番椒疫病 (Phytophthora blight) 由Phytophthora capsici L. 所引起，是全球番椒
生產的主要限制因子之一。已有學者利用不同基因型 (genotype) 的番椒品種/品系
做為鑑別寄主 (differential host)，鑑定病原菌的病原型，目前對番椒疫病菌病原型
的鑑定已普遍應用於抗病育種上。台灣自 2007 年陸續於茄子、番茄和番椒中分離
到番椒疫病菌 A2 配對型菌株。滅達樂是防治此病害的主要化學藥劑，在台灣以滅
達樂相關藥劑防治卵菌綱病原菌所造成的病害已超過 20 年的歷史。進行這個研究
的目的是1.) 要瞭解台灣番椒疫病菌株的生物特性，包括病原型、配對型的特性和
對滅達樂的感受性，2.) 篩選抗病品種，包括篩選抗病種原和評估亞蔬高級品系的
抗性。此研究利用四種不同基因型的番椒品種/品系 Early Calwonder、PBC 137、PBC
602 sel 和 PI 201234 做為鑑別寄主，以根部及培養土澆灌方式接種後，將自台灣
蒐集的番椒疫病菌共區分為 3 個不同的病原型，即病原型 1、2 和 3，目前在台
灣以病原型 3 的菌株為毒力最強且優勢的族群。病原型 3 的菌株在 PDA 和 V8
培養基平板上培養的生長速度都比病原型 1 和 2 緩慢。病原型 1 菌株的孢囊比
病原型 2 和 3 菌株的孢囊小而且圓。病原型 1 的孢囊平均為 43±3 × 33±2 μm，
長寬比值為 1.3±0.06；病原型 2 的孢囊平均為 50±6 × 35±5 μm，長寬比值為
1.4±0.09；病原型 3 的孢囊平均為 49±4 × 33±3 μm，長寬比值為 1.5±0.15。利用分
子檢測技術鑑定病原型，以 P. capsici 誘導蛋白設計引子對 (PcE/PcER) 進行聚合酶連鎖反應和增幅片段長度多形性分析 P. capsici 不同病原型之核酸多形性的結果，可將供試菌株明顯地區分為 2 個分子群，分子群 1 為病原型 1，分子群 2 為病原型 2 和 3。進行台灣番椒疫病菌株 A1 和 A2 配對型之特性比較，結果顯示A2 配對型菌株在 PDA 及 V8 平板上培養的生長速度皆比 A1 配對型菌株緩慢；A2 配對型菌株於 36℃ 高溫下仍能生長、孢囊較 A1 配對型的孢囊大 (A2 配對型的孢囊平均為 50±3 x 31±2 μm； A1 配對型的孢囊平均為 47±5 x 35±3 μm)
且狹長 (A2 配對型的長/寬比值為 1.6±0.07；A1 配對型的長/寬比值為 1.4±0.08)。
A2 配對型菌株只有病原型 2 及 3，常造成較嚴重的罹病度，而目前田間番椒疫病菌株以 A1 配對型為較優勢之族群。評估台灣番椒疫病菌對滅達樂的感受性之檢測結果，得知台灣大部分番椒疫病菌對滅達樂感受性屬敏感性 (68.7%)。另將所有菌株區分為 2008 年以前收集的菌株與 2008 當年收集的菌株進行比較，菌株對滅達
樂的抗性有大幅提升的趨勢，百分率由 3 % 提升至 43.4 %。測試三種病原型對滅達樂感受性，則以病原型 1 菌株 (66.7%) 較抗滅達樂。若另以 2008 年收集的菌株區分為 A1 和 A2 配對型菌株進行評估，55.3 % A1 配對型菌株具抗藥性，A2配對型菌株則只有 13.3 % 具抗藥性，這顯示 A2 配對型菌株對滅達樂較敏感。本
次篩選抗病種原之研究是由亞蔬-世界蔬菜中心種原組 (Genetic resources and seed unit; GRSU) 提供 5 個不同種 (Capsicum baccatum、C. chacoense、C. chinense、C.
frutescens 和 C. pubescens) 的野生番椒及一個栽培種 C. annuum 總共 785 個種原 (accession)。全部種原均以根部及培養土澆灌方法接種疫病菌進行篩選，獲得野生番椒 C. frutescens 的 1 個種原 TC05415 之抗病植株子代抗病百分率達94 %
以上。其次評估亞蔬高級番椒品系之抗病性，由亞蔬-世界蔬菜中心番椒育種組(pepper unit) 提供 75 個高級品系也同樣進行根部及培養土澆灌接種疫病菌，結果篩選獲得 6 個高級品系之子代(F8)，抗病百分率達 87.5 % 以上，其中 7 個子代抗病百分率仍維持在 100 %。在本試驗篩選得到的抗病種原，可以提供番椒育種單位作為抗病育種的親本，而篩選的高抗病性之高級品系則可經由區域試驗檢測其抗病穩定性及園藝性狀，以作為未來推廣達到防治疫病之目的。目前田間番椒疫病菌株以病原型3 為較優勢族群，2008 年番椒疫病菌A2 配對型菌株的出現對番椒所造成的影響到目前尚未完全了解，然而A2 配對型菌株較能耐高溫、產生較多的游走孢子、且造成較嚴重的罹病度，對台灣的番椒可能造成嚴重危害。A1 和A2 配對型菌株行有性繁殖，基因重組使其遺傳特性更趨複雜，將使未來防治這個病害更加得困難，顯示對病原菌族群變化的監視、加強抗病種原的篩選與培育抗病品種是刻不容緩的工作。Phytophthora blight of pepper (Capsicum spp.) caused by Phytophthora capsici L. is
one of the major limiting factor of pepper production worldwide. Several reports had
identified the existence of pathogenic specificity of P. capsici on peppers. Pathotypes are
determined by the pathogen reaction to a set of differential hosts. The identification of
the pathotype of P. capsici has been commonly used the breeding program for disease
resistance. Our previous studies indicated that only the A1 mating type isolates of P.
