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Molecular and Biochemical Characterization of Plant 1 MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF PLANT GROWTH PROMOTING RHIZOBACTERIA FOR ENHANCING CROP YIELD UMMAY AMARA 03-arid-120 Department of Soil Science & Soil and Water Conservation Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2 2015 MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF PLANT GROWTH PROMOTING RHIZOBACTERIA FOR ENHANCING CROP YIELD by UMMAY AMARA (03-arid-120) A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosop hy in Soil Science Department of Soil Science & Soil and Water Conservation Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi 3 Pakistan 2015 CERTIFICATION I hereby undertake that this research is an original one and no part of this thesis falls under plagiarism. If found otherwise, at any stage, I will be responsible for the consequences. Student’s Name: Ummay Amara Signature: ____________ Registration No: 03-arid-120 Date: ________________ Certified that contents and form of the thesis entitled “Molecular and Biochemical Characterization of Plant Growth Promoting Rhizobacteria for Enhancing Crop Yield” submitted by Ms. Ummay Amara have been found satisfactory for the requirement of the degree. Supervisor: ________________________ (Dr. Rifat Hayat) Co-Supervisor: _____________________ (Dr. Iftikhar Ahmed) Member: __________________________ (Prof. Dr. Safdar Ali) Member: __________________________ (Dr. Ghulam Shabbir) Chairperson: _____________________ 4 Dean: ____________________________ Director, Advanced Studies: ___________________________ 5 CONTENTS Page List of Tables viii List of Figures ix Acknowledgment x ABSTRACT xi 1. INTRODUCTION 1 2. REVIEW OF LITERATURE 5 2.1 ROLE OF PGPRS IN CROP PRODUCTION 6 2.2 MECHANISMS OF PLANT GROWTH PROMOTION 7 2.2.1 Biological Nitrogen Fixation 8 2.2.2 Solubilization of Phosphates 10 2.2.3 Phytohormones Production by PGPRs 16 2.2.3.1 Indole-3-acetic acid (IAA) production 17 2.2.3.2 1-Aminocyclopropane-1-carboxylate deaminase production 19 2.2.3.3 Cytokinin production 23 2.2.3.4 Gibberellins production 25 2.2.4 Increased Iron Uptake 26 2.3 APPROACHES TO DEVELOP PGPRS 29 6 3. MATERIALS AND METHODS 35 3.1 SURVEY FOR SAMPLES COLLECTION 35 3.2 EXPERIMENTAL SITE 35 3.3 ISOLATION AND PURIFICATION OF BACTERIA 35 3.4 PLANT GROWTH PROMOTING CHARACTERIZATION 36 3.4.1 Phosphate Solubilization 36 3.4.2 Production of Indole-3-Acetic Acid (IAA) 37 3.4.3 Production of Siderophore 37 3.4.4 nifH Gene Amplification 37 3.5 PHENOTYPIC AND BIOCHEMICAL CHARACTERIZATIO 37 3.6 CHEMOTAXONOMIC ANALYSIS 38 3.7 IDENTIFICATION USING MOLECULAR TECHNIQUE 40 3.7.1 Preparation of DNA Template 40 3.7.2 Amplification of 16S rRNA Gene by PCR 40 3.7.3 Purification of PCR Products 41 3.7.4 DNA Sequencing 41 3.7.5 Phylogenetic Analysis (Bioinformatics) 41 7 3.8 SCREENING UNDER GROWTH CHAMBER 42 3.9 EVALUATION UNDER POT AND FIELD CONDITIONS 42 3.10 PLANT ANALYSIS 43 15 3.11 ASSESSMENT OF N2-FIXATION BY NATURAL N 44 3.12 STATISTICAL ANALYSIS 44 4. RESULTS AND DISCUSSION 46 4.1 CHARACTERIZATION FOR PGP TRAITS 46 4.1.1 Indole-3-Acetic Acid (IAA) 46 4.1.2 Phosphate Solubilizations 47 4.1.3 nifH Gene Amplification 48 4.1.4 Siderophore Activity, Catalase, Oxidase and Gram Staining 49 4.2 IDENTIFICATION USING 16S rRNA GENE SEQUENCING 53 4.3 VALIDATION Of NCCP-231T (Kosakonia sp.) 53 4.3.1 Molecular Characterization 57 4.3.2 Characterization for Biochemical and PGP Traits 73 4.3.3 Biochemical and Phenotypic Description 73 8 4.3.4 Chemotaxonomic Characterization 76 4.4 GROWTH CHAMBER EXPERIMENT 82 4.4.1 Wheat Root and Shoot Growth 85 4.4.2 Soybean Root and Shoot Growth 87 4.5 BIOCHEMICAL CHARACTERIZATION OF PGPRs 91 4.6 GREENHOUSE EXPERIMENTS 94 4.6.1 Inoculation Effects of Selected PGPRs on Wheat 95 4.6.2 Inoculation Effects of Selected PGPRs on Soybean 101 4.7 FIELD EXPERIMENTS 105 4.7.1 Inoculation Effects with PGPRs on Growth of Wheat 105 4.7.1.1 Biomass yield 105 4.7.1.2 Grain yield 106 4.7.1.3 Total shoot N yield of wheat 107 4.7.2 Inoculation Effects with PGPRs on Growth of Soybean 109 4.7.2.1 Biomass yield 109 4.7.2.2 Grain yield 110 4.7.2.3 Total shoot N yield of soybean 110 9 4.7.2.4 Total N2-fixed by soybean 112 SUMMARY 114 LITERATURE CITED 119 APPENDICES 150 List of Tables Table No. Page 1. Effect of PGPRs on different crop and their responses 12 2. Phosphate solubilization by PGPRs and crop responses 21 3. Plant growth regulator releases by PGPRs 31 4. PGPRs isolated from different location 39 5. Characterization of PGPRs isolated from soybean and chickpea crop 50 6. Characterization of PGPRs isolated from wheat crop 54 7. Identification of PGPRs based on 16S rRNA gene sequence 58 8. Phenotypic and molecular identification of NCCP-231T 