BIOANTAGONISTIC ACTIVITY OF PLANT GROWTH PROMOTING RHIZOBACTERIA (PGPR) AGAINST MELOIDOGYNE JAVANICA FOR THE CONTROL OF ROOT KNOT DISEASE OF TOMATOES By JAVED ASGHAR TARIQ M.Sc. (Hons) Agri. A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN PLANT PATHOLOGY FACULTY OF AGRICULTURE UNIVERSITY OF AGRICULTURE FAISALABAD 2008 ACKNOLEDGEMENTS I acknowledge with deep respect and gratitude the sincere advice and inspiring gratitude extended by Dr. M. Inam-ul- Haq Associate Professor Department of Plant Pathology University of Agriculture Faisalabad for his supervision and guidance throughout the course of this study. I am also grateful to Dr. Fauzia Yusuf Hafiz Prof./Head of the Department of BioSciences under whose guidance the work has been completed. She not only provided me all the lab facilities but also encouraged me to complete this new project in Plant Microbiology Division the NIBGE as Co-Supervisor /Head Plant Microbiology Division NIBGE Faisalabad. Moreover, Dr. Shahbaz Talib Sahi, Associate Professor Department of Plant Pathology University of Agriculture Faisalabad. and also Dr. Mumtaz Khan, Associate Professor Institute of Horticultural Sciences, University of Agriculture Faisalabad and Dr. Safdar Ali Anwar HEC Foreign Professor Department of Plant Pathology are also highly acknowledged for their valuable suggestions. The utmost cooperation of all administration of NIBGE and especially my boss Ms Sumera Yasmeen Senior Scientist NIBGE and my colleges, Mr Aamir Mahmood, Zahoor ul Hassan, Mr Nadeem Hassan Ph.D scholars, Mr. Tariq Mehmood, Mr Tariq Shah PSA, Mr Naveed Ahmed SA-1,Mr Shahzad are also acknowledged. I am also thankful to Director NIBGE Dr. Zafar M. Khalid and Dr. Sohail Hamid, P.S/Head Plant Microbiology Division for their cooperation in completion of my degree. CONTENTS Sr.No. Title Page # Acknowledgement List of Tables List of Figures 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 5 Biological control 5 Mechanisms of biocontrol 8 Siderophores 9 Competition for nutrients 10 Parasitism 10 Plant growth promoting rhizobacteria and root knot nematode 12 Brief history of root knot nematodes ( Meloidogyne spp) 12 Symptoms 13 Life cycle 13 Over view of management of root knot nematodes 15 Management by use of resistant cultivars 15 Use of chemicals 17 The incorporation of organic material 19 Biological control by fungi 20 Biological control of root knot nematodes by using PGPR 22 Mode of action 23 Identification of bioantagonistic bacteria 24 3 MATERIALS AND METHODS 26 3.1 Collection of Root Samples 26 3.2 Sampling from tomato plants. 26 3.3 Extraction of egg masses from galled roots 27 3.4 Counting the number of nematode eggs 27 3.5 Quantitative estimation of nematodes in infested roots 27 3.6 Picking of nematodes 28 3.7 Killing of nematodes 28 3.8 Fixing of nematodes 28 3.9 Preparation of mounts (slides) 28 3.10 Processing of root samples 30 3.11 Identification of root knot nematode on the basis of perineal 31 pattern 3.12 Mass culturing of root-knot nematodes 31 3.13 Isolation of plant growth promoting rhizobacteria 31 3.14 Morphological characters of bacterial isolates 32 3.15 Studying growth promoting characteristics of plant growth 32 promoting rhizobacteria 3.16 Antagonistic activity of PGPRs against Meloidogyne javanicain- 34 vitro. 3.17 Detection for metabolites 34 3.18 Molecular identification of potent strains 36 3.19 Efficacy of Pspudomonas fluorescens (Rh37) and Bacillus subtilis 39 (Asr38) against Meloidogyne javanica under controlled conditions (Growth chamber) 3.20 Efficacy of Pspudomonas fluorescens (Rh37) and Bacillus subtilis 40 (Asr38) against Meloidogyne javanica in net house 4 RESULTS 42 4.1 Collection of samples from district Barkhan 42 4.2 Isolation and Morphological Identification of bacterial isolates. 42 4.3 Phosphate solubilization 45 4.4 Quantitative estimation of mobilized phosphate: 45 4.5 Phytohormone (IAA) production 45 4.6 Nematicidal activity of bacterial strains 45 4.7 Metabolite secretion 48 4.8 Effect of rhizobacterial isolates (Pseudomonas fluorescence and 56 Bacillus subtilus on the growth parameters & severity of root knot disease in tomato in controlled conditions (growth chamber) 4.9 Effect of rhizobacterial isolates (Pseudomonas fluorescence and 59 Bacillus subtilus on the growth parameters & severity of root knot disease in tomato in net house conditions 5 DISCUSSION 68 6 SUMMARY 74 7 LITERATURE CITED 76 LIST OF APPEDICES 99 LIST OF TABLES Sr.No. Title Page # 1 Collection of samples 43 2 Morphological characterization of PGPR 44 3 Characteristics of rhizobacteria for phosphate solubilization and 46 indole acetic acid production 4 Antagonistic activity of PGPRs against root knot nematodes in- 47 vitro 5 Detection of the ability of six selected rhizosphere bacteria 49 isolated from tomato for the production of hydrogen cyanide, chitinases and proteases 6a Results of molecular identification of bacteria, Sequence of 53 16sRNA gene of the antagonistic strain using M13 primer 6b Quantification of proteases 55 7 