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Characterization and Management of Myrothecium Roridum Associated with Myrothecium Leaf Spot Disease of Momordica Charantia L. (Bitter Gourd) in Punjab, Pakistan

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Characterization and Management of Myrothecium Roridum Associated with Myrothecium Leaf Spot Disease of Momordica Charantia L. (Bitter Gourd) in Punjab, Pakistan CHARACTERIZATION AND MANAGEMENT OF MYROTHECIUM RORIDUM ASSOCIATED WITH MYROTHECIUM LEAF SPOT DISEASE OF MOMORDICA CHARANTIA L. (BITTER GOURD) IN PUNJAB, PAKISTAN SUMERA NAZ INSTITUTE OF AGRICULTURAL SCIENCES UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN 2015 CHARACTERIZATION AND MANAGEMENT OF MYROTHECIUM RORIDUM ASSOCIATED WITH MYROTHECIUM LEAF SPOT DISEASE OF MOMORDICA CHARANTIA L. (BITTER GOURD) IN PUNJAB, PAKISTAN The thesis submitted to the University of the Punjab, Lahore in partial fulfillment of the requirement for the degree of doctor of philosophy in Agricultural Sciences (Plant Pathology) By Sumera Naz Supervisors Dr. Salik Nawaz Khan Dr. Ghulam Mohy-ud-Din INSTITUTE OF AGRICULTURAL SCIENCES UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN 2015 CERTIFICATE This is to certify that the research work entitled entitled “Characterization and management of Myrothecium roridum associated with Myrothecium leaf spot disease of Momordica charantia L. (bitter gourd) in Punjab, Pakistan” described in this thesis by Ms. Sumera Naz, is an original work of Ph.D. scholar and has been carried out under my direct supervision. I have personally gone through all the data, results, materials reported in the manuscript and certify their correctness and authenticity. I further certify that the material included in this thesis has not been used in part or full in a manuscript already submitted or in the process of submission in partial or complete fulfillment of the award of any other degree from any institution. I also certify that the thesis has been prepared under our supervision according to the prescribed format and endorse its evaluation for the award of Ph.D. degree through the official procedure of the University of the Punjab, Lahore, Pakistan. Here, thesis is in pure academic language and it is free from typos and grammatical errors. Supervisor: Dr. Salik Nawaz Khan Assistant Professor IAGS, PU, Lahore Co-supervisor: Dr. Ghulam Mohy-ud-Din Director/Plant Pathologist AARI, Jhang Road, Faisalabad Date: ____________ i DECLARATION CERTIFICATE This thesis entitled “Characterization and management of Myrothecium roridum associated with Myrothecium leaf spot disease of Momordica charantia L. (bitter gourd) in Punjab, Pakistan” which is being submitted for the award of degree of Ph.D. in the University of the Punjab does not contain any material which has been submitted for the award of Ph.D. degree in any University and, to the best of my knowledge and belief, neither does this thesis contain any material published or written previously by another person, except when due reference is made to the source in the text of the thesis. (Sumera Naz) Ph.D. Scholar ii Dedicated to My Beloved Parents, Mr. & Mrs. Muhammad Ilyas Chattha & Siblings Qaisar Ilyas (Late), Amara Ilyas & Yasir Ilyas Who are symbol of my strength & confidence, their prayers & love enabled me to reach this milestone in life iii ACKNOWLEDGMENTS All the praises and respects to Almighty Allah-the only creator of the Universe, and to His Holy Prophet Muhammad (Peace be upon him) who is forever beacon of knowledge and guidance for humanity as a whole. I have no words to express my deepest sense of gratitude to Almighty Allah, Who, in spite of copious obscurity and acute frustrations, enabled me to complete this thesis. First and foremost, I would like to express my profound gratitude to my respected research supervisors Dr. Salik Nawaz Khan, Assistant Professor, Institute of Agricultural Sciences, University of the Punjab, Lahore and Dr. Ghulam Mohy-ud-Din, Director Plant Pathology Section, Ayub Agriculture Research Institute, Faisalabad for their enthusiastic concern, valuable supervision and invaluable suggestions. Their guidance enabled me to complete my work successfully. I would like to extend my special thanks to Dr. M. Saleem Haider, Professor and Director, Institute of Agricultural Sciences, University of the Punjab, Lahore for his sincere facilitation without which completion of this work was not possible. I am thankful to Dr. Naureen Shahrukh, Assistant Professor and Incharge First Fungal Culture Bank of Pakistan, for critically reviewing the manuscript. The help rendered by all faculty members, especially Dr. Arshad Javaid and Dr. Sajid Ali, Institute of Agricultural Sciences, University of the Punjab, Lahore is gratefully acknowledged. I don’t find words to express my sincerest and limitless thanks to Dr. Muhammad Najeebullah, Ginger