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ANTIFUNGAL ACTIVITY and PHYTOCHEMICAL ANALYSIS of VARIOUS MEDICINAL PLANTS AGAINST PATHOGENIC FUNGI of TOMATO (Lycopersicon Esculentum L.)

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ANTIFUNGAL ACTIVITY and PHYTOCHEMICAL ANALYSIS of VARIOUS MEDICINAL PLANTS AGAINST PATHOGENIC FUNGI of TOMATO (Lycopersicon Esculentum L.) ANTIFUNGAL ACTIVITY AND PHYTOCHEMICAL ANALYSIS OF VARIOUS MEDICINAL PLANTS AGAINST PATHOGENIC FUNGI OF TOMATO (Lycopersicon esculentum L.) BY SOBIA CHOHAN M.Sc. (Hons.) Agri. (Plant Pathology) A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY In Plant Pathology DEPARTMENT OF PLANT PATHOLOGY FACULTY OF AGRICULTURAL SCIENCES AND TECHNOLOGY BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN 2015 1 In the name of Allah, Most gracious, most merciful. Read! And your Lord is the Most Generous, Who has taught the writing by the pen, Has taught man that which he knew not. (Al-Quran) DECLARATION OF WORK I hereby declare that contents of the thesis entitled “Antifungal activity and phytochemical analysis of various medicinal plants against pathogenic fungi of tomato (Lycopersicon esculentum L.)” are produce of my own research and no part has been previously submitted for any other degree. Furthermore, work reported in this manuscript does not carry any duplication of research accomplished by other researchers’ except where due reference or acknowledgement is made overtly in the text. Sobia Chohan 2 The Controller of Examinations Bahauddin Zakariya University, Multan. It is certify that the contents and form of the thesis submitted by Mrs. Sobia Chohan (2009-ag-191) have been found satisfactory and recommend that it may be processed for evaluation by the External Examiner (s) for the award of the degree. SUPERVISOR DR. RASHIDA ATIQ Associate Professor (Department of Plant Pathology) 3 Dedicated To my PARENTS, BROTHERS And HUSBAND ACKNOWLEDGEMENTS All the acclamations are for The Almighty Allah alone, the most merciful and the most compassionate, Who is the entire source of wisdom and all the knowledge endowed to mankind, Who enabled me to achieve this goal. I offer my heartfelt praise to Holy Prophet Muhammad (PBUH) the most perfect and dignified, glorious one among and of ever borne on the surface of the earth, who is forever model of guidance and knowledge for the humanity. Ph.D is a sojourn wherein gratification with dissatisfaction, boom with gloom prevails. But, its essence lies what you accomplished in the end and is fuelled by interdependence rather than dependence. Though it is simple to name all the people who lent their support for my endeavor but it is awesome job to gratify them. I am very appreciative and gratified to my Supervisor, Dr. Rashida Atiq, Chairperson Department of Plant Pathology, Faculty of Agricultural Sciences & Technology, BZU, Multan for her valuable suggestions, constructive criticism, guidance and encouragements throughout my work. While working under her, the scrupulous execution of her work has instilled in me a great sense of confidence to carry out my work objectively and successfully. Undoubtedly, words can't suffice in expressing my sincerest gratitude to Professor (Retd.) Dr. Sardar Muhammad Mughal - an eminent Plant Virologist/Plant Pathologist. His cordial behavior, humble attitude, cooperativeness and nobility are 4 some of the exquisite traits which I would certainly like to cherish and immolate as the genuine taught of this charismatic teacher. I could not repay what I achieved today and what I learnt from him. I pay obeisance to this greatest steward of mine to whom I owe all my achievements. The scientific competence in him has rendered my scientific endeavor and zeal more glorious. I am greatly indebted to Dr. Saeed Akhter, Chairman and Associate Professor of Department of Food Sciences, FAS & T, BZU, Multan for permitting me to avail the facilities for my work and rendering every sort of encouragement during my tenure. I overwhelmingly acknowledge the services rendered by Tariq Ismail and Amir Ismail, Lectures of Department of Food Sciences, for providing the spectral data for my compounds. It gives me immense pleasure to express my deep sense of gratitude and affable thanks are also extended to all my Department colleagues for their constant encouragement and valuable support. I would like to express my profound gratitude towards my friend Safina Naz, Assistant Professor of Horticulture. Working with coordination is more valuable than working in seclusion. I feel fortunate enough to have the company of my brilliant students Mirza Abid Mahmood (Lecturer, Plant Pathology, MNSUA, Multan), Miss Naila Akram and Syed Atif Hasan for their persistent help and unconditional support. My wholehearted gratitude goes to my laboratory staff for their cooperation and sincere help during lengthy and hard period of my research work. The author extends sincere thanks to Directorate of External Linkages, Bahauddin Zakaryia University, Multan for financial supports. 