitinase Protease Pectinase Lipase

RS-A-1 Bacillus anthracis ATCC 14578T 100 - - - - - RS-A-2 Bacillus aerophilus 28K T 100 - ++ ++ - + RS-A-3 Bacillus tequilensis 10b T 99 - - - - - RS-A-4 Pseudomonas plecoglossicida FPC951 T 96 - + +++ ++ + RS-A-5 Bacillus cereus ATCC 14579 T 100 - + - + - RS-A-6 Bacillus anthracis ATCC 14578 T 100 + + +++ +++ + RS-A-7 Bacillus cereus ATCC 14579 T 100 + + - +++ + RS-A-8 Bacillus anthracis ATCC 14578 T 98 + + + + + RS-A-10 Pseudomonas taiwanensis BCRC 17751 T 99 - ++ - ++ - RS-A-11 Pseudomonas taiwanensis BCRC 17751 T 100 + + +++ +++ + RS-A-12 Pseudomonas taiwanensis BCRC 17751 T 99 - - - + - RS-A-15 Enterobacter cloacae subsp. dissolvens LMG 96 - - ++ + - 2683 T RS-A-16 Pseudomonas taiwanensis BCRC 17751 T 100 - - - - - RS-A-18 Enterobacter cancerogenus LMG 2693 T 99 - - - - - Enzymatic activities were estimated by measuring the diameter of the clear zones for protease, pectinase and chitinase , yellowish zones for cellulase, and white ppt zones for lipase. Rating of enzymatic activity was determined as zone of halo formed around bacterial colonies: -Negative; +, < 3 mm; ++, between 4-5 mm; +++, between 6-7 mm.

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4.5. Functional characterization of isolated bacterial strains

4.5.1. Antifungal activities of pre sowing isolated bacterial strains

The pre sowing isolates (14 strains) were screened for antifungal activities. The antifungal activities of all the selected bacterial strains were determined using Fusarium oxysporum and Aspergillus niger as a target organism. Results showed that 7 out of 14 strains were found positive regarding antifungal activity showing clear zones around them. Among these 3 Strains RS-A-4, RS-A-5 and RS-A-10 showed antifungal activity against the tested strains Fusarium oxysporum. The strains which showed clear antifungal activity against Fusarium oxysporum were belong to genera Pseudomonas and Bacillus. Whereas five strains RS-A-1, RS-A-3, RS-A-5, RS-A-7 and RS-A-18 showed antifungal activity against the tested strains Aspergillus niger. Among these four strains belong to genus Bacillus while one strains which showed positive activity was from genus Enterobacter. Only one strains RS-A-5 showed positive activity against both the tested fungal strains Fusarium oxysporum and Aspergillus nige. While seven strains did not show any antifungal activity against the tested fungal strains (Table 4.8).

4.5.2. Antibacterial activities of pre sowing isolated bacterial strains

All the selected bacterial strains were assessed for antibacterial activity using disc diffusion method against various pathogenic bacteria such as Pseudomonas syringae and Xanthomonas axonopodis. The results were checked for any antibacterial activity at intervals of 24 hours, 48 hours and 72 hours respectively. All the 14 selected strains did not show any antibacterial activity against any of the targeted bacterial strains (Table 4.8).

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Table 4. 8: Antifungal and antibacterial activities of the pre sowing isolates.

Antifungal Activity Antibacterial Activity Closely related type strain % Strains Identity code Fusarium Aspergillus Pseudomonas Xanthomonas oxysporum niger syringae axonopodis RS-A-1 Bacillus anthracis ATCC 14578T 100 - + - - RS-A-2 Bacillus aerophilus 28K T 100 ------RS-A-3 Bacillus tequilensis 10b T 99 - + - - RS-A-4 Pseudomonas plecoglossicida FPC951 T 96 + - - - RS-A-5 Bacillus cereus ATCC 14579 T 100 + + - - RS-A-6 Bacillus anthracis ATCC 14578 T 100 - - - - RS-A-7 Bacillus cereus ATCC 14579 T 100 -- + - - RS-A-8 Bacillus anthracis ATCC 14578 T 98 ------RS-A-10 Pseudomonas taiwanensis BCRC 17751 T 99 + --- - - RS-A-11 Pseudomonas taiwanensis BCRC 17751 T 100 - - - - RS-A-12 Pseudomonas taiwanensis BCRC 17751 T 99 ------RS-A-15 Enterobacter cloacae subsp. dissolvens LMG 96 - -- - - 2683 T RS-A-16 Pseudomonas taiwanensis BCRC 17751 T 100 -- - - - RS-A-18 Enterobacter cancerogenus LMG 2693 T 99 - + - -

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4.6. Production of cell wall hydrolyzing enzymes by isolates collected at vegetative stage from GM and Non-GM maize rhizosphere

Bacterial isolates were collected at vegetative stage from GM and Non-GM maize rhizosphere. Total 19 bacterial strains were collected from rhizospheric soil at vegetative stages of one transgenic and tow non transgenic (cv Islamabad gold and Islamabad white) maize crop. Six strains were collected from GM maize while 10 from Islamabad white and 3 from Islamabad gold varieties rhizospheric. Total 19 rhizospheric bacterial strains were in vitro characterized for production of fungal cell wall hydrolyzing enzymes such as cellulase, chitinase, protease, pectinase and lipase (Table 4.9). The detail of each individual enzyme is given below.

4.6.1. Cellulase activities of the vegetative stage isolated bacterial strains

The GM and Non-GM maize vegetative stage collected rhizospheric isolates were in vitro screened for cellulase enzyme production. Results showed that 7 out of 19 isolates showed cellulase activities. Two out of six isolates RS-BGM-22 and RS-BGM-23 of GM maize rhizosphere which belong to genera Bacillus and Pseudomonas respectively were positive for cellulase activates (Table 4.9). Whereas 4 out of 10 isolate namely; RS-BIW- 26, RS-BIW-30, RS-BIW-31 and RS-BIW-34 of Non-GM maize rhizosphere (Variety; Islamabad white) were showed cellulase activities with zone diameter (< 3 mm) (Table 4.9). While 1 out of 3 isolate (Islamabad gold variety rhizosphere) showed positive activities. While the remaining 12 isolates were found negative and did not showed any cellulase activates. The isolates which showed cellulase activities were belong to genera Bacillus, Pseudomonas, Sphingomonas, Enterobacter, and Chryseobacterium.

4.6.2. Chitinase activities of the vegetative stage isolated bacterial strains

The vegetative stage isolate of one GM and two Non-GM maize rhizosphere were in vitro screen for chitinase activities. The chitinase activity was screened for all the 19 bacterial strains on TSA medium, in which carbon was the sole source in a defined medium having colloidal chitin. These isolates were screened to determine chitinase

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production. Each isolate was inoculated on colloidal chitin agar (CCA) and incubated at 28 ºC in the dark up to 7 days, zones of chitin clearing were observed around colonies. Colony zone diameters were measured (mm) and used to indicate the chitinase activity of each isolate. Results showed that total 6 out of 19 isolates were positive for chitinase production. Among these three isolates RS-BGM-22, RS-BGM-23 and RS-BGM-22 of GM maize were positive for chitinase activities. Where is 3 out of 10 strains of Islamabad white maize variety rhizosphere isolates showed chitinase activities while none of the Islamabad gold variety isolates showed any chitinase activities. The highest chitinase activities with zone diameter (4-5 mm) were recorded in isolate of one GM; RS-BGM-23 and Non-GM; RS-BIW-25 maize rhizosphere. The effective strains RS-BGM-23 and RS-BIW-25 with maximum chitinase activities (4-5 mm) were belong to genera

Pseudomonas and Chryseobacterium respectively. While the other strains RS-BGM-22, RS-BGM-24, RS-BIW-28 and RS-BIW-32 showed comparatively lower chitinase activities with zone diameter (<3 mm). While the remaining 13 isolates were found negative and did not show any chitinase activates (Table 4.9).

4.6.3. Protease activities of the vegetative stage isolated bacterial strains

The vegetative stage isolate of one GM and two Non-GM maize rhizosphere were in vitro screen for protease activities. The protease activities was screened for all the 19 bacterial strains on their respective medium with added skimmed milk. All the bacterial strains after three days inoculation on medium were screened for protease activities. Strains producing a clear zone around colony were assumed to have positive for protease production. Results showed that 10 out of 19 selected strains were proved to possess the protease activity. Among the effective strains three GM maize rhizosphere isolates RS- BGM-20, RS-BGM-22 and RS-BGM-23 which belong to genera Micrococcus, Bacillus and Pseudomonas respectively, showed protease activities. While 7 isolates (5 from IW and 2 from IG) of Non-GM maize rhizosphere showed protease activities. From the diameter of the zone it was concluded that 10 strains RS-BGM-20 and RS-BGM-22, RS- BGM-23, RS-BIW-25, RS-BIW-26, RS-BIW-28, RS-BIW-30, RS-BIW-32, RS-BIG-36 and RS-BIG-37 showed positive protease activities (Table 4.9). Among these strains RS- BGM-20 and RS-BGM-22 showed highest protease activity with zone diameter (6-7 128

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mm). Whereas strains RS-BIW-25, RS-BIW-26, RS-BIW-28, RS-BIW-32, RS-BIG-36 and RS-BIG-37 showed relatively higher protease activities with zone diameter (4-5 mm) respectively. While the isolates RS-BGM-23 and RS-BIW-30 showed relatively lower protease activities with zone diameter of (< 3 mm). While the remaining 9 isolate did not showed any protease activities (Table 4.9).

Comparatively the isolate of GM maize rhizosphere were more effective in production of protease enzyme as compared to Non-GM maize rhizosphere isolates.

4.6.4. Pectinase activities of the vegetative stage isolated bacterial strains

The vegetative stage isolates of one GM and two Non-GM maize rhizosphere were in vitro screen for pectinase activities. The pectinase activities was screened for all the 19 bacterial on pectinase screening agar medium (PSAM). Results showed that 13 out of 19 selected strains namely; RS-BGM-19 RS-BGM-20, RS-BGM-21, RS-BGM-22, RS- BIW-25, RS-BIW-27, RS-BIW-28, RS-BIW-30, RS-BIW-31, RS-BIW-32, RS-BIG-35, RS-BIG-36 and RS-BIG-37 showed pectinase activities. The GM maize rhizosphere isolate 4 out of 6 showed pectinase activities with different zone diameter. Whereas 9 out of 13 isolates of Non-GM maize rhizosphere were also effective in production of pectinase enzyme. Among these strains RS-BGM-22 showed highest protease activity with zone diameter (6-7 mm). Whereas strains RS-BGM-20, RS-BIW-25, RS-BIW-32 and RS-BIG-37 showed relatively higher pectinase activities with zone diameter (4-5mm) respectively. While the isolates RS-BGM-19 and RS-BGM-21, RS-BIW-27, RS-BIW-28, RS-BIW-30, RS-BIW-31, RS-BIG-35 and RS-BIG-36 showed relatively lower pectinase activities with zone diameter of (< 3 mm). While the remaining 6 isolate did not show any pectinase activities. Comparatively the isolates of GM maize rhizosphere were more effective in production of pectinase enzyme as compared to Non-GM maize rhizosphere isolates (Table 4.9).

4.6.5. Lipase activities of the vegetative stage isolated bacterial strains

The vegetative stage isolates of GM and Non-GM maize rhizosphere were in vitro screened for lipase activity. Total 19 strains from on GM and two Non-GM maize

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rhizosphere were isolated at vegetative stage and evaluated for enzymes production. Lipase activity was determined on TSB medium with 1% tween 20. Strains surrounded by white precipitation were considered positive for lipase production. Total 19 strains in which 6 strains of GM and 13 from Non-GM maize rhizosphere were screened on prescribed medium. Results showed that 14 out of 19 selected strains RS-BGM-19, RS- BGM-21, RS-BGM-22, RS-BGM-23, RS-BGM-24, RS-BIW-25, RS-BIW-26, RS-BIW- 29, RS-BIW-30, RS-BIW-31, RS-BIW-32, RS-BIW-33, RS-BIW-34, and RS-BIG-37 were proved to possess the protease activity. The GM maize rhizosphere isolates were effective in lipase production. Five out of six GM maize rhizosphere isolates showed lipase activity. whereas 9 out of 13 Non-GM maize rhizosphere isolates illustrated lipase activity. While five isolate did not show any lipase activity (Table 4.9).

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Table 4. 9: In vitro screening of GM and Non-GM maize rhizosphere isolates collected during vegetative stage for cell wall degrading enzymes.

% Cell wall degrading enzymatic activities Strains ID Closely related type strain Identity Variety Cellulase Chitinase Protease Pectinase Lipase

RS-BGM-19 Chryseobacterium indologenes LMG 8337T 98 - - - + + RS-BGM-20 Micrococcus antarcticus T2 T 99 - - +++ ++ RS-BGM-21 GM Enterobacter cloacae subsp. dissolvens LMG 2683T 99 - - - + + RS-BGM-22 Bacillus cereus ATCC 14579 T 100 + + +++ +++ + RS-BGM-23 Pseudomonas moorei RW10 T 99 + ++ + - + RS-BGM-24 Sphingobacterium multivorum IAM14316 T 98 - + - + RS-BIW-25 Non Chryseobacterium indologenes LMG 8337 T 98 - ++ ++ ++ + RS-BIW-26 GM Pseudomonas lini CFBP 5737 T 99 + - ++ - + RS-BIW-27 Ism. white Bacillus isronensis B3W22 T 98 - - - + - RS-BIW-28 variety Chryseobacterium indologenes LMG 8337v 98 - + ++ + - RS-BIW-29 Acinetobacter pittii LMG 1035 T 99 - - - - + RS-BIW-30 Sphingomonas koreensis JSS26 T 99 + - + + + RS-BIW-31 Enterobacter cloacae subsp. dissolvens LMG 2683 T 100 + - - + + RS-BIW-32 Pseudomonas lini CFBP 5737 T 99 - + ++ ++ + RS-BIW-33 Microbacterium oleivorans DSM 16091 T 99 - - - - + RS-BIW-34 Sphingomonas koreensis JSS26(T) 99 + - - - + RS-BIG-35 Non Aerococcus urinaeequi IFO 12173 T 97 - - - + - RS-BIG-36 GM Chryseobacterium hagamense RHA2-9 T 97 + - ++ + - RS-BIG-37 Ism. gold Enterobacter ludwigii DSM 16688 T 99 - - ++ ++ + variety Enzymatic activities were estimated by measuring the diameter of the clear zones for protease, pectinase and chitinase , yellowish zones for cellulase, and white ppt zones for lipase. Rating of enzymatic activity was determined as zone of halo formed around bacterial colonies: -, Negative; +, < 3 mm; ++, between 4-5 mm; +++, between 6-7 mm.

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4.7. Functional characterization of isolated bacterial strains

4.7.1. Antifungal activities of vegetative stage isolated bacterial strains

The antifungal activities was in vitro screened for 19 bacterial strains isolated from GM and Non-GM maize rhizosphere during vegetative stage. The antibacterial activity of the selected strains were assessed by disc method on TSA medium. The antifungal activities of all the selected bacterial strains were determined using Fusarium oxysporum and Aspergillus niger as a target organism. Results showed that 4 out of 19 strains were found positive regarding antifungal activity showing clear zones around them. The strains RS- BGM-20, RS-BIW-25, RS-BIW-32 and RS-BIG-35 were positive against Fusarium oxysporum while none of the strains showed any activity against Aspergillus niger. The strains RS-BGM-20, RS-BIW-25, RS-BIW-32 and RS-BIG-35 which were positive against Fusarium oxysporum belong to genera Micrococcus, Chryseobacterium, Pseudomonas and Aerococcus respectively. The results showed that 15 strains either from GM or Non-GM maize rhizosphere did not show any antifungal activity against the tested organism (Table 4.10).

4.7.2. Antibacterial activities of vegetative stage isolated bacterial strains

The antibacterial activities was in vitro screened for 19 bacterial strains isolated from GM and Non-GM maize rhizosphere during vegetative stage. All the selected bacterial strains were assessed for antibacterial activity using disc diffusion method against various pathogenic bacteria such as Pseudomonas syringae and Xanthomonas axonopodis. The results were checked for any antibacterial activity at intervals of 24-72 hours respectively. All the 19 selected strains either from GM or Non-GM maize rhizosphere did not show any antibacterial activity against any of the targeted bacterial strains (Table 4.10).

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Table 4. 10: Antifungal and antibacterial activities of the isolates from GM and Non-GM maize rhizosphere during vegetative stage isolates.

% Antifungal Activity Antibacterial Activity Strains ID Variety Closely related type strain Identity Fusarium Aspergillus Pseudomonas Xanthomonas oxysporum niger syringae axonopodis RS-BGM-19 GM Chryseobacterium indologenes LMG 8337T 98 - - - - RS-BGM-20 Micrococcus antarcticus T2 T 99 + - - RS-BGM-21 Enterobacter cloacae subsp. dissolvens 99 - - - - LMG 2683 T RS-BGM-22 Bacillus cereus ATCC 14579 T 100 - - - - RS-BGM-23 Pseudomonas moorei RW10 T 99 - - - - RS-BGM-24 Sphingobacterium multivorum IAM14316 T 98 - - - - RS-BIW-25 Non Chryseobacterium indologenes LMG 8337 T 98 + - - - RS-BIW-26 GM Pseudomonas lini CFBP 5737 T 99 - - - - RS-BIW-27 Ism. Bacillus isronensis B3W22 T 98 - - - - RS-BIW-28 white Chryseobacterium indologenes LMG 8337 T 98 - - - - RS-BIW-29 variety Acinetobacter pittii LMG 1035 T 99 - - - - RS-BIW-30 Sphingomonas koreensis JSS26 T 99 - - - - RS-BIW-31 Enterobacter cloacae subsp. dissolvens 100 - - - - LMG 2683 T RS-BIW-32 Pseudomonas lini CFBP 5737 T 99 + - - - RS-BIW-33 Microbacterium oleivorans DSM 16091 T 99 - - - - RS-BIW-34 Sphingomonas koreensis JSS26 T 99 - - - - RS-BIG-35 Ism. Aerococcus urinaeequi IFO 1217 T 97 + - - - RS-BIG-36 gold Chryseobacterium hagamense RHA2-9 T 97 - - - - RS-BIG-37 variety Enterobacter ludwigii DSM 16688 T 99 - - - -

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4.8. Production of cell wall hydrolyzing enzymes by isolates collected at harvesting stage from GM and Non-GM maize rhizosphere

Bacterial isolates were collected at harvesting stage from GM and Non-GM maize rhizosphere. Total 13 bacterial strains were collected from rhizospheric soil at harvesting stages of one transgenic and tow non transgenic (cv; Islamabad gold and Islamabad white) maize crop. four strains were collected from GM maize while three from Islamabad white and six from Islamabad gold varieties rhizospheric. Total 13 rhizospheric bacterial strains were in vitro characterized for production of fungal cell wall hydrolyzing enzymes such as cellulase, chitinase, protease, pectinase and lipase (Table 4.11). The detail of each individual enzyme is given below.

4.8.1. Cellulase activities of the harvesting stage isolated bacterial strains

The bacterial strains were collected from GM and Non-GM maize rhizosphere during harvesting stage and were in vitro screened for cellulase enzyme production. After two days of incubation and treatments of the individual plates with Congo red dye and NaCl solution, finally cellulase activity of the strains were estimated by measuring the yellowish brown halo around individual colonies. The results in table 4.11 showed that maximum isolates of the harvesting stage showed cellulase activity. From the diameter of the zone it was concluded that 9 strains RS-CGM-39, RS-CGM-41, RS-CGM-42, RS- CGM-43, RS-CIW-45, RS-CIW-46, RS-CIG-48, RS-CIG-49 and RS-CIG-51 showed positive cellulase activities. Among these all the 4 strains of GM maize rhizosphere were positive for cellulase production with zone diameter (< 3 mm). While 5 out of 9 strains of Non-GM maize also exhibited cellulase activity with same diameter (> 3 mm). While four strains did not show any cellulase activity. Comparatively the isolates of GM maize rhizosphere were more effctive in cellulase production (Table 4.11).

4.8.2. Chitinase activities of the harvesting stage isolated bacterial strains

The harvesting stage isolate of one GM and two Non-GM maize rhizosphere were in vitro screen for chitinase activities. The chitinase activity was screened for all the 13

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bacterial strains on TSA medium, in which carbon was the sole source in a defined medium having colloidal chitin. These isolates were screened to determine chitinase production. Each isolate was inoculated on colloidal chitin agar (CCA) and incubated at 28ºC in the dark up to 7 days, zones of chitin clearing were observed around colonies. Colony zone diameters were measured (mm) and used to indicate the chitinase activity of each isolate. Results showed that 6 out of 13 selected strains namely; RS-CGM-39, RS-CGM-43, RS-CIW-46, RS-CIG-48, RS-CIG-49 and RS-CIG-50 were positive for chitinase production. Among these strains RS-CGM-43 and RS-CIG-50 showed highest chitinase activity with zone diameter (6-7 mm).Whereas strains RS-CIW-46 and RS- CIG-49 showed relatively higher chitinase activities with zone diameter (4-5 mm) respectively. While the isolates RS-CGM-39 and RS-CIG-48 showed relatively lower chitinase activities with zone diameter of (< 3 mm). While the remaining 7 isolates did not show any chitinase activities. The effective strains RS-CGM-43 and RS-CIG-50 which produced maximum chitinase activity were belong to genera Bacillus and Pseudomonas. Two isolate of GM and four of Non-GM maize rhizosphere showed chitinase activity (Table 4.11).

4.8.3. Protease activities of the harvesting stage isolated bacterial strains

The harvesting stage isolate of one GM and two Non-GM maize rhizosphere were in vitro screen for protease activity. The protease activities was screened for all the 13 bacterial strains of harvesting stage on their respective medium with added skimmed milk. All the bacterial strains after three days inoculation on medium were screened for protease activities. Strains producing a clear zone around colony were assumed to have positive for protease production. Results showed that 9 out of 13 selected strains RS- CGM-39, RS-CGM-43, RS-CIW-46, RS-CIG-48, RS-CIG-49, RS-CIG-50, RS-CIG-51, RS-CIG-52, RS-CIG-53 were proved to possess the protease activity. Among these strains RS-CGM-39, RS-CIW-46, RS-CIG-48, RS-CIG-50 and RS-CIG-52 showed highest protease activity with zone diameter (6-7 mm). Whereas strains RS-CGM-43, RS-CIG-49, RS-CIG-51 and RS-CIG-53 showed relatively higher protease activities with zone diameter (4-5 mm) respectively. While 4 isolates did not show any protease activity. Two isolates RS-CGM-39 and RS-CGM-43 of GM maize rhizosphere were effective in 135

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protease production with zone diameter (6-7 mm; 4-5 mm,) respectively. Whereas most of the Non-GM maize rhizosphere isolates were capable of protease production (Table 4.11).

4.8.4. Pectinase activities of the harvesting stage isolated bacterial strains

The harvesting stage rhizospheric isolate of one GM and two Non-GM maize crop were in vitro screen for pectinase activity. Total 13 bacterial strains 4 from GM and 9 from Non-GM maize which were isolated at crop harvesting stage were evaluated for pectinase production. The results in table 4. 11 showed that 7 out of 13 bacterial strains RS-CGM- 39, RS-CGM-43, RS-CIW-45, RS-CIG-49, RS-CIG-50, RS-CIG-52 and RS-CIG-53 showed pectinase activity. Among these strains RS-CGM-43 and RS-CIG-53 showed highest protease activity with zone diameter (6-7 mm). Whereas strains RS-CGM-39, RS-CIG-50 and RS-CIG-52 showed relatively higher pectinase activities with zone diameter (4-5 mm) respectively. While the isolates RS-CIW-45 and RS-CIG-49, showed relatively lower pectinase activities with zone diameter of (< 3 mm). While the remaining 6 isolate did not show any pectinase activities. Comparatively the isolates of GM maize rhizosphere were more effective in production of pectinase enzyme as compared to Non- GM maize rhizosphere isolates (Table 4.11).

4.8.5. Lipase activities of the harvesting stage isolated bacterial strains

The harvesting stage isolates of GM and Non-GM maize rhizosphere were in vitro screened for lipase activity. Total 13 strains from on GM and two Non-GM maize rhizosphere were isolated at harvesting stage and evaluated for enzymes production. Lipase activity was determined on TSB medium with 1% tween 20. Strains surrounded by white precipitation were considered positive for lipase production. Results showed that maximum isolate of the harvesting stage showed lipase activity. All the 4 isolate RS- CGM-39, RS-CGM-41, RS-CGM-42 and RS-CGM-43 of GM maize rhizosphere were able to produced lipase enzyme. Whereas 7 out of 8 isolates of Non-GM maize rhizosphere produced lipase enzyme. Comparatively there is no significant variation in

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lipase enzyme production of the isolates of GM and Non-GM maize rhizosphere (Table 4.11).

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Table 4. 11: In vitro screening of GM and Non-GM maize rhizosphere isolates collected during harvesting stage for cell wall degrading enzymes.

