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VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ---------- ---------- GRADUATION THESIS TITLE: ISOLATION, IDENTIFICATION AND STUDY OF BIOLOGICAL CHARACTERISTICS OF THE PATHOGENIC FUNGI CURVULARIA LUNATA CAUSING LEAF SPOT TO BANANAS Hanoi - 2023
VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ---------- ---------- GRADUATION THESIS TITLE: ISOLATION, IDENTIFICATION AND STUDY OF BIOLOGICAL CHARACTERISTICS OF THE PATHOGENIC FUNGI CURVULARIA LUNATA CAUSING LEAF SPOT TO BANANAS STUDENT : NGO PHUONG HIEN STUDENT CODE : 637411 CLASS : K63-CNSHE FACULTY : BIOTECHNOLOGY MAJOR : MICROBIOLOGY INSTRUCTOR : Ph.D. NGO THU HUONG Assoc Prof. NGUYEN XUAN CANH Hanoi - 2023
COMMITMENT The research was conducted from 9/2022 to 2/2023 under the supervision of Ph.D. Ngo Thu Huong, Assoc Prof. Nguyen Xuan Canh at Department of Microbiotechnology – Faculty of Biotechnology – Vietnam National University of Agriculture. I hereby declare that the data and results of this study are honest and have not been published in any scientific research at home or abroad. References have been clearly cited. Any help during the implementation of this thesis has been appreciated. i i
ACKNOWLEDGEMENTS Firstly, I want to express my sincere gratitude to all of the Executive Board, teachers, and lecturers of the Faculty of Biotechnology, Vietnam National University of Agriculture for helping, encouraging and providing the best working environment for me to complete the thesis. Secondly, I would like to express my most sincere thank to Ph.D. Ngo Thu Huong, Assoc. Prof. Nguyen Xuan Canh for guiding and giving valuable advice during my research so that I could complete my thesis successfully. Thirdly, I would like to express my deep gratitude to the lecturers of the Department of Microbiotechnology, Assoc. Prof.Nguyen Van Giang, Msc. Tran Thi Hong Hanh, Msc. Nguyen Thanh Huyen, Msc. Tran Thi Dao, Ph.D. student Nguyen Thi Thu and researcher Duong Van Hoan, Ta Ha Trang for getting me through the difficulties during my final thesis course. My completion of this project could not have been accomplished without their devotion. Last but not least, I crave for expressing my appreciation to my beloved family members, my friends, and colleagues for their encouragement, motivation so that I could complete my graduation thesis. Sincerely, Hanoi, February 3rd, 2023 Sincerely Ngo Phuong Hien ii
CONTENTS COMMITMENT......................................................................................................i ACKNOWLEDGEMENTS .....................................................................................ii CONTENTS...........................................................................................................iii LIST OF TABLES .................................................................................................vi LIST OF FIGURES...............................................................................................vii LIST OF ABBREVIATIONS ...............................................................................viii ABSTRACT...........................................................................................................ix I. INTRODUCTION..............................................................................................1 II. LITERATURE REVIEW.................................................................................4 2.1. Overview of Bananas ................................................................................4 2.1.1. Bananas in food demanding.......................................................................4 2.1.2. Bananas as economic importance...............................................................4 2.1.3. Diseases causing the banana production loss...............................................5 2.1.3.1. Diseases from bacteria..............................................................................5 2.1.3.2. Diseases from virus...................................................................................2 2.1.3.3. Diseases from fungi...................................................................................3 2.2. Overview of Curvularia lunata..................................................................6 2.2.1. Overview of Curvularia species.................................................................6 2.2.2. Overview of Curvularia lunata..................................................................8 2.2.2.1. Morphology of Curvularia lunata ..............................................................8 2.2.2.2. Various hosts of Curvularia lunata ............................................................9 2.2.2.3. Infection mechanism of Curvularia lunata................................................12 III. MATERIALS AND METHODS ...................................................................14 3.1. Time and location for the research ...........................................................14 iii
3.2. Materials..............................................................................................14 3.3. Methods ...............................................................................................15 3.3.1. Samples collection method..........................................................................15 3.3.2. Isolating and subculturing method.........................................................16 3.3.3. Pathogenicity test..................................................................................17 3.3.4. Studying on some biological characteristics of the selected fungi strain...17 3.3.4.1. Studying on the morphological characteristics of conidia, mycelium and conidiophore of the selected fungi strain...................................................17 3.3.4.2. Studying on the culturing features of the pathogenic fungi in different medium ...............................................................................................................18 3.3.4.3. Screening the ability to produce extracellular enzyme cellulase of the selected fungi strain..............................................................................................18 3.3.5. Indentification of the selected fungi strain...............................................18 3.3.5.1. DNA extraction method.........................................................................18 3.3.5.2. Amplification, sequencing and analyzing ITS gene sequence of isolated fungi strain method ..........................................................................................20 3.3.6. Screening, detecting toxic genes of fungal strains .....................................21 IV. RESULTS AND DISCUSSION .....................................................................23 4.1. Results of samples collection, isolation and subculturing the pathogenic pathogenic fungi strain. ...........................................................................23 4.2. Pathogenicity test ...................................................................................25 4.3. Classification of pathogenic fungi strain HN 5.2. .....................................26 4.3.1. Amplification, sequencing and analyzing ITS gene sequence of fungi strain HN 5.2....................................................................................................26 4.3.2. Clg2P gene amplification........................................................................29 4.4. Screening the development of HN 5.2 strain in different medium .............29 4.5. Testing the ability to extract cellulase of HN 5.2 strain..............................32 iv
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