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Formulation of a Biofertilizer for Salt Tolerant Rice Grown in Khazan Soils, Using Salt Pan Bacteria

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Formulation of a Biofertilizer for Salt Tolerant Rice Grown in Khazan Soils, Using Salt Pan Bacteria Formulation of a biofertilizer for salt tolerant rice grown in khazan soils, using salt pan bacteria A Thesis submitted to Goa University for the Award of the Degree of DOCTOR OF PHILOSOPHY in BIOTECHNOLOGY By Amruta Bartakke Research Guide Prof. Savita Kerkar Goa University Taleigao, Goa June, 2018 Formulation of a biofertilizer for salt tolerant rice grown in khazan soils, using salt pan bacteria A Thesis submitted to Goa University for the Award of the Degree of DOCTOR OF PHILOSOPHY in BIOTECHNOLOGY By Amruta Bartakke Research Guide Prof. Savita Kerkar Goa University Taleigao, Goa June, 2018 Dedicated to…………. My darling daughter Scanned by CamScanner Scanned by CamScanner Scanned by CamScanner Acknowledgement I have great pleasure to express my deep and sincere gratitude to my guide Prof. Savita Kerkar, Head, Biotechnology department, who has devoted her valuable time for guiding me throughout this research. I am extremely thankful for her commitment, generosity, constant encouragement, patience and enthusiasm. I wish to acknowledge Prof. Varun Sahni (Vice Chancellor) and Dr. Satish Shetye (former V.C), Goa University for proving the necessary infrastructure to carry out my research. I would like to gratefully acknowledge the University Grants Commission, India for financial assistance through the NET-JRF/SRF fellowship. I express my gratitude to present and former Deans of Faculty of Life Sciences, Prof. M.K. Janarthanam, Prof. Saroj Bhosle and Prof. G. N. Nayak for their constant support. I am also grateful to my VC’s nominee, Prof. S. Krishnan for his advice and encouragement. I take this opportunity to thank Prof. Usha D Muraleedharan, former Head, Department of Biotechnology, Goa University, for being so caring and always supportive. I also express my profound thankfulness to Prof. U.M.X Sangodkar, Prof. Sanjeev C. Ghadi, Dr. Urmila Barros and Dr. Abhishek Mishra for their valuable support. I express my gratitude to Dr. N.P. Singh (former Director), Dr. E.B. Chakurkar (Director) and Dr. S.B. Barbuddhe (former HR), ICAR-CCARI, Goa for providing the necessary facilities to carry out a part of my research in the soil science laboratory. I owe my sincere gratitude to Dr. Gopal Mahajan (Scientist) for his valuable guidance, comments and suggestions. I also thank Dr. Ashish Latare (SRF), Ms. Ruenna, Ms. Poonam and Ms. Heena for their timely help in the laboratory and Mr. Vithoba and Mr. Siddhesh in the field work. I can’t forget to mention the cooperation extended by Mr. Madhav Kelkar (Asst. Director) Farmers’ Training Centre, Ela, Goa; Mr. Vishwas Shirodkar and Mr. Swapnil Sukhtankar of Ela farms, Goa for certain aspects of my research work. I express my sincere gratitude to Mr. Machindra Kauthankar for take so much interest in my work and permitting me to use his field for the trials of rice cultivation. I also appreciate the assistance and support of his helpers, Mr. Datta Gaokar, Mrs. Sitabai Gaokar and Mr. Mhattru Wandekar. I would also like to thank Mr. Shyam Narayan and Mr. Basavraj for rendering a helpful hand whenever required. I appreciate the ever willing and cheerful assistance of the non-teaching staff of our department. I express my thankfulness to Serrao, Martin, Ruby, Neelima, Tulsidas, Ulhas, Sameer, Rahul, Parijat, Amonkar and Sandhya. I wish to thank my labmates Imran, Judith, Preethi, Ruchira, Alisha, Parentho, Pingal, Nicola, Delicia, Sreekala, Manasi and Priti for the jovial working environment in the lab. A special mention of my seniors, Tonima, Kuldeep, Flory, Krupali, Rupesh, Poonam and Asha for their support. Words would not suffice to thank my dearest friends and fellow researchers Michelle, Samantha, Kirti, Priyanka, Shuvankar and Lillyanne. I appreciate the love, support and encouragement given by them at all times. My personal gratitude goes to Prof. S.J. Godse, a fatherly figure in my life, for his incessant help, support and encouragement. I owe my deepest gratitude to my parents, brothers and in-laws whose strong belief in me, constant motivation and blessings has brought me to this place. I am falling short of words to appreciate the sacrifice, encouragement and efforts put in by my husband Chaitanya for the completion of this research work. A special thanks to my daughter Kimaya for her affection and thoughtfulness. I appreciate the love and understanding of my friends. I also place on record, my sense of gratitude to one and all who directly or indirectly lent their helping hand completion of