Cell wall degrading enzymatic activities % Strains ID Variety Closely related type strain Identity Cellulase Chitinase Protease Pectinase Lipase

RS-CGM-39 Bacillus cereus ATCC 14579T 100 + + +++ ++ + RS-CGM-41 GM Enterobacter cloacae subsp. dissolvens 98 + - - - + LMG 2683 T RS-CGM-42 Enterobacter cloacae subsp. dissolvens 98 + - - - + LMG 2683 T RS-CGM-43 Bacillus aryabhattai B8W22 T 93 + +++ ++ +++ + RS-CIW-45 Microbacterium trichothecenolyticum IFO 98 + - - + + Ism. 15077 T RS-CIW-46 white Bacillus idriensis SMC 4352-2 T 99 + ++ +++ - + RS-CIW-47 variety Bordetella petrii DSM 12804 T 97 - - - - - RS-CIG-48 Bacillus cereus ATCC 14579 T 99 + + +++ - + RS-CIG-49 Ism. Bacillus cereus ATCC 14579 T 100 + ++ ++ + + RS-CIG-50 gold Pseudomonas stutzeri ATCC 17588 T 99 - +++ +++ ++ + RS-CIG-51 variety Flavobacterium sasangense YC6274 T 97 + - ++ - + RS-CIG-52 Bacillus cereus ATCC 14579 T 100 - - +++ ++ + RS-CIG-53 Bacillus cereus ATCC 14579 T 100 - - ++ +++ + Enzymatic activities were estimated by measuring the diameter of the clear zones for protease, pectinase and chitinase , yellowish zones for cellulase, and white ppt zones for lipase. Rating of enzymatic activity was determined as zone of halo formed around bacterial colonies: -, Negative; +, < 3 mm; ++, between 4-5 mm; +++, between 6-7 mm

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4.9. Functional characterization of isolated bacterial strains

4.9.1. Antifungal activities of harvesting stage isolated bacterial strains

The antifungal activities was in vitro screened for 13 bacterial strains isolated from GM and Non-GM maize rhizosphere during harvesting stage. The antibacterial activity of the selected strains were assessed by disc method on TSA medium. The antifungal activities of all the selected bacterial strains were determined using Fusarium oxysporum and Aspergillus niger as a target organism. Results showed that 4 out of 13 strains were found positive regarding antifungal activity showing clear zones around them. The strains RS- CGM-41, RS-CIG-48, RS-CIG-52 and RS-CIG-53 were positive against tested fungal strains. The strains RS-CGM-41, RS-CIG-48 and RS-CIG-53 which were positive against Fusarium oxysporum belong to genera Enterobacter and Bacillus respectively. Whereas the strains RS-CIG-52 which belong to genus Bacillus showed positive antifungal activity against both the tested fungal strains (Fusarium oxysporum and Aspergillus niger). The results showed that 9 strains either from GM or Non-GM maize rhizosphere did not show any antifungal activity against the tested organism (Table 4.12).

4.9.2. Antibacterial activities of harvesting stage isolated bacterial strains

The antibacterial activities was in vitro screened for 13 bacterial strains isolated from GM and Non-GM maize rhizosphere during harvesting stage. All the selected bacterial strains were assessed for antibacterial activity using disc diffusion method against various pathogenic bacteria such as Pseudomonas syringae and Xanthomonas axonopodis. The results were checked for any antibacterial activity at intervals of 24, 48 and 72 hours respectively. All the 13 selected strains either from GM or Non-GM maize rhizosphere did not show any antibacterial activity against any of the targeted bacterial strains (Table 4.12).

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Table 4. 12: . Antifungal and antibacterial activities of the isolates from GM and Non-GM maize rhizosphere during harvesting stage.

Antifungal Activity Antibacterial Activity % Strains ID Variety Closely related type strain Identity Fusarium Aspergillus Pseudomonas Xanthomonas

oxysporum niger syringae axonopodis RS-CGM-39 Bacillus cereus ATCC 14579T 100 - - - - RS-CGM-41 GM Enterobacter cloacae subsp. dissolvens 98 + - - - LMG 2683T RS-CGM-42 Enterobacter cloacae subsp. dissolvens 98 - - - - LMG 2683T RS-CGM-43 Bacillus aryabhattai B8W22T 93 - - - - RS-CIW-45 Microbacterium trichothecenolyticum IFO 98 - - - - Ism. 15077T RS-CIW-46 white Bacillus idriensis SMC 4352-2T 99 - - - - RS-CIW-47 variety Bordetella petrii DSM 12804 T 97 - - - - RS-CIG-48 Bacillus cereus ATCC 14579 T 99 + - - - RS-CIG-49 Ism. gold Bacillus cereus ATCC 14579 T 100 - - - - RS-CIG-50 variety Pseudomonas stutzeri ATCC 17588 T 99 - - - - RS-CIG-51 Flavobacterium sasangense YC6274 T 97 - - - - RS-CIG-52 Bacillus cereus ATCC 14579 T 100 ++ + - - RS-CIG-53 Bacillus cereus ATCC 14579 T 100 + - - -

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5. DISCUSSION

The use of GM maize still raises concerns about their impact on environment as well as soil ecosystem specially microbial diversity. The present study was aimed to evaluate the possible effects of GM maize, in comparison to the Non-GM maize varieties, on the structure and abundance of bacterial communities in the rhizosphere at three different stages (pre sowing, vegetative and harvesting). For this purpose, the bacterial communities associated with the rhizosphere of GM maize were compared by culture dependent and independent methodologies to the two Non-GM maize varieties. In soil, a large range of factors affect bacterial community including plant type, soil type, agricultural management regime and the interaction among these factors (Garbeva, 2004; Van et al., 1997). Plant type may influence the composition and structure of the root- associated microbial communities mainly via root exudation of sugars, organic acids, amino acids, proteins and other compounds. Such compounds can be used by the rhizosphere microorganisms as energy and carbon sources. Different plant have their own typical exudation pattern and microorganisms respond differently to the compounds released by the plant root. Different root exudates are expected to have specific effects on rhizosphere communities (Garbeva, 2004)

In the present research work culture dependent and independent methods were applied to analyze bacterial diversity associated with the GM and Non-GM maize rhizosphere at crop developmental stages, 16S rRNA gene sequence analysis was used to estimate the bacterial diversity in the maize rhizosphere. In culture dependent method total fifty three strains were isolated, eighteen from the pre sowing stage and were grouped to two major phyla’s namely Firmicutes and Proteobacteria. and five genera such as Bacillus, Salirhabdus, Klebsiella, Enterobacter and Pseudomonas. Our results were contradictory with the previous studies indicated that the rhizosphere population decreases as a plant matures (Brimecombe et al., 2006)

Whereas twenty strains from vegetative stage (GMB-7; BIW-10; BIG-3) were isolated and grouped to four major phyla’s namely Proteobacteria, Bacteroidetes, Firmicutes and

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Actinobacteria and eleven genera such as Pseudomonas, Acinetobacter, Enterobacter, Stenotrophomonas, Sphingomonas, Chryseobacterium, Sphingobacterium, Bacillus, Solibacillus, Aerococcus and Microbacterium.

Fifteen strains from harvesting stage (CGM-6;CIW-5; CIG-5) of GM and Non-GM maize rhizosphere belonged to four major phyla’s namely Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. Most frequently reported bacteria linked to the maize crop (Zea mays L.) are Firmicutes (Bacillus), γ-Proteobacteria (Pseudomonas, Enterobacter), β-Proteobacteria (Burkholderia), and Actinobacteria (Arthrobacter) (Pereira et al., 2011). Less frequent are Paenibacillus (Firmicutes), Achromobacter, Herbaspirillum (β-Proteobacteria), Erwinia (γ-Proteobacteria), Sphingomonas (α- Proteobacteria), other Actinobacteria, and Bacteroidetes (Brusetti et al., 2004a). In agreement with these previous surveys, within the Firmicutes group we identified Bacillus species from all the three stages of GM and Non-GM maize rhizosphere and Enterobacter and Pseudomonas were found as main genera within the γ-Proteobacteria. Bacteria from Bacillus genus have been reported as maize kernel endophytes (Rijavec et al., 2007) and have been isolated not only from maize (Chelius and Triplett, 2001a) but also from many different plants such as peas, potatoes, conifers, bananas, and bean (Elvira-Recuenco and van Vuurde, 2000).

The results showed that in pre sowing samples cultured analysis Bacillus and Pseudomonas were the major genera. Whereas during vegetative stage of GM and Non- GM maize rhizosphere Pseudomonas, Chryseobacterium, Enterobacter, Bacillus and Sphingomonas were the major genera but there were no prominent variation in bacterial population of GM maize rhizosphere with that of Non-GM maize rhizosphere. This study showed that rhizosphere bacterial diversity was not altered by the cultivation of GM maize crop. Other studies assessing culturable soil microbial communities indicated that bacterial communities in rhizospheres were not affected by varieties of either transgenic or Non transgenic crops cultivation (Chiarini et al., 1998). While at harvesting stage the major bacterial genera were Bacillus, Microbacterium and Enterobacter both in GM as well as in Non-GM maize rhizosphere. Genera of Bacillus and Enterobacter were pre dominate in harvesting stage of GM rhizosphere whereas Microbacterium, Pseudomonas

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DISCUSSION CHAPTER 5 and Flavobacterium were isolated from Non-GM maize rhizosphere. Our study coincide with the previous finding of Schmalenberger and Tebbe (2002). Glufosinate-resistant maize was previously shown not to affect the bacterial community. Similarly, we found that GM and Non-GM maize had no effect on rhizosphere bacterial communities and this is the first such report to our knowledge.

Normally it was reported that rhizosphere bacterial diversity, characterized by physiological and molecular approaches, differed between soils in which maize grown. This supports the findings of Girvan et al. (2003) who have reported that soil texture was the overriding factor in controlling soil bacterial diversity, regardless of the cropping system. Similarly, (Dalmastri et al., 1999) have reported the important role of soil texture on bacterial diversity in rhizospheres of field-grown maize at the genus level. The results of the cultured isolates from the soil prior to sowing of GM and Non-GM maize indicated that bacterial diversity was least dominant as compared to vegetative and harvesting stages. In the pre sowing stage Bacillus and Pseudomonas were the pre dominant genera which confirmed the previous findings of Pereira et al. (2009) who have reported that Bacillus and Pseudomonas were major genera isolated from 20-day-old maize seedlings. The variations in bacterial diversity increased with crop developmental stages may be attributed to increased root exudation as the bacterial communities differ due to changes in root exudate quantity and composition by the developmental stages of the host plant, which select different bacterial groups during root colonization (Brimecombe et al., 2001).

Soil bacterial communities can be affected by other factors like soil characteristics, environmental conditions and crop management strategies like crop rotation and residue removal (Govaerts et al., 2007; Salles et al., 2006). Rhizosphere bacterial communities are also known to be governed by the complex interactions driven by soil type, plant species (genotypes) and growth (Marschner et al., 2001a). In order to evaluate the bacterial diversity, we assess GM and Non-GM maize rhizosphere as well as bulk soil sample by direct pyrosequencing. The dynamics of the relative abundances in soil and the community structures of soil bacterial communities as a function of cultivar type and growth stage, using one GM and two Non-GM maize varieties in field trial.

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Total eight samples one from pre sowing and six from GM and Non-GM maize rhizosphere (vegetative and harvesting stages) while one sample were collected from the same field after one year interval, were analyzed through pyrosequencing for bacterial community composition. Bacterial diversity was assessed through pyrosequencing technology using 515F primer. Minimum 200 bp region of the 16S rRNA was sequenced and nominal 20,000 reads per sample were obtained. From pre sowing soil sample 19 phyla were identified, among these phyla 47% were Proteobacteria, 21% Bacteroidetes, 10% Actinobacteria, 7% Firmicutes, 4% each Planctomycetes and Verrucomicrobia, 2% Crenarchaeota. While phyla Fibrobacteres, Euryarchaeota, Tenericutes, Synergistetes, Nitrospirae, Cyanobacteria, Dictyoglomi, Aquificae, Spirochaetes and Chloroflexi contribute lowest percentage of total bacterial population. Indeed, a striking observation of this study was that the young plant stage stands out as being a unique determinator of community structure in the rhizosphere, as compared to the other two plant growth stages. This may indicate that the distribution of plants and belowground organisms is similarly structured, in contrast to distributions (Becker et al., 2008). On the basis of genera level distribution of bacterial flora, total 446 different genera were obtained while some genera were unclassified. Total 24 genera were found in higher percentage from pre sowing sample and contributed the maximum portion of bacterial population. Among the major genera 1.891% Nitrososphaera, 5.934% Chitinophaga, 4.77% Ectothiorhodospira, 2.193% Rhodoplanes, 1.516% Bacillus and 2.356% Nitrosovibrio contributed the significant portion of total bacterial flora of the pre sowing. Divergent community structures occurred in the young plant stage (Inceoglu et al., 2011)

Whereas at vegetative growth stage, the major phyla in the rhizosphere of GM and Non- GM maize were Proteobacteria (127%), Actinobacteria (58%), Bacteroidetes (41%), Firmicutes (21%), Planctomycetes (14%), Verrucomicrobia (10%) and Chloroflexi (3%). On the basis of genera level distribution of bacterial flora, total 470 genera were obtained from one GM and two Non-GM maize (IG; IW) rhizosphere. Among the major genera average 4.455% Rubrobacter (GM-3.383%; IG-4.211%; IW-5.77%), 4.538% Chitinophaga (GM-5.371%; IG-4.545%, IW-3.697%), 3.259% Nitrososphaera (GM- 2.566%; IG-2.514%, IW-4.698%), 2.262% Rhodococcus (GM-1.319%;IG-3.697%,IW-

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1.769%), 2.534% Hyphomicrobium (GM- 2.141%; IG- 2.773%, IW- 2.689%), 2.379% Rhodoplanes (GM-2.231%; IG-2.197%, IW-2.710%) and 2.825 % Ectothiorhodospira (GM-3.139%; IG-2.781%, IW-2.555%) were the dominant genera in both GM and Non- GM maize rhizosphere during vegetative stage. Whereas the vegetative and senescence stages revealed increasingly similar community structures (Inceoglu et al., 2011). While total seven major groups were found in higher percentage and contributed the maximum population during harvesting stage of GM and Non-GM maize rhizosphere.

At harvesting stage, the bacterial diversity in the rhizosphere of GM and Non-GM maize at phyla level represented Actinobacteria (36%), Proteobacteria (32%), Bacteroidetes (8%), Firmicutes (7%), each Crenarchaeota (4%), Planctomycetes (4%) and Verrucomicrobia (1%). Across the different bulk and rhizosphere soils, the senescence stage had the most stable evenness across all varieties and bulk soil (Inceoglu et al., 2011) Among the major genera average 5.857% Rubrobacter, 4.898% Streptomyces, 4.415% Nitrososphaera, 3.465% Nocardioides, 2.213% Rhodoplanes, 2.262% Hyphomicrobium, 2.1405% Bacillus and 2.347% Chitinophaga were found the major genera in GM and Non-GM maize rhizosphere during harvesting stage.

There were no significant variation in the population of bacteria (based on genera level distribution) in GM maize rhizosphere as compared to their counterpart Non-GM maize varieties rhizospheric soil. Soil bacterial diversity and functioning often mainly focus on the most dominant species. However, some rare species may have large effects on soil functioning, in spite of their low total biomass or rareness (Hooper et al., 2005). Here, pyrosequencing provided the unique opportunity to access the less abundant bacterial taxa under potato, as well as to compare their presence across the varieties at different growth stages.

Moreover, dominant members of particular phyla or classes were used as ecological indicators to reveal prevailing ecological behavior. Although it is rather implausible that a complete phylum would have shared ecological characteristics, our indicators revealed a dynamics. This was thought to reflect the plant growth stages and conditions in the corresponding bulk soils. Many of the Betaproteobacteria (Fierer et al., 2007) and

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Pseudomonas species (Smit et al., 2001) are known as typical copiotrophs (r strategists) which tend to be strongly favored under nutrient-rich conditions.

In our study, the impact of GM maize on rhizosphere bacterial community were not significantly differ from that of Non-GM lines. Thus, cultivar (genotypes) did not have any outstanding effect on bacterial community structure and abundance. Previously, Milling et al. (2004) investigated the effects of a transgenic potato (which produced tubers with an altered starch composition) on the composition of bacterial in the rhizosphere. They compared a parent, a GM line, and another Non-GM cultivar for three growing seasons. They did not observe any significant influence of the modification on the dominant members of the rhizosphere bacterial communities. Based on our results, we concluded that there were no consistent and considerable variation in population of bacterial community of GM and Non-GM maize rhizosphere

Attempts were made to determine the plant growth promoting efficiency of isolated rhizobacteria from soil prior to sowing of maize (GM and non-GM) crops and rhizosphere of GM and non-GM maize crops at vegetative and reproductive growth phases. The rhizobacteria were applied as seed inoculation at 106 cells/mL prior to sowing.

The rhizospheric soil has a greater diversity of bacterial communities which play important roles in nutrient cycling, plant growth promotion and suppression of pathogenic microbes. These beneficial rhizobacteria are known as plant growth promoting rhizobacteria (PGPR) (Munees and Mulugeta, 2013). During current studies, the rhizobacteria isolated from rhizosphere of GM and non-GM maize exhibited positive effects on growth and development of maize seedling measured in terms of shoot and root length, shoot and root dry weight. These results are in agreement with previous findings of Ahemad and Khan (2010) who have reported that growth in PGPR inoculated plants was higher than uninoculated ones. The higher shoot length and fresh and dry weights in BIW-31 and BIW-33 can be correlated to the greater production of IAA and phosphate solubilization by these isolates.

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The ability of a PGPR to solubilize and make available insoluble forms phosphorus to plants make their application more beneficent and interesting in agriculture systems. The use of phosphate solubilizing bacteria as inoculants increases the P uptake by plants (Chen et al., 2006). Similarly, the plant beneficial effects of PGPR have mainly been attributed to the production of phytohormones like IAA and nitrate reduction (Somers et al., 2004). During current studies, the improvement in plant growth due to PGPR inoculation may be attributed to their secretion of IAA, capacity of phosphate solubilization and increased activity of nitrate reductase. Among the various isolates, Pseudomonas taiwanensis isolated from soil prior to sowing of maize plants exhibited higher IAA production, phosphate solubilization and activity of nitrate reductase which resulted in higher shoot/root ratio and shoot fresh weight. Similarly, the isolate BGM-23 (Pseudomonas moorei), RS-CIG-50 (Pseudomonas stutzeri), RS-CGM-41 (Enterobacter cloacae subsp. dissolvens) and RS-CGM-42 (Enterobacter cloacae subsp. dissolvens) exhibited higher production of IAA which was followed by higher nitrate reductase activity and phosphate solubilization. These results are in confirmatory with those of Hassan et al. (2009) who found that rhizobacterial isolates were capable of producing IAA and improved root and shoot growth in wheat. Among the rhizobacterial isolates from rhizosphere soil and root cuttings of Piper nigrum (L), Pseudomonas was the most efficient phosphate solubilizer and improved plant growth and development (Ramachandran et al., 2007) as was also evidenced during current studies. Mirza et al. (2001) observed that Enterobacter isolates had potential of IAA production and improved the 55% increase in root biomass. The higher root length due to RS-CGM-42 (Enterobacter cloacae subsp. dissolvens) and RS-CGM-41 (Enterobacter cloacae subsp. dissolvens) inoculation may be attributed to the greater IAA production by this isolate because IAA is the phytohormones which plays crucial role in root development and root elongation (Rosangela et al., 2011). Moreover, the beneficial effects of PGPR on plant growth can also be attributed to the development of nitrate reductase activity which results in the production of higher biomass (Rahim et al., 2013) as was also common during present studies that inoculation of nitrate reductase producing rhizobacteria exhibited positive effects on shoot and root growth. Nitrate reductase is the first enzyme in the nitrate assimilation pathway and is considered as limiting factor of plant growth

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DISCUSSION CHAPTER 5 and development (Solomonson and Barber, 1990) which can be improved in plants by PGPR inoculation (El-Komy et al., 2003).

Siderophores are low-molecular-weight molecules secreted by microorganisms under iron limiting conditions (Crowley et al., 1991). Previous studies have shown that PGPR capable of phosphate solubilization and IAA production also produced siderophores (Tank and Saraf, 2010). During present studies, the isolates RS-A-1, RS-A-4, RS-A-6, RS-A-8, RS-A-10, RS-A-11, RS-A-12, RS-A-18 isolated from soil prior to sowing of GM and non-GM maize crops, RS-BIW-25 isolated during vegetative growth of maize and RS-CIG-48, RS-CIG-49, RS-CIG-50, RS-CIG-52, RS-CIG-53 isolated at harvesting stage of maize were capable of producing siderophores. Previous studies have shown that most of the siderophores producing bacteria are Gram-negative among which Pseudomonas and Enterobacter genera are common. Whereas, Bacillus and Rhodococcus genera are the Gram-positive bacteria having potential of siderophores production (Tian et al., 2009). Siderophores produced by Rhizobacteria scavenge iron in the rhizosphere starving pathogenic organisms of proper nutrition to mount an attack of the crop (Saharan and Nehra, 2011). The siderophores produced by Rhizobacteria not only act as biocontrol but also help in mitigating abiotic stresses. Previous studies have shown that PGPR capable of phosphate solubilization, siderophores and IAA production under saline conditions ameliorated the adverse effects of NaCl (2%) in tomato (Tank and Saraf, 2010). Sadeghi et al. (2012) demonstrated that bacterial isolate (C) had the potential of producing siderophores and served not only as biocontrol agent but also ameliorated salt stress in wheat plants.

Vast majority of the rhizobacteria act by displacing or antagonizing deleterious microorganisms and thus serve as biocontrol agents (Baker et al., 1985; Zheng et al., 2013). The beneficial effects of rhizobacteria on plants are also due to their inhibitory effects on soil borne pathogens (van Loon and Glick, 2004). Cellulase production and utilization of substrates available in the rhizospehere are important for controlling the rhizophere competence (Baker, 1991). During current studies, the cellulase activity was recorded for rhizobacterial stains RS-A-6, RS-A-7, RS-A-8 and RS-A-11 isolated from rhizospeheric soil during pre-sowing of GM and non-GM maize, RS-BGM-22, RS-BGM-

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23, RS-BIW-26, RS-BIW-30, RS-BIW-31 and RS-BIW-34 isolated from rhizopshere at vegetative growth stage of GM and non-GM maize, whereas RS-CGM-39, RS-CGM-41, RS-CGM-42, RS-CGM-43, RS-CIW-45, RS-CIW-46, RS-CIG-48, RS-CIG-49 and RS- CIG-51 isolated at harvesting stage of GM and non-GM maize. The beneficial rhizobacteria inhibit the growth of phytopathogens by the production of cell wall lytic enzymes like cellulose, chitinase etc (Kumar et al., 2012). The extracellular cell wall degrading enzymes are positively correlated with biocontrol abilities of the producing rhizobacteria (El-Tarabily, 2006; Singh et al., 1999a).

Identification of chitinase producing bacteria from rhizosphere soil is important for the isolation of bacteria that have antifungal activities. A vast majority of bacteria and fungi produce chitinase enzymes (Donderski and Trzebiatowska, 1999) which plays important role in the control of fungal diseases (Kamil et al., 2007). During current studies, maximum chitinase activity was shown by rhizobacteria belonging to genera Bacillus, Pseudomonas and Chryseobacterium, Chitinase inhibit fungal growth by hydrolyzing chitin which is a major component of the fungal cell wall. Moreover, chitinases are of great biotechnological importance for engineering of phytopathogenic resistant plants, their use as food and preservative agents for seeds (Dempsey et al., 1998; Kamil et al., 2007) isolated 400 isolates from rhizospheric soil in Egypt and tested them for chitinase production. They found that majority of the chitinase producing rhizobacteria belonged to genus Bacillus. Moreover, the members of the genus Bacillus have been previously reported for the production of chitinases (Schallmey et al., 2004).

Lipases are widely distributed among microorganisms and are of great industrial importance. They not only hydrolyse triglycerides to free fatty acids and glycerol but are also used in the production of foods, biodiesel, phramceuticals, textiles and detergents etc (Jaeger and Eggert, 2002; Saxena et al., 1999b). During current studies, majority of the lipase producing bacteria isolated from soil prior to sowing GM and Non-GM maize crops belonged to genus Bacillus. At vegetative growth stage, the strains RS-BGM-19, RS-BGM-21, RS-BGM-22, RS-BGM-23, RS-BGM-24, RS-BIW-25, RS-BIW-26, RS- BIW-29, RS-BIW-30, RS-BIW-31, RS-BIW-32, RS-BIW-33, RS-BIW-34, and RS-BIG- 37 were positive for lipase production. During harvesting growth stage, all the 4 isolate

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RS-CGM-39, RS-CGM-41, RS-CGM-42 and RS-CGM-43 of GM maize rhizosphere were able to produce lipase enzyme. Whereas 7 out of 8 isolates of Non-GM maize rhizosphere produced lipase enzyme. A large number of bacteria produce lipase which has capacity to hydrolyze triglycerides (Jaeger et al., 1994). The bacteria belonging to genera Achromobacter, Alcaligenes, Arthrobacter, Bacillus, Burkholderia, Chromobacterium and Pseudomonas are reported extensively for the production of extracellular lipases (Gupta et al., 2004). Among the different genera, the extracellular lipases produced by Pseudomonas and Bacillus are widely used in biotechnological applications (Beisson et al., 2000). It is inferred that bacteria associated with rhizosphere of GM and NON-GM maize can be exploited for commercial scale production of lipases.

Microbial proteases may play important role in infection of hosts by degrading the host’s protective barriers. Microbial proteases have been proposed as virulence factors in their pathogenesis against nematodes (Huang et al., 2004). Majority of the protease producing bacteria isolated from soil prior to sowing of maize crops belonged to genera Bacillus and Pseudomonas. At vegetative growth stage of GM and NON-GM maize, majority of the isolated rhizobacteria belonged to genera Micrococcus, Bacillus, Chryseobacterium and Pseudomonas. Whereas, at harvesting stage, majority of the protease producing rhizobacteria belonged to genus Bacillus. These results are in agreement with previous findings of Lian et al. (2007) who have reported that bacteria belonging to genus Bacillus are more efficient producer of proteases multiple Bacillus sp. can promote crop health by suppressing plant pathogens and pests through the production of antibiotic metabolites or directly through the stimulation of plant host defense before the occurrence of infection (Lian et al., 2007). It could be inferred that various Bacillus species which have capacity of protease production might contribute to their activity as biocontrol agents (Siddiqui et al., 2005; Siddiqui and Mahmood, 1999).

Pectolytic enzymes produced by non-pathogenic bacteria are essential in the decay of dead plant material and thus contribute in the recycling of carbon compounds in biosphere (Alana et al., 1989). Among the various bacteria isolated from soil prior to sowing of GM and Non-GM maize, two Bacillus RS-A-6 and RS-A-7 and one Pseudomonas strains RS-A-11 showed highest pectinase activity. The higher pectinase

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DISCUSSION CHAPTER 5 producing rhizobacteria isolated during vegetative growth stage of GM and Non-GM maize belonged to genera Bacillus, Pseudomonas, Enterobacter and Micrococcus. At harvesting growth stage of both GM and non Non-GM maize, Bacillus was dominant genus with maximum number of pectinase producing species. The pectinolytic enzymes play crucial role in root invasion by bacteria and thus play important role in plant microbe interaction (Hayat et al., 2010). Moreover, these pectinase producing bacteria isolated from rhizphosphere of GM and Non-GM maize contribute to the nutrient cycling by degrading the pectic compounds present in the cell wall of plants and thus contribute to the fertility of soil.