5 The driving forces for accomplishment of my challenge are my beloved parents who extended every care, incessant prayers, moral support and affection not only in this period but during my entire span of life. I pay my sincere obeisance to my cherished parents without whom it was near impossible to fulfill my goal. I could not forget the loving memories of my mother whose hands always rose for my success and my beloved husband whose everlasting enthusiasm bore fruit in the form of this dissertation. I am indebtly grateful to my brothers, sisters-in-law and my relatives who are the real guiding forces and moral boosters of my triumph. Last but not the least; I cannot overlook my little nephew Umair Bin Saqib for providing energy during my work. Sobia Chohan LIST OF CONTENT 6 Sr No . Description Page No. Acknowledgments i Contents iv List of Tables vii List of Figures ix List of Plates x Abbreviations xi CHAPTER 1: INTRODUCTION 1 1. THE TOMATO PLANT 1 1.1. Production 1 1.2. Nutritional value 2 2. FUNGAL DISEASES OF TOMATO AND THEIR MANAGEMENT 3 2.1. Damping off and Seed rot 4 2.2. Foliar diseases 4 2.2.1. Early blight 4 2.2.2. Late Blight 6 2.2.3. Anthracnose 7 2.2.4. Wilt and root rot 9 3. IMPLICATIONS OF CHEMICAL CONTROL AND SCOPE OF 11 MEDICINAL PLANTS 4. AIM OF INVESTIGATIONS 13 Objectives 14 CHAPTER 2: REVIEW OF LITERATURE 15 1. INTRODUCTION 15 2. PROCESSING OF PLANTS AND ISOLATION OF ANTIMICROBIAL 15 AGENTS 2.1 Selection of tissue 16 2.2 Choice of Solvents 16 2.3 Choice of extraction procedure 18 3. ASSESSMENT OF ANTIMICROBIAL SUSCEPTIBILITY TEST (AST) 19 3.1 Diffusion test 19 7 3.2 Dilution method 20 4. MAJOR ANTIMICROBIAL PHYTOHEMICALS 21 4.1 Phenolics and Polyphenolics 21 4.1.1. Tannins 23 4.1.2. Flavonoids 24 4.1.3. Coumarins 24 4.1.4. Terpenoids 25 4.2 Alkaloids 25 4.3 Saponins/Steroids 25 5. SECONDARY METABOLITES 26 6. MODE OF ACTION OF ANTIMICROBIAL PHYTOCHEMICALS 27 7. SOME ANTIFUNGAL PROPERTIES OF TARGET BOTANICALS 28 7.1 Garlic (Allium sativum ) 29 7.2 Neem (Azadirachta indica) 30 7.3 Ginger (Zingiber officinale) 31 7.4 Mhendi, Henna (Lawsonia inermis) 31 7.5 Tulsi (Ocimum sanctum, O. basilicum) 32 7.6 Turmeric (Curcuma longa) 32 7.7 Marygold (Tagetes minuta, T. erecta and T. patula) 33 7.8 Eucalyptus (Eucalyptus globules) 33 7.9 Chinaberry (Melia azedarach) 34 7.10 Ashoka tree (Polyalthia longifolia var. Pendula 34 7.11 Periwinkle (Catharanthus roseus) 35 7.12 Mango (Mangifera indica) 35 7.13 Lemon (Citrus limon) 35 7.14 Jambolan, Jaman (Syzgium cumini) 36 7.15 Moringa oleifera 36 8. BENEFICIAL IMPACTS OF ANTIMICROBIAL AGENTS 37 CHAPTER 3: MATERIALS AND METHODS 41 3.1. General methods 41 8 3.2. Sample Collection 42 3.3. Isolation of fungi 42 3.3.1. Isolation from plant tissues 42 3.3.2. Isolation from seed 43 3.4. Identification, maintenance and multiplication of fungi 43 3.5. Pathogenicity tests of fungi 44 3.6. Assessment of antifungal properties of plant extracts 45 3.7. Processing of tissue and Extraction 47 3.8. Phytochemical testing of plant materials 49 3.9. In vitro evaluation of Antifungal activity of plant extracts 50 3.9.1. Poisoned Food Technique 51 3.9.2. Agar well diffusion method 51 3.10. Spore germination inhibition test 52 3.11. In vivo tests of plant extracts and Topsin-M against early blight in 53 greenhouse 3.12. Field evaluation of plant extracts and Topsin-M against early blight 54 3.13. Statistical Analysis 55 CHAPTER 4. RESULTS 56 CHAPTER 5. DISCUSSION 116 CHAPTER 6. SUMMERY 126 CHAPTER 7. REFERENCES 130 APPENDICES 179 LIST OF TABLES 9 Sr. No. Tables Page No. 3.1. Plants used as sources of extract 50 4.1. Frequency % of fungi isolated from tomato seeds using blotter and Agar 57 plate methods 4.2. *Mean Frequency % of fungi isolated from various parts of tomato plant 59 and level of significance 4.3. Qualitative analysis of medicinal plants 63 4.4. Total Phenol Contents in aqueous and methanol extracts of plants 66 4.5. Antifungal effect of aqueous fresh plant extracts at different 72 concentrations against important tomato fungi (Percent growth reduction) 4.6. Antifungal effect of aqueous dry plant extracts at different 76 concentrations against important tomato fungi (Percent growth reduction) 4.7. Antifungal effect of methanol fresh plant extracts at different 86 concentrations against important tomato fungi (Percent growth reduction) 4.8. Antifungal effect of methanol dry plant extracts at different 90 concentrations against important tomato fungi (Percent growth reduction) 4.9. Antifungal activity of aqueous (fresh) plant extracts at different 98 concentrations against selective tomato fungi using agar well method 4.10. Antifungal activity of aqueous (dry) plant extracts at different concentrations 99 against selective tomato fungi using agar well method 4.11. Antifungal activity of methanol (fresh) plant extracts at different 100 concentrations against selective tomato fungi using agar well method 4.12. Antifungal activity of methanol (dry) plant extracts at different 101 concentrations against selective tomato fungi using agar well method 4.13.
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