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MORPHOLOGICAL and MOLECULAR IDENTIFICATIONS of Aspergillus Spp

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MORPHOLOGICAL and MOLECULAR IDENTIFICATIONS of Aspergillus Spp MORPHOLOGICAL AND MOLECULAR IDENTIFICATIONS OF Aspergillus spp. ISOLATED FROM ENCLOSED BUILDINGS IN PENINSULAR MALAYSIA WARDAH BINTI ABDUL RAHMAN UNIVERSITI SAINS MALAYSIA 2012 MORPHOLOGICAL AND MOLECULAR IDENTIFICATIONS OF Aspergillus spp. ISOLATED FROM ENCLOSED BUILDINGS IN PENINSULAR MALAYSIA by WARDAH BINTI ABDUL RAHMAN Thesis submitted in fulfillment of the requirement for the degree of Master of Science March 2012 i ACKNOWLEDGEMENTS “In the name of Allah, the Most Gracious and the Most Merciful” First and foremost, Alhamdulillah, all praise to Allah s.w.t. for giving me a continuous strength and health to do this project and writing the dissertation until it is completed. I would like to express my utmost gratitude to my supervisor, Prof Baharuddin bin Salleh, who has supported me throughout my study with his patience, enthusiasm and immense knowledge. Without his full guidance and steadfast encouragement, this study would have not been successful. Special appreciation is due to my co-supervisor, Dr. Latiffah bt Zakaria, who has offered so much advices and insights on my work especially in molecular aspects. Her unfailing supports and suggestions given in carrying out this project were very valuable. A big thank you to Dr. Reddy, a post-doctoral fellow, for his concern and also willingness in lending me such precious references for morphological identification of Aspergillus. I would like to express my sincere thanks to Mr. Kamaruddin for his help and technical assistance especially during the sampling period. Not to mention, Mr. Johari, Miss Jamilah and all staffs in School of Biological Science who have contributed and extended their valuable assistance in the preparation and completion of this study. I am also grateful for the two-year ii scholarship provided by Universiti Sains Malaysia, under USM Fellowship programme. In my daily work I have been blessed with a friendly and cheerful group of fellow colleagues from Plant Pathology Laboratories i.e. Nur Azliza, Nik Mohd Izham, Masratul Hawa, Hafizi, Hew Pui Yee, Nurul Farizah, Siti Norsyila, Farhana Nazira, Bintra Mailina, Nur Hazrati, Norlia, Nor Fazila, Darnetty and Leong Sau Kuen. Thank you so much for your support and help that make this dissertation possible. Not forgotten to Nur Farhana and Balkhis from Microbiology Laboratory who has patiently taught me to use the MEGA 4.0 software. I am also indebted to my parents, Abdul Rahman bin Saad and Aniroh bt Idris for their constant strength and encouragement given, even after I have entered a new stage of life of becoming a wife. Your trustworthy and love are all I need and thankfully, it grew deeper day by day. Last but not least, I am very grateful to have a lovely, encouraging and understanding husband, Muhammad Nuruddin bin Bashah. Your endless love, concern, patience and blessing did motivate and strengthen me in every moment. Thank you for walking through this path besides me. iii TABLE OF CONTENT Page ACKNOWLEDGEMENTS ii TABLE OF CONTENT iv LIST OF TABLES viii LIST OF FIGURES x LIST OF ABBREVIATIONS xii ABSTRAK xv ABSTRACT xvii CHAPTER ONE : INTRODUCTION 1 CHAPTER TWO : LITERATURE REVIEW 2.1 Indoor Air Quality (IAQ) 8 2.1.1 IAQ in Malaysia 9 2.2 Sick Building Syndrome (SBS) 10 2.2.1 Causes of SBS 11 2.2.2 Health Effects due to SBS 13 2.3 Bioaerosol 14 2.4 Aspergillus as Human Pathogen, Allergen and Mycotoxin 15 Producer 2.5 Taxonomy of the Genus Aspergillus 17 iv 2.6 Identification and Classification of Aspergillus Species 23 2.7 Morphological Characteristics 24 2.8 Molecular Characteristics 25 2.8.1 PCR-based Technique 25 2.8.2 Ribosomal DNA (rDNA) 26 2.8.3 Internal Transcribed Spacer (ITS) Region 27 CHAPTER THREE : GENERAL MATERIALS AND METHODS 3.1 Fungal Isolates 29 3.2 Fungal Sources 33 3.3 Sampling Techniques 33 3.3.1 Surface Sampling 33 3.3.2 Air Sampling 34 3.4 Culture Media 36 3.5 Sterilization 37 3.5.1 Surface Sterilization 37 3.5.2 Steam Sterilization 37 3.5.3 Dry Heat Sterilization 37 3.6 Single Spore Isolation Technique 38 3.7 Slide Culture Technique 38 3.8 Preservation and Storage of Cultures 40 3.8.1 Cold Storage 40 3.8.2 Drying 40 3.8.3 Oil Overlay 41 v 3.9 Identification of Aspergillus Species through Morphological 42 Characteristics 3.10 Molecular Analysis 43 3.10.1 DNA Extraction 43 3.10.2 DNA Detection 45 3.10.3 DNA Quantification 45 3.11 Polymerase Chain Reaction (PCR) 46 3.11.1 Optimization of PCR Reagents 46 3.11.2 Amplification of ITS Regions 47 3.11.3 DNA Purification 49 3.11.4 Sequence Analysis of ITS Regions 49 3.11.5 Phylogenetic Analysis of ITS Regions 51 CHAPTER FOUR : RESULTS 4.1 Isolates of Aspergillus Species 52 4.2 Morphological Identification of Aspergillus Species 53 4.2.1 Aspergillus niger (Tiegh.) 