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研究生: 何芷蔚
Hoh, Zhi Wei
論文名稱: 茄鐮孢菌複合種感染海龜之致病相關基因體學
The genomic basis of pathogenicity in Fusarium solani species complex infecting sea turtle
指導教授: 蔡怡陞
Tsai, Isheng Jason
口試委員: 鍾嘉綾
Chung, Chia-Lin
曾庸哲
Tseng, Yung-Che
陳穎練
Chen, Ying-Lien
陳可萱
Chen, Ko-Hsuan
蔡怡陞
Tsai, Isheng Jason
口試日期: 2022/05/05
學位類別: 博士
Doctor
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2022
畢業學年度: 110
語文別: 英文
論文頁數: 119
中文關鍵詞: 茄鐮孢菌海龜蛋鏈孢霉病基因體學動物致病菌
英文關鍵詞: Fusarium solani species complex (FSSC), Sea turtle egg fusariosis (STEF), Pathogenomics, Genome compartmentalisation, Animal pathogens
研究方法: 調查研究
DOI URL: http://doi.org/10.6345/NTNU202200458
論文種類: 學術論文
相關次數: 點閱:72下載:1
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  • Fungi from the genus Fusarium is a common pathogen that infects plants, human, and animals. In particular, some members from the Fusarium solani species complex (FSSC) were comparatively well-studied because the most impacted hosts were agriculturally important plants. In animals, fusariosis caused by FSSC was increasingly reported in domestic, captive and wild animals in recent decades. Whilst it is commonly reported as an opportunistic pathogen towards animals, most reports were only able to identify the disease causative FSSC members and describe its pathologic changes on the infected host. Sea turtle egg fusariosis (STEF) has been reported around the globe and has caused huge mortality in this endangered animal. However, little is known regarding the ecology and pathology of STEF. The establishment of hatchery is part of the conservation works to protect sea turtle eggs. Nonetheless, a particular hatchery practice – reusing sand for egg incubation for several nesting seasons – is to be suspected for inducing higher FSSC infection risk in incubating eggs. This study determined the microbial diversity and pathogen abundance of nest sand (Chapter 1) to find the association between used-nest sand and FSSC infection on eggs. Distinct microbiota and a higher relative abundance of FSSC were found in nest sand of sea turtle hatcheries that reuse the sand compared to the nesting beach and hatchery which did not reuse sand. This work emphasizes that stringency in hatchery management must be maintained for the efforts of conservation to not be in vain. Besides the field survey, this study further determines the genomic basis of pathogenicity in FSSC pathogens isolated from dead sea turtle eggs and various host types (Chapter 2). Highly contiguated assemblies of six FSSC strains were produced and compared, revealing a spectrum of conservation patterns in the FSSC chromosome which can be categorised into three compartments: core, fast-core (FC), and lineage-specific (LS). Each chromosome type varied in structural architectures, with FC and LS chromosomes containing a significantly higher proportion of repetitive elements than core chromosomes and enriched in functions related to pathogenicity and niche expansion. These findings provide evidence that genome compartmentalisation was the outcome of multi-speed evolution amongst FSSC chromosomes, which is in contrast to the commonly recognized “two-speed” genome concept in fungal pathogens. In addition to the classification of FSSC’s genomic characteristics, several experiments were conducted to observe the infection scenarios and described the transcriptome responses of FSSC pathogens and Chinese soft-shelled turtle Pelodiscus sinensis eggs post-inoculation (Chapter 3). The experiments demonstrated that F. falciforme and F. keratoplasticum can penetrate eggshells and colonise egg inclusions, indicating FSSC are opportunistic pathogens toward eggs and identified differentially genes also associated with plant pathogenicity including the most upregulated genes encoding the CFEM (Common in Fungal Extracellular Membrane) domain. The outcome of this dissertation should allow the gain of fundamental knowledge regarding the pathology behind FSSC on eggs, which represents the beginning of critical steps towards the management of epidemics to reduce disease occurrences in the wild and man-managed settings. Moreover, this study establishes genomic resources and an animal model for fungal pathogens of the trans-kingdom hosts.

