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Molecular Based Identification of Wood Decay Fungi

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Molecular Based Identification of Wood Decay Fungi Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 1-1-2008 Molecular Based Identification of oodW Decay Fungi from Two Field Sites in Mississippi Robert Joseph Bucci Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Bucci, Robert Joseph, "Molecular Based Identification of oodW Decay Fungi from Two Field Sites in Mississippi" (2008). Theses and Dissertations. 3181. https://scholarsjunction.msstate.edu/td/3181 This Graduate Thesis - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. MOLECULAR BASED IDENTIFICATION OF WOOD DECAY FUNGI FROM TWO FIELD SITES IN MISSISSIPPI By Robert Joseph Bucci A Thesis Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Forest Products in the Forest Products Department Mississippi State, Mississippi August 2008 MOLECULAR BASED IDENTIFICATION OF WOOD DECAY FUNGI FROM TWO FIELD SITES IN MISSISSIPPI By Robert Joseph Bucci Approved: Susan V. Diehl Walter Diehl Associate Professor of Forest Products Professor of Biological Sciences (Major Professor) (Committee Member) M. Lynn Prewitt Rubin Shmulsky Assistant Research Professor of Forest Products Department Head (Committee Member) Associate Professor of Forest Products (Graduate Coordinator of the Department of Forest Products) Darrel Nicholas George M. Hopper Professor of Forest Products (Dean of the College of Forest (Committee Member) Resources) Name: Robert Joseph Bucci Date of Degree: August 9, 2008 Institution: Mississippi State University Major Field: Forest Products Major Professor: Dr. Susan V. Diehl Title of Study: MOLECULAR BASED IDENTIFICATION OF WOOD DECAY FUNGI FROM TWO FIELD SITES IN MISSISSIPPI Pages in Study: 66 Candidate for Degree of Master of Science This study focused on isolating important wood decay fungi from two field sites located in Harrison County, MS, and Oktibbeha County, MS. Southern Yellow Pine samples of various types and treatments including: Cu8, CuOm, ACQ, PCP, proprietary organic biocide, and un-treated were collected, and fungal isolates were cultured. DNA was extracted from isolated fungal cultures and amplified by polymerase chain reaction (PCR). The internal transcribe spacer (ITS) region was sequenced, and fungal cultures were identified by comparison to sequences on GenBank using BLAST. A total of 68 fungal isolates were recovered and successfully identified from 196 samples. Thirteen basidiomycete isolates were identified, with Veluticeps fimbriata occurring most frequently. The white-rot ascomycete, Daldinia fissa was also common. Two sequence-specific oligonucleotide probes (SSOP) were designed using Lasergene® PrimerSelect software. Unsuccessful attempts were made to attach poly (dT) tails to the probes in order to cross link the probes to nylon membranes. Key words: DNA, polymerase chain reaction, ITS, BLAST, GenBank ACKNOWLEDGEMENTS The author would like to extend his gratitude to all those who aided in the undertaking of this study. I would like to thank the Mississippi State University, Forest Product Department for the privileged opportunity to progress my knowledge in the field of forest product to the Master of Science level. I would like to extend special gratitude to Dr. Susan V. Diehl, my major advisor, for her committed guidance throughout this study. Also, I extend thanks to all other members of my advisory committee, Dr. M. Lynn Prewitt, Dr. Darrel Nicholas and Dr. Walter Diehl, for their assistance throughout this study. And also a special thanks to Juliet Tang, Facilities Manager of the Life Sciences & Biotechnology Institute at Mississippi State University, for her support and expert knowledge in the field of molecular biology. I would also like to thank our student workers who helped with much of the lab work. My thanks are also extended to my loving family, especially my patient parents, whom have supported and tolerated me throughout my seven year college career. Thank you. ii TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................. ii LIST OF TABLES...............................................................................................................v LIST OF FIGURES ........................................................................................................... vi CHAPTER I. INTRODUCTION....................................................................................................1 II. LITERATURE REVIEW..........................................................................................3 Wood Preservation...................................................................................................4 Fungal Taxonomy ....................................................................................................6 Molecular Methods ..................................................................................................8 PCR..................................................................................................................10 DNA Sequencing .............................................................................................10 GenBank ..........................................................................................................11 SSOP Membranes ............................................................................................12 III. METHODS AND MATERIALS ............................................................................15 Site Description......................................................................................................15 Sampling Procedure...............................................................................................15 Sample Processing .................................................................................................17 DNA Extraction .....................................................................................................18 PCR Amplification.................................................................................................19 DNA Sequencing ...................................................................................................21 Probe Design..........................................................................................................23 Polymer dT Tailing Reaction.................................................................................23 Psoralen-Biotin Labeling Reaction........................................................................23 Preparation of Filters..............................................................................................24 Hybridization Reaction ..........................................................................................25 Detection Reaction.................................................................................................25 Autoradiography ....................................................................................................26 iii IV. RESULTS AND DISCUSSION.............................................................................27 Occurrence and Abundance of Fungal Isolates .....................................................27 Comparison of Identified Fungal Isolates from the Two Test Sites ......................34 Comparison of Preservative Treatments................................................................38 Comparison of Observed Decay and Isolated Decay Fungi ..................................40 BLAST Search Results ..........................................................................................44 Probe Design..........................................................................................................46 Poly dT Tailing Reaction.......................................................................................46 V. CONCLUSION ......................................................................................................48 Summary of Fungal Results...................................................................................48 Identification Membranes ......................................................................................52 Future Research .....................................................................................................53 LITERATURE CITED ......................................................................................................54 APPENDIX A. RAW DATA ..........................................................................................................60 iv LIST OF TABLES 1 Number of Each Sample Type and Treatment Collected from the Dorman Lake, MS and Saucier, MS Field Sites ....................................................16 2 Taxonomy of Zygomycetes Isolated..............................................................................28 3 Taxonomy of Basidiomycetes Isolated..........................................................................30 4 Taxonomy of Ascomycetes Isolated..............................................................................32 5 Occurrence and Abundance of Similar Fungal Species by Family from the Dorman Lake, MS and Saucier, MS Field Sites ..................................................................35
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