54 4.2.2 Aspergillus japonicus (Saito) 58 4.2.3 Aspergillus flavus (Link) 63 4.2.4 Aspergillus fumigatus (Fresen.) 68 4.2.5 Aspergillus sclerotiorum (Huber) 72 4.2.6 Aspergillus ochraceus (K. Wilh.) 77 4.3 Sequence Analysis of the ITS Regions 82 4.3.1 Size of PCR Product 82 vi 4.3.2 Sequence Alignment 83 4.3.3 Phylogenetic Analysis of ITS1 Region 86 4.3.3.1 Neighbour Joining Method 86 4.3.3.2 Maximum-Parsimony Method 90 4.3.4 Phylogenetic Analysis of ITS2 Region 93 4.3.3.1 Neighbour Joining Method 93 4.3.3.2 Maximum-Parsimony Method 96 CHAPTER FIVE : DISCUSSION 5.1 Morphological Identification 99 5.2 Phylogenetic Analysis of ITS Region 106 5.3 General Discussion 111 CHAPTER SIX : CONCLUSION 120 CHAPTER SEVEN : FUTURE RESEARCH 121 REFERENCES 122 APPENDICES 145 LIST OF PUBLICATIONS 168 vii LIST OF TABLES Tables Page 1.1 Predominant fungal genera isolated from various 3 enclosed buildings 2.1 Mycotoxins produced by Aspergillus and the related 17 illness effecting humans 2.2 Important taxonomic treatments and identification 19 manuals for the genus Aspergillus 2.3 Summary of Aspergillus current scheme for 21 identification 2.4 Sexual stages associated with the genus Aspergillus 23 3.1 List of Aspergillus isolates used in this study obtained 30 from indoor air and surface sampling methods in enclosed buildings through out Peninsular Malaysia 3.2 Concentration and volume of PCR reagents used in 48 PCR-ITS for Aspergillus species 3.3 PCR-ITS cycling condition used for amplification of 48 Aspergillus species 3.4 List of representative species with GenBank 51 accession number 4.1 Microscopic and macroscopic characteristics of A. 55 niger isolates 4.2 Microscopic and macroscopic characteristics of A. 59 japonicus isolates 4.3 Microscopic and macroscopic characteristics of A. 64 flavus isolates 4.4 Microscopic and macroscopic characteristics of A. 69 fumigatus isolates viii 4.5 Microscopic and macroscopic characteristics of A. 73 sclerotiorum isolates 4.6 Microscopic and macroscopic characteristics of A. 78 ochraceus isolates 4.7 Isolates of Aspergillus used and maximum sequence 84 similarity (%) of ITS1 and ITS2 regions ix LIST OF FIGURES Figures Page 2.1 Diagram of the ribosomal DNA (rDNA) repeat unit 26 and the location of ITS regions 3.1 Anderson single stage sampler with MEA plate inside 34 3.2 Side view of Aspergillus slide culture 39 3.3 Diagram of the conserved ITS1 and ITS2 regions of 47 rDNA and binding sites for primers ITS2, ITS3, ITS4 and ITS5 . 4.1 Locations of sample collection isolated from enclosed 52 buildings in Peninsular Malaysia 4.2 Percentage of six species of Aspergillus isolated from 53 enclosed buildings in Peninsular Malaysia 4.3 Microscopic characteristics of A. niger isolated from 56 enclosed buildings in Peninsular Malaysia 4.4 Colony appearance (I) and pigmentation (II) of A. 57 niger on different agar media 4.5 Microscopic characteristics of A. japonicus isolated 61 from enclosed buildings in Peninsular Malaysia 4.6 Colony appearance (I) and pigmentation (II) of A. 62 japonicus on different agar media 4.7 Microscopic characteristics of A. flavus isolated from 66 enclosed buildings in Peninsular Malaysia 4.8 Colony appearance (I) and pigmentation (II) of A. 67 flavus on different agar media 4.9 Microscopic characteristics of A. fumigatus isolated 70 from enclosed buildings in Peninsular Malaysia x 4.10 Colony appearance (I) and pigmentation (II) of A. 71 fumigatus on different agar media 4.11 Microscopic characteristics of A. sclerotiorum 75 isolated from enclosed buildings in Peninsular Malaysia 4.12 Colony appearance (I) and pigmentation (II) of A. 76 sclerotiorum on different agar media 4.13 Microscopic characteristics of A. ochraceus isolated 80 from enclosed buildings in Peninsular Malaysia 4.14 Colony appearance (I) and pigmentation (II) of A. 81 ochraceus on different agar media 4.15A PCR amplification products of the ITS1 region from 82 Aspergillus species isolated from enclosed buildings in Peninsular Malaysia 4.15B PCR amplification products of the ITS2 region from 83 Aspergillus species isolated from enclosed buildings in Peninsular Malaysia 4.16 Neighbour Joining tree generated from sequences of 89 ITS1 region of Aspergillus species isolated from enclosed buildings in Peninsular Malaysia 4.17 Most Parsimonious tree generated from sequences 92 of ITS1 region of Aspergillus species isolated from enclosed buildings in Peninsular Malaysia C 4.18 Neighbour Joining tree generated from sequences of 95 A Aspergillus I ITS2 region of species isolated from B enclosed buildings in Peninsular Malaysia 4.19 Most Parsimonious tree generated from sequences 98 of ITS2 region of Aspergillus species isolated from enclosed buildings in Peninsular Malaysia xi LIST OF ABBREVIATIONS 18S Small Ribosomal Subunit 28S Large Ribosomal Subunit ABPA Allergic Bronchopulmonary
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