    Acknowledgements i Abstract ii List of Tables vi List of Figures vii List of Abbreviations viii Chapter 1: Background 1 1.1 Emerging fungal diseases 1 1.2 Fungal infection on sea turtle eggs 2 1.3 Fusarium pathogenomics 4 1.4 Objectives 4 Chapter 2: Microbial diversity and pathogen abundance in sea turtle hatchery 7 2.1 Introduction 8 2.2 Methods and Materials 9 2.2.1 Site description and sampling 9 2.2.2 Fungal isolation and culture 11 2.2.3 Nucleic acid isolations, amplifications and sequencing 11 2.2.4 Multi-locus sequence typing of fungal isolates 12 2.2.5 Amplicon read processing of sand samples 12 2.2.6 Fusarium solani species complex species-level analyses 12 2.2.7 Microbiota analysis of sand samples 13 2.3 Results 14 2.3.1 Data characteristics 14 2.3.2 Distinct microbiota between beach and hatchery sands 15 2.3.3 Differentially abundant taxa in hatchery sands 18 2.3.4 Higher abundance of FSSC and P. boydii in reused sand 20 2.4 Discussion 21 2.5 Conclusion 23 Chapter 3: Comparative genomics of Fusarium solani species complex 25 3.1 Introduction 26 3.2 Methods and Materials 27 3.2.1 Fungal culturing conditions 27 3.2.2 Species identification of isolates 27 3.2.3 Nucleic acids isolation, genome and transcriptome sequencing 28 3.2.4 Genome assembly and annotations 28 3.2.5 Comparative genomic analyses 29 3.2.6 Core, fast-core and lineage-specific chromosomes assignment 30 3.3 Results 30 3.3.1 Genome characteristics of six sequenced FSSC isolates 30 3.3.2 Comparative genomics analyses 32 3.3.3 FSSC genomes are multi-compartmented 34 3.3.4 Distinct structural and biological features of each chromosome type 37 3.3.5 Evolutionary dynamic of genome compartmentalisation 38 3.4 Discussion 39 3.5 Conclusion 41 Chapter 4: Pathology and transcriptomic responses of Fusarium solani species complex during animal infection 42 4.1 Introduction 43 4.2 Methods and Materials 44 4.2.1 Fungal culturing conditions 44 4.2.2 Animal sample preparation 44 4.2.3 Host attraction assay 45 4.2.4 Histology during initial disease establishment 45 4.2.5 Animal inoculation with FSSC 46 4.2.6 Total RNA isolation, library preparation and sequencing 47 4.2.7 RNA-seq reads processing 48 4.2.8 Transcriptome analysis of FSSC pathogens 48 4.2.9 Transcriptome analysis of P. sinensis host 49 4.3 Results 49 4.3.1 FSSC pathogens are not attracted to turtle egg 49 4.3.2 Penetration and colonisation of FSSC pathogens on turtle egg 50 4.3.3 Gene expression pattern of FSSC infecting P. sinensis egg 53 4.3.4 Gene expression pattern of P. sinensis infected by FSSC 53 4.3.5 Transcriptome profile of FSSC pathogens infecting P. sinensis egg 55 4.3.6 Plant virulence genes were upregulated during animal infection 57 4.3.7 Differentially expressed genes of FSSC during animal infection are not associated with fast-core and lineage-specific chromosomes 61 4.3.8 Transcriptome profile of P. sinensis infected by FSSC 62 4.4 Discussion 63 4.5 Conclusion 64 Chapter 5: Conclusion 65 References 67 Appendices 81 Appendix A: Supplementary Materials for Chapter 2 81 Appendix B: Supplementary Materials for Chapter 3 89 Appendix C: Supplementary Materials for Chapter 4 105 Appendix D: Data Availability 119

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