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Studies on Rhizosphere Microflora of Mandarin Plants and Their Assessment As Potential Biocontrol Agents Against Root Diseases

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Studies on Rhizosphere Microflora of Mandarin Plants and Their Assessment As Potential Biocontrol Agents Against Root Diseases STUDIES ON RHIZOSPHERE MICROFLORA OF MANDARIN PLANTS AND THEIR ASSESSMENT AS POTENTIAL BIOCONTROL AGENTS AGAINST ROOT DISEASES THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN SCIENCE (BOTANY) UNDER THE UNIVERSITY OF NORTH BENGAL Su6mittea 6y Sri Kuldeep Rai, M.Sc. Professor B.N. Chakraborty and Professor U. Chakraborty DEPARTMENT OF BOTANY UNIVERSITY OF NORTH BENGAL DARJEELING, INDIA - 734013 2011 (, 30I J4"' 4 [ .. MW LJIL • Acknowledgement The significance of the destination reached cannot be of much relevance if the path traveled is not accounted for. The endeavor of undertaking research has been both an intrigumg and eventful process. Moreover, the path of exploration, irrespective of the abil ity of the explorer cannot be embarked upon single handedly. There 1s the need for assistance. teamwork and above all guidance. Hence at the outset I would like to express my gratitude to my supcrv1sors. Professor Bishwanath Chakraborty and Professor Usha C hakrab orty,Immuno-Phytopatholog~ Laboratory, Department of Botany. University of North Bengal. They not only guided me through the steps of the road map leading to the compilation of my thesis but time and agam provided me with tools and teachings that have been helpful in all the spheres of my being. I would also like to put forward my humble gratitude to Dr. Arnab Sen. Head, Department of Botany for providing me with the opportunity to utilize the space and resources of the Department. Moreover, I also want to convey my deep sense of appreciation to all the facult~ members of the department who in some way or the other provided me with amendable mput and insights during my endeavor. I also acknowledge the financial assistance granted by University Grants Commission under Special Assistance Programme. I take the liberty to thank all my Senior Research Scholars, for the help the) endowed me with, during the initial phases. Moreover, my hearty gratitude to field man Late. Mr Sudarshan Tirkcy for his contribution. I also convey my appreciation to Mr. Babul da for his assistance. I wou ld like to express my indebted gratefulness to Mr. Pannalal Dey, Mr. Kiran Sunar. Mr. Utanka Dey, Mr. Arka Pratim Chakraborty. Mrs Sanjita Alley, Miss Swcta Khati and all other scholars without whose support and involvement, attaining th1s milestone \\Ould ha\ e been impossible. Last but not the least I express my appreciation to my family and relatives for the moral support. However I earnestly would like to put across my thankfulness to my dear friends Mr B. K. Rai, Mr. R. Yonlc and Miss P. Ray for selflessly lending me constant support and motivation throughout my work. lmmuno- Phytopathology laboratory Department of Botany, N .B. U. Date : 14-07-201 1 (Kuldeep Rai) Contents 1. Introduction ...................................................................................... 1-6 2. Literature Review ............................................................................... 7-35 3. Material and Methods... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 36-64 3.1. Plant Material........................................................................ 36 3 .2. Fungal culture ...................................................................... .. 36 3 .3. Isolation of microorganisms from Mandarin rhizosphere... .. .. .. .. .. .. .. .. 3 8 3 .4. Isolation and Identification of AM spores from rltizosphere soil.............. 38 3.5 Screening of root for mycorrhizal infection... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 3 9 3.6 Inoculation techniques and disease assessment...·:·............................ 39 3.7. Assessment of mycelial growth.................................................... 40 3.7.1. Solid media .............................................................. 40 3.7.2. Liquid media ............................................................. 41 3.8. Biochemical tests of microorganisms .............................................. 41 3.8.1. Gram reaction ..................................................... : ..... 41 3.8.2. Endospore stain......................................................... 41 3.8.3. Catalase .................................................................. 41 3. 8. 4. Urea digestion... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 41 3.8.5. Caseinhydrolysis ....................................................... 41 3.8.6. Starch hydrolysis ........................ , ............................... 42 3.8.7. Indole test.. ............................................................... 42 3.8.8. Siderophore production ................................................ 