ISOLATION AND CHARACTERIZATION OF ENTOMOPATHOGENIC FUNGI AND THEIR EVALUATION AGAINST BEMISIA TABACI WAHEED ANWAR INSTITUTE OF AGRICULTURAL SCIENCES UNIVERSITY OF THE PUNJAB LAHORE 2016 ISOLATION AND CHARACTERIZATION OF ENTOMOPATHOGENIC FUNGI AND THEIR EVALUATION AGAINST BEMISIA TABACI A thesis submitted to the University of the Punjab in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Agriculture (Plant Pathology) By Waheed Anwar Supervisors Prof. Dr. Muhammad Saleem Haider Prof. Dr. Ahmad Ali Shahid INSTITUTE OF AGRICULTURAL SCIENCES UNIVERSITY OF THE PUNJAB LAHORE CERTIFICATE This is to certify that the research entitled “Isolation and characterization of entomopathogenic fungi and their evaluation against Bemisia tabaci” described in this thesis by Mr. Waheed Anwar, is an original work of the author and has been carried out under our direct supervision. We have personally gone through all the data, materials and results reported in the dissertation and certify their correctness and authenticity. We further certify that the materials included in this thesis has not been used in part or full in a dissertation already submitted or in the process of submission in partial or complete fulfillment of the award of any other degree from any institution. We also certify that the thesis has been prepared under our supervision according to the prescribed format and we endorse its evaluation for the award of Ph.D. degree through the official procedures of the University of the Punjab, Lahore, Pakistan. Here thesis is in pure academic language and it is free from typos and grammatical errors. SUPERVISORS Dr. Muhammad Saleem Haider Professor, Date: ____________ Dr. Ahmad Ali Shahid Professor, Date: ____________ DECLARATION CERTIFICATE This thesis which is being submitted for the degree of Ph.D. in the University of the Punjab, Lahore, Pakistan 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 faith, 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. Waheed Anwar Ph.D. Scholar Institute of Agricultural Sciences University of the Punjab, Lahore, Pakistan Dedicated To My Beloved PARENTS ACKNOWLEDGMENTS All praises are for Almighty (The most affectionate, the most merciful), and Holy Prophet Muhammad (May God bless and peace be upon him).I bow before Almighty Allah in deep thankfulness that His infinite wisdom and mercy, granted me enough strength to complete this critique. I thank from the core of heart to Holy Prophet Muhammad (May God bless and peace be upon Him) forever a torch of guidance and knowledge for humanity as a whole. I would like to express the deepest appreciation to my Supervisor Prof. Dr. Muhammad Saleem Haider, Institute of Agricultural Sciences, University of the Punjab, Lahore, who has the attitude and the substance of a genius: he continually and convincingly conveyed a spirit of adventure in regard to research and scholarship, and an excitement in regard to teaching. Without his guidance and persistent help this dissertation would not have been possible. I wish to express my deep sense of gratitude to my co supervisor Prof. Dr. Ahmad Ali Shahid, Center of Excellence in Molecular Biology, University of the Punjab, Lahore, for his skillful advices, sincere co-operation, and learned guidance, keen interest, help and superb suggestions. I found him very generous, supportive and always available in time of need. A great debt of gratefulness and sincere thanks are extended toward Dr. Zia Ur Rehman and Dr. Usman Hameed, Research Associates, Institute of Agricultural Sciences, University of the Punjab, Lahore, for their support, kind attitude, endless help and best wishes during the accomplishment of this research work. I am very thankful to Higher Education Commission of Pakistan (HEC) for providing financial support to perform partial research work in Brown’s Lab, School of Plant Sciences, University of Arizona, Arizona, USA under international Research Support Initiative Program (IRSIP) for six months. My very special thanks to Prof. Dr. Judith K. Brown, School of Plant Sciences, University of Arizona, Arizona, USA for their kind help, accommodative behaviour and suggestions during IRSIP fellowship. I express my sincere thanks to Dr. Uzma Bashir and Dr. Noureen Akhtar, The First Fungal Culture Bank of Pakistan (FCBP), IAGS, University of the Punjab, Lahore, Pakistan for their constructive suggestions, timely helping and cooperation during identification of fungal Isolates. I am also thankful to Prof. Dr. Ashraf Randhawa (Rtd.), Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan for their his valuable input during the compilation of final draft. I am also grateful to