ABSTRACT CHARACTERIZATION OF RHIZOSPHERIC ACTINOMYCETES OF MAJOR CROP PLANTS AND THEIR PLANT GROWTH PROMOTING PROPERTIES UNDER JHUM FIELDS OF MIZORAM A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY MARCY D. MOMIN MZU REGISTRATION NO: 5005 of 2011 PH.D REGISTRATION NO: MZU/PH.D/1021 OF 31.05.2017 DEPARTMENT OF FORESTRY SCHOOL OF EARTH SCIENCES AND NATURAL RESOURCES MANAGEMENT 2020 CHARACTERIZATION OF RHIZOSPHERIC ACTINOMYCETES OF MAJOR CROP PLANTS AND THEIR PLANT GROWTH PROMOTING PROPERTIES UNDER JHUM FIELDS OF MIZORAM BY MARCY D. MOMIN DEPARTMENT OF FORESTRY SUPERVISOR Dr. S. K. TRIPATHI SUBMITTED IN PARTIAL FULLFILMENT OF THE DEGREE OF PHILOSOPHY IN FORESTRY OF MIZORAM UNIVERSITY MIZORAM i DECLARATION I Miss Marcy D. Momin, hereby declare that the subject matter of this thesis is the record of work done by me, that the contents of this thesis did not form basis of the award of any previous degree to me or to do the best of my knowledge to anybody else, and that the thesis has not been submitted by me for any research degree in any other University/Institute. This is being submitted to the Mizoram University for the degree of Doctor of Philosophy in the Department of Forestry. (Marcy D. Momin) (Head) (Supervisor) ii MIZORAM UNIVERSITY Department of Forestry Aizawl – 796004 Prof. S.K. Tripathi Fax: 0389-2330394 Email: [email protected] Mob:09436353773 CERTIFICATE This is to certify that the thesis entitled “Characterization of rhizospheric actinomycetes from major crop plants and their plant growth promoting properties under jhum fields of Mizoram” submitted to the Mizoram University, Aizawl for the award of the degree of Doctor of Philosophy in Forestry is the original work carried out by Miss Marcy D. Momin (Reg. No. MZU/Ph.D./1021 of 31.05.2017) under my supervision. I further certified that the thesis is the result of his own investigation and neither the thesis as a whole nor any part of it was submitted earlier to any University or Institute for the award of any degree. The candidate has fulfilled all the requirements laid down in the Ph.D. regulations of the Mizoram University. His passion oriented hard work for the completion of the research is to be duly appreciated. Date: Prof. S.K. Tripathi Place: (Supervisor) iii TO WHOM IT MAY COMCERN This is to certify that Miss Marcy D. Momin, a Ph.D Scholar, Registration No. MZU/Ph.D./1021 of 31.05.2017 has worked on the thesis entitled “Characterization of rhizospheric actinomycetes from major crop plants and their plant growth promoting properties under jhum fields of Mizoram”. He has fulfilled all criteria the mandatory publication. It is also certified that the scholar has been admitted in the department through an entrance test followed by an interview as per the regulation 2016. Head iv Acknowledgements I owe my deepest thanks and heartfelt gratitude to Dr. S. K. Tripathi Supervisor, Department of Forestry, Mizoram University, Aizawl, Mizoram, for his excellent guidance, assistance and sharing his precious treasure of knowledge throughout my Ph. D research. I express my heartiest gratefulness to Dr. Abhrajyoti Ghosh and his laboratory team, Dept. of Biochemistry, Bose Institute, Kolkata. I also extend my heartfelt thanks to Dr. Awadhesh Kumar and his laboratory team, Dept. of Horticulture and Dept. of Botany, Mizoram University. I am equally thankful to my parents and family who strongly encouraged and supports in my research. At last I am very thankful to Dr. Ngangbam Somen and Dr. Keshav Upadhyay, Miss Shreyosee Ghosh, Department of Forestry, Mizoram University and Mr. Abhijeet Mohanto, Dept. of Botany, Mizoram University and the other faculty members and my laboratory mates, who have shown great co-operation and helping all necessary things during my research, spared their valuable time and gave me constant support and necessary help. Place: Aizawl, Mizoram Marcy D. Momin Date: 02.11.2020 v List of abbreviations and symbols % Percent ºC Degree Celsius g Gram ml Millilitre m Metre mm Millimeter µm Micrometer µl Microlitre µg/ml Microgram per millilitre µg Microgram mM Millilimeters cm2 Centimeter Square w/v Weight/Volume rpm Revolutions per minute O.D. Optical Density nm Nanometer Mins Minutes CFU Colony Forming Unit CAS Chrome Azurol S P Phosphorus K Potassium vi N Nitrogen SOC Soil Organic Carbon NaCl Sodium Chloride DMSO Dimethyl sulfoxide ISP2 International StreptomycesProject 2 ISP1 International Streptomyces Project 1 IAA Indole-3-acetic acid SCA Starch Casein Agar CSM Cross Streak Media L-Tryptophan Levorotatory form of tryptophan KNO3 Potassium nitrate KH2PO4 Potassium dihydrogen phosphate MgSO4.7H2O Magnesium sulphate CaCO3 Calcium Carbonate FeSO4.7H2O Ferrous sulfate heptahydrate K2HPO4 