Botanist, Vegetable Research Institute, AARI, Faisalabad and Malik Muhammad Fiaz, Ex-Director General, Pest Warning & Quality Control of Pesticides, Punjab, Lahore, without their kind cooperation it was almost impossible to initiate the project. I also thank Phytopathology Group Leader and Dr. Jaffargholi Imani, Institute of Phytopathology and Applied Zoology, Justus-Liebig- University, Giessen, Germany for their contributions to host pathogen interaction studies. I am grateful to the Higher Education Commission, Pakistan, who provided two fellowships for completion of this degree and funding for the research under Indigenous 5000 PhD Fellowship Program (Batch VII November, 2011-October, 2015) and International Research Support Initiative Program (IRSIP January-July, 2014). Thanks are due to Ms. Faiza, Mr. Amjad, and Mr. Iqbal, Lab. Attendants; Mr. Asif Nadeem, Composer, for supporting me in the lab. I also wish to express my appreciation to all those who helped me in one way or the other in completion of my thesis especially Ms. Shumaila Farooq, Mr. Waheed Akram, Ms. Saba Ghazanfar, Ms. Hina Nazli, Ms. Rabia Akram and PhD fellows. Last but not least, I am most gratifying to my Parents, for their prayers, support, and encouragement throughout my educational journey. I have no words to convey my love and affection to my sister, Ms. Amara Ilyas and brother, Mr. Yasir Ilyas for their precious love to me and for their patience. Sumera Naz iv TABLE OF CONTENTS Title Page # Certificate i Declaration certificate ii Dedications iii Acknowledgments iv Table of contents v List of figures x List of plates xii List of tables xiii List of research papers published from thesis xiv Abbreviations xv Summary xvii 1. Introduction 1.1. Momordica charantia 1 1.1.1 Plant taxonomy 1 1.1.2 Plant description 2 1.1.3 Nutritional value 2 1.1.4 Medicinal value 4 1.1.5 Industrial value 5 1.2. Crop statistics in Pakistan 6 1.3. State Policies 6 1.4. Production constraints 7 1.5. Pathological constraints 7 1.6. Common fungal diseases 7 1.7. Review of literature 8 1.8. Genus Myrothecium 8 1.9. Myrothecium roridum taxonomy 10 1.9.1. Geographical distribution and host range 11 1.9.2. History of Myrothecium leaf spot disease of bitter gourd in 12 Pakistan 1.9.3. Aggressiveness behavior 12 1.9.4. Germplasm screening for resistance 13 v 1.9.5. Management Strategies 14 1.9.5.1. Intercropping 14 1.9.5.2. Plant aqueous extract 14 1.9.5.3. Chemical control 15 1.9.5.4. IPM strategies 15 Objectives 16 2. Materials and Methods 2.1. Geographical distribution of Myrothecium roridum in Punjab, Pakistan 17 2.1.1. Survey 17 2.1.2. Disease assessment 19 2.1.3. Specimen collection 21 2.1.4. Isolation of associated Fungi 21 2.1.5. Identification of fungus 21 2.1.6. Application of Koch‟s postulates 22 2.1.7. Culture authentication 22 2.2. Characterization of Myrothecium roridum 22 2.2.1. Evaluation of virulence of isolates 22 2.2.2. Morphological studies 23 2.2.3. Physiological studies 25 2.2.3.1. Optimization of nutrients 26 2.2.3.2. Optimization of temperature 26 2.2.3.3. Optimization of pH 26 2.2.3.4. Effect of photoperiod 26 2.2.3.4. Optimization of culture age & Virulence 27 Assessment 2.2.4. Genetic variation studies 27 2.2.4.1. Preparation of solution 27 2.2.4.2. Extraction protocol 28 2.2.4.3. Estimation of Extracted DNA 28 2.2.4.4. DNA Quality Analysis through Agarose Gel 29 electrophoresis 2.2.4.5. Random Amplification of Polymorphic DNA 29 (RAPD) Analysis 2.2.4.5.1. Random Primer Screening 29 vi 2.2.4.5.2. Amplification reaction 31 2.2.4.5.3. RAPD Temperature Cycling Conditions 31 2.2.4.5.4. Analysis of Amplified DNA Fragments 31 2.2.4.5.5. RAPD data analysis 32 2.3. Development pattern of Myrothecium roridum within host leaf 32 and root tissues 2.3.1. WGA-AF 488 staining 32 2.3.1.1. Preparation of ½ MS medium 32 2.3.1.2. Preparation of 1 X PBS buffer 33 2.3.1.3. WGA-AF 488 33 2.3.1.4. Preparation of spore suspension 33 2.3.1.5. Preparation of leaf and root samples 34 2.3.1.6. Staining 34 2.3.2. Transmission electron microscopy 34 2.3.2.1. Sample preparation 34 2.3.2.2. Fixation 35 2.3.2.3. TEM analysis 35 2.4. In vivo Screening of bitter gourd germplasm 35 2.4.1. Soil sterilization 36 2.4.2. Susceptibility reaction in pot under natural environmental 36 conditions 2.4.3. Susceptibility reaction in field under natural 36 environmental conditions 2.5. Disease management strategies 37 2.5.1. Efficacy of plant aqueous extracts against MLS 37 2.5.1.1.Collection of plants 37 2.5.1.2. Preparation of aqueous extract 37 2.5.1.3. In vitroexperiment 37 2.5.1.4. In vivo experiment 38 2.5.2. Intercropping of aromatic plants 38 2.5.3. Efficacy of commercial fungicides against MLS disease 40 2.5.3.1. Commercial Fungicides 40 2.5.3.1.1. In vitro experiment 40 2.5.3.1.2. In vivo experiment 40 2.5.3.2. Technical grade fungicides 41 2.5.3.2.1.
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