The use of antagonistic microorganisms may be an alternative to fungicides for the control of postharvest diseases. Out of 14 rhizobacterial strains isolated prior to sowing of maize crop, 7 were positive for antifungal activity. Fusarium oxysporum was observed susceptible to rhizobacteria belonging to genera Pseudomonas and Bacillus. The antifungal activity against Aspergillus niger was more common among rizobacterial isolates belonging to genus Bacillus. Only one strain of Enterobacter showed antifungal activity against Aspergillus niger. Similarly, at vegetative growth stage of maize, rhizobacterial isolates belonging to genera Micrococcus, Chryseobacterium, Pseudomonas and Aerococcus were having antifungal activity against Fusarium oxysporum. At harvesting stage, out of total 13 isolates, 4 isolates belonging to genera Enterobacter and Bacillus were observed positive for antifungal activity against Fusarium oxysporum. Lambert et al. (1987) found that 37 rhizobacterial isolates from rhizosphere of maize at different growth stages exhibited antifungal activity and inhibited the growth of pathogenic fungi.

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CONCLUSION

Based on the results obtained in our studies, the predominant bacterial groups in the rhizosphere of GM and Non-GM maize were Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Planctomycetes; other groups that were consistently found, although at lower abundance, were Fibrobacteres, Thermi, Euryarchaeota, Tenericutes, Synergistetes and Thermotogae. There was no indication of consistent bacterial diversity in the rhizosphere of GM and Non-GM maize as well as in bulk soil sample. Through culture method we find change in bacterial diversity of GM and Non-GM maize rhizosphere at different growth stages as compared to bulk soil samples. But in uncultured approach no significant variations were observed in bacterial diversity, and relative abundances of taxa at different growth stages of GM and Non-GM maize rhizosphere. We did not observe changes in the bacterial population at different growth stages which cannot be attributed to genetic modification of maize with respect to control (Non-GM). Presence of plants (either GM or Non-GM maize) as well as plant growth stages did not affect diversity of rhizosphere bacterial community. Although the type of strains have significant influence on their functional characterization rather than its source of isolation either from bulk soil or from GM and Non-GM maize rhizosphere. It is inferred that bacterial diversity have no correlation with transgenic crop cultivation. Some of the isolated strains have great potential for plant growth promotion characterization and can also be used as biocontrol agents against different plant diseases. Results are from only one transgenic and two Non transgenic maize varieties thus, more detailed studies on diversity of rhizosphere bacterial community are necessary to confirm these effects.

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6. REFERENCES

Acosta-Martıineza, V., Dowdb, S., Sunc, Y., and Allend, V. (2008). Tag-encoded pyrosequencing analysis of bacterial diversity in a single soil type as affected by management and land use. Soil Biol Biochem 40, 2762-2770.

Ahemad, M., and Khan, M. S. (2010). Ameliorative effects of Mesorhizobium sp. MRC4 on chickpea yield and yield components under different doses of herbicide stress. Pestic Biochem Phys 98, 183-190.

Alana, A., Gabilondo, A., Hernando, F., Moragues, M. D., Dominguez, J. B., Llama, M. J., and Serra, J. L. (1989). pectin lyase production by a Penicillium-Italicum Strain. Appl Environ Microbiol 55, 1612-1616.

Amand, P. C. S., Skinner, D. Z., and Peaden, R. N. (2000). Risk of alfalfa transgene dissemination and scale-dependent effects. Theor Appl Genet 101, 10-114.

Amann, R. I., Ludwig, W., and Schleifer, K. H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59, 143-169.

Antoun, H., and Kloepper, J. W. (2001). Plant growth-promoting rhizobacteria (PGPR), in: Encyclopedia of Genetics. Brenner, S. and Miller, J.H., eds., Academic Press, N.Y, 1477-1480.

Antoun, H., Beauchamp, C. J., Goussard, N., Chabot, R., and Lalande, R. (1998). Potential of Rhizobium and Bradyrhizobium species as plant growth promoting rhizobacteria on non-legumes: Effect on radishes (Raphanus sativus L.). Plant Soil 204, 57-67.

Arshad, M., Shaharoona, B., and Mahmood, T. (2008). Inoculation with plant growth promoting rhizobacteria containing ACC-deaminase partially eliminates the

153

REFERENCES CHAPTER 6

effects of water stress on growth, yield and ripening of Pisum sativum L. . Pedosphere 18, 611-620.

Arzanesh, M. H., Alikhani, H. A., Khavazi, K., Rahimian, H. A., and Miransari, M. (2011). Wheat (Triticum aestivum L.) growth enhancement by Azospirillum sp. under drought stress. World J Microb Biot 27, 197-205.

Aseri, G. K., Jain, N., Panwar, J., Rao, A. V., and Meghwal, P. R. (2008). Biofertilizers improve plant growth, fruit yield, nutrition, metabolism and rhizosphere enzyme activities of pomegranate (Punica granatum L.) in Indian Thar Desert. Sci Hortic 117, 130-135.

Atkinson, D., and Watson, C. A. (2000). The beneficial rhizosphere: a dynamic entity. Appl Soil Ecol 15, 99-104.

Badri, D. V., Quintana, N., El Kassis, E. G., Kim, H. K., Choi, Y. H., Sugiyama, A., Verpoorte, R., Martinoia, E., Manter, D. K., and Vivanco, J. M. (2009). An ABC transporter mutation alters root exudation of phytochemicals that provoke an overhaul of natural soil microbiota. Plant Physiol 151, 2006-2017.

Bais, H. P., Park, S. W., Weir, T. L., Callaway, R. M., and Vivanco, J. M. (2004). How plants communicate using the underground information superhighway. Trends Plant Sci 9, 26-32.

Baker, C. J., Stavely, J. R., and Mock, N. (1985). Biocontrol of bean rust by bacillus- subtilis under field conditions. Plant Dis 69, 770-772.

Baker, R. (1991). Diversity in Biological-Control. Crop Prot 10, 85-94.

Bakker, P. A. H. M., Raaijmakers, J. M., Bloemberg, G. V., Hofte, M., Lemanceau, P., and Cooke, M. (2007). New perspectives and approaches in plant growth- promoting rhizobacteria research. Eur J Plant Pathol 119, 241-242.

154

REFERENCES CHAPTER 6

Baldwin, F. L. (2000). Transgenic crops: a view from the US extension service. Pest Manag Sci 56, 584-585.

Barber, S. (1999). Transgenic plants: ®eld testing and commercialization including a consideration of novel herbicide resistance rape (Brassica napus L.). In: Lutman PLW ed. Gene flow and agriculture relevance for transgenic crops. Nottingham. Major Design and Production, 3-12.

Bartsch, D., and Schuphan, I. (2002). Lessons we can learn from ecological biosafety research. J Biotechnol 98, 71-77.

Bashan, Y., De-Bashan, L. E. (2005). Bacteria/plant growth-promotion. In: Hillel, D.(Ed.), Encyclopedia of Soils in the Environment,. Elsevier, Oxford, UK 1, 103- 115.

Bates, S. L., Zhao, J.-Z., Roush, R. T., and Shelton, A. M. (2005). Insect resistance management in GM crops: past, present and future. Nat Biotechnol 23, 57-62.

Becker, R., Behrendt, U., Hommel, B., Kropf, S., and Ulrich, A. (2008). Effects of transgenic fructan-producing potatoes on the community structure of rhizosphere and phyllosphere bacteria. FEMS Microbiol Ecol 66, 411-25.

Beisson, F., Tiss, A., Riviere, C., and Verger, R. (2000). Methods for lipase detection and assay: a critical review. Eur J Lipid Sci Tech102, 133-153.

Berdy, J. (1995). Are actinomycetes exhausted as a source of secondary metabolites? Proceedings of the 9th International Symposium on the Biology of Actinomycetes Part 1. Allerton Press, New York, 3-23.

Berg, G., Krechel, A., Ditz, M., Sikora, R. A., Ulrich, A., and Hallmann, J. (2005a). Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi. FEMS Microbiol Ecol 51, 215-229.

155

REFERENCES CHAPTER 6

Berg, G., Opelt, K., Zachow, C., Lottmann, J., Gotz, M., Costa, R., and Smalla, K. (2006). The rhizosphere effect on bacteria antagonistic towards the pathogenic fungus Verticillium differs depending on plant species and site. FEMS Microbiol Ecol 56, 250-261.

Berg, G., Roskot, N., Steidle, A., Eberl, L., Zock, A., and Smalla, K. (2002). Plant- dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants. Appl Environ Microbiol 68, 3328- 3338.

Berg, G., Zachow, C., Lottmann, J., Gotz, M., Costa, R., and Smalla, K. (2005b). Impact of plant species and site on rhizosphere-associated fungi antagonistic to Verticillium dahliae kleb. Appl Environ Microbiol 71, 4203-4213.

Bhattacharyya, P. N., and Jha, D. K. (2012). Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J Microb Biot 28, 1327-1350.

Binladen, J., Gilbert, M. T., Bollback, J. P., Panitz, F., Bendixen, C., and Nielsen, R. (2007). The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing. PLoS One 2, 197.

Bloemberg, G. G. V., and Lugtenberg, B. J. J. (2001). Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Curr Opin Plant Biol4, 343-350.

Bowen, G. D., and Rovira, A. D. (1991). The rhizosphere," Plant roots-the hidden half/Ed. Marcel Dekker, Inc., New York. 629-641

Brake, J., and Vlachos, D. (1998). Evaluation of transgenic event 176 "Bt" corn in broiler chickens. Poult Sci 77, 648-653.

Bressan, M., Roncato, M. A., Bellvert, F., Comte, G., Haichar, F. E., Achouak, W., and Berge, O. (2009). Exogenous glucosinolate produced by Arabidopsis thaliana has

156

REFERENCES CHAPTER 6

an impact on microbes in the rhizosphere and plant roots. ISME Journal 3, 1243- 1257.

Brimecombe, M. J., De Leij, F. A. A. M., and Lynch, J. M. (2001). Nematode community structure as a sensitive indicator of microbial perturbations induced by a genetically modified Pseudomonas fluorescens strain. Biol Fert Soils 34, 270-275.

Brimecombe, M., F. de Leij, and J. Lynch. (2006). The effect of root exudates on rhizosphere microbial populations. CRC Press, Boca Raton, FL., 95-140.

Brusetti, L., Francia, P., Bertolini, C., Pagliuca, A., Borin, S., Sorlini, C., Abruzzese, A., Sacchi, G., Viti, C., Giovannetti, L., Giuntini, E., Bazzicalupo, M., and Daffonchio, D. (2004a). Bacterial communities associated with the rhizosphere of transgenic Bt 176 maize (Zea mays) and its non transgenic counterpart. Plant Soil 266, 11-21.

Buchenauer, H. (1998). Biological control of soil-borne diseases by rhizobacteria. J Plant Dis Protect 105, 329-348.

Buee, M., De Boer, W., Martin, F., van Overbeek, L., and Jurkevitch, E. (2009). The rhizosphere zoo: An overview of plant-associated communities of microorganisms, including phages, bacteria, archaea, and fungi, and of some of their structuring factors. Plant Soil 321, 189-212.

Buyer, J. S., Roberts, D. P., and Russek-Cohen, E. (2002). Soil and plant effects on microbial community structure. Can J Microbiol 48, 955-964.

Cappuccino, J. G., and Sherman, N. (1996). Microbiology: A Laboratory Manual. The Benjamin/Cummings Publishing Company, Inc. New York.

Castaldini, M., Turrini, A., Sbrana, C., Benedetti, A., Marchionni, M., Mocali, S., Fabiani, A., Landi, S., Santomassimo, F., Pietrangeli, B., Nuti, M. P., N., M., and Giovannetti, M. (2005). Impact of Bt corn on rhizospheric and soil eubac-terial

157

REFERENCES CHAPTER 6

communities and on beneficial symbiosis in experimental microcosms. Appl. Environ. Microbiol 71, 6719-6729.

Cattelan, A., Hartel, P., and Furhmann, F. (1999). Screening for plant growth promoting rhizobacteria to promote early soybean growth. Soil Sci Soc Am J 63, 1670-1680.

Cavan, G., Biss, P., and Moss, S. R. (1998). Herbicide resistance and gene flow in wild- oats (Avena fatua and Avena sterilis ssp. ludoviciana). Ann Appl Biol 133, 207- 217.

Chelius, M. K., and Triplett, E. W. (2001). The diversity of archaea and bacteria in association with the roots of Zea mays L. Microb Ecol 41, 252-263.

Chen, Y. P., Rekha, P. D., Arun, A. B., Shen, F. T., Lai, W. A., and Young, C. C. (2006). Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Appl Soil Ecol 34, 33-41.

Cherry, J. R., and Fidantsef, A. L. (2003). Directed evolution of industrial enzymes: an update. Curr. Opin. Biotechnol 14, 438-443.

Chiarini, L., Bevivino, A., Dalmastri, C., Nacamulli, C., and Tabacchioni, S. (1998). Influence of plant development, cultivar and soil type on microbial colonization of maize roots. Appl Soil Ecol 8, 11-18.

Chow, M. L., Radomski, C. C., McDermott, J. M., Davies, J., and Axelrood, P. E. (2002). Molecular characterization of bacterial diversity in Lodgepole pine (Pinus contorta) rhizosphere soils from British Columbia forest soils differing in disturbance and geographic source. FEMS Microbiol Ecol 42, 347-357.

Colwell, R. R. (1997). Microbial diversity: the importance of exploration and conservation. J. Indust. Microbiol. Biotech 18, 302-307.

158

REFERENCES CHAPTER 6

Cowan, D. A. (2000). Microbial genomes - the untapped resource. Trends. Biotechnol 18, 14-16.

Cowan, D., Meyer, Q., Stafford, W., Muyanga, S., Cameron, R., and Wittwer, P. (2005). Metagenomic gene discovery: past, present and future. Trends Biotechnol 23, 321-329.

Cowgill, S. E., Bardgett, R. D., Kiezebrink, D. T., and Atkinson, H. J. (2002). The Ef-fect of transgenic nematode resistance on non-target organisms in the potato rhizosphere. J. Appl. Ecol 39, 915-932.

Crawford, D. L., Doyle, J. D., Wang, Z., Hendricks, C. W., Bentjen, S. A., Bolton Jr, H., Frederickson, J. K., and Bleakley, B. H. (1993). Effects of a lignin peroxidase- expressing recombinant, Streptomyces lividans TK23.1, on biogeochemical cycling and the numbers and activities of microorganisms in soil. Appl. Environ. Microbiol 59, 508-518.

Crawley, M. J., Brown, S. L., Hails, R. S., Kohn, D. D., and Rees, M. (2001). Biotechnology - transgenic crops in natural habitats. Nature 409, 682-683.

Crowley, D. E., Y.C. Wang, C.P.P. Reid, and Szaniszlo, P. J. (1991). Siderophore-iron uptake mechanisms by microorganisms and plants. Plant Soil 130, 179-198.

Cruden, D. L., and Markovetz, A. J. (1979). Carboxymethyl cellulose decomposition by intestinal bacteria of cockroaches. Appl. Environ. Microbiol 38, 369-372.

Curl, E. A., and Truelove, B. (1986). The rhizosphere. Springer, New York.

Da Mota, F. F., Gomes, E. A., and Seldin, L. (2008). Auxin production and detection of the gene coding for the auxin efflux carrier (AEC) protein in Paenibacillus polymyxa. J Microbiol 56, 275-264.

Dale, P. J., Clarke, B., and Fontes, E. M. G. (2002). Potential for the environmental impact of transgenic crops (vol 20, pg 567, 2002). Nat Biotechnol 20, 843-843.

159

REFERENCES CHAPTER 6

Dalmastri, C., Chiarini, L., Cantale, C., Bevivino, A., and Tabacchioni, S. (1999). Soil type and maize cultivar affect the genetic diversity of maize root-associated Burkholderia cepacia populations. Microb Ecol 38, 273-284.

Daniel, R. (2005). The soil metagenome, a rich resource for the discovery of novel natural products. Curr. Opin. Biotechnol 15, 199-204.

Davis, F. B., Mousa, S. A., O'Connor, L., Mohamed, S., Lin, H. Y., Cao, H. J., and Davis, P. J. (2004). Proangiogenic action of thyroid hormone is fibroblast growth factor-dependent and is initiated at the cell surface. Circ Res 94, 1500-1506.

Dekleva, M. L., Titus, J. A., and Strohl, W. R. (1985). Nutrient effects on anthracycline production by Streptomyces peucetius in a defined medium. Can. J. Microbiol 31, 287-294.

Dell’Amico, E., Cavalca, L., and Andreoni, V. (2008). Improvement of Brassica napus growth under cadmium stress by cadmium-resistant rhizobacteria. Soil Biol Biochem 40, 74-84.

Delmont, T. O., Robe, P., Cecillon, S., Clark, I. M., Constancias, F., Simonet, P., Hirsch, P. R., and Vogel, T. M. (2011). Accessing the soil metagenome for studies of microbial diversity. Appl. Environ. Microbiol 77, 1315-1324.

Demain, A. L. (2000). Small bugs, big business: The economic power of microbe. Biol. Adv. Oxford 18, 499-514.

Dempsey, D. A., Silva, H., and Klessig, D. F. (1998). Engineering disease and pest resistance in plants. Trends Microbiol 6, 54-61.

Dharmatilake, A. J., and Bauer, W. D. (1992). Chemotaxis of rhizobium melilotitowards nodulation gene inducing compounds from alfalfa. Appl. Environ. Microbiol 58, 1153-1158

160

REFERENCES CHAPTER 6

Di Giovanni, G. D., Watrud, L. S., Seidler, R. J., and Widmer, F. (1999). Comparison of parental and transgenic alfalfa rhizosphere bacterial communities using Biolog GN metabolic fingerprinting and enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). Microbial Ecol 37, 129-139.

DiCello, F., Bevivino, A., Chiarini, L., Fani, R., Paffetti, D., Tabacchioni, S., and Dalmastri, C. (1997). Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages. Appl Environ Microbiol 63, 4485-4493.

Dilfuza, E. (2010). Colonization of tomato roots by some potentially human-pathogenic bacteria and their plant-beneficial properties. EurAsia J BioSci 4, 112-118.

Domingo, J. L. (2007). Toxicity studies of genetically modified plants: a review of the published literature. Crit Rev Food Sci Nutr 47, 721-33.

Donderski, W., and Trzebiatowska, M. (1999). Chitinase activity production by planktonic, benthic and epiphytic bacteria inhabiting the moty bay of the Jeziorak Lake (Poland). Pol. J. Environ. Stud 8(4) 215-220.

Donegan, K. K., Palm, C. J., Fieland, V. J., Porteous, L. A., Ganio, L. M., Schaller, D. L., Bucau, L. Q., and Seidler, R. J. (1995). Changes in levels, species and DNA fingerprints of soil microorganisms associated with cotton expressing the Bacillus thuringiensis var. kurstaki endotoxin. Appl. Soil Ecol 2, 111-124. .

Dowd, S. E., Callaway, T. R., Wallcott, R. D., Sun, Y., McKeehan, T., Hagevoort, R. G., and Edrington, T. S. (2008a). Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Microbiology 8, 125.

Dowd, S. E., Sun, Y., Wolcott, R. D., and Carroll, J. A. (2008b). Novel bacterial diagnostic and microbiome evaluation tool. Foodborne Pathogens and Disease. in press.

161

REFERENCES CHAPTER 6

Drogue, B., Dore, H., Borland, S., Wisniewski-Dye, F., and Prigent-Combaret, C. (2012). Which specificity in cooperation between phytostimulating rhizobacteria and plants? Res Microbiol 163, 500-510.

Dunfield, K. E., and Germida, J. J. (2003). Seasonal changes in the rhizosphere microbial communities associated with field-grown genetically modified canola (Brassica napus). Appl Environ Microbiol 69, 7310-7318.

Dunne, C., Moenne-Loccoz, Y., McCarthy, J., Higgins, P., Powell, J., Dowling, D. N., and O'Gara, F. (1998). Combining proteolytic and phloroglucinol-producing bacteria for improved biocontrol of Pythium-mediated damping-off of sugar beet. Plant Pathology 47, 299-307.

Eastman, K., and Sweet, J. (2002). Environmental Issue Report No. 28. Genetically Modified Organisms (GMOs): The significance of gene flow through pollen transfer. European Environment Agency.

El-Komy, H. M., Hamdia, M. A., and El-Baki, G. K. A. (2003). Nitrate reductase in wheat plants grown under water stress and inoculated with Azospirillum spp. Biol Plantarum 46, 281-287.

Ellis, R. J., Thompson, I. P. and Bailey, M. J. (1999). Temporal fluctuations in the pseudomonad population associated with sugar beet leaves. FEMS Microb. Ecol 28, 345-356.

Ellstrand, N. C. (2001). When transgenes wander, should we worry? . Plant Physiol 125, 1543-1545.

Ellstrand, N. C., Prentice, H. C., and Hancock, J. F. (1999). Gene flow and introgression from domesticated plants into their wild relatives. Annu Rev Ecol Syst 30, 539- 563.

El-Tarabily, K. A. (2006). Rhizosphere-competent isolates of streptomycete and non- streptomycete actinomycetes capable of producing cell-wall-degrading enzymes

162

REFERENCES CHAPTER 6

to control Pythium aphanidermatum damping-off disease of cucumber. Can J Bot 84, 211-222.

Elvira-Recuenco, M., and van Vuurde, J. W. L. (2000). Natural incidence of endophytic bacteria in pea cultivars under field conditions. Can J Microbiol 46, 1036-1041.

Ervin, D., Welsh, R., Batie, S. S., and Carpentier, C. L. (2003). Towards an ecological systems approach in public research for environmental regulation of transgenic crops. Agric. Ecosyst. Env. 99, 1–14.

Escher, N., Kach, B., and Nentwig, W. (2000). Decomposition of transgenic bacillus thuringensismaize by microorganisms and woodlice porcello scaber (Crustacea:Isopoda). Basic Appl. Entomol 1, 161-169.

Fang, M., Motavalli, P. P., Kremer, R. J., and Nelson, K. A. (2007). Assessing changes in soil microbial communities and carbon mineralization in Bt and non-Bt corn residue-amended soils. Appl. Soil Ecol 37, 150-160.

Faostat (2006). http://faostat.fao.org (accessed June 16, 2006).

Fernadez-Cornejo, J., and McBride, W. (2000). Genetically engineered crop for pest management in US agriculture: farm level effects. Resource Economics Division, Economic Research Service. USDA, Agricultural Report, No. 786.

Ferreira, L. H. P. L., Molina, J. C., Brasil, C., and Andrade, G. (2003). Evaluation of Bacillus thuringiensis bioinsecticidal protein effects on soil microorganisms. Plant Soil 256, 161-168.

Fierer, N., Bradford, M. A., and Jackson, R. B. (2007). Toward an ecological classification of soil bacteria. Ecology 88, 1354-1364.

163

REFERENCES CHAPTER 6

Filion, M., Hamelin, R. C., Bernier, L., and St-Arnaud, M. (2004). Molecular profiling of rhizosphere microbial communities associated with healthy and diseased black spruce (Picea mariana) seedlings grown in a nursery. Appl Environ Microbiol 70, 3541-3551.

Filion. M (2008). Do transgenic plants affect rhizobacteria populations? Microb. Biotechnol 1, 463-75.

Frankowski, J., Lorito, M., Scala, F., Schmid, R., Berg, G., and Bahl, H. (2001). Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48. Arch Microbiol 176, 421-426.

Garbeva, P., Van, VJA., Van, EJD., (2004). Microbial diversity in soil: selection of microbial populations by plant and soil type and applications for disease suppressiveness. Annu Rev Phytopathol 42, 243-270.

Garland, J. L. (1997). Analysis and interpretation of community-level physiological profiles in microbial ecology. FEMS Microbiol. Ecol 24, 289-300.

Germida, J. J., and Siciliano, S. D. (2001). Taxonomic diversity of bacteria associated with the roots of modern, recent and ancient wheat cultivars. Biol Fert Soils 33, 410-415.

Giovannetti, M., & Avio, L. (2002). Biotechnology of arbuscular mycorrhizas.Appl mycol biotechnol, 2, 275-310.

Girvan, M. S., Bullimore, J., Pretty, J. N., Osborn, A. M., and Ball, A. S. (2003). Soil type is the primary determinant of the composition of the total and active bacterial communities in arable soils. Appl Environ Microbiol 69, 1800-1809.

Glass, A. D. M. (1989). Plant Nutrition: An Introduction to Current Concepts. Jones and Bartlett Publishers, Boston, MA, USA.

164

REFERENCES CHAPTER 6

Glick, B. R. (2012). Plant growth-promoting bacteria: mechanisms and applications. Scientifca, 1-15.

Glick, B. R., Cheng, Z., Czarny, J., and Duan, J. (2007). Promotion of plant growth by ACC deaminase-producing soil bacteria. Eur J Plant Pathol 119, 329-339.

Glick, B. R., Patten, C. L., Holguin, G., and Penrose, D. M. (1999). Biochemical and genetic mechanisms used by plant growth promoting bacteria. London: Imperial College Press.

Gochnauer, M. B., Mccully, M. E., and Labbe, H. (1989). Different populations of bacteria associated with sheathed and bare regions of roots of field-grown maize. Plant Soil 114, 107-120.

Gomoryova, E., Vass, D., Pichler, V., and Gomory, D. (2009). Effect of alginite amendment on microbial activity and soil water content in forest soils. Biologia (Bratislava) 64, 585-588.

Gordon, S. A., and Weber, R. P. (1951). Colorimetric estimation of indoleacetic. Plant Physiol Biochem 26, 192–195.

Goss, J. (1968). Development, physiology and biochemistry of corn and wheat pollen. Bot. Rev 34 1597–1602.

Govaerts, B., Mezzalama, M., Unno, Y., Sayre, K. D., Luna-Guido, M., Vanherck, K., Dendooven, L., and Deckers, J. (2007). Influence of tillage, residue management, and crop rotation on soil microbial biomass and catabolic diversity. Appl Soil Ecol 37, 18-30.

Graner, G., Persson, P., Meijer, J., and Alstrom, S. (2003). A study on microbial diversity in different cultivars of Brassica napus in relation to its wilt pathogen, Verticillium longisporum. FEMS Microbiol Lett 29, 269–276.

165

REFERENCES CHAPTER 6

Grayston, S. J., Wang, S., Campbell, C. D., and Edwards, A. C. (1998). Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biol. Biochem 30, 369-378.

