TABLE OF CONTENTS CHAPTER NO. TITLE PAGE NO. LIST OF TABLES LIST OF FIGURES 1 INTRODUCTION AIM AND SCOPE OF THE PRESENT INVESTIGATION 7

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1 viii TABLE OF CONTENTS ABSTRACT LIST OF TABLES LIST OF FIGURES iii xxiii xxviii 1 INTRODUCTION AIM AND SCOPE OF THE PRESENT INVESTIGATION 7 2 LITERATURE REVIEW AN OVERVIEW OF TEA TEA CULTIVATION IN SOUTHERN INDIA BIOTIC AND ABIOTIC FACTORS FOR TEA PLANT GROWTH ROLE OF FERTILIZERS IN TEA PLANTS PLANT GROWTH REGULATORS IN TEA PLANTS TEA DISEASES RED ROOT DISEASE OF TEA PLANTS PLANT GROWTH PROMOTING RHIZOBACTERIA: AN ALTERNATIVE APPROACH FOR CHEMICAL FERTILIZERS MECHANISM OF PLANT GROWTH PROMOTING RHIZOBACTERIA AND ITS SIGNIFICANCE 16

2 ix 2.10 FLUORESCENT PSEUDOMONAS AS PLANT GROWTH PROMOTING RHIZOBACTERIA AND BIOCONTROL AGENTS Pseudomonas fluorescens as Plant Growth Regulator and Phosphate solubilizer Siderophore and Hydrogen Cyanide Production by Pseudomonas fluorescens Antibiosis and Production of Antimicrobial Metabolites Induced Systemic Resistance Bioformulation of Pseudomonas fluorescens 21 3 MATERIALS AND METHODS SURVEY OF RED ROOT ROT DISEASE Collection of Infected Root Samples ISOLATION, BIOCHEMICAL 23 CHARACTERIZATION AND MOLECULAR IDENTIFICATION OF PSEUDOMONAS SPP. FROM TEA SOILS Site of soil collection Edaphic and Environmental Analysis of Soil Samples Determination of various Soil Edaphic Parameters Estimation of Total Organic Carbon Estimation of Total Nitrogen 25

3 3.2.6 Estimation of available Phosphorus and Exchangeable Potassium Determination of Exchangeable Calcium, Sodium and Magnesium Isolation of Pseudomonas spp. from Tea Soil Biochemical Characterization of Pseudomonas spp Molecular Identification of Pseudomonas spp Isolation of genomic DNA from Pseudomonas fluorescens strains Amplification, sequencing and RAPD analysis of DNA of Pseudomonas fluorescens strains SDS PAGE analysis of protein pattern of Pseudomonas fluorescens strains EFFECTS OF BIOTIC AND ABIOTIC FACTORS x

4 xi Effects of different Carbon, Nitrogen, Amino acids and Vitamin Sources on Growth of Pseudomonas fluorescens Strains Effects of different ph, Temperature, Light sources and NaCl Concentration on the growth of Pseudomonas fluorescens Strains Antibiotic Resistance of Pseudomonas fluorescen Strains IN VITRO TESTS FOR PLANT GROWTH PROMOTING TRAITS AND ANTAGONISTIC ACTIVITIES OF PSEUDOMONAS FLUORESCENS STRAINS Estimation of Indole acetic acid Detection of Indole acetic acid in Thin Layer Chromatography Estimation of Gibberellic Acid Siderophore Production Phosphate Solubilisation Hydrogen Cyanide Production Ammonia Production Salycylic Acid Production 34

5 xii Extraction of Root Proteins and SDS-PAGE Analysis Dual Culture Technique Screening and Assay of Hydrolytic Enzymes Efficiency of crude culture filtrate of Pseudomonas fluorescens against Poria hypolateritia through Food Poisoned Technique Solvent Extraction of Crude Metabolites of Pseudomonas fluorescens VP5 strain against Poria hypolateritia by Agar well Diffusion Method Characterization of Secondary Metabolites of Pseudomonas fluorescens VP5 Strain through Gas Chromatography- Mass Spectrometry Method Sensitivity of selected Pseudomonas fluorescens Strains to Fungicides Shelf -life of Pseudomonas fluorescens VP5 Strain in different Formulations GREENHOUSE EXPERIMENTS Preparation of Nursery Sleeves for Pathogen Induction and Treatment Application Assessment of Disease Incidence and Disease Reduction Measurement of Plant Growth Parameters 44