Dipotassium phosphate FeCl3 Iron(III) chloride HClO4 Perchloric acid Ca3 (PO4)2 Calcium phosphate Fe Iron Zn Zinc FePO4 Iron(III) phosphate K-solubilizing bacteria Potassium solubilizing bacteria vii T Treatment C Control ANOVA Analysis of Variance LSD Least Significant Difference SA Surface Area AD Average Diameter AL Average Length FW Fresh Weight DW Dry Weight SE Standard Error AM Aerial Mycelium SM Substrate Mycelium PGP Plant growth-promoting PGPR Plant growth-promoting rhgizobacteria KVK Krishi Vigyan Kendra DNA Deoxyribonucleic acid 16S rRNA 16S ribosomal RNA PCR Polymerase Chain Reaction Tag-polymerase Thermus aquaticus Polymerase MEGA Molecular Evolutionary Genetics Analysis Blast N Basic Local Alignment Search Tool for Nucleotides viii NCBI National Center for Biotechnology Information ix CONTENTS Page numbers Inner cover page i Declaration ii Supervisor’s Certificate iii HOD Certificate iv Acknowledgements v List of abbreviations and vi-ix symbols Contents CHAPTER 1: INTRODUCTION 1-18 1.1.The rhizosphere: structure and function 1-2 1.2.Characteristics of actinomycetes 3-5 1.3.Functions of actinomycetes in the rhizosphere 5-6 1.4.Rhizospheric soil characteristics 7 1.5.Soil fertility in shifting cultivation and actinomycetes 8 1.6.North-Eastern (NE) region 8-9 1.7.Objectives of the study 9 References 10-18 CHAPTER 2: REVIEW OF LITERATURE 19-46 2.1.Microorganisms and their applications 19 i 2.2.General characteristics of actinomycetes 19-20 2.3.Isolation of actinomycetes from soil 20-21 2.4.Identification of actinomycetes 21 2.5. Plant growth promoters 22 2.5.1. Indole acetic acid (IAA) 22-23 2.5.2. Phosphate solubilization 23-24 2.5.3. Siderophores 24-25 2.5.4. Nitrogen fixation 25-26 2.5.5. Enzyme production 26 2.6. Rhizosphere actinomycetes 26-27 2.7. Plant-microbe-interaction 27-28 2.8.Physico-chemical soil characteristics 28-31 2.9. Significance of study 31 References 32-46 CHAPTER 3: MATERIALS AND METHODS 47-55 3.1. Study sites and the collection of samples 47 3.2. Isolation of rhizosphere actinomycetes from soil samples 48-49 3.3. Morphological and Microscopic characterization 49 3.4. In-vitro screening of rhizosphere actinomycetes isolates 49 for plant-growth promoting properties ii 3.4.1. Screening for phosphate solubilization 49 3.4.2. Indole-3-acetic acid (IAA) production 49-50 3.4.3. Siderophore production 50 3.4.4. Ammonia production 50 3.4.5. Nitrogen fixation 50 3.4.6. Amylase production 50 3.4.7. Catalase production 51 3.5. In-vivo screening of actinomycetes for PGP potential 51 3.5.1. Preparation of culture (AB832) 51 3.5.2. Pot experiment 51 3.5.3. Rhizospheric soil characteristics 52 3.6. Genomic DNA extraction, amplification of 16S r RNA gene and sequencing 52 3.7. Phylogenetic analysis 53 References 54-55 CHAPTER 4: RESULTS 56-82 4.1. Isolation of rhizopsheric actinomycetes 56 4.2. Characterization and identification of rhizospheric actinomycetal isolates 57-58 4.3. In-vitro screening of the isolated rhizospheric actinomycetes for plant-growth promoting potential 69 4.3.1. Phosphate solubilization 69 iii 4.3.2. Ammonia production 69 4.3.3. Siderophore production 70 4.3.4. Indole-3-acetic acid (IAA) production 70 4.3.5. Nitrogen fixation 72 4.3.6. Catalase test 71 4.3.7. Amylase production 72 4.4. In-vivo assessment of the selected rhizospheric actinomycetes 73 4.4.1. Seed germination assay 74 4.4.2. Effect of AB832 on plant growth promotion of beans 74-76 4.4.3. Effect of AB832 on plant growth promotion of maize 76-78 4.4.4. Effect of AB832 on rhizospheric soil properties 78-79 4.4.5. Analysis of rhizospheric soil properties of the study area 79-80 4.5. Identification of PGP potential actinomycetes strains by 16S rRNA gene sequencing 80-81 4.6. Phylogenetic analysis of PGP potential actinomycetes strains 81-82 CHAPTER 5: DISCUSSION 83-116 5.1. Problems of soil fertility in shifting cultivation and the role of actinomycetes 83 5.2. Actinomycetes association with plant rhizosphere and their ecological importance 83-84 5.3. Isolation of rhizospheric actinomycetes from crop plants of shifting cultivation 84-85 5.4. In-vitro screening of plant growth-promoting properties of the isolates 86 iv 5.4.1. Phosphate solubilization 87 5.4.2. Ammonia production 87-88 5.4.3. Siderophore production 88 5.4.4. IAA production 88 5.4.5. Nitrogen fixation 89 5.4.6. Amylase and catalase production 89-90 5.5. In-vivo assessment of the selected rhizospheric actinomycetes 90-91 5.5.1. Seed germination assay 91 5.5.2. Effect of root and shoot development 91-93 5.5.3. Effect on rhizospheric soil properties 93-95 5.6. Rhizospheric soil properties analysis of the study area 95 5.6.1. Nitrogen (N) 95-96 5.6.2. Phosphorus (N) 96-97 5.6.3. Potassium (K) 97-98 5.6.4.