Gremion, F., Chatzinotas, A., and Harms, H. (2003). Comparative 16 S rDNA and 16 S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabol-ically active bacteria in heavy metal-contaminated bulk and rhizosphere soil. Environ Microbiol 5, 896-907.

Griffiths, B. S., Geoghegan, I. E., and Robertson, W. M. (2000). Testing genetically engineered potato, producing the lectins GNA and Con A, on non-target soil organisms and processes. J. Appl. Ecol 37, 159-170

Gu, Y.-H., and Mazzola, M. (2003 ). Modification of fluorescent pseudomonad community and control of apple replant disease induced in a wheat cultivar- specific manner. Appl. Soil Ecol 24, 57-72

Gupta, R., Gupta, N., and Rathi, P. (2004). Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotechnol 64, 763-781.

Hack H, G. H., Klemke T, Klose R, Meier U, Stauss R, Witzenberger A. (1993). Phanologische entwicklungsstadien der Kartoffel (Solanum tuberosum L.) Nachrbl Dtsch Pflanzenschutzd. Nachrbl Dtsch Pflanzenschutzd 45, 11-19.

Halfhill, M. D., Richards, H. A., and Mabon, S. A. (2001). Expression of GFP and Bt transgenes in Brassica napus and hybridization with Brassica rapa. Theor Appl Genet 103, 659-667.

Hammond, B., Dudek, R., Lemen, J., and Nemeth, M. (2004). Results of a 13 week safety assurance study with rats fed grain from glyphosate tolerant corn. Food Chem Toxicol 42, 1003-1014.

166

REFERENCES CHAPTER 6

Hartwig, U. A., Joseph, C. M., and Phillips, D. A. (1991). Flavonoids released naturally from alfalfa seeds enhance growth rate of Rhizobium meliloti. Plant Physiol 95, 797-803.

Hassan, E., Hossein, A. A., and Abolfazl A., A. (2009). Evaluation of plant growth hormones production (iaa) ability by iranian soils rhizobial strains and effects of superior strains application on wheat growth indexes. World Appl Sci J 6 (11), 1576-1584

Hawes, M. C. ( 1990). Living plant cells released from the root cap: a regulator of microbial populations in the rhizosphere. Plant Soil 129, 19-27.

Hayat, R., Ali, S., Amara, U., Khalid, R., and Ahmed, I. (2010). Soil beneficial bacteria and their role in plant growth promotion: a review. Ann Microbiol 60, 579-598.

Hendricks, C. W., Doyle, J. D., and Hugley, B. (1995). A New Solid Medium for Enumerating Cellulose Utilizing Bacteria in Soil. Appl. Environ. Microbiol 61, 2016-2019.

Hentschel, U., Schmid, M., Wagner, M., Fieseler, L., Gernert, C., and Hacker, J. (2001). Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the mediterranean sponges aplysina aerophoba and aplysina cavernicola. FEMS Microbiol Ecol 35, 305-312.

Hernández-León, R., Martínez-Trujillo, M., Valencia-Cantero, E., & Santoyo, G. (2012). Construction and characterization of a metagenomic DNA library from the rhizosphere of wheat (Triticum aestivum). Phyton (Buenos Aires), 81, 133-137.

Hertenberger, G., Zampach, P., and Bachmann, G. (2002). Plant species affect the concentration of free sugars and free amino acids in different types of soil. JPNSS 165, 557-565.

167

REFERENCES CHAPTER 6

Hilbeck, A., Baumgartner, M., Freid, P. M., and Bigler, F. (1998). Effects of Bacillus thuringiensis corn-fed prey on mortality and development time of immature Chrysoperla carnae. Environmental Entomology 27, 480-487.

Hooper, D. U., Chapin, F. S., Ewel, J. J., Hector, A., Inchausti, P., Lavorel, S., Lawton, J. H., Lodge, D. M., Loreau, M., Naeem, S., Schmid, B., Setala, H., Symstad, A. J., Vandermeer, J., and Wardle, D. A. (2005). Effects of biodiversity on ecosystem functioning: A consensus of current knowledge. Ecological Monographs 75, 3-35.

Huang, J. K., Rozelle, S., Pray, C., and Qf, W. (2002). Plant biotechnology in China. Science 295, 674-7.

Huang, X. W., Zhao, N. H., and Zhang, K. Q. (2004). Extracellular enzymes serving as virulence factors in nematophagous fungi involved in infection of the host. Res Microbiol 155, 811-816.

Hutchison, W. D., Burkness, E. C., Mitchell, P. D., Moon, R. D., Leslie, T. W., Fleischer, S. J., Abrahamson, M., Hamilton, K. L., Steffey, K. L., Gray, M. E., Hellmich, R. L., Kaster, L. V., Hunt, T. E., Wright, R. J., Pecinovsky, K., Rabaey, T. L., Flood, B. R., and Raun, E. S. (2010). Areawide suppression of european corn borer with bt maize reaps savings to non-bt maize growers. Science 330, 222-225.

Inceoglu, O., Abu Al-Soud, W., Salles, J. F., Semenov, A. V., and van Elsas, J. D. (2011). Comparative analysis of bacterial communities in a potato field as determined by pyrosequencing. PLoS One 6.

Innerebner, G., Knief, C., and Vorholt, J. A. (2011). Protection of Arabidopsis thaliana against leaf-pathogenic Pseudomonas syringae by Sphingomonas Strains in a controlled model system. Appl Environ Microbiol 77, 3202-3210.

Jaeger, K. E., and Eggert, T. (2002). Lipases for biotechnology. Curr Opin Biotech 13, 390-397.

168

REFERENCES CHAPTER 6

Jaeger, K. E., Ransac, S., Dijkstra, B. W., Colson, C., Vanheuvel, M., and Misset, O. (1994). Bacterial Lipases. FEMS Microbiol Rev 15, 29-63.

James, C. (2012). Global Status of Commercialized Biotech/GM Crops. ISAAA Brief ISAAA: Ithaca, NY. 44.

Kamil, Z., Rizk, M., Saleh, M., and Moustafa, S. (2007). Isolation and identification of rhizosphere soil chitinolytic bacteria and their potential in antifungal biocontrol. GJMS 2 (2), 57-66.

Kim, K. Y., Jordan, D., and McDonald, G. A. (1998). Enterobacter agglomerans, phosphate solubilizing bacteria, and microbial activity in soil: Effect of carbon sources. Soil Biol Biochem 30, 995-1003.

Kirk, J. L., Beaudette, L. A., Hart, M., Moutoglis, P., Klironomos, J. N., Lee, H., and Trevors, J. T. (2004). Methods of studying soil microbial diversity. J. Microbiol. Methods 58, 169-188

Kling, J. (1996). Could transgenic supercrops one day breed superweeds? Science 274, 180-181.

Kloepper, J. W., & Schroth, M. N. (1978, August). Plant growth-promoting rhizobacteria on radishes. In Proceedings of the 4th international conference on plant pathogenic bacteria (Vol. 2, pp. 879-882).

Koshella, J., and Stotzky, G. (2002). Larvicidal toxins from bacillus thuringensis subsp. Kurstaki, morrisoni (strain tenebrionis), and israelensis have no microbicidal or microblastic activity against selected bacteria, fungi and algae in vitro. Can. J. Microbiol 48, 262-267.

Kowalchuk, G. A., Buma, D. S., de Boer, W., Klinkhamer, P. G., and Van Veen, J. A. (2002). Effects of above-ground plant species composition and diversity on the diversity of soil-borne microorganisms. Antonie Van Leeuwenhoek, 81(1-4), 509- 520.

169

REFERENCES CHAPTER 6

Kowalchuk, G. A., Stienstra, A. W., Heilig, G. H. J., Stephen, J. R., and Woldendorp, J. W. (2000). Changes in the community structure of ammonia-oxidizing bacteria during secondary succession of calcareous grasslands. Env Microbio 2, 99-110.

Kumar, D. P., Anupama P. D., Rajesh K. S., R., Thenmozhi, A., Nagasathya, N., and Paneerselvam A., T., (2012). Evaluation of extracellular lytic enzymes from indigenous Bacillus isolates. J. Microbiol. Biotech. Res. 2 (1), 129-137.

Lam, H. M., Coschigano, K. T., Oliveira, I. C., MeloOliveira, R., and Coruzzi, G. M. (1996). The molecular-genetics of nitrogen assimilation into amino acids in higher plants. Annu Rev Plant Physiol Plant Mol Biol 47, 569-593.

Lambert, B., Leyns, F., Vanrooyen, L., Gossele, F., Papon, Y., and Swings, J. (1987). Rhizobacteria of maize and their antifungal activities. Appl Environ Microbiol 53, 1866-1871.

Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., and Higgins, D. G. (2007). Clustal W and clustal X version 2.0. Bioinformatics 23, 2947-2948.

Lavigne, C., Klein, E. K., and Couvet, D. (2002). Using seed purity data to estimate an average pollen mediated gene flow from crops to wild relatives. Theor Appl Genet 104, 139-145.

Lee, S. H., Ka, J. O., and Cho, J. C. (2008). Members of the phylum Acidobacteria are dominant and metabolically active in rhizosphere soil. FEMS Microbiol Lett 285, 263-269.

Lefol, E., Fleury, A., and Darmency, H. (1996). Gene dispersal from transgenic crops. II. Hybridization between oilseed rape and the wild hoary mustard. Sex Plant Reprod 9, 189-196.

170

REFERENCES CHAPTER 6

Leimanis, S., Hamels, S., Naze, F., Mbella, G. M., Sneyers, M., Hochegger, R., Broll, H., Roth, L., Dallmann, K., Micsinai, A., La Paz, J. L., Pla, M., Brunen-Nieweler, C., Papazova, N., Taverniers, I., Hess, N., Kirschneit, B., Bertheau, Y., Audeon, C., Laval, V., Busch, U., Pecoraro, S., Neumann, K., Rosel, S., van Dijk, J., Kok, E., Bellocchi, G., Foti, N., Mazzara, M., Moens, W., Remacle, J., and Van Den Eede, G. (2008). Validation of the performance of a GMO multiplex screening assay based on microarray detection. Eur Food Res Technol 227, 1621-1632.

Lian, L. H., Tian, B. Y., Xiong, R., Zhu, M. Z., Xu, J., and Zhang, K. Q. (2007). Proteases from Bacillus: a new insight into the mechanism of action for rhizobacterial suppression of nematode populations. Lett Appl Microbiol 45, 262- 269.

Liu, B., Zeng, Q., Yan, F., Xu, H., and Xu, C. (2004). Effects of transgenic plants on soil microorganisms. Plant Soil 271, 1-13.

Losey, J. E., Rayor, L. S., and Carter, M. E. (1999). Transgenic pollen harms monarch larvae. Nature 399, 214.

Lynch, J. M. (1990). Introduction: some consequences of microbial rhizosphere competence for plant and soil. The rhizosphere., 1-10.

Lynch, J. M., and Whipps, J. M. (1990). Substrate ﬂow in the rhizosphere. Plant Soil 129, 1-10.

Maloney, P. E., Van Bruggen, A. H. C., and Hu, S. (1997). Bacterial community structure in relation to the carbon environments in lettuce and tomato rhizosphere and in bulk soil. Microb. Ecol 34, 109-117.

Mantelin, S., and Touraine, B. (2004). Plant growth-promoting bacteria and nitrate availability impacts on root development and nitrate uptake. J. Exp. Bot 55, 27- 34.

171

REFERENCES CHAPTER 6

Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A., Berka, J., Braverman, M. S., Chen, Y. J., Chen, Z. T., Dewell, S. B., Du, L., Fierro, J. M., Gomes, X. V., Godwin, B. C., He, W., Helgesen, S., Ho, C. H., Irzyk, G. P., Jando, S. C., Alenquer, M. L. I., Jarvie, T. P., Jirage, K. B., Kim, J. B., Knight, J. R., Lanza, J. R., Leamon, J. H., Lefkowitz, S. M., Lei, M., Li, J., Lohman, K. L., Lu, H., Makhijani, V. B., McDade, K. E., McKenna, M. P., Myers, E. W., Nickerson, E., Nobile, J. R., Plant, R., Puc, B. P., Ronan, M. T., Roth, G. T., Sarkis, G. J., Simons, J. F., Simpson, J. W., Srinivasan, M., Tartaro, K. R., Tomasz, A., Vogt, K. A., Volkmer, G. A., Wang, S. H., Wang, Y., Weiner, M. P., Yu, P. G., Begley, R. F., and Rothberg, J. M. (2005). Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376-380.

Marilley, L., and Aragno, M. (1999). Phylogenetic diversity of bacterial communities differing in degree of proximity of Lolium perenne and Trifolium repens roots. Appl Soil Eco l13, 127-136.

Markewitz, D., and Richter, D. D. (1998). The bio in aluminum and silicon geochemistry. Biogeochemistry 42, 235-252.

Marschner, P., Yang, C. H., Lieberei, R., and Crowley, D. E. (2001). Soil and plant specific effects on bacterial community composition in the rhizosphere. Soil Biol Biochem 33, 1437-1445.

Mathesius, U., Schlaman, H. R. M., Spaink, H. P., Sautter, C., Rolfe, B. G., and Djordjevic, M. A. (1998). Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides. Plant J 14, 23-34.

172

REFERENCES CHAPTER 6

Mayak, S., Tirosh, T., and Glick, B. R. (2004). Plant growth-promoting bacteria confer resistance in tomato plants to salt stress. Plant Physiol Biochem 42, 565-572.

McCaig, A. E., Glover, L. A., and Prosser, J. I. (1999). Molecular analysis of bacterial community structure and diversity in unimproved and improved upland grass pastures. Appl Environ Microbiol 65, 1721-1730.

Medina, M. J. H., Gagnon, H., Piché, Y., Ocampo, J. A., García Garrido, J. M., and Vierheilig, H. (2003). Root colonization by arbuscular mycorrhizal fungi is affected by the salicylic acid content of the plant. Plant Sci 164, 993-998.

Mendes, R., Kruijt, M., de Bruijn, I., Dekkers, E., van der Voort, M., Schneider, J. H. M., Piceno, Y. M., DeSantis, T. Z., Andersen, G. L., Bakker, P. A. H. M., and Raaijmakers, J. M. (2011). Deciphering the rhizosphere microbiome for disease- suppressive bacteria. Science 332, 1097-1100.

Mendoza, C., Viteri, F. E., Lonnerdal, B., Young, K. A., Raboy, V., and Brown, K. H. (1998). Effect of genetically modified, low-phytic acid maize on absorption of iron from tortillas. Am. J. Clin. Nutr.68, 1123-1127.

Meyer, J. B., Lutz, M. P., Frapolli, M., Pechy-Tarr, M., Rochat, L., Keel, C., Defago, G., and Maurhofer, M. (2010). Interplay between wheat cultivars, biocontrol pseudomonads, and soil. Appl Environ Microbiol 76, 6196-6204.

Milling, A., Smalla, K., Maidl, F. X., Schloter, M., and Munch, J. C. (2004). Effects of transgenic potatoes with an altered starch composition on the diversity of soil and rhizosphere bacteria and fungi. Plant Soil 266, 23-39.

Mirza, M. S., Ahmad, W., Latif, F., Haurat, J., Bally, R., Normand, P., and Malik, K. A. (2001). Isolation, partial characterization, and the effect of plant growth- promoting bacteria (PGPB) on micro-propagated sugarcane in vitro. Plant Soil, 237(1), 47-54.

173

REFERENCES CHAPTER 6

Munees, A., and Mulugeta, K. (2013). Mechanisms and applications of plant growth promoting rhizobacteria: Current perspective. J King Saud Univ Sci In Press.

Munkvold, G. P., Hellmich, R. L., and Rice, L. G. (1999). Comparison of fumonisin concentrations in kernels of transgenic Bt maize hybrids and nontransgenic hybrids. Plant Dis 83, 130-138.

Nautiyal, C. S. (1999). An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Lett,170(1), 265-270.

Nijhuis, E. H., Maat, M. J., Zeegers, I. W. E., Waalwijk, C., and Van Veen, J. A. (1993). Selection of bacteria suitable for introduction into the rhizosphere of grass. Soil Biol Biochem 25, 885-895.

Nonnenmann, M. W., Bextine, B., Dowd, S. E., Gilmore, K., and Levin, J. L. (2010). Culture-independent characterization of bacteria and fungi in a poultry bioaerosol using pyrosequencing: A New Approach. J Occup Environ Hyg.7, 693-699.

Oger, P., Petit, A., and Dessaux, Y. (1997). Genetically engineered plants producing opines alter their biological environment. Nat. Biotechnol 15, 369-372.

Okubara, P. A., and Bonsall, R. F. (2008). Accumulation of Pseudomonas-derived 2,4- diacetylphloroglucinol on wheat seedling roots is influenced by host cultivar. Biol. Control 46, 322-331.

Ortiz, R. (1998). Critical role of plant biotechnology for the genetic improvement of food crops: perspectives for the next millennium. Electron J Biotechno, 1(3), 16-17.

Osullivan, D. J., and Ogara, F. (1992). Traits of fluorescent pseudomonas spp involved in suppression of plant-root pathogens. Microbiol Rev 56, 662-676.

Patten, C. L., and Glick, B. R. (2002). The role of bacterial indoleacetic acid in the development of the host plant system. Appl. Environ. Microbiol 68, 3795–3801.

174

REFERENCES CHAPTER 6

Pereira, P., Ez, F., Rosenblueth, M., Etcheverry, M., and Nez-Romero, E. (2011). Analysis of the bacterial diversity associated with the roots of maize (zea mays l.) Through culture-dependent and culture-independent methods. ISRN Ecology , 225-241.

Pereira, P., Nesci, A., and Etcheverry, M. (2009). Impact of two bacterial biocontrol agents on bacterial and fungal culturable groups associated with the roots of field- grown maize. Lett Appl Microbiol 48, 493-499.

Perez-Miranda, S., Cabirol, N., George-Tellez, R., Zamudio-Rivera, L. S., and Fernandez, F. J. (2007). O-CAS, a fast and universal method for siderophore detection. J Microbiol Methods 70, 127-131.

Pfender, W. F., and Wootke, S. L. (1988). Microbial communities of Pyrenophora- infested wheat straw as examined by multivariate analysis. Microb. Ecol 15, 95- 113.

Philip, J. D., Belinda, C., and Eliana, M. G. F. (2002). Potential for the environmental impact of transgenic crops. Nature. Biotechnol, 567-574.

Pilet, P. E., and Saugy, M. (1987). Effect on Root-Growth of Endogenous and Applied IAA and ABA a critical reexamination. Plant Physiol 83, 33-38.

Prakash, C. S. (2001). The genetically modified crop debate in the context of agricultural evolution. Plant Physiol126, 8-5.

Rahim, N., Abbas, M., Hoshang, N., Saber, S., and Akbar, O. (2013). Effect of plant growth promoting rhizobacteria (PGPR) on reduction nitrogen fertilizer application in rapeseed (Brassica napus L.). Middle East J Sci Res 14 (2), 213- 220.

Raju, E. V. N., and Divakar, G. (2013). Screening and isolation of pectinase producing bacteria from various regions in bangalore. IJRPBS 4 (1), 151-154.

175

REFERENCES CHAPTER 6

Ramachandran, K., Srinivasan, V., Hamza, S., and Anandaraj, M. (2007). Phosphate solubilizing bacteria isolated from the rhizosphere soil and its growth promotion on black pepper (Piper nigrum L.) cuttings. In "First International Meeting on Microbial Phosphate Solubilization" (E. Velázquez and C. Rodríguez-Barrueco, eds.), Vol. 102, pp. 325-331. Springer Netherlands.

Ramette, A., LiPuma, J. J., and Tiedje, J. M. (2005). Species abundance and diversity of Burkholderia cepacia complex in the environment. Appl Environ Microbiol 71, 1193-1201.

Rashid, S., Charles, T. C., and Glick, B. R. (2012). Isolation and characterization of new plant growth-promoting bacterial endophytes. Appl Soil Ecol 61, 217-224.

Redmond, J. W., Batley, M., Djordjevic, M. A., Innes, R. W., Kumpel, P. L., and Rolfe, B. G. (1986). Flavones induce expression of nodulation genes in Rhizobium. Nature 323 632-635.

Reichenbach, H. (2001). Myxobacteria, producers of novel bioactive substances. J. Ind. Microbiol. Biotechnol 27, 149-156.

Rengel, Z. (2002). Genetic control of root exudation. Plant Soil 245, 59-70.

Rengel, Z., Ross, G., and Hirsch, P. (1998). Plant genotype and micronutrients status influence colonization of wheat roots by soil bacteria. J. Plant Nutr 21, 99-113.

Renwick, A., Campbell, R., and Coe, S. (1991). Assesment of In vivo screening systems for potential biocontrol agents of Gaeumannomyces graminis. Plant Pathol J 40, 524-532.

Rifkin, J. (1998). The biotech century. Victor Gollanz, London, 272 pp.

Rijavec, T., Lapanje, A., Dermastia, M., and Rupnik, M. (2007). Isolation of bacterial endophytes from germinated maize kernels. Can J Microbiol 53, 802-808.

176

REFERENCES CHAPTER 6

Rodriguez, H., and Fraga, R. (1999). Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol Adv 17, 319-339.

Roesch, L. F., Fulthrope, R. R., Riva, A., Casella, G., Hadwin, A. K. M., Kent, A. D., Daroub, S. M., Camargo, F. A. O., Farmerie, W. G., and Triplett, E. W. (2007b). Pyrosequencing enumerates and contrasts soil microbial diversity. ISME J 1, 283– 290.

Rosangela, N. I. K., Luciano, T. K., Simone, C. P., José, C. B., Maria, T. O. L., Jackson, M., and Eliana, G. M. L. (2011). Phosphorus solubilizing and IAA production activities in plant growth promoting rhizobacteria from brazilian soils under sugarcane cultivation. J Eng Appl Sci 7 (11), 1446-1454.

Sadeghi, A., Karimi, E., Dahaji, P., Javid, M., Dalvand, Y., and Askari, H. (2012). Plant growth promoting activity of an auxin and siderophore producing isolate of Streptomyces under saline soil conditions. World J Microb Biot 28, 1503-1509.

Saha, N., Ulrich, A., Becker, R., and Wirth, S. (2007). Assessing the changes in bacterial diversity in rhizosphere and phyllosphere of transgenic and non-transgenic potato plant. PTCB 17, 87-95.

Saharan, B., and Nehra, V. (2011). Plant growth promoting rhizobacteria: a critical review. LSMR-21, 1-30.

Salles, J. F., van Elsas, J. D., and van Veen, J. A. (2006). Effect of agricultural management regime on Burkholderia community structure in soil. Microb Ecol 52, 267-279.

Santi, C., Bogusz, D., and Franche, C. (2013). Biological nitrogen fixation in non-legume plants. Ann Bot 111, 743-767.

Saxena, D., and Stoztky, G. (2001). Bacillus thuringiensis(bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earth-worms,

177

REFERENCES CHAPTER 6

nematodes, protozoa, bacteria, and fungi in soil. Soil Biol. Biochem 33, 1225- 1230.

Saxena, D., Flores, S., and Stotzky, G. (1999). Insecticidal toxin in root exudates from Bt corn. Nature 402-480.

Saxena, R. K., Ghosh, P. K., Gupta, R., Davidson, W. S., Bradoo, S., and Gulati, R. (1999). Microbial lipases: Potential biocatalysts for the future industry. Curr Sci 77, 101-115.

Schallmey, M., Singh, A., and Ward, O. P. (2004). Developments in the use of Bacillus species for industrial production. Can J Microbiol 50, 1-17.

Scheffler, J. A., Parkinson, R., and Dale, P. J. (1995). Evaluating the effectiveness of isolation distances for field plots of oilseed rape (brassica-napus) using a herbicide-resistance transgene as a selectable marker. Plant Breeding 114, 317- 321.

Schmalenberger, A., and Tebbe, C. C. (2002). Bacterial community composition in the rhizosphere of a transgenic, herbicide-resistant maize (Zea mays) and comparison to its non-transgenic cultivar Bosphore. FEMS Microbiol Ecol 40, 29-37.

Schwyn, B., and Neilands, J. B. (1987). Universal chemical-assay for the detection and determination of siderophores. Anal Biochem 160, 47-56.

Scott, S. E., and Wilkinson, M. J. (1999). Low probability of chloroplast movement from oilseed rape (Brassica napus) into wild Brassica rapa. Nat Biotechnol 17, 390- 392.

Semenov, A. M., Van Bruggen, A. H. C., and Zelenev, V. V. (1999 ). Moving waves of bacterial populations and total organic carbon along roots of wheat. Microbiol Ecol 37, 116-128.

178

REFERENCES CHAPTER 6

Siddiqui, I. A., Haas, D., and Heeb, S. (2005). Extracellular protease of Pseudomonas fluorescens CHA0, a biocontrol factor with activity against the root-knot nematode Meloidogyne incognita. Appl Environ Microbiol 71, 5646-5649.

Siddiqui, Z. A., and Mahmood, I. (1999). Role of bacteria in the management of plant parasitic nematodes: A review. Bioresour Technol 69, 167-179.

Singh, B. K., Munro, S., Potts, J. M., and Millard, P. (2007). Influence of grass species and soil type on rhizosphere microbial community structure in grassland soils. Appl Soil Ecol 36, 147-155.

Singh, P. P., Shin, Y. C., Park, C. S., and Chung, Y. R. (1999). Biological control of Fusarium wilt of cucumber by chitinolytic bacteria. Phytopathology 89, 92-99.

Smalla, K., Wieland, G., Buchner, A., Zock, A., Parzy, J., Kaiser, S., Roskot, N., Heuer, H., and Berg, G. (2001). Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Appl Env Microbiol 67, 4742-4751.

Smibert, R., and Krieg, N. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology. Edited by Gerhardt P, Murray RGE, Wood WA, Krieg NR. American Society of Microbiology. Washington DC, 607-654.

Smit, E., Leeflang, P., Gommans, S., van den Broek, J., van Mil, S., and Wernars, K. (2001). Diversity and seasonal fluctuations of the dominant members of the bacterial soil community in a wheat field as determined by cultivation and molecular methods. Appl Environ Microbiol 67, 2284-91.

Snow, A. (2002). Transgenic crops, why gene flow matters. Nat Biotechnol 20, 542.