6 xiii Analysis of Macroelements contents in Foliage Analysis of Leaf Biochemical Constituents RED ROOT ROT DISEASE MANAGEMENT THROUGH FIELD TRAIL EXPERIMENTS Field Location Selection of Fungicides and Botanicals Preparation of Bioformulations Disease assessment Nutrient Status and Analysis of Soil Edaphic Parameters on Poria hypolateritia Infested Tea Soils Soil Dehydrogenase and Leaf Nitrate Reductase Activity Preparation of Leaf Material for Enzyme Extract Assay of Phenolics and Phenolic Defensive Enzymes Assay of Peroxidase, Polyphenol oxidase and Catalse Assay of Chitinase, -1,3-glucanase and Protease Enzyme Activity Green Leaf Yield Assessment Estimation of root carbohydrate Response of Plant Growth Parameters 53

7 xiv Physiological Variations Determination of Biochemical Constituents Estimation of total chlorophyll and carotenoids Estimation of total sugars and total nitrogen Estimation of total lipid Estimation of total proteins Estimation of total amino acids Estimation of total polyphenols Estimation of total catechins Tea Quality Statistical Analysis 57 4 RESULTS SURVEY OF RED ROOT ROT DISEASE IN TEA PLANTATIONS Influence of Environmental Parameters on the Population Density of Poria hypolateritia Obtained from Southern Indian Tea Plantations Correlation of Population Density of Poria hypolateritia with Environmental Factors Population density of Poria hypolateritia in Anamallais district 63

8 xv Population density of Poria hypolateritia in Central Travancore district Population density of Poria hypolateritia in High Range district Population density of Poria hypolateritia in Nilgiris districts Population density of Poria hypolateritia in Koppa district ISOLATION, BIOCHEMICAL CHARACTERIZATION AND MOLECULAR IDENTIFICATION OF PSEUDOMONAS SPP. FROM SOUTHERN INDIA TEA SOILS Influence of Edaphic and Environmental Parameters of Tea Soil Samples Enumeration of Pseudomonas spp. from Tea Soil Samples Impact of Seasonal Variation on the Population Dynamics of Pseudomonas spp Correlation of Bacterial Population Density with Edaphic and Environmental Factors Morphological and Biochemical Characterization of Pseudomonas spp Molecular Identification of Pseudomonas spp. 81

9 xvi Protein Profile of Pseudomonas fluorescens Strains BIOTIC AND ABIOTIC FACTORS AFFECTING GROWTH OF PSEUDOMONAS FLUORESCENS STRAINS Standardization of Media for Mass Multiplication of Pseudomonas fluorescens strains Growth Kinetics Study of Pseudomonas fluorescens strains Growth Trend of Pseudomonas fluorescens Strains in the Tea Ecosystem Influence of different Carbon Sources on the growth of Pseudomonas fluorescens Strains Impact of different Nitrogen Sources on the growth of Pseudomonas fluorescens Strains Determination of the Abilty to Utilize different Amino Acids and Vitamins Sources by Pseudomonas fluorescens Strains Effect of different ph and Temperature Regimes on the growth of Pseudomonas fluorescens Strains Influence of different Light Sources on the growth of Pseudomonas fluorescens Strains 94

10 xvii Growth response of Pseudomonas fluorescens Strains at different of Sodium Chloride Concentration Plasmid Mediated Antibiotic Resistance Pattern of Pseudomonas fluorescens Strains Isolated from Tea Rhizosphere Soils PLANT GROWTH PROMOTING TRAITS AND ANTAGONISTIC ACTIVITIES OF PSEUDOMONAS FLUORESCENS STRAINS Study of Plant Growth Promoting Traits Exhibited by Pseudomonas fluorescens Strains under in vitro Conditions Phosphatase Enzymes Produced by Pseudomonas fluorescens Strains from Southern India Pathogen Isolation and Identification Micromorphology of Poria hypolateritia Protein Profiles of Healthy and Infected Roots Antifungal Activity of Pseudomonas fluorescens Strains against Poria hypolateritia Dual culture technique Inhibitory effect of bacterial strains on fungal hyphal morphology Hydrolytic enzymes secreted by Pseudomonas fluorescens strains 111

11 xviii Effects of crude culture filtrates of Pseudomonas fluorescens strains on growth of Poria hypolateritia Assessment of solvent extracts of crude metabolites of Pseudomonas fluorescens VP5 strain against Poria hypolateritia using an agar well diffusion method Gas Chromatography Mass Spectrometry Analysis of Secondary Metabolites Produced by Pseudomonas fluorescens VP5 Strain Compatibility of Fungicidal Resistance of Pseudomonas fluorescens Strains Screening of suitable Carriers for Pseudomonas fluorescens VP5 Strain EVALUATION OF FUNGICIDES AND BIOCONTROL AGENTS FOR CONTROL OF PORIA HYPOLATERITIA UNDER GREENHOUSE CONDITION Management of Red Root Rot Infection by Applying Fungicides and Biocontrol Agents on Nursery Grown Tea Plants Rooting Systems of Tea Clone in Response to the Application of Fungicides and Biocontrol Agents 120

