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Identification of Yeast and Yeast-Like Fungal Species in the Upper Midwest Using Physiological and Dna Sequence Data" (2008)

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Identification of Yeast and Yeast-Like Fungal Species in the Upper Midwest Using Physiological and Dna Sequence Data Northern Michigan University NMU Commons All NMU Master's Theses Student Works 2008 IDENTIFICATION OF YEAST AND YEAST- LIKE FUNGAL SPECIES IN THE UPPER MIDWEST USING PHYSIOLOGICAL AND DNA SEQUENCE DATA Scott rE ic Danneman Northern Michigan University Follow this and additional works at: https://commons.nmu.edu/theses Recommended Citation Danneman, Scott rE ic, "IDENTIFICATION OF YEAST AND YEAST-LIKE FUNGAL SPECIES IN THE UPPER MIDWEST USING PHYSIOLOGICAL AND DNA SEQUENCE DATA" (2008). All NMU Master's Theses. 376. https://commons.nmu.edu/theses/376 This Open Access is brought to you for free and open access by the Student Works at NMU Commons. It has been accepted for inclusion in All NMU Master's Theses by an authorized administrator of NMU Commons. For more information, please contact [email protected],[email protected]. IDENTIFICATION OF YEAST AND YEAST-LIKE FUNGAL SPECIES IN THE UPPER MIDWEST USING PHYSIOLOGICAL AND DNA SEQUENCE DATA By Scott Eric Danneman THESIS Submitted to Northern Michigan University In partial fulfillment of the requirements For the degree of Master of Science Graduate Studies Office 2008 SIGNATURE APPROVAL FORM This thesis by Scott Danneman is recommended for approval by the student’s thesis committee in the Department of Biology and by the Dean of Graduate Studies. Committee Chair: Dr. Donna Becker Date First Reader: Dr. Jill Leonard Date Second Reader: Dr. Alec Lindsay Date Third Reader: Dr. Alan Rebertus Date Department Head: Dr. Neil Cumberlidge Date Dean of Graduate Studies: Cynthia Prosen Date OLSON LIBRARY NORTHEN MICHIGAN UNIVERSITY THESIS DATA FORM In order to catalog your thesis properly and enter a record in the OCLC international bibliographic data base, Olson Library must have the following requested information to distinguish you from others with the same or similar names and to provide appropriate subject access for other researchers. NAME: Danneman Scott Eric DATE OF BIRTH: 01/11/1983 ABSTRACT IDENTIFICATION OF YEAST AND YEAST-LIKE FUNGAL SPECIES IN THE UPPER MIDWEST USING PHYSIOLOGICAL AND DNA SEQUENCE DATA By Scott Eric Danneman Yeasts are unicellular fungi that play key biological roles and are useful in many human applications from producing alcohol to serving as model organisms. They are found as free living organisms or as symbionts of animals and plants. An estimated 1% of the total extant species have been described. The objectives of this research focused on isolating and identifying yeast and yeast-like fungal species from the Upper Midwest using phenotypic tests and DNA sequencing. A total of 32 fungi showing single cell growth were isolated from plants, soil and compost from around the Upper Midwest. The isolates were purified and examined for morphological traits such as colony color, and cell characteristics. The isolates were tested physiologically in their abilities to utilize carbon and nitrogen compounds. A section of the large subunit rDNA was sequenced and compared to the same sequence from known species. Sequencing of the internal transcribed spacer regions of some of the isolates was also performed and compared to sequences from known species. A total of 14 species were identified with the most common species being Aureobasidium pullulans. Phylogenetic analyses showed the isolates as having diverse ancestry with isolates belonging to both the Ascomycetes and the Basidiomycetes. Two new species were found, one belonging to the genus Cryptococcus and another belonging to a new genus. i Copyright by Scott Eric Danneman 2008 ii DEDICATION Dedicated to my parents Gene and Deb Danneman, Sierra Boyle, and all those people from Marquette, MI that have helped me every step of the way on my education journeys. iii ACKNOWLEDGMENTS I would like to thank Dr. Donna Becker for being a great mentor throughout my years at Northern Michigan University. She supported and shared my passions on every occasion. She helped mold my mind into becoming the person I am. Her assistance in nearly every aspect of my graduate student years was irreplaceable. Dr. Alec Lindsay with his inspiring character taught me nearly everything I know about evolution and DNA. His questions and lessons taught me how to approach scientific questions. His assistance was integral to the molecular techniques used in my thesis work. Dr. Alan Rebertus opened my eyes to the world of botany. His passion for botany shaped the way I think about and deal with plants. His insight has been vital to the completion of numerous projects. Dr. Jill Leonard was a torch that fanned the flames of my passions. She was always there to offer insight into every idea I had. Her helpful questioning added to every project. I would like to thank my parents Gene and Deb Danneman for raising me to become the person I am. They have been there every step of the way and pushed me along when I needed it. iv Sierra Boyle my girlfriend, and best friend, supported me every step of the way with everything that she does. Her moral support during difficult times was all that was needed to get me back to work. I would like to thank Suzie Piziali, Jingfang Niu, and Dave Erickson for all their logistical support. Suzie helped me figure out problems as they rose and without her nothing would have been accomplished. Jing was a constant source of good ideas and always helped me find the supplies I needed. Dave helped me dig through the piles of chemicals to find what I needed. For monetary support I would like to thank the Excellence in Education Grant. This thesis follows the bibliographic format prescribed by Mycological Research. v TABLE OF CONTENTS LIST OF TABLES viii LIST OF FIGURES ix INTRODUCTION 1 CHAPTER 1: LITERATURE REVIEW 3 WHAT IS A YEAST? 