179

REFERENCES CHAPTER 6

Sogin, M. L., Morrison, H. G., Huber, J. A., Mark Welch, D., Huse, S. M., and Neal, P. R. (2006). Microbial diversity in the deep sea and the underexplored ‘rare biosphere’. Proc Natl Acad Sci USA 103, 12115-12120.

Solomonson, L. P., and Barber, M. J. (1990). Assimilatory nitrate reductase - functional- properties and regulation. Annu Rev Plant Physiol Plant Mol Biol 41, 225-253.

Somers, E., Vanderleyden, J., and Srinivasan, M. (2004). Rhizosphere bacterial signalling: A love parade beneath our feet. Crit Rev Microbiol 30, 205-240.

Sorensen, J. (1997). The rhizosphere as a habitat for soil microorganisms, In J. D. van Elsas, J. T. Trevors, and E. M. H. Wellington (ed.), Modern soil microbiology. Marcel Dekker, Inc., New York, N.Y. 21-45

Spaepen, S., & Vanderleyden, J. (2011). Auxin and plant-microbe interactions. Cold Spring Harb Perspect Biol, 3(4).

Stafford, W. H. L., Baker, G. C., Brown, S. A., Burton, S. G., and Cowan, D. A. (2005). Bacterial diversity in the rhizosphere of Proteaceae species. Environ Microbiol 7, 1755-1768.

Stefan, M., Munteanu, N., Stoleru, V., and Mihasan, M. (2013). Effects of inoculation with plant growth promoting rhizobacteria on photosynthesis, antioxidant status and yield of runner bean. Rom. Biotechnol. Lett. 18(2), 8132-8143.

Strobel, G., and Daisy, B. (2003). Bioprospecting for microbial endophytes and their natural products. Microb. Mol. Biol. Rev 67, 491-502.

Sylvia, D. M., and Chellemi, D. O. (2001). Interactions among root-inhabiting fungi and their implications for biological control of root pathogens. Adv Agron 73 1-33.

180

REFERENCES CHAPTER 6

Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596-1599.

Tank, N., and Saraf, M. (2010). Salinity-resistant plant growth promoting rhizobacteria ameliorates sodium chloride stress on tomato plants. J. Plant Interact 5, 51-58.

Teixeira, L. C. R. S., Peixoto, R. S., Cury, J. C., Sul, W. J., Pellizari, V. H., Tiedje, J., and Rosado, A. S. (2010b). Bacterial diversity in rhizosphere soil from Antarctic vascular plants of Admiralty Bay, maritime Antarctica. The ISME Journal 4, 989- 1001.

Tesfaye, M., Dufault, N. S., Dornbusch, M. R., Allan, D. L., Vance, C. P., and Samac, D. A. (2003). Influence of enhanced malate dehydrogenase expression by alfalfa on diversity of rhizobacteria and soil nutrient availability. Soil Biol. Biochem 35, 1103-1113.

Teshima, R., Watanabe, T., Okunuki, H., Isuzugawa, K., Akiyama, H., Onodera, H., Imai, T., Toyoda, M., and Sawada, J. (2002). Effect of subchronic feeding of genetically modified corn (CBH351) on immune system in BN rats and B10A mice. J Food Hyg Soc Jpn 43, 273-279.

Thevissen, K., Warnecke, D. C., Francois, I. E. J. A., Leipelt, M., Heinz, E., Ott, C., Zahringer, U., Thomma, B. P. H. J., Ferket, K. K. A., and Cammue, B. P. A. (2004). Defensins from insect and plants interact with fungal glucosylceramides. J. Biol. Chem 279, 3900-3905.

Tian, F., Ding, Y. Q., Zhu, H., Yao, L. T., and Du, B. H. (2009). Genetic diversity of siderophore-producing bacteria of tobacco rhizosphere. Braz J Microbiol 40, 276- 284.

Torsvik, V., and Ovreas, L. (2006). Microbial phylogeny and diversity in soil. In: J. D. van-Elsas J. K. Jansson and J. T Trevors. (Eds.), Modern Soil Microbiology. CRC Press, Boca Raton, FL, USA 23-54.

181

REFERENCES CHAPTER 6

Torsvik, V., Ovreas, L., & Thingstad, T. F. (2002). Prokaryotic diversity--magnitude, dynamics, and controlling factors. Sci. Signal. 296, 1060-1064.

Turrini, A., Sbrana, C., Nuti, M. P., Pietrangeli, B., and Giovannetti, M. (2004). Development of a model system to assess the impact of genetically modified corn and eggplant on arbuscular mycorrhizal fungi. Plant Soil 266, 69-75.

Uren, N. C. (2001). "Types, amounts, and possible functions of compounds released into the rhizosphere by soil-grown plants," The Rhizosphere/Ed. Marcel Dekker, New York.

Uroz, S., Buee, M., Murat, C., Frey-Klett, P., and Martin, F. (2010). Pyrosequencing reveals a contrasted bacterial diversity between oak rhizosphere and surrounding soil. EnvirMicrobiol Rep 2, 281-288.

Van Loon, L., and Glick, B. (2004). Increased plant fitness by rhizobacteria. In: Sandermann H (ed) Molecular ecotoxicology of plants,. Springer, Berlin 170, 177-205.

Velazquez-Sepulveda, I., Orozco-Mosqueda, M., Prieto-Barajas, C., and Santoyo, G. (2012). Bacterial diversity associated with the rhizosphere of wheat plants (Triticum aestivum): Toward a metagenomic analysis. J Exp Bot . 81, 81-87.

Vessey, J. K. (2003a). Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 255, 571-586.

Watt, M., Hugenholtz, P., White, R., and Vinall, K. (2006). Numbers and locations of native bacteria on field-grown wheat roots quantified by fluorescence in situ hybridization (FISH). Environ Microbiol 8, 871-884.

Watt, M., McCully, M. E., and Kirkegaard, J. A. (2003). Soil strength and rate of root elongation alter the accumulation of Pseudomonas spp. and other bacteria in the rhizosphere of wheat. Funct Plant Bio 30, 483-491.

182

REFERENCES CHAPTER 6

Weinert, N., Piceno, Y., Ding, G. C., Meincke, R., Heuer, H., Berg, G., Schloter, M., Andersen, G., and Smalla, K. (2011). PhyloChip hybridization uncovered an enormous bacterial diversity in the rhizosphere of different potato cultivars: many common and few cultivar-dependent taxa. FEMS Microbiol Ecol 75, 497-506.

Westgate, M., Lisazo, J., and Batchelor, W. (2003). Quantitative relationship between pollen-shed density and grain yield in maize. Crop Sci 43 934-942.

Whipps, J. M. (2001b). Microbial interactions and biocontrol in the rhizosphere. J Exp Bot 52, 487-511.

Whitman, W. B., Coleman, D. C., and Weibe, W. J. (1998). Prokaryotes: the unseen majority. Proc. Natl. Acad. Sci. USA 95, 6578-6583.

Wu, W. X., Ye, Q. F., Min, H., Duan , X. J., and Jin, W. M. (2004). Bt transgenic rice straw affects the culturable microbiota and dehydrogenase and phosphatase activities in a flooded paddy soil. Soil Biol. Biochem 36, 289-295.

Yang, C. H., and Crowley, D. E. (2000). Rhizosphere microbial community structure in relation to root location and plant iron nutritional status. Appl. Environ. Microbiol 66, 345-351.

Zhang, Q., Zhang, Z. Y., Lin, S. Z., Lin, Y. Z., & Yang, L. (2005). Assessment of rhizospheric microorganisms of transgenic Populus tomentosa with cowpea trypsin inhibitor (CpTI) gene. J Forest, 7(3), 28-34.

Zheng, M., Shi, J. Y., Shi, J., Wang, Q. G., and Li, Y. H. (2013). Antimicrobial effects of volatiles produced by two antagonistic Bacillus strains on the anthracnose pathogen in postharvest mangos. Biol Control 65, 200-206.

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Appendix 1 Pyrosequencing based bacterial diversity of GM and Non GM maize rhizosphere Appendix 1-A. Pyrosequencing-based assessment of bacterial community ,phylum level distribution of 16S rRNA sequences from GM and Non GM maize rhizosphere at different developmental stages .

A prime AA' Stage B Stage C*** Phylum A% % *Ave% GMB% IGB% IWB% **Ave% GMC% IGC% IWC% Ave% Fibrobacteres 0.086 0.025 0.056 0.072 0.045 0.014 0.044 0.007 0 0.007 0.005 Thermi 0 0.005 0.003 0.014 0.006 0.004 0.008 0.007 0.032 0.007 0.015 Actinobacteria 9.639 17.719 13.679 15.769 21.266 21.222 19.419 30.654 29.777 49.577 36.669 Crenarchaeota 1.891 7.293 4.592 2.57 2.528 4.698 3.265 3.408 5.753 4.163 4.441 Planctomycetes 3.935 4.115 4.025 4.318 4.739 5.35 4.802 4.921 4.813 3.634 4.456 Euryarchaeota 0.059 0.505 0.282 0.23 0.393 0.19 0.271 0.304 0.155 0.111 0.190 Tenericutes 0.023 0.044 0.034 0.009 0.048 0.025 0.027 0.003 0.064 0.021 0.029 Proteobacteria 46.891 43.938 45.415 44.087 43.204 40.434 42.575 35.96 34.82 26.308 32.363 Synergistetes 0.081 0.049 0.065 0.09 0.073 0.07 0.078 0.095 0.059 0.093 0.082 Nitrospirae 0.081 0.206 0.144 0.036 0.059 0.074 0.056 0.145 0.208 0.086 0.146 Firmicutes 6.724 10.402 8.563 6.875 6.433 8.163 7.157 8.629 7.375 5.655 7.220 Bacteroidetes 20.826 8.421 14.624 16.862 13.305 11.466 13.878 8.342 10.593 6.108 8.348 Cyanobacteria 0.19 0.647 0.419 0.163 0.298 0.3 0.254 0.608 0.731 0.571 0.637 Dictyoglomi 0.05 0.069 0.060 0.036 0.07 0.06 0.055 0.037 0.048 0.032 0.039 Acidobacteria 1.164 0.515 0.840 0.998 0.525 0.458 0.660 0.48 0.608 0.196 0.428 Deferribacteres 0 0.015 0.008 0 0.008 0 0.003 0 0.005 0.007 0.004 Aquificae 0.018 0.034 0.026 0.009 0.022 0.032 0.021 0.007 0.005 0.004 0.005 Spirochaetes 0.023 0.034 0.029 0.018 0.039 0.032 0.030 0.024 0.037 0.014 0.025 Chloroflexi 0.884 0.956 0.920 0.709 0.885 1.47 1.021 1.321 0.763 0.343 0.809 Thermotogae 0.081 0.064 0.073 0.054 0.045 0.06 0.053 0.047 0.075 0.061 0.061 Verrucomicrobia 3.745 1.56 2.653 3.803 3.267 2.837 3.302 2.037 1.996 1.528 1.854 Others 3.606 3.384 3.495 3.275 2.731 3.042 3.016 2.965 2.081 1.474 2.173 185

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Appendix 1-B. Pyrosequencing-based assessment of bacterial community , family level distribution of 16S rRNA sequences from GM and Non GM maize rhizosphere at different developmental stages .

A AA" Stage B Stage C*** Family A% prime% *Ave% GMB% IGB% IWB% **Ave% GMC% IGC% IWC% Ave% Rhizobiaceae 0.912 0.721 0.8165 0.953 0.919 0.871 0.914333 1.415 1.761 1.514 Alteromonadaceae 0.05 0.128 0.089 0.041 0.051 0.07 0.054 0.111 0.064 0.054 0.076 Fibrobacteraceae 0.086 0.025 0.056 0.072 0.045 0.014 0.044 0.007 0 0.007 0.005 Aurantimonadaceae 0 0 0.000 0 0.003 0.004 0.002 0.01 0 0.011 0.007 Pelobacteraceae 0.366 0.284 0.325 0.267 0.292 0.194 0.251 0.169 0.091 0.096 0.119 Nocardioidaceae 0.803 1.888 1.3455 1.798 3.891 2.154 2.614333 6.424 5.961 6.997 6.460667 Burkholderiaceae 0.532 0.27 0.401 0.497 0.438 0.363 0.433 0.375 0.208 0.228 0.270 Rhodocyclaceae 1.137 0.687 0.912 1.084 0.958 0.705 0.916 0.696 0.459 0.428 0.528 Neisseriaceae 0 0.015 0.008 0.005 0 0.004 0.003 0.007 0 0.004 0.004 Bartonellaceae 0.014 0.005 0.010 0.014 0 0.004 0.006 0.027 0.005 0 0.011 Spirulinaceae 0.018 0.015 0.017 0.018 0.045 0.109 0.057 0.027 0.037 0.018 0.027 Paenibacillaceae 0.135 0.24 0.188 0.253 0.169 0.278 0.233 0.226 0.31 0.261 0.266 Hyphomicrobiaceae 4.468 4.723 4.596 5.104 5.95 6.26 5.771 6.059 5.673 4.216 5.316 Xanthobacteraceae 0.009 0.025 0.017 0.018 0.017 0.011 0.015 0.024 0.021 0.014 0.020 Actinosynnemataceae 0.068 0.407 0.238 0.149 0.112 0.289 0.183 0.338 0.326 0.196 0.287 Myxococcaceae 0.311 0.814 0.563 0.312 0.402 0.395 0.370 0.372 0.326 0.189 0.296 Rhodobacteraceae 0.307 0.51 0.409 0.47 0.413 0.419 0.434 0.419 0.491 0.353 0.421 Shewanellaceae 0.005 0 0.003 0.018 0.008 0.004 0.010 0.024 0 0 0.008 Thermodesulfobiaceae 0.005 0.005 0.005 0 0 0.007 0.002 0.003 0 0.004 0.002 Caulobacteraceae 0.059 0.078 0.069 0.163 0.104 0.063 0.110 0.138 0.133 0.061 0.111 Peptococcaceae 0.46 0.167 0.314 0.316 0.211 0.187 0.238 0.165 0.16 0.121 0.149 Ferroplasmaceae 0.005 0.025 0.015 0.005 0.011 0 0.005 0.007 0.005 0 0.004 Pseudanabaenaceae 0.068 0.083 0.076 0.05 0.107 0.085 0.081 0.294 0.294 0.107 0.232 186

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Dermabacteraceae 0.014 0.029 0.022 0.009 0.025 0.021 0.018 0.02 0.043 0.007 0.023 Bradyrhizobiaceae 1.047 0.721 0.884 0.772 1.363 1.59 1.242 1.415 1.82 1.339 1.525 Promicromonosporaceae 0.005 0.069 0.037 0.172 0.042 0.021 0.078333 2.158 1.553 1.664 1.791667 Sulfobacillaceae 0.32 0.741 0.531 0.262 0.36 0.282 0.301 0.375 0.203 0.125 0.234 Propionibacteriaceae 0.027 0.005 0.016 0.014 0.017 0 0.010 0.007 0.016 0.004 0.009 Geodermatophilaceae 0.302 0.79 0.546 0.425 0.405 0.606 0.479 0.415 1.206 1.014 0.878 Aerococcaceae 0.005 0.01 0.008 0.005 0.006 0.004 0.005 0.014 0 0.011 0.008 Thermoactinomycetaceae 0.329 0.255 0.292 0.244 0.16 0.243 0.216 0.149 0.133 0.107 0.130 Glycomycetaceae 0 0.005 0.003 0.014 0.011 0.004 0.010 0.03 0.027 0.014 0.024 Lachnospiraceae 0.104 0.221 0.163 0.104 0.081 0.113 0.099 0.118 0.048 0.057 0.074 Desulfobacteraceae 1.403 0.947 1.175 1.586 1.323 0.994 1.301 0.665 0.635 0.45 0.583333 Aquificaceae 0.018 0.015 0.017 0.005 0.017 0.032 0.018 0.003 0 0.004 0.002 Syntrophomonadaceae 0.027 0.108 0.068 0.018 0.022 0.035 0.025 0.044 0.011 0.021 0.025 Veillonellaceae 0.009 0.01 0.010 0.014 0.006 0.007 0.009 0.014 0 0 0.005 Rikenellaceae 0.027 0.034 0.031 0.005 0 0.011 0.005 0 0.016 0.004 0.007 Prevotellaceae 0 0 0.000 0.005 0 0.011 0.005 0 0.011 0 0.004 Chamaesiphonaceae 0 0.034 0.017 0.005 0 0 0.002 0.014 0.101 0.046 0.054 Phormidiaceae 0.041 0.392 0.217 0.036 0.034 0.039 0.036 0.054 0.069 0.307 0.143 Coriobacteriaceae 0.018 0.02 0.019 0.027 0.003 0.007 0.012 0 0.005 0.007 0.004 Clostridiaceae 0.347 2.084 1.216 0.384 0.596 0.532 0.504 1.155 0.726 0.407 0.763 Solibacteraceae 0.361 0.338 0.350 0.267 0.199 0.233 0.233 0.365 0.395 0.107 0.289 Cryptosporangiaceae 0.005 0.01 0.008 0.009 0.014 0.018 0.014 0.014 0.053 0.018 0.028 Moraxellaceae 2.049 1.481 1.765 1.879 1.739 1.505 1.708 0.787 0.763 0.639 0.730 Porphyromonadaceae 0.447 0.074 0.261 0.063 0.062 0.116 0.080 0.017 0.011 0.007 0.012 Halomonadaceae 0.208 0.083 0.146 0.122 0.11 0.137 0.123 0.108 0.096 0.082 0.095 Chloroflexaceae 0.181 0.231 0.206 0.19 0.376 0.356 0.307 0.22 0.197 0.118 0.178 Crenotrichaceae 0.023 0.02 0.022 0.009 0.006 0.028 0.014 0.02 0.005 0 0.008 187

APPENDICES

Cyclobacteriaceae 0.063 0.059 0.061 0.018 0.056 0.204 0.093 0.071 0.091 0.025 0.062 Erysipelotrichaceae 0.018 0.044 0.031 0 0.031 0.004 0.012 0.003 0.032 0.014 0.016 Flavobacteriaceae 1.241 0.348 0.795 0.781 0.517 0.814 0.704 0.138 0.133 0.068 0.113 Nocardiopsaceae 0 0.015 0.008 0.018 0.006 0.028 0.017 0.017 0.171 0.021 0.070 Deferribacteraceae 0 0.015 0.008 0 0.008 0 0.003 0 0.005 0.007 0.004 Hydrogenothermaceae 0 0.02 0.010 0.005 0.006 0 0.004 0.003 0.005 0 0.003 Magnetococcaceae 0.099 0.088 0.094 0.081 0.022 0.085 0.063 0.02 0.011 0.007 0.013 Dietziaceae 0.063 0 0.032 0.014 0.02 0.011 0.015 0 0 0 0.000 Anaerobaculaceae 0.014 0 0.007 0.018 0.003 0.004 0.008 0 0 0 0.000 Micromonosporaceae 1.403 2.531 1.967 1.676 1.57 1.769 1.672 2.172 1.542 2.731 2.148333 Eubacteriaceae 0.014 0.015 0.015 0.014 0.006 0.004 0.008 0.003 0 0 0.001 Halothiobacillaceae 0.122 0.034 0.078 0.099 0.104 0.074 0.092 0.037 0.064 0.054 0.052 Oscillochloridaceae 0.09 0.069 0.080 0.108 0.124 0.19 0.141 0.098 0.075 0.086 0.086 Kineosporiaceae 0.027 0.044 0.036 0.041 0.006 0.021 0.023 0.003 0.059 0.025 0.029 Syntrophobacteraceae 0.108 0.284 0.196 0.108 0.155 0.109 0.124 0.334 0.085 0.054 0.158 Koribacteraceae 0.803 0.177 0.490 0.732 0.326 0.226 0.428 0.111 0.208 0.089 0.136 Methylophilaceae 0.104 0.029 0.067 0.23 1.815 0.226 0.757 0.037 0.139 0.011 0.062 Flammeovirgaceae 0.199 0.074 0.137 0.185 0.197 0.116 0.166 0.155 0.133 0.096 0.128 Deinococcaceae 0 0.005 0.003 0.014 0.006 0.004 0.008 0.003 0.032 0.007 0.014 Oceanospirillaceae 0.032 0.078 0.055 0.032 0.034 0.032 0.033 0.027 0.053 0.025 0.035 Methanomicrobiaceae 0.027 0.211 0.119 0.041 0.18 0.035 0.085 0.057 0.016 0.014 0.029 Sinobacteraceae 0.979 0.441 0.710 0.763 1.183 0.571 0.839 0.405 0.294 0.121 0.273 Piscirickettsiaceae 1.616 0.608 1.112 0.935 1.337 0.892 1.055 0.74 0.374 0.168 0.427 Campylobacteraceae 0.05 0.034 0.042 0.059 0.039 0.039 0.046 0.03 0.027 0.018 0.025 Alcaligenaceae 0.113 0.078 0.096 0.117 0.135 0.099 0.117 0.095 0.08 0.054 0.076 Actinomycetaceae 0.352 0.152 0.252 0.117 0.096 0.074 0.096 0.02 0.075 0.032 0.042 Sporolactobacillaceae 0.005 0.02 0.013 0.023 0.02 0.021 0.021 0.01 0.011 0.004 0.008 188

APPENDICES

Thermotogaceae 0.081 0.064 0.073 0.054 0.045 0.06 0.053 0.047 0.075 0.061 0.061 Acidimicrobiaceae 0.862 1.918 1.390 1.473 1.75 1.724 1.649 2.111 1.644 1.26 1.671667 Enterobacteriaceae 0.736 0.554 0.645 0.772 0.449 0.684 0.635 0.338 0.331 0.228 0.299 Legionellaceae 0.014 0.005 0.010 0.018 0.014 0.011 0.014 0.017 0 0.007 0.008 Cellulomonadaceae 0 0.054 0.027 0.045 0.025 0.049 0.040 0.03 0.075 0.054 0.053 Hyphomonadaceae 0.027 0.074 0.051 0 0.073 0.004 0.026 0.03 0.005 0 0.012 Saprospiraceae 2.437 0.353 1.395 1.875 1.039 0.73 1.215 0.165 0.235 0.1 0.167 Turicibacteraceae 0.027 0.201 0.114 0.072 0.045 0.081 0.066 0.088 0.075 0.039 0.067 Sphingobacteriaceae 0.826 0.113 0.470 0.533 0.517 0.324 0.458 0.172 0.288 0.921 0.460 Rhodobiaceae 0.176 0.137 0.157 0.185 0.202 0.493 0.293 0.22 0.165 0.161 0.182 Brucellaceae 0.09 0.049 0.070 0.068 0.025 0.053 0.049 0.007 0.037 0.014 0.019 Intrasporangiaceae 0.099 0.26 0.180 0.262 0.402 0.293 0.319 0.426 0.395 0.696 0.506 Methanobacteriaceae 0.005 0.02 0.013 0.009 0.011 0.014 0.011 0.007 0 0.004 0.004 Beijerinckiaceae 0.009 0.029 0.019 0.005 0.028 0.014 0.016 0.034 0.08 0.054 0.056 Staphylococcaceae 0.28 0.083 0.182 0.262 0.146 0.243 0.217 0.118 0.112 0.064 0.098 Alcanivoracaceae 0.014 0.005 0.010 0.027 0.07 0.014 0.037 0.014 0.005 0.004 0.008 Dehalobacteriaceae 0 0 0.000 0.005 0 0.004 0.003 0.007 0.005 0.004 0.005 Desulfuromonadaceae 0.018 0.02 0.019 0 0.008 0.004 0.004 0.003 0 0.004 0.002 Thermomonosporaceae 0.171 0.603 0.387 0.203 0.23 0.476 0.303 0.429 0.352 0.778 0.520 Acidithiobacillaceae 0.153 0.201 0.177 0.095 0.157 0.247 0.166 0.236 0.256 0.171 0.221 Micrococcaceae 0.257 0.628 0.443 0.551 0.559 0.832 0.647 2.003 2.236 18.161 7.467 Synergistaceae 0.068 0.049 0.059 0.072 0.07 0.067 0.070 0.095 0.059 0.093 0.082 Bacteroidaceae 0.063 0.02 0.042 0.018 0.025 0.004 0.016 0.017 0.016 0.007 0.013 Corynebacteriaceae 0.014 0.034 0.024 0.023 0.017 0.035 0.025 0.007 0.016 0 0.008 Pseudomonadaceae 0.894 0.907 0.901 0.723 1.096 0.652 0.824 0.608 1.617 1.207 1.144 Dictyoglomaceae 0.05 0.069 0.060 0.036 0.07 0.06 0.055 0.037 0.048 0.032 0.039 Spartobacteriaceae 1.137 0.383 0.760 1.08 1.208 1.043 1.110 0.844 0.544 0.536 0.641 189