12 xix Influence of Fungicides and Biocontrol Agents on Plant growth Promotion of UPASI-9 Tea Clone Impact of Fungicides and Bioformulations on Plant Growth Parameters Analysis of Macronutrient status in Leaves of Tea Plants Treated with Fungicides and Biocontrol Agents Impact of Fungicides and Biocontrol Treatments on Biochemical Constituents of Foliage Population Dynamics of Biocontrol Agents in Pathogen Infested Soil Samples of Nursery Sleeves containing tea plants EVALUATION OF INDIGENOUS PSEUDOMONAS FLUORESCENS AGAINST RED ROOT ROT DISEASE OF TEA PLANTS UNDER FIELD CONDITIONS Influence of Fungicides, Botanicals and Biocontrol Agents on Nutrient Status and Analysis of Soil Edaphic Parameters on Red Root Infested Soils Impact of Application of Fungicides and Biocontrol agents on Soil Dehydrogenase and Leaf Nitrate Reductase Activity in Treated and Untreated Poria hypolateritia -Infected Plants 135

13 xx Phenolics and Phenolic Defensive Enzymes in Treated and Untreated Red Root Rot Infected Tea Plants Differences in Redox Defensive Enzymes between Treated and Untreated Red Root Rot Infected Tea Plants Stimulation of Pathogen-Related Enzymes in Treated and Untreated Red Root Rot Infected Tea Plants Red Root Rot Disease Protection and Green Leaf Yield Responses of Treated Plants to Fungicides and Biocontrol Agents Physiological Responses of Treated Plants to Fungicides and Biocontrol Agents Effects of Fungicides and Biocontrol Agents Application on Biochemical Parameters Analysis of Tea Quality Parameters between Treated and Untreated Flush Shoots Population Dynamics of Biocontrol Agents in field soil DISCUSSION RED ROOT ROT INCIDENCE IN SOUTHERN INDIA TEA PLANTATIONS 153

14 xxi 5.2 NUTRIENT STATUS AND POPULATION DENSITY OF FLUORESCENT PSEUDOMONAS SPP. IN TEA SOIL OF SOUTHERN INDIA MORPHOLOGICAL, AND BIOCHEMICAL CHARACTERISTICS, AND MOLECULAR IDENIFICATION OF PSEUDOMONAS SPP EFFECT OF BIOTIC AND ABIOTIC FACTORS AFFECTING GROWTH OF PSEUDOMONAS FLUORESCENS STRAINS IN VITRO PLANT GROWTH PROMOTING TRAITS OF PSEUDOMONAS FLUORESCENS STRAINS IN VITRO ANTAGONISTIC ACTIVITIES OF PSEUDOMONAS FLUORESCENS STRAINS EVALUATION OF EFFECTS OF INDIGENOUS PSEUDOMONAS FLUORESCENS STRAIN AGAINST PORIA HYPOLATERITIA UNDER GREENHOUSE CONDITION EVALUATION EFFECTS OF INDIGENOUS PSEUDOMONAS FLUORESCENS STRAIN AGAINST RED ROOT ROT DISEASE OF TEA PLANTS UNDER FIELD CONDITIONS 167

15 xxii 6 CONCLUSIONS AND FUTURE WORK 171 APPENDIX REFERENCES 181 LIST OF PUBLICATIONS 198 CURRICULUM VITAE 199

16 xxiii LIST OF TABLES TABLE NO. TITLE PAGE NO. 2.1 List of fungal root diseases and causal agents in tea plants Treatment details of greenhouse experiments Treatment details of field experiments Mean red root rot incidence scores for tea clones and seedlines in different districts, from a survey of the disease carried out in tea plantations of southern India (PDI: %) Classification of susceptibility of different tea clones and seedlines to red root rot, assessed in a survey of tea plantations in different planting districts of southern India Average climatic parameters, and Poria hypolateritia soil populations, in different tea growing districts of southern India Analysis of correlation coefficients between environmental parameters and population densities of Poria hypolateritia in Anamallais and Central Travancore tea planting districts Analysis of correlation coefficients between environmental parameters and population densities of Poria hypolateritia in High Range and Nilgris planting districts 66