3 THE YEAST LIFE CYCLES 4 THE IMPORTANCE OF YEAST 6 IDENTIFICATION OF YEAST SPECIES AND SPECIES CONCEPTS 13 CHAPTER 2: ISOLATION AND MORPHOLOGICAL/BIOCHEMICAL TESTING OF YEAST-LIKE ISOLATES 26 ABSTRACT 26 INTRODUCTION 26 MATERIALS AND METHODS 27 RESULTS 30 DISCUSSION 37 CHAPTER 3: SEQUENCING OF rDNA GENES FROM ISOLATES FOR SPECIES IDENTIFICATION 39 ABSTRACT 39 INTRODUCTION 39 vi MATERIALS AND METHODS 40 RESULTS 44 DISCUSSION 64 CHAPTER 4: FINAL SPECIES DETERMINATIONS: COMBINING PHENOTYPIC AND SEQUENCE DATA 71 ABSTRACT 71 INTRODUCTION 71 MATERIALS AND METHODS 72 RESULTS 73 DISCUSSION 97 BIBLIOGRAPHY 107 vii LIST OF TABLES Table 2.1: Morphological and Physiological Results 35 Table 3.1: Sequence Lengths 46 Table 3.2: Summary of LSU rDNA BLAST Results 47 Table 3.3: Summary of ITS1, 5 8S and ITS2 rDNA BLAST Results 51 Table 4.1: Identification of Positive Control 74 Table 4.2: Identification of Isolate Y3A 75 Table 4.3: Identification of Isolate Y4 75 Table 4.4: Identification of Isolate Y9 76 Table 4.5: Identification of Isolate Y10 77 Table 4.6: Identification of Isolate Y12 78 Table 4.7: Identification of Isolates Y16 and Y17A 79 Table 4.8: Identification of Isolate Y18 80 Table 4.9: Identification of Isolate Y22 81 Table 4.10: Identification of Isolate Y35 82 Table 4.11: Identification of Isolates Y1, Y2, Y3B, Y8, Y14, Y17B, Y17C, Y19, Y23, Y26, Y29, Y30, Y32, Y34, and Y37 85 Table 4.12: Identification of Isolate Y25 87 Table 4.13: Identification of Isolate Y27 88 Table 4.14: Identification of Isolates Y5, Y13, Y20, Y28, and Y31 89 Table 4.15: Environments Sampled and Species Isolated 104 viii LIST OF FIGURES Figure 1.1: Negative stain of budding yeast cells of the yeast Cryptococcus wieringae 4 Figure 1.2: Asexual and sexual life cycles of Saccharomyces cerevisiae 6 Figure 1.3: Primer map of region of interest showing genes of interest and primers utilized to amplify those genes 23 Figure 2.1: Colony of Y13 and Y20 on PDA as seen under a dissecting scope 32 Figure 2.2: Red diffusing chemical produced by isolate Y17A as seen under a dissecting scope 33 Figure 2.3: Photo micrograph of Y20 showing thick walled pseudohyphae 33 Figure 2.4: Photo micrograph of Y20 showing transition from yeast colony (lower right) to pseudohyphae (middle) to true hyphae (upper left) 34 Figure 2.5: Photo micrograph of Y20 grown in liquid medium 34 Figure 3.1: Sample gel image generated from amplification of the D1/D2 regions of the LSU rDNA 44 Figure 3.2: Sample gel image generated from the amplification of the ITS1, 5 8S and ITS2 regions of the rRNA 50 Figure 3.3: Legend for icons found in Figures 3.4 and 3.5 that represent the various isolate environments 52 Figure 3.4: The phylogenetic placement of all isolates collected that were Ascomycetes 53 Figure 3.5: The phylogenetic placement of all isolates collected that were Basidiomycetes 54 Figure 3.6: Phylogenetic placement of isolates Y5, Y13 and Y28 using the LSU regions 56 Figure 3.7: Phylogenetic placement of isolates Y5, Y13 and Y28 using the ITS regions 57 Figure 3.8: Neighbor-joining tree of isolates Y5, Y13 and Y28 58 Figure 3.9: Phylogenetic placement of isolate Y35 using LSU data 60 Figure 3.10: Phylogenetic placement of isolate using ITS data 61 ix Figure 3.11: Neighbor-joining tree of isolate Y35 based on LSU data 62 Figure 3.12: Neighbor-joining tree of isolate Y35 based on ITS data 63 Figure 4.1: Colony characteristics under a dissecting scope (Left) and cell/bud characteristics at 1000X (Right) of Y35 after two weeks on PDA 83 Figure 4.2: Cell and budding characteristics of Y35 at 1000X grown on PDA after two weeks (Left) and on malt yeast extract plus dextrose agar (MYDA) after three days (Right) 83 Figure 4.3: Isolate colonies closely matching to Aureobasidium pullulans as seen with a dissecting scope grown on PDA 86 Figure 4.4: Colonies of Y5 and Y13 as seen under a dissecting scope 91 Figure 4.5: Cell and bud characteristics of Y5 and Y13 92 Figure 4.6: Darkly pigmented pseudo-hyphae of Y5 as seen at 400X magnification 93 Figure 4.7: Hyphae of Y5 showing basal conidiogenesis as seen at 400X magnification 94 Figure 4.8: Hyphae structures of Y5 95 Figure 4.9: Characteristics of the hyphae of Y5 directly below a colony (looking up from bottom) as seen at 100X magnification 96 Figure 4.10: Fine aerial hyphae protruding <1 mm off the top of a 4 month old colony of Y5 grown on PDA as seen at 100X magnification 97 x INTRODUCTION Fungi that grow and reproduce in a single cellular state are commonly termed yeasts.
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