APPENDICES

Microthrixaceae 0.077 0.093 0.085 0.081 0.135 0.095 0.104 0.088 0.037 0.068 0.064 Nostocaceae 0.063 0.123 0.093 0.045 0.11 0.067 0.074 0.22 0.219 0.089 0.176 Psychromonadaceae 0.063 0.059 0.061 0.181 0.003 0.042 0.075 0.027 0 0 0.009 Nitrospiraceae 0.077 0.206 0.142 0.036 0.051 0.074 0.054 0.142 0.192 0.086 0.140 Xanthomonadaceae 1.097 0.642 0.870 1.346 0.792 0.775 0.971 0.875 1.382 0.753 1.003 Isosphaeraceae 0.036 0.02 0.028 0.032 0.022 0.035 0.030 0.034 0.053 0.036 0.041 Pseudonocardiaceae 0.203 0.505 0.354 0.316 0.464 0.532 0.437 0.719 0.72 0.375 0.605 Exiguobacteraceae 0 0.015 0.008 0.005 0.006 0.028 0.013 0.02 0.005 0.014 0.013 Caldicellulosiruptoraceae 0.068 0.059 0.064 0.149 0.07 0.102 0.107 0.041 0.059 0.032 0.044 Polyangiaceae 1.71 2.447 2.079 1.418 1.152 1.604 1.391 1.385 1.211 0.639 1.078333 Planctomycetaceae 0.713 1.03 0.872 0.772 0.885 0.916 0.858 0.986 0.784 0.682 0.817 Methylocystaceae 0.487 0.275 0.381 0.425 0.155 0.352 0.311 0.091 0.043 0.079 0.071 Bogoriellaceae 0 0.01 0.005 0 0.003 0 0.001 0 0.037 0.032 0.023 Bacillaceae 1.918 3.149 2.534 2.182 1.871 3.098 2.384 2.817 2.62 2.192 2.543 Frankiaceae 0.041 0.029 0.035 0.018 0.037 0.025 0.027 0.081 0.048 0.064 0.064 Acholeplasmataceae 0.005 0 0.003 0.009 0.017 0.021 0.016 0 0.032 0.007 0.013 Opitutaceae 1.39 0.78 1.085 2.073 0.997 0.98 1.350 0.645 0.923 0.718 0.762 Francisellaceae 0.081 0.054 0.068 0.099 0.067 0.07 0.079 0.068 0.096 0.071 0.078 Gordoniaceae 0.014 0.01 0.012 0.068 0.014 0.039 0.040 0.003 0.011 0 0.005 Planococcaceae 1.151 1.388 1.270 1.314 1.407 1.875 1.532 1.635 1.772 1.417 1.608 Pseudoalteromonadaceae 0.014 0.01 0.012 0 0.008 0.007 0.005 0.003 0 0.004 0.002 Microbacteriaceae 0.181 0.387 0.284 0.529 0.331 0.451 0.437 0.351 1.147 1.582 1.026667 Acetobacteraceae 0.578 0.231 0.405 0.212 0.152 0.134 0.166 0.22 0.411 0.286 0.306 Spirochaetaceae 0.023 0.029 0.026 0.014 0.039 0.028 0.027 0.024 0.037 0.014 0.025 Geobacteraceae 0.068 0.128 0.098 0.072 0.166 0.056 0.098 0.108 0.091 0.014 0.071 Ectothiorhodospiraceae 5.785 6.258 6.0215 4.102 3.79 3.433 3.775 1.891 2.183 1.892 1.988667 Ruminococcaceae 0.781 0.525 0.653 0.524 0.511 0.222 0.419 0.456 0.267 0.143 0.289 190

APPENDICES

Phyllobacteriaceae 0.153 0.221 0.187 0.298 0.317 0.275 0.297 0.334 0.347 0.307 0.329 Sphingomonadaceae 2.044 1.329 1.687 4.21 1.832 1.812 2.618 2.192 1.622 1.614 1.809333 Nautiliaceae 0.005 0 0.003 0 0.008 0 0.003 0.003 0.016 0 0.006 Thermobaculaceae 0.158 0.52 0.339 0.104 0.132 0.317 0.184 0.388 0.112 0.093 0.198 Chromatiaceae 1.34 0.77 1.055 1.021 1.197 0.895 1.038 0.703 0.374 0.286 0.454 Mycobacteriaceae 0.528 1.045 0.787 1.238 1.079 1.378 1.232 1.986 1.339 0.757 1.360667 Enterococcaceae 0.23 0.201 0.216 0.239 0.225 0.215 0.226 0.132 0.139 0.132 0.134 Desulfomicrobiaceae 0.149 0.078 0.114 0.154 0.104 0.085 0.114 0.044 0.053 0.032 0.043 Methylococcaceae 0.596 1.157 0.877 0.705 0.857 0.761 0.774 0.841 0.704 0.618 0.721 Cystobacteraceae 0.361 0.996 0.679 0.547 0.539 0.719 0.602 0.679 0.47 0.375 0.508 Syntrophaceae 0.081 0.132 0.107 0.095 0.169 0.095 0.120 0.078 0.107 0.071 0.085 Carnobacteriaceae 0.072 0.039 0.056 0.054 0.031 0.074 0.053 0.074 0.064 0.054 0.064 Thiotrichaceae 1.832 1.422 1.627 1.391 1.674 1.431 1.499 0.959 0.662 0.743 0.788 Methylobacteriaceae 0.596 1.334 0.965 0.669 0.671 0.938 0.759333 0.963 1.334 1.01 1.102333 Bdellovibrionaceae 0.027 0.029 0.028 0.027 0.006 0 0.011 0.014 0.016 0.011 0.014 Roseiflexaceae 0.095 0.113 0.104 0.163 0.138 0.254 0.185 0.125 0.128 0.046 0.100 Nitrosomonadaceae 3.493 2.276 2.885 2.259 1.961 2.323 2.181 1.712 1.564 1.039 1.438333 Symbiobacteriaceae 0.149 0.132 0.141 0.063 0.053 0.081 0.066 0.081 0.069 0.064 0.071 Flexibacteraceae 8.222 4.34 6.281 6.694 5.203 4.726 5.541 4.644 5.886 3.035 4.521667 Gemmataceae 0.144 0.275 0.210 0.145 0.211 0.3 0.219 0.23 0.368 0.186 0.261 Family Unspecified 9.061 5.15 7.106 8.822 8.161 6.732 7.905 5.076 5.651 3.317 4.681 Heliobacteriaceae 0.045 0.02 0.033 0.05 0.022 0.025 0.032 0.037 0.043 0.007 0.029 Streptosporangiaceae 0.077 0.128 0.103 0.081 0.149 0.159 0.130 0.159 0.112 0.211 0.161 Methanosarcinaceae 0.023 0.25 0.137 0.176 0.191 0.137 0.168 0.23 0.133 0.093 0.152 Herpetosiphonaceae 0.361 0.025 0.193 0.145 0.112 0.352 0.203 0.49 0.251 0 0.247 Halanaerobiaceae 0.009 0.029 0.019 0 0 0.018 0.006 0.003 0 0.011 0.005 Saccharospirillaceae 0.009 0.005 0.007 0 0.008 0.004 0.004 0.003 0 0 0.001 191

APPENDICES

Thermoanaerobacteraceae 0.081 0.343 0.212 0.09 0.183 0.145 0.139 0.54 0.187 0.15 0.292 Rhodothermaceae 0.009 0.039 0.024 0.054 0.017 0.063 0.045 0.02 0.027 0.011 0.019 Listeriaceae 0.086 0.137 0.112 0.14 0.14 0.155 0.145 0.125 0.24 0.129 0.165 Aeromonadaceae 0.018 0.01 0.014 0.018 0.028 0.028 0.025 0.101 0.016 0.036 0.051 Oxalobacteraceae 0.153 0.387 0.270 0.23 0.23 0.152 0.204 0.135 0.187 0.278 0.200 Nannocystaceae 0.46 0.397 0.429 0.235 0.118 0.211 0.188 0.128 0.139 0.114 0.127 Pirellulaceae 1.995 1.653 1.824 1.811 2.101 2.383 2.098333 2.27 2.289 1.603 2.054 Cardiobacteriaceae 0.018 0.02 0.019 0 0 0 0.000 0.027 0 0.007 0.011 Cenarchaeaceae 0 0.005 0.003 0.005 0.014 0 0.006 0.064 0 0 0.021 Rubrobacteraceae 2.518 4.183 3.351 3.383 4.211 5.77 4.455 6.471 6.414 4.687 5.857333 Nocardiaceae 0.226 0.029 0.128 1.427 3.72 1.946 2.364 0.257 0.624 0.503 0.461 Verrucomicrobiaceae 1.218 0.392 0.805 0.65 1.053 0.814 0.839 0.54 0.528 0.275 0.448 Helicobacteraceae 0.005 0.015 0.010 0 0.003 0.004 0.002 0.014 0.005 0.007 0.009 Lactobacillaceae 0.005 0.005 0.005 0.009 0.014 0.014 0.012 0 0.021 0.004 0.008 Desulfovibrionaceae 1.313 1.192 1.253 1.328 1.039 0.98 1.116 0.554 0.518 0.371 0.481 Brevibacteriaceae 0 0 0.000 0 0.003 0.004 0.002 0 0 0 0.000 Haliangiaceae 0.826 0.682 0.754 0.583 0.298 0.557 0.479 0.284 0.406 0.118 0.269 Nitrososphaeraceae 1.891 7.288 4.590 2.566 2.514 4.698 3.259 3.33 5.753 4.163 4.415333 Leptospirillaceae 0.005 0 0.003 0 0.008 0 0.003 0.003 0.016 0 0.006 Desulfobulbaceae 0.271 0.436 0.354 0.257 0.242 0.356 0.285 0.216 0.432 0.211 0.286 Natranaerobiaceae 0 0.015 0.008 0 0.003 0 0.001 0.003 0 0 0.001 Rhodospirillaceae 3.159 3.933 3.546 2.652 2.506 2.851 2.669667 3.871 3.143 2.453 3.155667 Alicyclobacillaceae 0.032 0.137 0.085 0.032 0.022 0.035 0.030 0.135 0.016 0.029 0.060 Erythrobacteraceae 0.686 0.397 0.542 0.669 0.365 0.451 0.495 0.301 0.331 0.189 0.274 Comamonadaceae 0.379 0.324 0.352 0.501 0.511 0.367 0.460 0.22 0.165 0.264 0.216 Streptomycetaceae 1.286 1.839 1.5625 1.59 1.916 2.383 1.963 3.908 3.522 7.611 5.013667 Others 3.691 3.512 3.6015 3.442 2.998 3.151 3.197 3.114 2.204 1.557 2.291667 192

APPENDICES

Total % 99.9865 99.9902 99.9819 99.9663 99.9789 99.9595 99.984 99.9929

193

APPENDICES

Appendix 1-C. Pyrosequencing-based assessment of bacterial community , genus level distribution of 16S rRNA sequences from GM and Non GM maize rhizosphere at different developmental stages .

Stage B Stage C A AA' **Ave GMC ***Ave Genus A% prime% *Ave% GMB% IGB% IWB% % % IGC% IWC% % Microbulbifer 0.018 0.029 0.024 0.005 0.011 0.004 0.007 0.003 0.005 0.014 0.007 Rhodovibrio 0.095 0.226 0.161 0.163 0.16 0.169 0.164 0.274 0.219 0.157 0.217 Ornithinicoccus 0 0 0.000 0 0 0 0.000 0.014 0.005 0.011 0.010 Oscillospira 0.158 0.196 0.177 0.104 0.138 0.06 0.101 0.162 0.048 0.046 0.085 Polaromonas 0.005 0 0.003 0.005 0.028 0.011 0.015 0 0.011 0.007 0.006 Nitrobacter 0.023 0.005 0.014 0.009 0.003 0.004 0.005 0 0 0.004 0.001 Singulisphaera 0.014 0.005 0.010 0.009 0.006 0.011 0.009 0.007 0.027 0.007 0.014 Erwinia 0.032 0.01 0.021 0.014 0.02 0.014 0.016 0.017 0.016 0.014 0.016 Thermoactinomyces 0 0.015 0.008 0.005 0.006 0.007 0.006 0.007 0.005 0.007 0.006 Streptomyces 1.241 1.8 1.521 1.549 1.891 2.323 1.921 3.83 3.405 7.458 4.898 Ralstonia 0.005 0.01 0.008 0.05 0.022 0.014 0.029 0.017 0.011 0.05 0.026 Promicromonospora 0 0.034 0.017 0.005 0.025 0 0.010 2.135 1.526 1.635 1.765 Chryseobacterium 0.113 0.098 0.106 0.113 0.065 0.053 0.077 0.034 0.043 0.011 0.029 Terribacillus 0 0.029 0.015 0.009 0.006 0 0.005 0.01 0.011 0.007 0.009 Bulleidia 0.005 0.044 0.025 0 0.003 0.004 0.002 0.003 0.027 0.014 0.015 Erythrobacter 0.361 0.113 0.237 0.203 0.087 0.123 0.138 0.041 0.027 0.021 0.030 Thiocystis 0 0.005 0.003 0.009 0.003 0.004 0.005 0.01 0.005 0 0.005 Brochothrix 0.077 0.113 0.095 0.126 0.107 0.141 0.125 0.108 0.208 0.1 0.139 Blastopirellula 1.038 1.113 1.076 1.558 1.492 1.716 1.589 1.392 1.307 1.125 1.275 Blastopirellula 1.038 1.113 1.076 1.558 1.492 1.716 1.589 1.392 1.307 1.125 1.275 Syntrophus 0.054 0.069 0.062 0.041 0.067 0.021 0.043 0.024 0.037 0.025 0.029

194

APPENDICES

Pseudoalteromonas 0.014 0.01 0.012 0 0.008 0.007 0.005 0.003 0 0.004 0.002 Jeotgalicoccus 0.014 0.015 0.015 0.018 0.008 0.014 0.013 0.007 0.011 0.004 0.007 Chloroflexus 0.181 0.231 0.206 0.19 0.376 0.356 0.307 0.22 0.197 0.118 0.178 Desulfatibacillum 0.171 0.147 0.159 0.217 0.242 0.176 0.212 0.111 0.107 0.096 0.105 Ectothiorhodospira 4.77 4.576 4.673 3.139 2.781 2.555 2.825 1.439 1.66 1.482 1.527 Desulfomicrobium 0.149 0.078 0.114 0.154 0.104 0.085 0.114 0.044 0.053 0.032 0.043 Symbiobacterium 0.149 0.132 0.141 0.063 0.053 0.081 0.066 0.081 0.069 0.064 0.071 Methanosarcina 0.023 0.25 0.137 0.176 0.191 0.137 0.168 0.23 0.133 0.093 0.152 Magnetococcus 0.099 0.088 0.094 0.081 0.022 0.085 0.063 0.02 0.011 0.007 0.013 Leptospirillum 0.005 0 0.003 0 0.008 0 0.003 0.003 0.016 0 0.006 Thiorhodovibrio 0.009 0.049 0.029 0.032 0 0.021 0.018 0 0 0.007 0.002 Knoellia 0.077 0.226 0.152 0.244 0.393 0.271 0.303 0.399 0.363 0.578 0.447 Rhodoplanes 2.193 1.957 2.075 2.231 2.197 2.710 2.379 2.374 2.556 1.71 2.213 Pandoraea 0.054 0.054 0.054 0.068 0.087 0.046 0.067 0.081 0.069 0.05 0.067 Desulfobacterium 0.023 0.039 0.031 0.041 0.045 0.042 0.043 0.01 0.043 0.011 0.021 Bryantella 0 0 0.000 0 0.003 0.007 0.003 0.003 0 0.004 0.002 Rhodovulum 0.158 0.211 0.185 0.23 0.14 0.166 0.179 0.193 0.251 0.104 0.183 Oscillatoria 0.041 0.039 0.040 0.032 0.014 0.014 0.020 0.02 0.011 0.011 0.014 Enterobacter 0.005 0 0.003 0.014 0.017 0.007 0.013 0.003 0.005 0 0.003 Arthrospira 0 0.01 0.005 0.005 0.003 0.007 0.005 0.01 0.005 0.007 0.007 Dermabacter 0 0.005 0.003 0 0.006 0.007 0.004 0 0 0.004 0.001 Methyloversatilis 0.343 0.304 0.324 0.267 0.27 0.194 0.244 0.247 0.155 0.168 0.190 Pelomonas 0.005 0.02 0.013 0.018 0.02 0.014 0.017 0.017 0.005 0.018 0.013 Halorhodospira 0.447 0.51 0.479 0.357 0.57 0.321 0.416 0.186 0.165 0.089 0.147 Caldicellulosiruptor 0.068 0.059 0.064 0.149 0.07 0.102 0.107 0.041 0.059 0.032 0.044 Methylocystis 0.018 0.025 0.022 0.005 0.014 0.018 0.012 0.024 0.011 0.011 0.015 Mycobacterium 0.528 1.045 0.787 1.238 1.079 1.378 1.232 1.986 1.339 0.757 1.361 195

APPENDICES

Reinekea 0.009 0.005 0.007 0 0.008 0.004 0.004 0.003 0 0 0.001 Moryella 0.009 0.005 0.007 0.027 0.003 0.014 0.015 0 0 0.007 0.002 Thiohalospira 0.099 0.333 0.216 0.122 0.031 0.099 0.084 0.02 0.107 0.071 0.066 Thermonema 0.014 0.005 0.010 0.018 0.003 0.021 0.014 0.01 0.021 0.011 0.014 Lysinibacillus 0.393 0.451 0.422 0.352 0.292 0.578 0.407 0.557 0.427 0.453 0.479 Thiobacillus 0.104 0.49 0.297 0.19 0.048 0.173 0.137 0.041 0.091 0.125 0.086 Propionicimonas 0.009 0.054 0.032 0.032 0.039 0.021 0.031 0.054 0.075 0.068 0.066 Geobacillus 0.302 0.333 0.318 0.271 0.214 0.374 0.286 0.351 0.272 0.171 0.265 Brevundimonas 0.005 0.005 0.005 0.05 0.034 0.014 0.033 0.02 0.021 0.004 0.015 Archangium 0.311 0.795 0.553 0.375 0.494 0.645 0.505 0.591 0.443 0.346 0.460 Francisella 0.081 0.054 0.068 0.099 0.067 0.07 0.079 0.068 0.096 0.071 0.078 Couchioplanes 0.077 0.26 0.169 0.122 0.208 0.106 0.145 0.226 0.075 0.568 0.290 Hymenobacter 0.063 0.186 0.125 0.072 0.09 0.056 0.073 0.041 0.027 0.05 0.039 Legionella 0.014 0.005 0.010 0.018 0.014 0.011 0.014 0.017 0 0.007 0.008 Pseudonocardia 0.086 0.27 0.178 0.136 0.183 0.215 0.178 0.209 0.181 0.164 0.185 Pseudoxanthomonas 0.014 0.005 0.010 0.023 0.042 0.004 0.023 0.017 0.048 0 0.022 Caulobacter 0.032 0.025 0.029 0.054 0.031 0.018 0.034 0.02 0.027 0.025 0.024 Achromatium 0.208 0.098 0.153 0.104 0.079 0.148 0.110 0.041 0.053 0.054 0.049 Amaricoccus 0 0.083 0.042 0.036 0.042 0.011 0.030 0.054 0.032 0.025 0.037 Actinobaculum 0.311 0.152 0.232 0.095 0.084 0.07 0.083 0.017 0.075 0.029 0.040 Methylocaldum 0.068 0.167 0.118 0.095 0.073 0.141 0.103 0.081 0.229 0.064 0.125 Haloanella 0.113 0.01 0.062 0.005 0.003 0.014 0.007 0.007 0 0.004 0.004 Streptosporangium 0.068 0.088 0.078 0.063 0.121 0.106 0.097 0.125 0.08 0.136 0.114 Geitlerinema 0 0.078 0.039 0.005 0 0.007 0.004 0.007 0 0.004 0.004 Desemzia 0.018 0 0.009 0.005 0 0.007 0.004 0 0 0 0.000 Corynebacterium 0.014 0.034 0.024 0.023 0.017 0.035 0.025 0.007 0.016 0 0.008 Kribbella 0.068 0.074 0.071 0.113 0.483 0.166 0.254 1.476 0.886 2.063 1.475 196

APPENDICES

Nannocystis 0.027 0.206 0.117 0.036 0.02 0.011 0.022 0.03 0.016 0.025 0.024 Novosphingobium 0.032 0.005 0.019 0.172 0.034 0.007 0.071 0.138 0.016 0.036 0.063 Lactobacillus 0.005 0.005 0.005 0.009 0.014 0.014 0.012 0 0.021 0.004 0.008 Anabaenopsis 0.023 0.054 0.039 0.014 0.017 0.018 0.016 0.041 0.043 0.036 0.040 Marinilactibacillus 0 0 0.000 0 0.003 0 0.001 0 0.005 0.014 0.006 Opitutus 1.39 0.78 1.085 2.073 0.997 0.98 1.350 0.645 0.923 0.718 0.762 Syntrophomonas 0.027 0.108 0.068 0.018 0.022 0.035 0.025 0.044 0.011 0.021 0.025 Geodermatophilus 0.23 0.559 0.395 0.321 0.303 0.458 0.361 0.25 0.678 0.471 0.466 Burkholderia 0.298 0.162 0.230 0.28 0.278 0.204 0.254 0.196 0.08 0.096 0.124 Sphingobacterium 0.068 0.01 0.039 0.032 0.053 0.025 0.037 0.01 0.037 0.007 0.018 Hyphomicrobium 1.593 1.726 1.660 2.141 2.773 2.689 2.534 2.827 2.156 1.803 2.262 Halomonas 0.19 0.064 0.127 0.104 0.09 0.134 0.109 0.091 0.096 0.071 0.086 Hydrogenophaga 0.041 0.015 0.028 0.045 0.059 0.004 0.036 0 0.011 0.007 0.006 Nocardiopsis 0 0 0.000 0 0.006 0.014 0.007 0.007 0.021 0.004 0.011 Cellulosimicrobium 0.005 0.02 0.013 0.014 0.017 0.021 0.017 0.024 0.027 0.029 0.027 Oscillochloris 0.09 0.069 0.080 0.108 0.124 0.19 0.141 0.098 0.075 0.086 0.086 Thiothrix 1.399 1.04 1.220 1.048 1.273 1.022 1.114 0.716 0.486 0.514 0.572 Thiothrix 1.399 1.04 1.220 1.048 1.273 1.022 1.114 0.716 0.486 0.514 0.572 Spirochaeta 0.023 0.029 0.026 0 0.039 0.021 0.020 0.02 0.005 0.011 0.012 Methylibium 0.122 0.029 0.076 0.172 0.143 0.113 0.143 0.054 0.155 0.061 0.090 Porphyrobacter 0.32 0.284 0.302 0.465 0.275 0.324 0.355 0.253 0.304 0.168 0.242 Brevibacillus 0 0.039 0.020 0.005 0.017 0.014 0.012 0.003 0.021 0.011 0.012 Aeromicrobium 0.144 0.186 0.165 0.267 0.405 0.307 0.326 0.544 0.8 0.364 0.569 Chthoniobacter 1.137 0.383 0.760 1.08 1.208 1.043 1.110 0.844 0.544 0.536 0.641 Roseateles 0.054 0.034 0.044 0.041 0.045 0.046 0.044 0.027 0.032 0.004 0.021 Sediminicola 0.068 0 0.034 0.027 0.025 0.004 0.019 0.003 0 0 0.001 Halothermothrix 0.009 0.029 0.019 0 0 0.018 0.006 0.003 0 0.011 0.005 197

APPENDICES

Xanthobacter 0.005 0 0.003 0 0.003 0.004 0.002 0.007 0.005 0 0.004 Lewinella 2.365 0.333 1.349 1.829 1.011 0.684 1.175 0.165 0.229 0.096 0.163 Magnetospirillum 0.014 0.02 0.017 0.018 0.011 0.018 0.016 0.024 0.011 0.007 0.014 Allochromatium 0.009 0.015 0.012 0 0.003 0.011 0.005 0.024 0 0 0.008 Shuttleworthia 0 0 0.000 0.005 0.003 0.004 0.004 0.003 0.005 0 0.003 Massilia 0.023 0.231 0.127 0.054 0.076 0.039 0.056 0.014 0.043 0.064 0.040 Riemerella 0.451 0.108 0.280 0.375 0.118 0.106 0.200 0.017 0.016 0.018 0.017 Pimelobacter 0.059 0.093 0.076 0.126 0.301 0.148 0.192 0.392 0.374 0.214 0.327 Blautia 0.018 0 0.009 0.009 0.014 0.004 0.009 0 0 0 0.000 Frateuria 0.041 0.01 0.026 0.032 0.042 0.039 0.038 0.02 0.032 0.014 0.022 Cyclobacterium 0.005 0.015 0.010 0 0.006 0.007 0.004 0.02 0.053 0.004 0.026 Afifella 0.054 0.054 0.054 0.059 0.037 0.063 0.053 0.071 0.037 0.029 0.046 Micrococcus 0 0.005 0.003 0.005 0.006 0.018 0.010 0.003 0.005 0.007 0.005 Verrucosispora 0.014 0.083 0.049 0.036 0.014 0.032 0.027 0.037 0.027 0.014 0.026 Blastococcus 0.072 0.231 0.152 0.104 0.101 0.148 0.118 0.165 0.528 0.543 0.412 Sporolactobacillus 0.005 0.02 0.013 0.023 0.02 0.021 0.021 0.01 0.011 0.004 0.008 Blastochloris 0.113 0.083 0.098 0.063 0.126 0.109 0.099 0.057 0.101 0.043 0.067 Campylobacter 0.05 0.034 0.042 0.059 0.039 0.039 0.046 0.03 0.027 0.018 0.025 Solimonas 0.081 0.02 0.051 0.059 0.084 0.092 0.078 0.03 0.027 0.014 0.024 Saccharomonospora 0.027 0.005 0.016 0.041 0.022 0.028 0.030 0.03 0.037 0.029 0.032 Methylocella 0 0.005 0.003 0.005 0.003 0 0.003 0 0.011 0.004 0.005 Ethanoligenens 0.063 0.069 0.066 0.059 0.098 0.039 0.065 0.118 0.021 0.004 0.048 Rhodopirellula 0.009 0.025 0.017 0 0.028 0 0.009 0.01 0.011 0.004 0.008 Luteimonas 0 0 0.000 0.005 0 0.007 0.004 0 0 0.004 0.001 Rheinheimera 0.023 0.01 0.017 0.018 0.028 0.021 0.022 0.165 0.016 0.029 0.070 Aeromonas 0.018 0.01 0.014 0.018 0.028 0.028 0.025 0.101 0.016 0.036 0.051 Rhodobaca 0.081 0.064 0.073 0.05 0.084 0.074 0.069 0.034 0.027 0.021 0.027 198

