葡萄座腔菌科 (Botryosphaeriaceae) 涵蓋許多重要的植物病原真菌，其下的毛球雙胞菌屬 (Lasiodiplodia) 包含許多農業重要植物病原真菌，其物種具有多寄主並廣泛分布於各種環境中。L. theobromae是Lasiodiplodia屬內重要且致病力強的物種，多變的形態使其被認為包含許多隱蔽性物種，為一複合群 (complex)。在台灣，對於Lasiodiplodia屬的物種調查、遺傳多樣性和致病力差異缺乏整合性研究。因此，本研究針對台灣有關感染果樹作物的Lasiodiplodia屬病原真菌物種多樣性進行調查、評估遺傳多樣性、遺傳結構劃定及致病力差異評估。調查結果發現數種Lasiodiplodia物種在其果樹物種中的新感染記錄。本研究結合四組基因片段進行Lasiodiplodia屬的物種鑑定分析，鑑定結果包含六種Lasiodiplodia物種：L. brasiliensis、L. hormozganensis、L. pseudotheobromae、L. rubropurpurea、L. theobromae 及 L. iranensis。於後利用16組具多型性且可跨物種之微衛星基因座進行遺傳分析，驗證Lasiodiplodiaa屬的物種劃界並評估其遺傳多樣性及遺傳分化，劃定遺傳結構及基因型群。分析結果顯示，六種Lasiodiplodia屬的物種和本研究劃定的L. theobromae四個基因型群（G1-G4）具有高遺傳多樣性及低基因交流，並存在與寄主相關的遺傳分化及致病力差異。此外，本研究所劃定的L. theobromae之基因型群顯示了其寄主植物與病原真菌的共演化關係。此研究結合族群遺傳學及植物病理研究資訊，對於在台灣存在的Lasiodiplodia屬的物種之遺傳和致病力差異提供關鍵見解，可供相關的病害防治及管理策略參考資源。

The genus Lasiodiplodia contains many agricultural plant pathogens. The type species L. theobromae is multi-host and considered a species complex. In Taiwan, the species survey, genetic diversity and the aggressiveness variation of the Lasiodiplodia have never been accurately studied. Therefore, this study investigated Lasiodiplodia species associated with various fruit species. Several Lasiodiplodia species are new records in fruit species. Fungal identification was accomplished with phylogenetic analyses based on combined four sequence data. Six Lasiodiplodia species were identified: L. brasiliensis, L. hormozganensis, L. pseudotheobromae, L. rubropurpurea, L. theobromae and L. iranensis. Sixteen high-efficiency transferable microsatellite markers were used to test Lasiodiplodia species delimitation and examine genetic diversity. Six Lasiodiplodia species and four identified genetic groups (G1-G4) of L. theobromae with high genetic diversity, low gene flow, recombination, host-associated differentiation, and aggressiveness variation, indicating powerful evolutionary potential in Taiwan. Moreover, the genetic group pattern expressed host-pathogen interaction's co-evolutionary dynamics. This study provided critical insights into the genetic and pathogenicity variation of Lasiodiplodia species and would be helpful for related pathogen disease management.