17 xxiv TABLE NO. TITLE PAGE NO. 4.6 Analysis of correlation coefficient between environmental parameters and population density of Poria hypolateritia in Koppa tea planting district Mean edaphic parameters measured for different tea growing districts in southern India, and population densities of Pseudomonas spp. isolated from soils in these districts Mean environmental parameters and population densities of Pseudomonas spp. isolated from tea plantations in different districts of southern India Numbers and designations of Pseudomonas spp. isolated from soil samples collected from different locations of southern Indian tea plantations Analysis of correlation coefficients of edaphic parameters in the Valparai tea plantation district with the population density of Pseudomonas spp Analysis of correlation coefficients of environmental parameters in the Valparai tea planting district with the population density of Pseudomonas spp Morphological features of Pseudomonas spp. isolated from southern Indian tea soils Characteristic features of Pseudomonas isolates obtained from different southern Indian tea plantations Accession numbers of Pseudomonas fluorescens strains submitted to NCBI, Maryland, USA Mean optical density measurements for seven Pseudomonas fluorescens strains growing in liquid media containing different tea plant and tea soil extracts 89

18 xxv TABLE NO. TITLE PAGE NO Mean minimum inhibitory concentrations of antibiotic resistance profile of Pseudomonas fluorescens strains isolated from southern Indian tea rhizosphere soils Mean amounts of indole acetic acid and gibberellic acid produced by different Pseudomonas fluorescens strains Mean potential plant growth promoting characteristics measured from broth cultures of different Pseudomonas fluorescens strains Mean sizes of red root rot lesions for Poria hypolateritia isolates, from different tea plantation districts Mean radial growth of three Poria hypolateritia isolates on PDA plates for different periods Culture characteristics of VP03 Poria hypolateritia isolate grown for different periods in potato dextrose agar plates Mean linear growth (mm) of Poria hypolateritia colonies in dual culture plates with different Pseudomonas fluorescens strains Mean specific activity of hydrolytic enzymes from Pseudomonas fluorescens strains on the third day of culture Mean linear growth of Poria hypolateritia in PDA plates amended with crude extracts from Pseudomonas fluorescens broth cultures Solvent extraction of crude metabolites of Pseudomonas fluorescens VP5 against Poria hypolateritia by agar well diffusion method 115

19 xxvi TABLE NO. TITLE PAGE NO Mass spectral analysis of data and names, formulae, molecular masses and potential activities of secondary metabolites produced by Pseudomonas fluorescens VP5 strain Mean colony forming units of different Pseudomonas fluorescens strains assessed at different ppm concentration in six different fungicides Mean numbers of colony forming units of Pseudomonas fluorescens VP5 in six different formulations during storage, assessed at different time intervals (days) after inoculation Mean proportions of disease infection on UPASI-9 tea plants inoculated with Poria hypolateritia and treated with different fungicides and biocontrol bioformulations Mean parameters of root development for tea clone UPASI-9, for plants treated with different fungicides and bioformulations. Soil EC and ph values are also presented for the different treatments Mean tea plant parameters for plants grown in soil infested with Poria hypolateritia then treated with different fungicides or biocontrol treatments Mean nutrient contents in leaves of tea plants grown in soil infested with Poria hypolateritia after treatment with different fungicides or biocontrol treatments Mean contents of biochemical constituents of tea leaves from plants treated with different fungicides and biocontrol agents 131

20 xxvii TABLE NO. TITLE PAGE NO Different mean field soil parameters after treatment with different fungicides or biocontrol treatments. The field soil was naturally infested with Poria hypolateritia Mean disease incidences and tea plant yield parameters for field-grown tea plants treated with different fungicides and biocontrol treatments. Yield is for the period Mean plucking surface areas, numbers of plucking points, leaf moisture and internodal lengths for field-grown tea plants treated with different fungicides or biocontrol treatments, for tea bushes grown during the period Mean physiological parameters in mother leaves of field-grown tea plants, treated with different fungicides and biocontrol treatments, for the period Mean biochemical composition components measured in field-grown tea leaves from plants treated with different fungicides and biocontrol treatments, for the period during Mean tea quality parameters for product harvested from field-grown tea plants treated with different fungicides or biocontrol treatments, for the period Assessments and mean values of tea liquor characteristics from tea harvested from field-grown tea plants treated with different fungicides or biocontrol treatments, for the period