APPENDICES

Cryptosporangium 0.005 0.01 0.008 0.009 0.014 0.018 0.014 0.014 0.053 0.018 0.028 Rubrobacter 2.518 4.183 3.351 3.383 4.211 5.77 4.455 6.471 6.414 4.687 5.857 Anoxybacillus 0 0.015 0.008 0.014 0.008 0.042 0.021 0.02 0.021 0.011 0.017 Coprothermobacter 0.005 0.005 0.005 0 0 0.007 0.002 0.003 0 0.004 0.002 Subtercola 0.009 0.025 0.017 0.027 0.008 0.035 0.023 0.01 0.021 0.032 0.021 Pedobacter 0.758 0.103 0.431 0.501 0.464 0.3 0.422 0.162 0.251 0.914 0.442 Aphanizomenon 0 0.005 0.003 0 0.008 0 0.003 0.003 0.005 0 0.003 Granulicatella 0.05 0.029 0.040 0.045 0.025 0.042 0.037 0.057 0.043 0.021 0.040 Hyphomonas 0 0.015 0.008 0 0.006 0 0.002 0 0.005 0 0.002 Ruminococcus 0.551 0.211 0.381 0.343 0.267 0.113 0.241 0.135 0.176 0.082 0.131 Chromobacterium 0 0.015 0.008 0.005 0 0.004 0.003 0.007 0 0.004 0.004 Acinetobacter 0.221 0.172 0.197 0.108 0.169 0.197 0.158 0.088 0.091 0.093 0.091 Candidatus Rhodoluna 0 0.02 0.010 0.005 0.003 0.004 0.004 0 0 0 0.000 Rhodoferax 0 0 0.000 0.005 0.003 0 0.003 0 0 0 0.000 Anaerobaculum 0.014 0 0.007 0.018 0.003 0.004 0.008 0 0 0 0.000 Pseudomonas 0.763 0.844 0.804 0.592 0.955 0.589 0.712 0.402 0.651 1.16 0.738 Oxalobacter 0.005 0 0.003 0.009 0.017 0 0.009 0.007 0.005 0.082 0.031 Congregibacter 0.059 0.078 0.069 0.122 0.228 0.081 0.144 0.108 0.043 0.029 0.060 Caldanaerobacter 0.009 0.245 0.127 0.027 0.098 0.067 0.064 0.375 0.069 0.057 0.167 Limnohabitans 0 0 0.000 0.009 0.003 0.004 0.005 0.003 0 0.004 0.002 Azospirillum 2.017 1.687 1.852 1.423 1.149 1.205 1.259 2.064 1.046 0.750 1.287 Phenylobacterium 0.023 0.049 0.036 0.059 0.039 0.032 0.043 0.098 0.085 0.032 0.072 Oceanicaulis 0.018 0 0.009 0 0.006 0 0.002 0.014 0 0 0.005 Flexibacter 2.225 1.721 1.973 1.427 1.188 1.748 1.454 1.547 1.905 0.900 1.451 Herpetosiphon 0.361 0.025 0.193 0.145 0.112 0.352 0.203 0.49 0.251 0 0.247 Wautersiella 0.023 0.01 0.017 0.005 0.008 0 0.004 0.003 0 0 0.001 Pontibacter 0.027 0.044 0.036 0.009 0.034 0.021 0.021 0.034 0.021 0.007 0.021 199

APPENDICES

Luteibacter 0.023 0.005 0.014 0.059 0.008 0.021 0.029 0.024 0.027 0.007 0.019 Caloramator 0.063 0.083 0.073 0.036 0.056 0.053 0.048 0.064 0.005 0.011 0.027 Enterococcus 0.23 0.196 0.213 0.23 0.219 0.201 0.217 0.115 0.139 0.129 0.128 Shewanella 0.005 0 0.003 0.018 0.008 0.004 0.010 0.024 0 0 0.008 Novispirillum 0 0.005 0.003 0.005 0.003 0.004 0.004 0.003 0 0.011 0.005 Myxococcus 0.072 0.132 0.102 0.05 0.107 0.078 0.078 0.132 0.107 0.068 0.102 Prevotella 0 0 0.000 0.005 0 0.011 0.005 0 0.011 0 0.004 Microlunatus 0.027 0.005 0.016 0.014 0.017 0 0.010 0.007 0.016 0.004 0.009 Virgisporangium 0.203 0.49 0.347 0.307 0.287 0.388 0.327 0.5 0.107 0.143 0.250 Planomicrobium 0.05 0.049 0.050 0.041 0.042 0.074 0.052 0.068 0.064 0.05 0.061 Desulfotomaculum 0.199 0.034 0.117 0.099 0.107 0.12 0.109 0.091 0.096 0.071 0.086 Pedomicrobium 0.126 0.579 0.353 0.172 0.23 0.152 0.185 0.135 0.235 0.146 0.172 Serratia 0.208 0.128 0.168 0.348 0.053 0.197 0.199 0.03 0.027 0.043 0.033 Gemmata 0.144 0.275 0.210 0.145 0.211 0.3 0.219 0.23 0.368 0.186 0.261 Cylindrospermopsis 0.005 0 0.003 0.005 0 0 0.002 0 0 0.007 0.002 Halobacillus 0.032 0.029 0.031 0.023 0.025 0.049 0.032 0.014 0.005 0.011 0.010 Georgenia 0 0.01 0.005 0 0.003 0 0.001 0 0.037 0.032 0.023 Methylobacterium 0.596 1.334 0.965 0.669 0.671 0.938 0.759 0.963 1.334 1.01 1.102 Janibacter 0.009 0.034 0.022 0.018 0.003 0.021 0.014 0.01 0.021 0.1 0.044 Trichodesmium 0 0 0.000 0 0.02 0 0.007 0.003 0 0.011 0.005 Nevskia 0.018 0.01 0.014 0.014 0.034 0.011 0.020 0.003 0 0.004 0.002 Bartonella 0.014 0.005 0.010 0.014 0 0.004 0.006 0.027 0.005 0 0.011 Rhodocyclus 0.086 0.025 0.056 0.117 0.124 0.063 0.101 0.037 0.069 0.021 0.042 Micromonospora 0.483 0.809 0.646 0.542 0.447 0.666 0.552 0.726 0.56 1.499 0.928 Agrococcus 0 0 0.000 0 0 0.007 0.002 0.003 0 0.004 0.002 Desulfobulbus 0.244 0.348 0.296 0.239 0.211 0.331 0.260 0.193 0.422 0.203 0.273 Microscilla 0 0 0.000 0.009 0 0 0.003 0.003 0 0 0.001 200

APPENDICES

Actinokineospora 0.05 0.172 0.111 0.099 0.031 0.18 0.103 0.101 0.123 0.054 0.093 Brachymonas 0.077 0.029 0.053 0.032 0.025 0.042 0.033 0.01 0.027 0.011 0.016 Coxiella 0 0 0.000 0 0.003 0 0.001 0.003 0 0.004 0.002 Listeria 0.009 0.025 0.017 0.014 0.034 0.014 0.021 0.017 0.032 0.029 0.026 Stenotrophomonas 0.041 0.039 0.040 0.023 0.017 0.042 0.027 0.081 0.149 0.121 0.117 Paenibacillus 0.059 0.118 0.089 0.095 0.07 0.099 0.088 0.081 0.181 0.189 0.150 Thalassobacillus 0.032 0.025 0.029 0.018 0.039 0.018 0.025 0.037 0.011 0.025 0.024 Crenothrix 0.023 0.02 0.022 0.009 0.006 0.028 0.014 0.02 0.005 0 0.008 Frigoribacterium 0 0.015 0.008 0 0.011 0.014 0.008 0.007 0.043 0.039 0.030 Sulfurimonas 0.005 0.015 0.010 0 0.003 0.004 0.002 0.014 0.005 0.007 0.009 Acidithiobacillus 0.153 0.201 0.177 0.095 0.157 0.247 0.166 0.236 0.256 0.171 0.221 Mechercharimyces 0.302 0.206 0.254 0.208 0.107 0.159 0.158 0.098 0.117 0.05 0.088 Plesiocystis 0.433 0.191 0.312 0.199 0.098 0.201 0.166 0.098 0.123 0.089 0.103 Aneurinibacillus 0.009 0.015 0.012 0 0.003 0.011 0.005 0.014 0.011 0.007 0.011 Rhizobium 0.113 0.167 0.140 0.167 0.205 0.289 0.220 0.605 0.816 0.764 0.728 Methylomicrobium 0.005 0.005 0.005 0.014 0.003 0 0.006 0.037 0.005 0.004 0.015 Kaistella 0.041 0.02 0.031 0 0.022 0.014 0.012 0.01 0 0.004 0.005 Neptunomonas 0.005 0.015 0.010 0.005 0 0 0.002 0 0.011 0.018 0.010 Sporosarcina 0.027 0.069 0.048 0.045 0.039 0.053 0.046 0.047 0.048 0.082 0.059 Bacillus 1.516 2.643 2.080 1.802 1.52 2.52 1.947 2.287 2.209 1.924 2.140 Halolactibacillus 0.014 0.005 0.010 0.005 0.008 0.014 0.009 0.014 0.016 0.004 0.011 Desulfovibrio 1.313 1.192 1.253 1.328 1.039 0.980 1.116 0.554 0.518 0.371 0.481 Oceanobacillus 0.032 0.044 0.038 0.018 0.031 0.049 0.033 0.057 0.032 0.032 0.040 Schlegelella 0.054 0.093 0.074 0.059 0.126 0.046 0.077 0.054 0.032 0.021 0.036 Dokdonella 0.162 0.01 0.086 0.176 0.096 0.074 0.115 0.01 0.112 0.025 0.049 Sphingobium 0.086 0.029 0.058 0.072 0.022 0.042 0.045 0.122 0.08 0.032 0.078 Ferroplasma 0.005 0.025 0.015 0.005 0.011 0 0.005 0.007 0.005 0 0.004 201

APPENDICES

Sphingomonas 0.848 0.544 0.696 1.626 0.643 0.613 0.961 0.895 0.39 0.361 0.549 Methylophaga 1.516 0.481 0.999 0.885 1.292 0.821 0.999 0.692 0.315 0.143 0.383 Turicibacter 0.027 0.201 0.114 0.072 0.045 0.081 0.066 0.088 0.075 0.039 0.067 Nisaea 0.005 0.005 0.005 0.005 0.008 0.018 0.010 0.003 0.005 0.004 0.004 Nitrosococcus 1.3 0.682 0.991 0.962 1.157 0.839 0.986 0.503 0.352 0.25 0.368 Candidatus Pelagibacter 0.018 0.005 0.012 0.018 0.02 0.018 0.019 0.007 0.005 0.021 0.011 Alistipes 0.027 0.034 0.031 0.005 0 0.011 0.005 0 0.016 0.004 0.007 Methanoculleus 0.027 0.211 0.119 0.041 0.18 0.035 0.085 0.057 0.016 0.014 0.029 Phaeospirillum 0.014 0.181 0.098 0 0.09 0.032 0.041 0.037 0.011 0.029 0.026 Baumannia 0 0.025 0.013 0 0 0 0.000 0.007 0.011 0.011 0.010 Macrococcus 0.009 0 0.005 0 0.011 0.011 0.007 0.014 0.005 0.004 0.008 Thermobaculum 0.158 0.52 0.339 0.104 0.132 0.317 0.184 0.388 0.112 0.093 0.198 Leptonema 0 0.005 0.003 0.005 0 0.004 0.003 0 0 0 0.000 Nitrosomonas 0.311 0.113 0.212 0.149 0.16 0.109 0.139 0.084 0.101 0.046 0.077 Bosea 0.018 0.078 0.048 0.077 0.067 0.113 0.086 0.128 0.117 0.075 0.107 Cystobacter 0.032 0.181 0.107 0.167 0.031 0.056 0.085 0.081 0.016 0.018 0.038 Hylemonella 0.014 0.02 0.017 0.027 0.02 0.028 0.025 0.017 0 0.021 0.013 Lautropia 0 0 0.000 0.005 0.003 0.011 0.006 0.017 0.011 0.004 0.011 Methanobacterium 0 0.015 0.008 0.005 0.006 0.004 0.005 0.003 0 0.004 0.002 Pelotomaculum 0.081 0.064 0.073 0.072 0.037 0.025 0.045 0.027 0.021 0.014 0.021 Vagococcus 0 0 0.000 0.009 0 0.007 0.005 0.007 0 0 0.002 Rhodopseudomonas 0.027 0.005 0.016 0.005 0.028 0.088 0.040 0.027 0.059 0.046 0.044 Lentzea 0.009 0.103 0.056 0.032 0.051 0.095 0.059 0.206 0.187 0.114 0.169 Cycloclasticus 0.05 0.044 0.047 0.032 0.028 0.046 0.035 0.02 0.043 0.014 0.026 Azohydromonas 0.113 0.078 0.096 0.117 0.115 0.099 0.110 0.095 0.08 0.054 0.076 Bacteroides 0.063 0.039 0.051 0.018 0.028 0.004 0.017 0.041 0.016 0.007 0.021

202

APPENDICES

Chitinophaga 5.934 2.457 4.196 5.371 4.545 3.697 4.538 2.472 3.106 1.464 2.347 Ammoniphilus 0.068 0.064 0.066 0.149 0.079 0.155 0.128 0.118 0.096 0.039 0.084 Desulfuromonas 0.018 0.02 0.019 0 0.008 0.004 0.004 0.003 0 0.004 0.002 Candidatus Phytoplasma 0.005 0 0.003 0.009 0.017 0.021 0.016 0 0.032 0.007 0.013 Xanthomonas 0.054 0.01 0.032 0.023 0.006 0.085 0.038 0.189 0.251 0.189 0.210 Amycolatopsis 0.032 0.064 0.048 0.018 0.121 0.049 0.063 0.098 0.112 0.05 0.087 Planctomyces 0.713 1.03 0.872 0.772 0.885 0.916 0.858 0.986 0.784 0.682 0.817 Pleomorphomonas 0.036 0.029 0.033 0.027 0.008 0.049 0.028 0.007 0.011 0.029 0.016 Maricaulis 0 0.049 0.025 0 0.051 0 0.017 0.01 0 0 0.003 Parabacteroides 0.135 0.005 0.070 0.027 0.039 0.028 0.031 0.003 0.011 0 0.005 Halothiobacillus 0.122 0.034 0.078 0.099 0.104 0.074 0.092 0.037 0.064 0.054 0.052 Marinibacillus 0 0.015 0.008 0 0.011 0 0.004 0.003 0 0 0.001 Nitrospira 0.077 0.206 0.142 0.036 0.051 0.074 0.054 0.142 0.192 0.086 0.140 Herminiimonas 0.005 0.015 0.010 0.014 0.02 0.011 0.015 0.007 0.016 0.011 0.011 Ensifer 0.411 0.353 0.382 0.542 0.464 0.388 0.465 0.513 0.555 0.5 0.523 Microbacterium 0.023 0.049 0.036 0.104 0.025 0.039 0.056 0.014 0.037 0.054 0.035 Haererehalobacter 0.014 0.015 0.015 0.009 0.008 0.004 0.007 0.003 0 0.011 0.005 Frankia 0.041 0.029 0.035 0.018 0.037 0.025 0.027 0.081 0.048 0.064 0.064 Alicyclobacillus 0.032 0.137 0.085 0.032 0.022 0.035 0.030 0.135 0.016 0.029 0.060 Kocuria 0.005 0.005 0.005 0 0 0.011 0.004 0 0.005 0.368 0.124 Inquilinus 0.009 0.025 0.017 0.014 0.034 0.028 0.025 0.017 0.048 0.025 0.030 Afipia 0.009 0 0.005 0 0.006 0.004 0.003 0.003 0 0.007 0.003 Segetibacter 1.358 0.510 0.934 1.260 1.127 0.649 1.012 0.449 0.63 0.371 0.483 Alkalilimnicola 0.072 0.078 0.075 0.068 0.121 0.067 0.085 0.051 0.053 0.032 0.045 Mesonia 0.005 0 0.003 0.018 0.003 0.018 0.013 0 0 0 0.000 Candidatus Aquirestis 0.072 0.02 0.046 0.045 0.028 0.046 0.040 0 0.005 0.004 0.003

203

APPENDICES

Nostoc 0.023 0.054 0.039 0.005 0.073 0.046 0.041 0.165 0.149 0.032 0.115 Anabaena 0.014 0.01 0.012 0.023 0.011 0.004 0.013 0.01 0.021 0.014 0.015 Rapidithrix 0.005 0.005 0.005 0.009 0.008 0.004 0.007 0.003 0.011 0.007 0.007 Pseudaminobacter 0.005 0.005 0.005 0.009 0.017 0.007 0.011 0.003 0 0 0.001 Porphyromonas 0.293 0.069 0.181 0.027 0.014 0.078 0.040 0.014 0 0.004 0.006 Dichelobacter 0.018 0.02 0.019 0 0 0 0.000 0.027 0 0.007 0.011 Solibacillus 0.014 0.044 0.029 0.023 0.011 0.032 0.022 0.091 0.027 0.025 0.048 Methanobrevibacter 0.005 0.005 0.005 0.005 0.006 0.011 0.007 0.003 0 0 0.001 Collimonas 0.108 0.054 0.081 0.099 0.073 0.039 0.070 0.041 0.053 0.043 0.046 Ramlibacter 0.077 0.103 0.090 0.099 0.067 0.063 0.076 0.044 0.005 0.039 0.029 Hydrocarboniphaga 0.961 0.432 0.697 0.75 1.149 0.56 0.820 0.402 0.294 0.118 0.271 Pantoea 0.041 0.025 0.033 0.027 0.039 0.06 0.042 0.044 0.08 0.021 0.048 Trabulsiella 0.307 0.235 0.271 0.239 0.264 0.271 0.258 0.196 0.144 0.096 0.145 Methylosinus 0.433 0.221 0.327 0.393 0.132 0.285 0.270 0.061 0.021 0.039 0.040 Kurthia 0.041 0.039 0.040 0.023 0.048 0.063 0.045 0.057 0.101 0.068 0.075 Catellatospora 0.284 0.206 0.245 0.271 0.278 0.215 0.255 0.253 0.331 0.125 0.236 Salinicoccus 0.212 0.034 0.123 0.149 0.045 0.13 0.108 0.054 0.048 0.021 0.041 Alkalibacterium 0.005 0.01 0.008 0.005 0.003 0.025 0.011 0.017 0.016 0.018 0.017 Bradyrhizobium 0.97 0.633 0.802 0.682 1.259 1.382 1.108 1.256 1.644 1.207 1.369 Marinomonas 0.027 0.064 0.046 0.027 0.034 0.032 0.031 0.027 0.043 0.007 0.026 Microtetraspora 0 0.005 0.003 0 0.003 0.007 0.003 0.01 0.005 0 0.005 Actinomadura 0.167 0.348 0.258 0.185 0.219 0.448 0.284 0.385 0.347 0.739 0.490 Desulfotalea 0.027 0.083 0.055 0.018 0.031 0.025 0.025 0.024 0.011 0.007 0.014 Saccharopolyspora 0.054 0.137 0.096 0.099 0.126 0.18 0.135 0.375 0.336 0.129 0.280 Kineosporia 0.027 0.044 0.036 0.041 0.006 0.021 0.023 0.003 0.053 0.025 0.027 Sporanaerobacter 0 0.02 0.010 0.018 0.02 0.014 0.017 0.007 0.011 0.004 0.007 Geobacter 0.068 0.128 0.098 0.072 0.166 0.056 0.098 0.108 0.091 0.014 0.071 204

APPENDICES

Thiomicrospira 0.045 0.064 0.055 0.018 0.017 0.025 0.020 0.014 0.011 0.011 0.012 Hirschia 0.009 0.01 0.010 0 0.011 0.004 0.005 0.007 0 0 0.002 Mesorhizobium 0.063 0.137 0.100 0.194 0.18 0.166 0.180 0.24 0.267 0.2 0.236 Isosphaera 0.023 0.015 0.019 0.023 0.017 0.025 0.022 0.027 0.027 0.029 0.028 Paracoccus 0.023 0.034 0.029 0.068 0.076 0.056 0.067 0.044 0.075 0.061 0.060 Roseiflexus 0.095 0.113 0.104 0.163 0.138 0.254 0.185 0.125 0.128 0.046 0.100 Rhodococcus 0.176 0.02 0.098 1.319 3.697 1.769 2.262 0.233 0.614 0.489 0.445 Marinobacter 0.023 0.088 0.056 0.036 0.034 0.067 0.046 0.091 0.059 0.039 0.063 Algoriphagus 0.054 0.029 0.042 0.014 0.051 0.173 0.079 0.041 0.037 0.018 0.032 Roseomonas 0.578 0.226 0.402 0.208 0.152 0.13 0.163 0.216 0.4 0.278 0.298 Acidovorax 0.036 0.034 0.035 0.072 0.051 0.07 0.064 0.037 0.011 0.021 0.023 Anaeromyxobacter 0.217 0.564 0.391 0.226 0.239 0.236 0.234 0.176 0.171 0.075 0.141 Skermanella 0.587 1.491 1.039 0.772 0.871 1.138 0.927 1.118 1.537 1.228 1.294 Stigmatella 0.018 0.02 0.019 0.005 0.014 0.018 0.012 0.007 0.011 0.011 0.010 Brachybacterium 0.014 0.025 0.020 0.009 0.02 0.014 0.014 0.02 0.043 0.004 0.022 Devosia 0.442 0.378 0.410 0.497 0.624 0.599 0.573 0.665 0.624 0.514 0.601 Pelobacter 0.366 0.284 0.325 0.267 0.292 0.194 0.251 0.169 0.091 0.096 0.119 Agrobacterium 0.05 0.025 0.038 0.077 0.115 0.095 0.096 0.176 0.192 0.125 0.164 Methylobacillus 0.023 0.015 0.019 0.045 0.205 0.12 0.123 0.027 0.139 0.007 0.058 Leucothrix 0.199 0.28 0.240 0.23 0.306 0.219 0.252 0.189 0.112 0.175 0.159 Tropheryma 0 0 0.000 0 0.006 0.007 0.004 0.003 0.005 0.007 0.005 Tissierella 0.023 0.02 0.022 0.032 0.017 0.007 0.019 0.027 0.048 0.014 0.030 Martelella 0.009 0 0.005 0 0.003 0.004 0.002 0.003 0 0.014 0.006 Phyllobacterium 0.077 0.069 0.073 0.095 0.104 0.092 0.097 0.088 0.064 0.089 0.080 Loktanella 0 0.015 0.008 0.005 0 0.011 0.005 0 0.005 0.004 0.003 Brevibacterium 0 0 0.000 0 0.003 0.004 0.002 0 0 0 0.000 Sejongia 0.036 0.01 0.023 0.036 0.011 0.004 0.017 0 0.005 0.004 0.003 205

APPENDICES

Sulfurihydrogenibium 0 0.02 0.010 0.005 0.006 0 0.004 0.003 0.005 0 0.003 Thioalkalivibrio 0.293 0.27 0.282 0.226 0.239 0.219 0.228 0.155 0.107 0.093 0.118 Nocardia 0.05 0.01 0.030 0.108 0.022 0.176 0.102 0.024 0.011 0.014 0.016 Actinoplanes 0.271 0.476 0.374 0.217 0.216 0.296 0.243 0.304 0.304 0.264 0.291 Rhodospirillum 0.397 0.27 0.334 0.248 0.146 0.215 0.203 0.294 0.208 0.236 0.246 Aminobacter 0.005 0 0.003 0 0.008 0 0.003 0 0.016 0.004 0.007 Azonexus 0.027 0.029 0.028 0.059 0.006 0.025 0.030 0.014 0 0.029 0.014 Acidimicrobium 0.862 1.918 1.390 1.473 1.750 1.724 1.649 2.111 1.644 1.26 1.672 Agromyces 0.095 0.191 0.143 0.285 0.225 0.275 0.262 0.203 0.934 1.296 0.811 Psychroflexus 0.014 0.015 0.015 0.005 0.045 0.497 0.182 0.007 0.011 0.004 0.007 Streptacidiphilus 0.009 0 0.005 0.005 0.006 0 0.004 0.044 0.021 0.104 0.056 Janthinobacterium 0 0.02 0.010 0.014 0.011 0.014 0.013 0.01 0.016 0.043 0.023 Alcanivorax 0.014 0.005 0.010 0.027 0.07 0.014 0.037 0.014 0.005 0.004 0.008 Actinocorallia 0.005 0.245 0.125 0.014 0.011 0.028 0.018 0.024 0.005 0.025 0.018 Adlercreutzia 0.005 0.02 0.013 0.005 0 0.004 0.003 0 0 0.007 0.002 Treponema 0 0 0.000 0.014 0 0.007 0.007 0.003 0.032 0.004 0.013 Kaistobacter 1.069 0.75 0.910 2.322 1.124 1.145 1.530 1.027 1.131 1.182 1.113 Actinotalea 0 0.034 0.017 0.032 0.02 0.039 0.030 0.017 0.021 0.032 0.023 Staphylococcus 0.045 0.034 0.040 0.095 0.081 0.088 0.088 0.044 0.048 0.036 0.043 Tetrasphaera 0.014 0 0.007 0 0.006 0 0.002 0.003 0.005 0.007 0.005 Prosthecobacter 1.209 0.387 0.798 0.646 1.051 0.807 0.835 0.54 0.528 0.275 0.448 Nautilia 0 0 0.000 0 0.003 0 0.001 0.003 0.005 0 0.003 Thiohalomonas 0.429 0.951 0.690 0.321 0.635 0.363 0.440 0.591 0.347 0.239 0.392 Nocardioides 0.384 0.868 0.626 0.967 2.073 1.061 1.367 3.29 3.453 3.652 3.465 Leptothrix 0.018 0 0.009 0 0.003 0.018 0.007 0.003 0 0.004 0.002 Acetivibrio 0.005 0.039 0.022 0.014 0.006 0.011 0.010 0.037 0.021 0.007 0.022 Veillonella 0.005 0.005 0.005 0.005 0 0.007 0.004 0 0 0 0.000 206

