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研究生:蔡宜珍
研究生(外文):Ichen Tsai
論文名稱:探討臺灣茶輪斑病相關擬盤多毛孢類群真菌之多樣性與病原性
論文名稱(外文):Diversity and pathogenicity of pestalotioid species associated with grey blight disease of tea (Camellia sinensis) in Taiwan
指導教授:歐海仁
指導教授(外文):Hiran A. Ariyawansa
口試委員:曾顯雄鍾嘉綾羅南德
口試委員(外文):Shean-Shong TzeanChia-Lin ChungRoland Kirschner
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:193
中文關鍵詞:假擬盤多毛孢屬新種新擬盤多毛孢屬親緣關係分析擬盤多毛孢屬
外文關鍵詞:Neopestalotiopsisnovel speciesPestalotiopsisphylogenetic analysesPseudopestalotiopsis
DOI:10.6342/NTU202000371
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茶 (Camellia sinensis (L.) O. Kuntze) 是廣泛被栽植於全球熱帶與亞熱帶地區的嗜好料類特用作物。製茶時多使用嫩葉,因此茶的葉部病害顯得至關重要。由擬盤多毛孢類群真菌導致的輪斑病,是世界上最具危害性的茶樹病害之一。傳統上,擬盤多毛孢類群真菌被以分生孢子中間三個細胞的顏色分布型式進行分類。Maharachchikumbura等人於2014年依據分子鑑定與形態特徵,從擬盤多毛孢屬 (Pestalotiopsis) 中另立二新屬,分別為假擬盤多毛孢屬 (Pseudopestalotiopsis) 與新擬盤多毛孢屬 (Neopestalotiopsis)。即便現在臺灣已有不少前人研究可作為茶樹真菌病害相關知識奠基,茶輪斑病相關真菌的多樣性、致病性,及分子生物學系統基礎仍有待釐清。本研究聚焦於擬盤多毛孢類群真菌,目標為探究導致臺灣茶輪斑病的病原真菌種類與特性。研究期間,於臺灣17處茶區調查採集共獲98株擬盤多毛孢類群真菌株。利用單對與結合的基因序列片段 (ITS, tub2, tef1-α) 進行比對分析與親緣關係鑑定,配合形態特徵分類依據,本研究中,多數菌株的分類地位已被確知,且所探討的98株真菌分別隸屬於於前述三屬中。一新種於本研究中被發現,並命名為Pseudopestalotiopsis annellata。此外,本研究發現共五種臺灣新紀錄種:Ps. chinensis、Ps. camelliae-sinensis、P. camelliae、P. yanglingensis以及P. trachicarpicola,同時為首次於臺灣茶樹上發現這些菌種。我們也歸納本研究中最頻繁自茶分得的菌種是Ps. chinensis。病原性試驗結果指出,本試驗所使用的23菌株中有22株確能感染具傷口的茶樹葉片,所致病斑大小於異種有程度上的差別,且葉部病斑形態與茶園中自然發生的病斑特徵相似。依據本研究在親緣鑑定與病原性測試的結果,我們認為Ps. chinensis 與 Ps. camelliae-sinensis 可能是在臺灣茶園中造成茶輪斑病的主要真菌類群。我們亦於研究中發現,目前被廣泛應用於擬盤多毛孢類群真菌親緣分析的數對基因座並無法揭開新擬盤多毛孢屬內菌種的親緣關係。本研究是臺灣首次深入探討致茶輪斑病的擬盤多毛孢類群真菌的多樣性與病原性。
Camellia sinensis (L.) O. Kuntze, commonly known as tea, is widely cultivated in tropical and subtropical areas around the world. Tea is produced with young shoots of tea plants, therefore, the foliar diseases are of great concern. Grey blight disease caused by pestalotioid fungi is known as one of the most destructive tea diseases on earth. Traditionally, the taxonomy of pestalotioid taxa was mainly based on conidial morphology such as colour intensities of the median cells, and classified mainly in Pestalotiopsis and Pestalotia. However, species of Pestalotiopsis were recently reviewed by Maharachchikumbura et al. (2014), and two novel genera, Pseudopestalotiopsis and Neopestalotiopsis, were segregated from Pestalotiopsis. Even though several studies have provided the groundwork for the fungal diseases associated with C. sinensis in Taiwan, the knowledge related to the pathogenicity, diversity and molecular systematics causing grey blight remained unresolved. This study was conducted to identify and characterize the causal agent(s) of the grey blight disease on tea plants. A total of 98 pestalotioid fungi associated with symptomatic leaves of C. sinensis from 17 tea fields in Taiwan were investigated. Based on single and concatenated DNA sequences (ITS, tub2, tef1-α) phylogenies together with morphology, we resolved most of the pestalotioid species used in this study. The study revealed seven well-classified taxa, and seven ambiguous clades in three genera, i.e. Pseudopestalotiopsis, Pestalotiopsis, and Neopestalotiopsis. One novel species in Pseudopestalotiopsis, Pseudopestalotiopsis annellata, was introduced. Ps. chinensis, Ps. camelliae-sinensis, P. camelliae, P. trachicarpicola and P. yanglingensis were reported for the first time from C. sinensis in Taiwan. Ps. chinensis was the species most frequently isolated from C. sinensis in this study. Results of pathogenicity assessments exhibited that, with wound inoculation, 22 out of the 23 isolates in this study were pathogenic on tea leaves, and pathogenicity among species varied from obvious to minute. The induced foliar symptoms were comparable to those occurring under natural conditions in the field. Overall, Ps. chinensis, and Ps. camelliae-sinensis were identified as the major pathogens associated with grey blight disease of tea in Taiwan. Furthermore, our data suggested that the currently broadly used loci in Pestalotiopsis and allied genera do not steadily deliver constant and adequate resolution in tree topologies, especially for Neopestalotiopsis. This is the first study of the diversity and pathogenicity of pestalotioid fungi as causal agents of tea grey blight disease in Taiwan.
ACKNOWLEDGEMENTS I
摘要 III
ABSTRACT IV
LIST OF TABLES IX
LIST OF FIGURES X
CHAPTER ONE: LITERATURE REVIEW 1
Historical outline 1
Molecular studies and modern classification 3
Life cycle 6
Ecology and importance of pestalotioid taxa 7
Tea and grey blight disease 8
CHAPTER TWO: PHYLOGENY AND TAXONOMY 12
Introduction 12
Materials and methods 14
Fungal isolates and morphological examination 14
DNA extraction and PCR amplification 17
Phylogenetic analyses 18
Results 20
Field surveys and fungal isolations 20
Phylogeny 21
Taxonomy 35
Discussion 60
CHAPTER THREE: PATHOGENICITY EVALUATION 63
Introduction 63
Materials and methods 65
Pathogenicity tests 65
Growth rate 68
Statistical analysis 68
Results 68
Pathogenicity 68
Growth rate 74
Discussion 78
GENERAL CONCLUSIONS 82
REFERENCES 84
APPENDIX 97
Appendix A: Supplementary data 97
A1. Sequences of phylogenetically representative isolates included in this study. 97
A2. Details of isolates included in the phylogenetic analyses. 131
A3. Single-gene phylogenetic trees 174
A4. Supplementary figures of pathogenicity test 189
Appendix B: Media and protocols 190
B1. Medial compositions and preparations 190
B2. PCR protocols 192
Appendix C: List of field owners and farmers 193
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