Performance of plant growth and Meloidogyne javanica on 62 tomatoes when rhizobacterial isolates were applied in growth chamber 8 Performance of plant growth and Meloidogyne javanica on 65 tomatoes when rhizobacterial isolates were applied in nethouse LIST OF FIGURES Fig.No. Title Page # 1 HCN detection 50 2 Chitinase detection producer strain 51 3 Protease detection 52 LIST OF APPENDICES Appendix Title Page # # 1 Analysis of variance showing effect of bacterial isolates their 99 modes of applications on plant height of tomatoes under controlled conditions 2 Analysis of variance showing effect of bacterial isolates their 100 modes of applications on fresh weight of shoot of tomatoes under controlled conditions 3 Analysis of variance showing effect of bacterial isolates their 101 modes of applications on dry weight of shoot of tomatoes under controlled conditions 4 Analysis of variance showing effect of bacterial isolates their 102 modes of applications on fresh weight of root of tomatoes under controlled conditions 5 Analysis of variance showing effect of bacterial isolates their 103 modes of applications on dry weight of root tomatoes under controlled conditions 6 Analysis of variance showing effect of bacterial isolates their 104 modes of applications on number of juveniles/250 gram of soil tomatoes under controlled conditions 7 Analysis of variance showing effect of bacterial isolates their 105 modes of applications on number of juveniles /1 gram fresh weight of root of tomatoes under controlled conditions 8 Analysis of variance showing effect of bacterial isolates their 106 modes of applications on egg masses of tomatoes under controlled conditions 9 Analysis of variance showing effect of bacterial isolates their 107 modes of applications on gall index of tomatoes under controlled conditions 10 Analysis of variance showing effect of bacterial isolates their 108 modes of applications on plant height of tomatoes under net house conditions 11 Analysis of variance showing effect of bacterial isolates their 109 modes of applications on fresh weight of shoot of tomatoes under net house conditions 12 Analysis of variance showing effect of bacterial isolates their 110 modes of applications on dry weight of shoot of tomatoes under net house conditions 13 Analysis of variance showing effect of bacterial isolates their 111 modes of applications on fresh weight of root tomatoes under net house conditions 14 Analysis of variance showing effect of bacterial isolates their 112 modes of applications on dry weight of root of tomatoes under net house conditions 15 Analysis of variance showing effect of bacterial isolates their 113 modes of applications on number of juveniles/250 g of soil under net house conditions 16 Analysis of variance showing effect of bacterial isolates their 114 modes of applications on number of juveniles/1 gram fresh weight of root of tomatoes under net house conditions 17 Analysis of variance showing effect of bacterial isolates their 115 modes of applications on number of egg masses of tomatoes under net house conditions 18 Analysis of variance showing effect of bacterial isolates their 116 modes of applications on gall index of tomatoes under net house conditions To The Controller of Examinations, University of Agriculture, Faisalabad We, the supervisory committee, certify that contents and form of thesis submitted by Mr. Javed Asghar Tariq, Reg. No. 92-ag-1411, have been found satisfactory and recommend that it is processed for evaluation by the external examiner (s) for the award of degree. SUPERVISORY COMMITTEE CHAIRMAN (Dr. M. Inam-ul-Haq) CO-SUPERVISOR (Dr. Fauzia Yusuf Hafeez) MEMBER (Dr. Shahbaz Talib Sahi) MEMBER (Dr. M. Mumtaz Khan) CHAPTER-1 INTRODUCTION In order to meet the challenges of providing food to the ever increasing population of Pakistan, there is an urgent need to boost crop yield. Among the many different crops grown in Pakistan, the importance of tomatoes (Lycopersicum esculentum Mill ) as a food crop can not be over emphasized. It has the highest amount of vitamins among all the vegetables, has anticancerrous properties, and is good for diabetic patients. Its fruit can be used either as such or as tomato products such as juice and sauce, etc. It is cultivated over an area of 4528519 hac with the production of 124748282 million tones on global level. In Pakistan it is cultivated over an area of 46.2 thousand hac with the production of 468.1 thousand tones giving yield of 10.1 tones per hac. (Statistical year book of Pakistan, 2005-2006). This yield is quite low as compared to other developing countries like Iran, India, and Bangladesh. There are number of reasons for this low production of tomatoes in Pakistan, with pathogenic nematodes and other soilborne diseases constituting the most important limiting factors. Nematodes are tiny worms, lower invertebrates, highly diversified perhaps the most numerous multicellular animals on the earth. The nematode is a probably one of the oldest existing life forms dating back millions of years.