APPENDICES

Nitrosospira 0.826 0.49 0.658 0.42 0.34 0.529 0.430 0.324 0.299 0.25 0.291 Escherichia 0.095 0.069 0.082 0.045 0.022 0.056 0.041 0.01 0.011 0.014 0.012 Rubrivivax 0 0.005 0.003 0.014 0.022 0.007 0.014 0.017 0.011 0.004 0.011 Anaerostipes 0 0.005 0.003 0 0.006 0.018 0.008 0.02 0 0.011 0.010 Methylococcus 0.063 0.005 0.034 0.041 0.011 0.014 0.022 0.003 0.011 0.004 0.006 Sorangium 0.095 0.231 0.163 0.158 0.098 0.243 0.166 0.145 0.203 0.121 0.156 Candidatus Aquiluna 0.054 0.069 0.062 0.068 0.048 0.049 0.055 0.078 0.085 0.068 0.077 Ochrobactrum 0.041 0.049 0.045 0.054 0.014 0.028 0.032 0 0.032 0.014 0.015 Thermomonas 0.605 0.515 0.560 0.745 0.503 0.43 0.559 0.426 0.651 0.328 0.468 Salicola 0 0 0.000 0 0.011 0 0.004 0.01 0 0 0.003 Piscirickettsia 0.005 0.02 0.013 0 0 0 0.000 0.014 0.005 0 0.006 Aerococcus 0.005 0.01 0.008 0.005 0.006 0 0.004 0.014 0 0.004 0.006 Thermobifida 0 0.015 0.008 0.018 0 0.014 0.011 0.01 0.149 0.018 0.059 Aurantimonas 0 0 0.000 0 0.003 0.004 0.002 0.01 0 0.011 0.007 Labrys 0.005 0.025 0.015 0.018 0.014 0.007 0.013 0.017 0.016 0.014 0.016 Chamaesiphon 0 0.034 0.017 0.005 0 0 0.002 0.014 0.101 0.046 0.054 Elizabethkingia 0.059 0 0.030 0.036 0.028 0.021 0.028 0.003 0.005 0.004 0.004 Pseudochrobactrum 0.05 0 0.025 0.014 0.011 0.025 0.017 0.007 0.005 0 0.004 Tistrella 0.023 0.025 0.024 0.005 0.034 0.025 0.021 0.037 0.053 0.007 0.032 Paucimonas 0.009 0.01 0.010 0.009 0.006 0 0.005 0 0.005 0 0.002 Thermobispora 0.005 0.029 0.017 0.023 0.011 0.06 0.031 0.007 0.053 0.004 0.021 Eubacterium 0 0.015 0.008 0.005 0.011 0.004 0.007 0 0 0 0.000 Cryocola 0 0.005 0.003 0.014 0 0.011 0.008 0.003 0 0.021 0.008 Polynucleobacter 0 0 0.000 0 0 0 0.000 0 0 0 0.000 Natronobacillus 0 0.005 0.003 0.005 0 0.011 0.005 0.024 0.005 0.004 0.011 Carboxydothermus 0.068 0.034 0.051 0.041 0.048 0.06 0.050 0.061 0.085 0.079 0.075 Tannerella 0.018 0 0.009 0.009 0.008 0.011 0.009 0 0 0.004 0.001 207

APPENDICES

Gordonia 0.014 0.01 0.012 0.068 0.014 0.039 0.040 0.003 0.011 0 0.005 Ureibacillus 0.605 0.721 0.663 0.831 0.961 1.068 0.953 0.787 1.105 0.718 0.870 Chitinimonas 0.068 0.02 0.044 0.041 0.028 0.056 0.042 0.061 0.032 0.014 0.036 Heliobacterium 0.045 0.02 0.033 0.05 0.022 0.025 0.032 0.037 0.043 0.007 0.029 Variovorax 0.072 0.005 0.039 0.126 0.107 0.085 0.106 0.034 0.064 0.114 0.071 Corallococcus 0.023 0.118 0.071 0.036 0.056 0.081 0.058 0.064 0.048 0.046 0.053 Natranaerobius 0 0.015 0.008 0 0.003 0 0.001 0.003 0 0 0.001 Desulfobacca 0.027 0.064 0.046 0.054 0.101 0.074 0.076 0.054 0.069 0.046 0.056 Sphingosinicella 0 0 0.000 0.009 0.008 0.004 0.007 0 0 0.004 0.001 Fibrobacter 0.086 0.025 0.056 0.072 0.045 0.014 0.044 0.007 0 0.007 0.005 Thermoanaerobacter 0.005 0.064 0.035 0.023 0.037 0.018 0.026 0.105 0.032 0.014 0.050 Thermanaerovibrio 0.068 0.049 0.059 0.072 0.07 0.067 0.070 0.095 0.059 0.093 0.082 Haliangium 0.826 0.682 0.754 0.583 0.298 0.557 0.479 0.284 0.406 0.118 0.269 Nitrosopumilus 0 0.005 0.003 0.005 0.014 0 0.006 0.064 0 0 0.021 Eggerthella 0.005 0 0.003 0.005 0.003 0.004 0.004 0 0.005 0 0.002 Phormidium 0 0.01 0.005 0 0 0.007 0.002 0.003 0.053 0.253 0.103 Glycomyces 0 0.005 0.003 0.014 0.011 0.004 0.010 0.03 0.027 0.014 0.024 Acetobacterium 0.014 0 0.007 0 0.003 0 0.001 0.003 0 0 0.001 Plesiomonas 0.018 0 0.009 0.027 0.003 0.004 0.011 0 0 0 0.000 Azoarcus 0.005 0.005 0.005 0.023 0.014 0.014 0.017 0.007 0 0 0.002 Flavobacterium 0.23 0.074 0.152 0.126 0.135 0.06 0.107 0.041 0.037 0.018 0.032 Pseudidiomarina 0.005 0 0.003 0 0 0.004 0.001 0.003 0 0 0.001 Isoptericola 0 0.01 0.005 0.136 0 0 0.045 0 0 0 0.000 Gramella 0.059 0.005 0.032 0.023 0.045 0.011 0.026 0 0.005 0.004 0.003 Bdellovibrio 0.027 0.029 0.028 0.027 0.006 0 0.011 0.014 0.016 0.011 0.014 Microcoleus 0 0.255 0.128 0.005 0 0.004 0.003 0.01 0 0.025 0.012 Echinicola 0.005 0.015 0.010 0.005 0 0.025 0.010 0.01 0 0.004 0.005 208

APPENDICES

Herbaspirillum 0.005 0.059 0.032 0.032 0.028 0.049 0.036 0.057 0.048 0.036 0.047 Sulfobacillus 0 0.005 0.003 0.005 0.008 0.007 0.007 0.01 0 0 0.003 Clostridium 0.320 2.050 1.185 0.37 0.565 0.508 0.481 1.128 0.726 0.411 0.755 Psychromonas 0.063 0.059 0.061 0.181 0.003 0.042 0.075 0.027 0 0 0.009 Cellulomonas 0 0.02 0.010 0.014 0.006 0.011 0.010 0.014 0.053 0.021 0.029 Leptolyngbya 0.068 0.083 0.076 0.05 0.107 0.085 0.081 0.294 0.294 0.107 0.232 Thioploca 0.027 0.005 0.016 0.009 0.017 0.042 0.023 0.014 0.011 0 0.008 Beijerinckia 0.009 0.025 0.017 0 0.025 0.014 0.013 0.034 0.069 0.05 0.051 Persicobacter 0.171 0.059 0.115 0.149 0.183 0.085 0.139 0.122 0.101 0.079 0.101 Spirulina 0.018 0.015 0.017 0.018 0.045 0.109 0.057 0.027 0.037 0.018 0.027 Saccharothrix 0.009 0.132 0.071 0.014 0.025 0.014 0.018 0.03 0.016 0.014 0.020 Candidatus Koribacter 0.803 0.177 0.490 0.732 0.326 0.226 0.428 0.111 0.208 0.089 0.136 Methylomonas 0 0.059 0.030 0.014 0.037 0.035 0.029 0.057 0.016 0.011 0.028 Candidatus Cardinium 0 0 0.000 0 0 0 0.000 0.02 0.005 0 0.008 Cupriavidus 0.108 0.025 0.067 0.054 0.014 0.025 0.031 0.003 0.005 0.014 0.007 Winogradskyella 0.014 0 0.007 0.005 0.003 0.014 0.007 0.01 0.011 0 0.007 Chelativorans 0.005 0.01 0.008 0 0.003 0.007 0.003 0.003 0 0.004 0.002 Dechloromonas 0 0 0.000 0.009 0.011 0 0.007 0.007 0 0.004 0.004 Salinispora 0.072 0.206 0.139 0.181 0.121 0.067 0.123 0.125 0.139 0.118 0.127 Psychrobacter 0.144 0.113 0.129 0.185 0.205 0.145 0.178 0.064 0.112 0.057 0.078 Candidatus Portiera 0.005 0.005 0.005 0.009 0 0 0.003 0.003 0 0 0.001 Sphingopyxis 0.009 0 0.005 0.009 0 0 0.003 0.01 0.005 0 0.005 Thermaerobacter 0.32 0.736 0.528 0.257 0.351 0.275 0.294 0.365 0.203 0.125 0.231 Thermotoga 0.072 0.059 0.066 0.054 0.042 0.056 0.051 0.041 0.075 0.054 0.057 Virgibacillus 0.014 0.02 0.017 0.018 0.02 0.028 0.022 0.027 0.037 0.025 0.030 Rhodobium 0.122 0.083 0.103 0.126 0.166 0.43 0.241 0.149 0.128 0.132 0.136 Alkaliphilus 0.009 0.02 0.015 0.009 0.003 0.007 0.006 0.014 0.016 0.011 0.014 209

APPENDICES

Microbispora 0 0.02 0.010 0.005 0.008 0.021 0.011 0.003 0 0.007 0.003 Thermocrinis 0.018 0.015 0.017 0.005 0.017 0.032 0.018 0.003 0 0.004 0.002 Candidatus Nitrososphaera 1.891 7.288 4.590 2.566 2.514 4.698 3.259 3.33 5.753 4.163 4.415 Thermosipho 0.009 0.005 0.007 0 0.003 0.004 0.002 0.007 0 0.007 0.005 Rubellimicrobium 0.005 0.01 0.008 0.009 0.011 0.032 0.017 0.03 0.032 0.018 0.027 Dyella 0.068 0.01 0.039 0.099 0.02 0.007 0.042 0.003 0.011 0.011 0.008 Thauera 0.668 0.324 0.496 0.61 0.534 0.409 0.518 0.385 0.235 0.207 0.276 Desulfosporosinus 0.176 0.064 0.120 0.145 0.067 0.039 0.084 0.041 0.037 0.032 0.037 Chondromyces 1.616 2.217 1.917 1.26 1.053 1.36 1.224 1.24 1.009 0.518 0.922 Desulfitobacterium 0.005 0.005 0.005 0 0 0.004 0.001 0.007 0.005 0.004 0.005 Planifilum 0.023 0.034 0.029 0.032 0.045 0.074 0.050 0.041 0.011 0.046 0.033 Renibacterium 0.009 0.034 0.022 0.018 0.042 0.025 0.028 0.142 0.139 0.914 0.398 Candidatus Solibacter 0.361 0.338 0.350 0.267 0.199 0.233 0.233 0.365 0.395 0.107 0.289 Arthrobacter 0.23 0.535 0.383 0.506 0.5 0.744 0.583 1.753 2.071 0 1.275 Leucobacter 0 0.005 0.003 0.023 0.011 0.018 0.017 0.03 0.027 0.043 0.033 Candidatus Microthrix 0.077 0.093 0.085 0.081 0.135 0.095 0.104 0.088 0.037 0.068 0.064 Syntrophobacter 0.108 0.284 0.196 0.108 0.155 0.109 0.124 0.334 0.085 0.054 0.158 Laceyella 0.005 0 0.003 0 0.003 0.004 0.002 0.003 0 0.004 0.002 Erythromicrobium 0.005 0 0.003 0 0.003 0.004 0.002 0.007 0 0 0.002 Pannonibacter 0.005 0.005 0.005 0.005 0.006 0.004 0.005 0.007 0 0.011 0.006 Deinococcus 0 0.005 0.003 0.014 0.006 0.004 0.008 0.003 0.032 0.007 0.014 Citricoccus 0.014 0.049 0.032 0.023 0.011 0.035 0.023 0.105 0.016 0.107 0.076 Dietzia 0.063 0 0.032 0.014 0.02 0.011 0.015 0 0 0 0.000 Alkanindiges 0.46 0.231 0.346 0.235 0.416 0.352 0.334 0.199 0.267 0.175 0.214 Methylotenera 0.081 0.015 0.048 0.185 1.61 0.106 0.634 0.01 0 0.004 0.005 Coprococcus 0.036 0.132 0.084 0.036 0.022 0.025 0.028 0.02 0.016 0.007 0.014

210

APPENDICES

Marmoricola 0.14 0.613 0.377 0.294 0.59 0.451 0.445 0.669 0.374 0.635 0.559 Lysobacter 0.09 0.039 0.065 0.163 0.051 0.06 0.091 0.098 0.101 0.046 0.082 Azorhizophilus 0.122 0.064 0.093 0.126 0.112 0.056 0.098 0.169 0.763 0.043 0.325 Nitrosovibrio 2.356 1.672 2.014 1.689 1.461 1.685 1.612 1.304 1.163 0.743 1.070 Raoultella 0.014 0.025 0.020 0.032 0.017 0.025 0.025 0.007 0.016 0.018 0.014 Nonomuraea 0.009 0.015 0.012 0.014 0.017 0.025 0.019 0.02 0.027 0.068 0.038 Azotobacter 0.009 0 0.005 0.005 0.028 0.007 0.013 0.037 0.203 0.004 0.081 Exiguobacterium 0 0.015 0.008 0.005 0.006 0.028 0.013 0.02 0.005 0.014 0.013 Shinella 0.329 0.177 0.253 0.167 0.132 0.095 0.131 0.118 0.197 0.111 0.142 Salmonella 0.009 0 0.005 0.014 0.014 0.025 0.018 0.02 0.021 0.004 0.015 Moraxella 1.223 0.966 1.095 1.351 0.95 0.811 1.037 0.436 0.294 0.314 0.348 Labrenzia 0.032 0.078 0.055 0.054 0.037 0.056 0.049 0.041 0.027 0.111 0.060 Rhodothermus 0.009 0.039 0.024 0.054 0.017 0.063 0.045 0.02 0.027 0.011 0.019 Flexithrix 0.009 0.005 0.007 0.009 0.003 0.007 0.006 0.02 0 0 0.007 Akkermansia 0.009 0.005 0.007 0.005 0.003 0.007 0.005 0 0 0 0.000 Mitsuokella 0.005 0.005 0.005 0 0.006 0 0.002 0 0 0 0.000 Kitasatospora 0.036 0.039 0.038 0.041 0.025 0.06 0.042 0.034 0.096 0.064 0.065 Methylosphaera 0.46 0.922 0.691 0.542 0.733 0.571 0.615 0.662 0.443 0.536 0.547 Dictyoglomus 0.05 0.069 0.060 0.036 0.07 0.06 0.055 0.037 0.048 0.032 0.039 Others 4.907 3.634 4.271 4.129 4.055 3.768 3.984 3.831 2.296 1.54 2.556 Where samples represent as: A - pre sowing soil samples; A'-soil sample from same field after one year; GMB -vegetative stage GM maize rhizosphere sample; IGB - vegetative stage Non GM maize variety (IG) rhizosphere; IWB - vegetative stage Non GM maize variety (IW) rhizosphere; GMC - harvesting stage GM maize rhizosphere sample; IGC - harvesting stage Non GM maize variety (IG) rhizosphere; IWC - harvesting stage Non GM maize variety (IW) rhizosphere. whereas IG stand for Islamabad gold and IW for Islamabad white (name of maize varieties). Rhizosphere soil were pyrosequencing-based analyzed (200bp 515F ) Above then 20,000 reads/sample were got and analyzed for bacterial identification. Total 21 phyla,184 families and 469 genera were identified and their percent representation is indicated . (200bp 515F bacterial diversity assays)

211

APPENDICES

* AA' Ave%- represent average percent of pre sowing and after one year interval samples

** Stage B Ave%- represent average percentage of vegetative stage samples ( One GM and Two Non GM maize rhizosphere)

*** Stage C Ave%- average percentage of harvesting stage samples ( One GM and Two Non GM maize rhizosphere)

212

APPENDICES

Appendix: 2A-1 Eztaxon classification of isolated bacterial strains pre sowing stage

S. No Isolates Total Number of % isolates

1 Bacillus cereus 3 17 2 Pseudomonas taiwanensis 4 22 3 Enterobacter cloacae subsp. 1 5 4 Bacillus anthracis 5 28 5 Bacillus tequilensis 2 11 6 Pseudomonas plecoglossicida 1 5 7 Bacillus aerophilus 1 6 8 Enterobacter cancerogenus 1 6

213

APPENDICES

Appendix: 2A-2 RDP base classification of the pre sowing isolates

Isolate Phylum Class Order Family genus

RS-A-1 Bacillus anthracis RS-A-2 Bacillus aerophilus RS-A-3 Bacillus tequilensis RS-A-5 Bacillus cereus RS-A-6 Firmicutes Bacilli Bacillales Bacillaceae Bacillus Bacillus anthracis RS-A-7 Bacillus cereus RS-A-8 Bacillus anthracis RS-A-9 Bacillus tequilensis RS-A-11 Pseudomonas taiwanensis RS-A-17 Bacillus cereus RS-A-13 Salirhabdus Bacillus anthracis RS-A-15 Enterobacteri Enterobacteri Klebsiella Enterobacter cloacae RS-A-18 Gammaprot ales aceae Enterobacter Enterobacter cancerogenus RS-A-4 eobacteria Pseudomonas Proteobacteria Pseudomona Pseudomona plecoglossicida RS-A-10 dales daceae Pseudomonas Pseudomonas taiwanensis RS-A-12 Pseudomonas taiwanensis RS-A-14 Pseudomonas taiwanensis RS-A-16 Pseudomonas taiwanensis

214

APPENDICES

Appendix: 2B-1 Genus based Eztaxon classification of isolates from GM and non GM maize Rhizosphere

S. No Genus Total Number of % isolates

1 Chryseobacterium 4 19 2 Micrococcus 1 5 3 Enterobacter 3 14 4 Bacillus 2 9 5 Pseudomonas 5 24 6 Sphingobacterium 1 5 7 Acinetobacter 1 5 8 Sphingomonas 2 9 9 Microbacterium 1 5 10 Aerococcus 1 5

215

APPENDICES

Appendix: 2B-2 RDP base classification of the isolated from GM and non GM maize Rhizosphere

Isolate Phylum Class Order Family Genus Species RS-BGM-23 Pseudomonas moorei

RS-BIW-26 Pseudomonas lini

RS-BIW-32 Pseudomonadaceae Pseudomonas Pseudomonas lini Pseudomonada RS-BIG-38 Pseudomonas cremoricolorata les RS-BIW-29 Moraxellaceae Acinetobacter Acinetobacter pittii

RS-BGM-21 Enterobacter cloacae Gammaprote Enterobacteria RS-BIW-31 Enterobacter cloacae obacteria les Enterobacteriaceae Enterobacter RS-BIG-37 Proteobacteri Enterobacter ludwigii a Xanthomonada RS-BGM-24A Xanthomonadaceae Stenotrophomonas Pseudomonas hibiscicola les RS-BIW-34 Alphaproteo Sphingomonad Sphingomonadacea Sphingomonas koreensis Sphingomonas RS-BIW-30 bacteria ales e Sphingomonas koreensis RS-BGM-19 Chryseobacterium indologenes RS-BIW-25 Chryseobacterium indologenes Flavobacteria Flavobacteriales Flavobacteriaceae Chryseobacterium RS-BIW-28 Chryseobacterium indologenes RS-BIG-36 Bacteroidete Chryseobacterium hagamense s Sphingobacter Sphingobacteria RS-BGM-24 Sphingobacteriaceae Sphingobacterium Sphingobacterium multivorum ia les RS-BGM-22 Bacillaceae Bacillus Bacillus cereus Bacillales RS-BIW-27 Bacilli Planococcaceae Solibacillus Bacillus isronensis Firmicutes RS-BIG-35 Lactobacillales Aerococcaceae Aerococcus Aerococcus urinaeequi RS-BGM-20 Actinobacteri Micrococcus antarcticus Actinobacteria Actinomycetales Micrococcineae Microbacterium RS-BIW-33 a Microbacterium oleivorans

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APPENDICES

Appendix: 2C-1 Genus based Eztaxon classification of isolated isolates from GM and non GM maize Rhizosphere harvesting stage

Total Number of % S. No Genus isolates

1 Bacillus 7 44 2 Enterobacter 2 13 3 Microbacterium 4 25 4 Bordetella 1 6 5 Pseudomonas 1 6 6 Flavobacterium 1 6

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APPENDICES

Appendix: 2C-2 RDP base classification of the isolates from GM and non GM maize Rhizosphere during harvesting stage isolates

Isolate Phylum Class Order Family Genus Species

RS-CGM-39 Bacillus cereus RS-CGM-40 Bacillus cereus RS-CGM-43 Bacillus aryabhattai RS-CIW-46 Firmicutes Bacilli Bacillales Bacillaceae Bacillus Bacillus idriensis RS-CIG-48 Bacillus cereus RS-CIG-49 Bacillus cereus RS-CIG-52 Bacillus cereus RS-CIG-53 Bacillus anthracis RS-CGM-41 Enterobacte Enterobacte Enterobacter Enterobacter cloacae RS-CGM-42 Proteobacteri Gammapro riales riaceae Enterobacter cloacae RS-CIG-50 a teobacteria Pseudomon Pseudomon Pseudomonas Pseudomonas stutzeri adales adaceae RS-CIW-47 Betaproteo Burkholderi Alcaligenac Bordetella Bordetella petrii bacteria ales eae RS-CGM-43A Actinobacteri Actinobact Actinomycet Microbacter Microbacteriu Microbacterium hominis RS-CIW-44 a eria ales iaceae m Microbacterium hominis RS-CIW-45 Microbacterium trichothecenolyticum RS-CIG-51 Bacteroidetes Flavobacte Flavobacter Flavobacter Flavobacteriu Flavobacterium sasangense ria iales iaceae m

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APPENDICES

Appendix 3 The GenBank (NCBI) accession number allotted for 16S rRNA sequence of the isolates

S/No Strain ID NCBI Gene Bank Closely related type strain % 16S accession number rRNA identity First stage ( Before sowing transgenic and non transgenic maize crop in the field trial) 1 RS-A-1 KC430942 Bacillus cereus ATCC 14579T 100 2 RS-A-2 KC430943 Bacillus stratosphericus 41KF2aT 100 3 RS-A-3 KC430944 Bacillus tequilensis 10bT 99 4 RS-A-4 KC430945 Pseudomonas taiwanensis BCRC 17751T 96 5 RS-A-5 KC430946 Bacillus cereus ATCC 14579T 100 6 RS-A-6 KC430947 Bacillus cereus ATCC 14579T 100 7 RS-A-7 KC430948 Bacillus cereus ATCC 14579T 100 8 RS-A-8 KC430949 Bacillus cereus ATCC 14579T 98 9 RS-A-9 KC430950 Bacillus tequilensis 10bT 99 10 RS-A-10 KC430951 Pseudomonas taiwanensis BCRC 17751T 99 11 RS-A-11 KC430952 Bacillus anthracis ATCC 14578T 100 12 RS-A-12 KC430953 Pseudomonas taiwanensis BCRC 17751T 99 13 RS-A-13 KC430954 Bacillus cereus ATCC 14579T 84 14 RS-A-14 KC430955 Pseudomonas taiwanensis BCRC 17751T 99 15 RS-A-15 KC430956 Enterobacter cloacae subsp. dissolvens LMG 2683T 96 16 RS-A-16 KC430957 Pseudomonas taiwanensis BCRC 17751T 100 17 RS-A-17 KC430958 Bacillus cereus ATCC 14579T 99 18 RS-A-18 KC430959 Enterobacter cancerogenus LMG 2693T 99 Second stage (During vegetative stage strains isolation from transgenis and non transgenic maize rhizosphere) 19 RS-BGM-19 KC430960 Chryseobacterium indologenes LMG 8337T 98 20 RS-BGM-20 KC430961 Micrococcus antarcticus T2T 99

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APPENDICES

21 RS-BGM-21 KC430962 Enterobacter cloacae subsp. dissolvens LMG 2683T 99 22 RS-BGM-22 KC430963 Bacillus cereus ATCC 14579T 100 23 RS-BGM-23 KC430964 Pseudomonas moorei RW10T 99 24 RS-BGM-24 KC430965 Sphingobacterium multivorum IAM14316T 98 25 RS-BIW-25 KC430966 Chryseobacterium indologenes LMG 8337T 98 26 RS-BIW-26 KC430967 Pseudomonas lini CFBP 5737T 99 27 RS-BIW-27 KC430968 Bacillus isronensis B3W22T 98 28 RS-BIW-28 KC430969 Chryseobacterium indologenes LMG 8337T 98 29 RS-BIW-29 KC430970 Acinetobacter pittii LMG 1035T 99 30 RS-BIW-30 KC430971 Sphingomonas koreensis JSS26T 99 31 RS-BIW-31 KC430972 Enterobacter cloacae subsp. dissolvens LMG 2683T 100 32 RS-BIW-32 KC430973 Pseudomonas lini CFBP 5737T 98 33 RS-BIW-33 KC430974 Microbacterium oleivorans DSM 16091T 99 34 RS-BIW-34 KC430975 Sphingomonas koreensis JSS26T 99 35 RS-BIG-35 KC430976 Aerococcus urinaeequi IFO 12173 T 97 36 RS-BIG-36 KC430977 Chryseobacterium hagamense RHA2-9T 97 37 RS-BIG-37 KC430978 Enterobacter ludwigii DSM 16688T 99 38 RS-BIG-38 KC430979 Pseudomonas parafulva AJ 2129T 100 Third stage (During harvesting stage strains isolation from transgenis and non transgenic maize rhizosphere) 39 RS-CGM-39 KC430980 Bacillus cereus ATCC 14579T 100 40 RS-CGM-40 KC430981 Bacillus cereus ATCC 14579T 100 41 RS-CGM-41 KC430982 Enterobacter cloacae subsp. dissolvens LMG 2683T 98 42 RS-CGM-42 KC430983 Enterobacter cloacae subsp. dissolvens LMG 2683T 98 43 RS-CGM-43 KC430984 Bacillus aryabhattai B8W22T 93 44 RS-CIW-44 KC430985 Microbacterium hominis IFO 15708T 97 45 RS-CIW-45 KC430986 Microbacterium trichothecenolyticum IFO 15077T 98 46 RS-CIW-46 KC430987 Bacillus idriensis SMC 4352-2T 99 47 RS-CIW-47 KC430988 Bordetella petrii DSM 12804T 97 48 RS-CIG-48 KC430989 Bacillus cereus ATCC 14579T 99

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APPENDICES

49 RS-CIG-49 KC430990 Bacillus cereus ATCC 14579T 100 50 RS-CIG-50 KC430991 Pseudomonas stutzeri ATCC 17588T 99 51 RS-CIG-51 KC430992 Flavobacterium sasangense YC6274T 97 52 RS-CIG-52 KC430993 Bacillus cereus ATCC 14579T 100 53 RS-CIG-53 KC430994 Bacillus cereus ATCC 14579T 100

Appendix 4: Physicochemical characteristics of field soil used for growing of GM and non GM maize crops Chemical and physical properties Texture class Loam pH 7.9 E.C 3.91 dS.m-1 Soil Organic matter 1.19 % Available phosphorous 22.5 µg/g DW Saturation 38 % K+ 130 µg/g DW

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