75 9. Biochemical characteristics of NCCP-231T 77 10. Characterization of NCCP-231T isolated for PGP traits 81 11. Cellular fatty acid composition and G+C content of strain NCCP-231T 83 12. Effect of PGPRs possessing high plant growth promoting activity 86 10 13. Effect of PGPRs possessing high plant growth promoting activity 90 14. Biochemical characterization of selected PGPRs by using API 20E 92 15. Biochemical characterization of PGPRs by using API ZYM 93 16. Antibiotic resistant test of PGPRs 96 17. Effect on root and shoot growth of wheat by inoculation of PGPRs 99 18. Effect on root and shoot growth of soybean by inoculation of PGPRs 103 19. Effect on yield of wheat as affected by inoculation of PGPRs 108 20. Effect on yield of soybean as affected by inoculation of PGPRs 111 List of Figures Fig. No. Page 1. Soil phosphorus mobilization and immobilization by PSBs 18 2. Biosynthetic Pathway of IAA Synthesis in Bacteria 24 3. Mechanism to decrease level of ethylene in the root of plants 27 4. Phylogenetic tree of 231, A45, A84 and A85 belonging to Kosakonia 61 5. Phylogenetic tree of A52 belonging to the genus Burkholderia 62 6. Phylogenetic tree of A6 belonging to the genus Arthrobacter 63 7. Phylogenetic tree of A81 belonging to the genus Microbacterium 64 8. Phylogenetic tree of A2, A4 and A5 belonging to the genus Acinetobacter 65 11 9. Phylogenetic tree of A15, A18, A64 and A72 belonging to Psychrobacter 66 10. Phylogenetic tree of A25, A27, A29, A48, A49, A56, A58, A59 and A78 67 11. Phylogenetic tree of A44, A53 and A63 belonging to genus the Serratia 68 12. Phylogenetic tree of A42 and A88 belonging to the genus Pseudomonas 69 13. Phylogenetic tree of A28 belonging to the genus Serratia 70 14. Phylogenetic tree of A35 and A46 belonging to the genus Staphylococcus 71 15. Phylogenetic tree of A61 and A62 belonging to the genus Enterobacter 72 16. Phylogenetic tree of NCCP-231T 74 17. Polar lipid profile of NCCP-231T 84 18. Bacterial antibiotic resistant activity of selected PGPRs. 97 ACKNOWLEDGEMENTS I am thankful to Almighty Allah for blessing me good health, strength and perseverance needed to complete this study. I offer my humble gratitude from the core of my heart to the Holy Prophet Hazrat Muhammad (SAWW) who is forever a torch of guidance for the humanity as a whole. I pay my thanks and respect to my supervisor, Dr. Rifat Hayat, who guided me at each and every step of research. I am also very thankful to members of my supervisory committee, Dr. Iftikhar Ahmed, Prof. Dr. Safdar Ali and Dr. Ghulam 12 Shabbir, for their valuable suggestions and constructive criticism during the study. I wish to extend special thanks to Dr. Wei Xiao, Dr. Yong-Xia Wang and Prof. Dr. Xiao-Long Cui, Yunnan institute of Microbiology, Yunnan University, Kunming, China for helping me to have an in-depth understanding of soil microbiology. Heartiest thanks and deep appreciations are due to my affectionate sisters Shumaila Bilal, Faiza jabeen, Azeema kokab, Brother Abdul Mannan, my best friend Rabia Khalid and other family members for their prayers and sincere support during the study. Last but not least, I am thankful to my husband Mr. Abdul Hadi and my son Abdul Wali Khan for their love and friendly cooperation in my life. (Ummay Amara) 13 ABSTRACT Plant-microbe interaction in the rhizosphere is the determinant of soil fertility and plant health. The beneficial bacteria residing in the surroundings of roots play a vital role in stimulating the plant growth. Soil bacteria are being utilized in agriculture for long times for their ability to improve plant nutrition. The objective of proposed study was to identify the potent PGPRs by using molecular tagging of 16S rRNA gene sequencing. The PGPRs were to be isolated from rhizospheric soil of wheat and soybean. Potential PGPRs were applied alone and in combination with varying rates of fertilizers to determine their impact on yield of crops under controlled and field conditions. For this purpose, extensive survey had been carried out for the collection of rhizospheric soil of legumes (soybean and chickpea) grown in farmer’s field at Pothwar (Rawalpindi, Attock & Chakwal) and for wheat from the districts of Rawalpindi, Attock, Chakwal, Haripur, Abbottabad and Gilgit. Dilution plate technique was use by using Tryptic Soya Agar (TSA; Difco) as nutrient media for the isolation of rhizospheric soil bacteria. More than eighty bacterial strains were isolated and characterized for plant growth promoting (PGP) properties like auxin i.e. indole acetic acid (IAA), P-solubilization, siderophore, and nifH gene amplification. For biochemical characterization of potential isolates, API kits (API ZYM and API 20E) were used.
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