my friends and fellows including, Abdul Wahid, M. Javed Iqbal, Rashid Alvi, Sidra Mushtaq, Muhammad Tariq Manzoor, Azmat Ullah Khan, Muhammad Ali, Hamid Mushtaq, Sehrish Iftikhar, Asim Javed, Hafeez Ul Haq, Jawad Shah, Mehwish Rauf, Amna Ali, Raheela Hafeez especially Kiran Nawaz, my Seniors especially Dr. Hafiz Muhammad Azhar, Dr. Nasir Subhani and Dr. Sajid Ali, and all staff members of IAGS including Muhammad Sajjad Gujjar and Abdul Raffy for their assistance and good desires throughout my research work. With a great sense of respect and honour I wish to express my deep sense of gratitude to my parents, Brothers and Sister for their support at various steps of this study. Their moral and financial support, sincere wishes and encouragement remained a great source for strengthening for education for whole span of this study. Their love and concern in me can be remembered but can never be returned fully. I am nothing without their prayers, cares and shares for my success. Waheed Anwar CONTENTS Sr. no Contents Page No. Certificate Declaration certificate Dedications Acknowledgments Table of contents List of tables i List of figures ii List of plates vii Abbreviations viii Summary ix 1 INTRODUCTION AND LITERATURE REVIEW 1 1.1 Bemisia tabaci 1 1.2 Host range of Bemisia tabaci 2 1.3 Biotypes of Bemisia tabaci 3 1.4 Historical outbreaks of Bemisia tabaci 5 1.5 Types of damage caused by Bemisia tabaci 7 1.6 Pest management strategies for Bemisia tabaci 8 1.6.1 Cultural control 8 1.6.2 Chemical control 9 1.6.3 Biological control 10 1.7 Entomopathogenic Fungi 13 1.8 Classification of Entomopathogenic Fungi 13 1.8.1 Phylum Oomycota 14 1.8.2 Phylum Chytridiomycota 14 1.8.3 Phylum Zygomycota 14 1.8.4 Phylum Ascomycota and Deuteromycota 14 1.9 Classification of fungi at phylogenetic level 15 1.9 Molecular Approach for identification of Entomopathogenic Fungi 17 1.10 Infection mechanism of Entomopathogenic Fungi 18 1.11 Distribution of Entomopathogenic Fungi 20 1.12 Pathogenicity of Entomopathogenic Fungi 21 1.13 Chitinases 25 1.13.1 Fungal Chitinases 27 1.14 Applications of Chitinases 27 1.15 Pathogenicity of Fungal Chitinases 28 1.16 Plants Transformation 30 1.17 Virus Induced gene silencing 30 1.17.1 Vectors for viral induced gene silencing 31 2 METHODOLOGY 34 2.1 Isolation and morphological characterization of fungi 34 2.1.1 Survey and sampling 34 2.1.2 Isolation of fungi 35 2.1.3 Morphological characterization of fungi 36 2.2 Molecular characterization of fungi 36 2.2.1 Phylogenetic analysis 37 2.3 Occurrence and Diversity 37 2.4 Virulence bioassay of entomopathogenic fungi against Bemisia tabaci 38 2.4.1 Maintenance of Bemisia tabaci 38 2.4.2 Spore suspension 39 2.4.3 Spore counting 39 2.4.4 Spore viability 40 2.4.5 Designing of virulence bioassay experiment 40 2.4.6 Data analysis 41 2.5 Isolation and characterization of chitinase genes from 42 entomopathogenic fungi 2.5.1 Primer designing for the fungal chitinases 42 2.5.2 Isolation of Chit1, Chit2 from M. anisopliae and Chit1from 44 Beauveria and T. longibrachiatum 2.5.2.1 Ligation 44 2.5.2.2 Transformation in E. coli (DH5α) 44 2.5.2.3 Plasmid isolation 45 2.4.2.4 Restriction digestions 45 2.4.2.5 Sequencing and homology analysis 46 2.5.3 Characterization of Chitinase Genes 46 2.5.3.1 Homology modeling and validation 46 2.6 Expression of Chitinase Open Reading Frame (ORF’s) in Cotton 47 Plants 2.6.1 Obtaining of a Virus Induced Gene Silencing (VIGS) vector 47 2.6.2 Isolation of chitinases ORF’s 47 2.6.3 Cloning into ρGEM-T easy 48 2.6.4 Construction of VIGS-Chit recombinant plasmids 48 2.6.5 Particle bombardment 48 2.7 Transgenic Expression Analysis 49 2.7.1 RNA extraction 49 2.7.2 cDNA 50 2.8 Chitinase Assay 50 2.8.1 Enzyme assay 51 2.8.2 Calculations 52 2.9 Virulence bioassay of chitinase transgenic plants against B. tabaci 52 03 RESULTS 53 3.1 Sampling and Morphological characterization of insect-associated 53 fungi 3.1.1 Isolation of fungi 52 3.1.2 Morphological characterization of insect-associated fungi 55 3.2 Molecular characterization and phylogenetic analysis of insect 60 associated fungi 3.2.1 Phylogenetic analysis of rDNA-ITS region of Penicillium sp. 61 3.2.2 Phylogenetic analysis of rDNA-ITS region of Beauveria sp. 61 3.2.3 Phylogenetic analysis of rDNA-ITS region of Metarhizium 64 anisopliae 3.2.4 Phylogenetic analysis of rDNA-ITS region of Alternaria sp. 64 3.2.5 Phylogenetic analysis of rDNA-ITS region of Acremonium sp. 67 3.2.6 Phylogenetic analysis of rDNA-ITS region of Clonostachys rosea 67 3.2.7 Phylogenetic analysis of rDNA-ITS region of Cladosporium sp. 70 3.2.8 Phylogenetic analysis of rDNA-ITS region of Trichoderma sp.