42 3.8.9. Chitinase production .................... :.............................. 42 3.8.10. CellulaseProductiou ................................................... 42 3.8.11. Proteaseproduction ................................................... 42 3.8.12. H2S production ......................................................... 42 3. 9. In vitro testing of rhizosphere microorganisms for antagonism toward~ root pathogen .......................................................................... 43 3.9.1 Fungi ........................................................................ 43 3.9.1.1. Solid medium ................................................ 43 3.9.2 Bacteria ..................................................................... 43 3.9.2.1. Solid medium ................................................ 43 3.9.2.2. Liquid medium ............. : ................................ 43 3 .I 0. Scanning Electron Microscopic observation of selected beneficial microorganisms ....................................................................... 44 3.11. Assessment of bacterial growth .................................................... 44 3.12. In vitro Screening and Evaluation of phosphate solubilizing activity of isolated microorganisms ........................................................................ 45 3.12.1. Screening ................................................................ .45 ii 3.12.2. Evaluation ............................................................... 45 3.13. Application ofPGPR ........................... .... :. ................................ 46 3.13.1. Soil drench ............................................................... 46 3.13.2.Foliarspray .............................................................. 46 3 .14. Inocula preparation and application of biocontrol fungi ........................ 46 3.14.1. Al\1F ...................................................................... 46 3.14.2. BCA ...................................................................... 47 3.15. Extraction and estimation of soluble proteins ................................... 47 3.15.1. Mycelia ............................. ." .................................... 47 3.15.2. Root.. .................................................................... 47 3.15.3.Estimationofproteincontent ·····:···································· 48 3.16. SDS-PAGE analysis of soluble proteins ......... :.............................. 48 3.16 .I. Preparation of stock solution........................................ 48 3.16.2. Preparation of gel... .................... , . 49 3.16.3. Sample preparation ..................................................... 50 3.16.4. Electrophoresis ......................................................... 50 3.16.5. Fixing and staining ..................................................... 50 3.17.1nuuunological studies ............................................................. 50 3.17.1. Preparation of antigen ........................................... :.... 50 3.17.1.1. Fungal antigen............................................ 50 3.17.1.2. Root antigen............................................. 51 3.17.2. Raising ofpolyclonal antibodies.................................. 51 3.17.2.1. Rabbits and their maintenance......................... 51 3.17.2.2. lnununization . .. .. 51 3.17.2.3. Bleeding................................................. 51 3.17.3. PurificationoflgG................................................... 52 3.17.3.1 Precipitation ................ ."............................. 52 3.17.3.2 Colwnnpreparation...................................... 52 3.17.3.3 Fraction collection....................................... 52 3.17.4. Inununological assays.............................................. 53 3.17.4.1. Agar gel double diffusion............................... 53 3.17.4.1.1. Preparation ofagarose slides............. 53 3.17.4.1.2. Diffusion..................................... 53 3.17.4.1.3. Washing, staining and drying ofslides... 53 3.17.4.2. Plate trapped antigen coated (PTA) -ELISA.......... 54 3 .17.4.3 .Dot inununobinding assay... 54 3.17.4.4. Western blot analysis ..... : ... ... ... ... .. ... .. ... .... 55 3.17.4.5. Fluorescence antibody staining and microscopy... 55 3.17.4.5.1.. Fungal mycelia . .. 55 3.17.4.5.2.Cross section of mandarin roots and leaves 55 iii 3.18. Isolation of genomic DNA . 56 3 .18.1. Preparation of genomic DNA extraction buffer... 56 3.18.2. Genomic DNA extraction ............. :............................ 56 3.18.3. Purification of genomic DNA....................................... 57 3 .18.4. Measurement of DNA concentration using Spectrophotometry 57 3.18.5. Agarose gel eletrophoresis to check DNA quality............... 57 3.18.5.1. Preparation of DNA samples for electrophoresis... 57 3.18.5.2. Run gel electrophoresis for
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