21 xxviii LIST OF FIGURES FIGURE NO. TITLE PAGE NO. 1.1 Symptoms of red root rot Fungicides and biocontrol agents treated UPASI- 9 tea clone grown on nursery sleeves Analysis of correlation coefficients between environmental parameters and population density of Poria hypolateritia in Anamallais tea planting district (Response surface methodology analysis) Analysis of correlation coefficients between environmental parameters and population density of Poria hypolateritia in High Range tea planting district (Response surface methodology analysis) Analysis of correlation coefficients between environmental parameters and population density of Poria hypolateritia in Koppa tea planting district (Response surface methodology analysis) Seasonal variation of soil population densities of Pseudomonas spp. in southern Indian tea plantations A Cultural characteristics of representative Pseudomonas strains obtained from different agroclimatic zones of southern Indian tea plantations B Micrographs of cells Pseudomonas spp (a) Agarose gel showing genomic DNA banding patterns of six Pseudomonas strains (b) Amplified PCR products of six Pseudomonas strains isolated from southern India tea plantations 82

22 xxix FIGURE NO. TITLE PAGE NO. 4.9(A) RAPD gel patterns of Pseudomonas fluorescens isolated from southern Indian tea plantations (B) Dendrogram showing the genetic relationship among six Pseudomonas fluorescens strains based on RAPD analysis Protein profile of Pseudomonas fluorescens strains Mean populations of different Pseudomonas fluorescens strains grown on different broth media Optical density measurements for selected six Pseudomonas fluorescens strains growing in King s B medium Mean optical density values for Pseudomonas fluorescens strains growing in liquid media containing different carbon sources Mean optical density values for Pseudomonas fluorescens strains growing on liquid media containing different nitrogen sources Mean optical density values for Pseudomonas fluorescens strains growing on liquid media containing different amino acids and vitamins Mean optical density values for Pseudomonas fluorescens strains growing on liquid media at different ph and temperature Mean optical density values for Pseudomonas fluorescens strains growing on liquid media at different light sources Mean optical density values for Pseudomonas fluorescens strains growing on liquid media containing different sodium chloride concentration 95

23 xxx FIGURE NO. TITLE PAGE NO In vitro plant growth promoting traits exhibited by Pseudomonas fluorescens strains Thin layer chromotography of indole acetic acid production by different Pseudomonas fluorescens strains detected by Salkowski reagent Mean amounts of acid and alkaline phosphatases produced by different Pseudomonas fluorescens strains Poria hypolateritia infected various parts of tea root system Isolation of pathogen from infected tea root Growth patterns of Poria hypolateritia in solid medium Micromorphology of Poria hypolateritia observed under microscope Mean mycelium dry weight for three Poria hypolateritia isolates (VP03, MP01 and KP05) growing for different periods in liquid medium SDS-PAGE analysis of protein profiles from roots of healthy and Poria hypolateritia infected tea plants Dual culture plates of Poria hypolateritia (left hand white colonies) and different Pseudomonas fluorescens strains (VP5 to MTCC; right hand streaks) at 7, 14, 21 days after inoculation of plates. Three control plates (no Pseudomonas fluorescens) are shown at the extreme right Probable action of cell wall degrading enzymes on mycelium of Poria hypolateritia in dual cultures Zones of inhibition around Pseudomonas fluorescens colonies on plates testing for different hydrolytic enzymes 112

24 xxxi FIGURE NO. TITLE PAGE NO Mean amounts of different hydrolytic enzymes produced by different Pseudomonas fluorescens strains Gas chromatography mass spectrometry chromatogram of secondary metabolites produced by Pseudomonas fluorescens VP5 for antifungal activity Mean root and shoot parameters for UPASI-9 tea plants grown in soil infested with Poria hypolateritia, after treatment with different fungicides or potential biocontrol agents Mean plant dry weight, plant strength, total number branches and leaves for UPASI-9 tea plants grown in soil infested with Poria hypolateritia, after treatment with different fungicides or potential biocontrol agents Mean proportions of amino acids (a), polyphenol and catechin (b) in tea leaves from plants treated with fungicides and biocontrol agents Mean populations of biocontrol organisms in tea soil samples during Mean total soil microbial activity (mg CO 2 produced; a) and mean leaf nitrate reduction (µmol of NO 2 /g FW/h; b), after different treatments were applied to field soil infested with Poria hypolateritia Mean amounts of phenolics (a), trans cinnamic acid (b) and P-coumaric acid (c) in leaves from red root infected tea plants after treatment with different fungicides and biocontrol agents 138

25 xxxii FIGURE NO. TITLE PAGE NO Mean values of the redox defensive enzymes peroxidase (a), polyphenol oxidase (b) and catalase (c) in leaves from red root tea plants after treatment with different fungicides and biocontrol treatments Mean values of pathogen related enzymes chitinase (a), -1,3- glucanse (b) and protease (c) in leaves from red root infected tea plants after treatment with different fungicides and biocontrol treatments Mean numbers of individual biocontrol agents in field soils sampled at different periods after treatment with three different biocontrol agents 151