論文審定書 i
Acknowledgements ii
摘要 iii
Abstract iv
List of contents v
List of Figures xii
List of Tables xx
CHAPTER 1 – INTRODUCTION1
1.1Introduction1
1.2 Botryosphaeriaceae fungi3
1.2.1 The systematics of Botryosphaeriaceae4
1.2.2 The genera of Botryosphaeriaceae and sexual reproduction evidence5
1.3 The genus Lasiodiplodia7
1.3.1 Taxonomy of Lasiodiplodia9
1.3.2 Confusing taxonomy problem of Lasiodiplodia18
1.3.3 Disease symptoms and co‐infection of Lasiodiplodia22
1.3.4 The reports in Taiwan24
1.4 Lasiodiplodia theobromae (Pat.) Griffon & Maubl.26
1.4.1 Taxonomy for Lasiodiplodia theobromae27
1.4.2 Morphology and cultural characteristics28
1.4.3 Conidia dispersal ability of L. theobromae29
1.5 Population genetic studies of Lasiodiplodia31
1.5.1 Molecular identification studies of Lasiodiplodia31
1.5.2 Population genetic studies among Lasiodiplodia species32
1.5.3 Microsatellite genotyping34
1.6 Variability for aggressiveness37
1.7 Rationale, goals and objectives41
1.7.1 Rationale41
1.7.2 Goals42
1.7.3 Objectives43
CHAPTER 2 – STARTING MATERIALS AND METHODS45
2.1 Disease symptoms and fungal collection45
2.1.1 Fungal isolation50
2.1.2 Morphological characteristics51
2.2 DNA extraction55
CHAPTER 3 – MULTILOCUS SPECIES DELIMITATION AND THE CRYPTIC DIVERSITY OF THE LASIODIPLODIA SPECIES IN TAIWAN56
3.1 Introduction56
3.2 Goals and objectives58
3.2.1 Goals58
3.2.2 Objectives58
3.3 Materials and Methods59
3.3.1 PCR and sequencing59
3.3.1.1 sequence assembly61
3.3.2 Multigene phylogenetic analysis62
3.3.2.1 Neighbor-joining method62
3.3.2.2 Maximum Parsimony method63
3.3.2.3 Maximum likelihood method63
3.3.2.4 Bayesian inference method64
3.3.2.5 Haplotype network construction64
3.3.2.6 Cryptic species diversity of Lasiodiplodia65
3.3.3 Assessment of nucleotide diversity66
3.3.3.1 Sequence nucleotide diversity analysis66
3.3.3.2 Recombination and linkage disequilibrium66
3.3.4 Demographic history67
3.3.5 Genetic differentiation analysis68
3.3.6 Genetic structure analysis69
3.3.6.1 STRUCTURE analysis69
3.3.6.2 GENELAND analysis71
3.4 Results73
3.4.1 Fungal isolates73
3.4.2 Molecular diagnosis and phylogenetic reconstruction75
3.4.3 Cryptic species diversity of Lasiodiplodia87
3.4.4 Nucleotide diversity, recombination and linkage disequilibrium90
3.4.5 The hierarchical analysis approaches to assess grouping factor94
3.4.6 Population structure98
3.4.6.1 Cluster analysis of the Lasiodiplodia species98
3.4.6.2 Cryptic genetic groups delimitation of L. theobromae103
3.4.7 Demographic parameter estimation105
3.5 Discussion and conclusion108
3.5.1 New occurrence records of Lasiodiplodia species in Taiwan108
3.5.2 The inconsistent phylogeny of Lasiodiplodia species111
3.5.3 Comments on species delimitation and taxonomy115
3.5.4 The genetic diversity of Lasiodiplodia species117
3.5.5 Recombination, demographic and divergence time of Lasiodiplodia118
3.5.6 Cryptic genetic diversity and structure of Lasiodiplodia121
CHAPTER 4 – FINE-SCALE POPULATION GENETIC STRUCTURE FOR LASIODIPLODIA THEOBROMAE IN TAIWAN124
4.1 Introduction124
4.2 Goals and objectives127
4.2.1 Goals127
4.2.2 Objectives127
4.3 Materials and Methods129
4.3.1 Microsatellite genotyping129
4.3.2 Assessment of transferability, gene diversity and recombination130
4.3.3 Genetic differentiation analysis132
4.3.4 Genetic structure analysis133
4.3.4.1 Principal coordinate analysis (PCoA)133
4.3.4.2 STRUCTURE analysis133
4.3.4.3 Discriminant Analysis of Principal Components (DAPC) analysis136
4.3.4.4 GENELAND analysis137
4.3.5 Estimation of multilocus SSR genotype evolutionary relationships138
4.3.6 Isolation-with-migration analyses138
4.3.7 Variation in pathogenicity140
4.4 Results142
4.4.1 Polymorphism of microsatellite Markers142
4.4.2 Genetic diversity and inbreeding146
4.4.3 The hierarchical analysis approaches to assess the grouping factor150
4.4.4 Patterns of genetic structure156
4.4.4.1 Cluster analysis of Lasiodiplodia species156
4.4.4.2 Cryptic genetic groups delimitation of Lasiodiplodia theobromae168
4.4.4.3 Multilocus genotype (MLG) evolutionary relationships176
4.4.5 Demographic parameter estimation180
4.4.5 Variation in pathogenicity187
4.5 Discussion and conclusion188
4.5.1 Strategies for the rapid identification of Lasiodiplodia cryptic species with high-efficiency microsatellite marker188
4.5.2 Genetic diversity and recombination189
4.5.3 Patterns of genetic structure and the factors that drive the genetic differentiation of Lasiodiplodia theobromae193
4.5.4 Genetic differentiation with a high proportion of shared alleles of Lasiodiplodia species196
4.5.4.1 The degree of gene flow197
4.5.4.2 The directional selection200
4.5.4.3 Shared ancestral variation201
4.5.5 Variation in pathogenicity202
CHAPTER 5 – COMPREHENSIVE DISCUSSION AND CONCLUSIONS206
5.1 Discussion206
5.1.1 The diversity of Lasiodiplodia species from fruit crops in Taiwan206
5.1.2 The genetic diversity and recombination208
5.1.3 Shared ancestral variation and directional selection of Lasiodiplodia212
5.1.5 Host-associated differentiation214
5.2 Conclusions and future work220
References222
Appendices277

郭章信 (1998) Botryodiplodia theobromae引起的萊豆苗莖枯病。植物保護學會刊 40：315-327。
黃巧雯, 張詠婷, 趙若茵, 黃晉興 (2019) 由 Lasiodiplodia theobromae 引起之百香果果腐病。台灣農業研究 68：333-340。
蘇俊峯,