this thesis. Last, but surely not the least, I would like the Almighty for bestowing me with the physical and mental capacity to carry out this research work. Amruta Bartakke Contents Abbreviations List of tables List of figures List of plates CHAPTER 1. Introduction 1 - 7 CHAPTER 2. Review of literature 8 - 30 2.1 Soil salinity 2.2 Coastal saline soils 2.2.1 Khazan lands 2.2.2 Rice cultivation in coastal saline soils 2.3 Biofertilizers 2.3.1 Plant growth promoting rhizobacteria 2.3.2 Biofertilizers for saline soils 2.4 Formulations of biofertilizers CHAPTER 3. Materials and Methods 31 - 65 3.1. Sampling site 3.2. Sampling of saltpan biofilm 3.3. Analysis of saltpan water 3.4. Bacteriological analysis of the biofilm 3.4.1. Acridine orange direct counts (AODC) 3.4.2. Culturable Bacterial counts 3.4.2.1.Aerobic counts 3.4.2.2.Anaerobic counts 3.4.2.3.Sulphate reducing bacterial (SRB) counts 3.5. Isolation of halotolerant biofilm bacteria 3.6. Maintenance and storage of isolates 3.7. Screening of biofilm bacteria for plant growth promoting activity 3.7.1. Indole acetic acid production 3.7.2. Phosphate solubilization 3.7.2.1.Plate assay 3.7.2.2.Quantitative assay 3.7.3. Production of extracellular enzymes 3.7.3.1.Amylases 3.7.3.2.Proteases 3.7.3.3.Lipases 3.7.3.4.Cellulases 3.7.3.5.Pectinases 3.7.4. Nitrogen fixation 3.7.5. Ammonia production 3.7.6. HCN production 3.8. Assessing the biocompatibility between the bacterial isolates 3.9. In vitro synergistic effect of selected consortia of bacteria 3.10. Identification of bacterial isolates 3.10.1. Cell morphology and Gram character 3.10.2. Spore staining 3.10.3. Motility test 3.10.4. Salt tolerance 3.10.5. Catalase test 3.10.6. Oxidase test 3.10.7. Cell size 3.10.8. Biochemical tests 3.10.9. Carbohydrate utilization tests 3.10.10. Metabolic fingerprinting 3.10.11. Antibiotic susceptibility test 3.10.12. Molecular identification 3.11. Preparation of bacterial suspension for in vitro and in vivo assays 3.12. Seed germination and root elongation assay 3.13. Pot trial 3.13.1. Collection of Khazan soil from different fields 3.13.2. Physicochemical parameters of soil 3.13.2.1. Soil pH and electrical conductivity 3.13.2.2. Soil organic carbon 3.13.2.3. Soil available nitrogen 3.13.2.4. Soil available phosphorous 3.13.2.5. Exchangeable calcium and magnesium 3.13.2.6. Exchangeable cations 3.13.2.7. CaCl2 extractable sulphur 3.13.2.8. Soil available boron 3.13.2.9. Soil available micronutrients 3.13.3. Collection and pre-processing of soil pot trial 3.13.4. Preparation of bacterial inoculants 3.13.5. Design of pot experiment 3.13.6. Analysis of growth parameters of rice 3.13.7. Analysis of soil parameters 3.13.7.1. Physicochemical properties 3.13.7.2. Biological properties 3.13.7.2.1. Basal soil respiration 3.13.7.2.2. Soil microbial biomass carbon (Cmb) 3.13.7.2.3. Metabolic quotient and fraction of the SOC as SMBC (CmbSOC) 3.13.7.2.4. Dehydrogenase activity 3.13.7.2.5. Phosphatase activity 3.13.7.2.6. Urease activity 3.14. Formulation of biofertilizers 3.14.1. Large scale production of bacterial suspension 3.14.2. Preparation of carrier for biofertilizers 3.14.3. Preparation of biofertilizers 3.14.4. Shelf life of biofertilizers 3.15. In situ field trial of prepared biofertilizer on rice (Oryza sativa L. var. Korgut) 3.15.1. Design of field trial 3.15.2. Analysis of physicochemical and biological properties of soil 3.15.3. Determination of yield 3.15.3.1. Grain yield 3.15.3.2. Straw yield 3.15.3.3. Harvest index 3.15.4. Determination of plant growth and yield components 3.15.5. Analysis of the grains 3.15.5.1. Gel consistency 3.15.5.2. Carbohydrate content 3.15.5.3. Protein content 3.15.5.4. Water uptake 3.16. Statistical analyses of data CHAPTER 4. Results 66 -143 4.1 Characteristics of the biofilm 4.2 Analysis of saltpan water 4.3 Bacteriological analysis of the biofilm 4.3.1 Acridine orange direct counts 4.3.2 Culturable bacterial counts 4.4 Isolation of halotolerant biofilm bacteria 4.5 Screening of biofilm bacteria for plant growth promoting activity 4.6 Biocompatibility between the bacterial isolates 4.7 In vitro synergistic effect of selected consortia of bacteria 4.8 Identification of selected bacterial isolates 4.9 Preparation of bacterial suspension for in vitro and in vivo assays 4.10 Seed germination and root elongation assay 4.11 Pot trial 4.11.1 Selection of Khazan soil 4.11.2 Pot trial with rice – Oryza sativa cv. CSR 27 4.11.2.1 Effect of bacterial inoculation and organic amendment on plant growth 4.11.2.2 Effect of bacterial inoculation and organic amendment on chemical properties of soil 4.11.2.3 Effect of bacterial inoculation and organic amendment on biological activity of soil 4.11.3 Pot trial with rice – Oryza sativa var.
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