capsici was found in Taiwan befoe 2007. The A2 mating type isolates were found on
tomato, eggplant and pepper since 2007. Metalaxyl related chemicals have been used in
the control of many different oomycete pathogens more than 20 years in Taiwan. The
objectives of this study are: i.) To understand the characteristics of P. capsici on pepper in
Taiwan including the characterizations of pathotype, mating type and in vitro assessment
of metalaxyl sensitivity and ii.) To screen the resistance variety from wild peppers and to
evaluate the resistance of AVRDC advanced lines for disease control. Four pepper
varieties/lines, Early Calwonder, PBC 137, PBC 602 sel and PI 201234, were used as
indicator plants to classify all of P. capsici isolates into three pathotypes, type1, 2 and 3.
Pathotype 3 showed the highest virulence and was the predominant population in Taiwan.
The growth rate of pathotpe 3 was slowler than pathotype 1 and 2 on both PDA and V8
plates. Pathotype 1 isolates produced small and round sporangia (43±3 × 33±2 μm, L/B:
1.3±0.06). The sporangia of pathotype 2 were 50±6 × 35±5 μm, L/B: 1.4±0.09, while
those of pathotype 3 were 49±4 × 33±3 μm, L/B: 1.5±0.15. Pathotype 1 was significantly
different from pathotype 2 and 3 based on PCR polymorphisms using primers from P.
capsici elicitin and AFLP analysis. The comparison of the difference of morphology,
zoospore productivity, growth rate, pathogenicity between A1 and A2 mating type isolates of P. capsici showed that the growth rate of the A2 mating type isolates was slower than A1 mating type in both PDA and V8 plates. The A2 mating type isolates produced larger and oval sporangia (50±3 x 31±2 um; L/B: 1.6), and grew even at 36℃.
These isolates caused more severe disease because they belonged to either pathotype 2 or 3. A1 mating type was the predominant type in Taiwan. In vitro assessment of metalaxyl sensitivity of P. capsici isolates indicated that 68.7 % of the isolates were classified as sensitive. The only 3 % isolates collected before 2008 were resistant to metalaxyl, while 43.4 % of those collected in 2008 were resistant to metalaxyl. Most pathotype 1 isolates (66.7 % resistant) were resistant to metalaxyl than pathotype 2 and 3 isolates (7.0 % and 15.1 % respectively). More A2 mating type isolates (13.3% resistant) were sensitive to
metalaxyl than A1 isolates (55.3% resistant). Resistance screening of wild germplasms collected from GRSU (Genetic resources and seed unit) of AVRDC and evaluation of resistance in AVRDC advanced lines by root-drench noculation were conducted. One wild accessions of TC05415 in Capsicum frutescens showing resistant percentage higher
than 94 % were selected. Six progeny lines (F8) of AVRDC advanced lines exhibited
resistant percentage higher than 87.5% and 7 progeny lines of AVRDC advanced lines
exhibiting 100 % resistance were selected. The resistant accessions can be used as the
resistant parent in the breeding program for disease resistance. Those resistant advanced
lines will be evaluated for durable resistance and horticultural traits for the future
extension. The impact of the occurrence of the A2 mating type on peppers is not yet clear
in Taiwan because it first appeared only in 2008. However, the A2 mating type isolates
with tolerance to high temperature and ability to produce more zoospores as well as the
genetic variation followed by sexual recombination between A1 and A2 mating types may lead to the difficulties of disease control in the future. Therefore, it's important to monitor the population shift of P. capsici and develop the resistant varieties of pepper for the effective control of phytophthora blight.中文摘要 .....................................................................................................................i
英文摘要 ....................................................................................................................iv
目錄 ...........................................................................................................................vii
表次目錄......................................................................................................................xi
圖次目錄......................................................................................................................xii
第一章：前人研究 ....................................................................................................1
引用文獻 ......................................................................................................5
第二章：台灣番椒疫病菌 (Phytophthora capsici) 生物特性之探討 ...................9
中文摘要 .....................................................................................................................9
英文摘要 ...................................................................................................................12
前言 ...........................................................................................................................15
材料和方法 ...............................................................................................................19
供試菌株的蒐集 ..............................................................................................19
菌株種類的鑑定 ..............................................................................................19
供試植株的準備 ..............................................................................................21
游走孢子接種源的製備與接種 ......................................................................21
I. 病原型 (pathotype) 特性之探討 ......................................................................21
病害等級的調查與病原型的鑑定 ..................................................................22
不同病原型之培養特性比較 ..........................................................................22
1. 在 PDA 及 V8 培養基平板之菌落形態與生長速度 ..........................22
2. 孢囊大小與形狀之比較 ..........................................................................22
3. 溫度對游走孢子產量之影響 ..................................................................23
利用分子檢測技術分析不同病原原型 P. capsici 之核酸多形性 ...............23
1. 聚合酶連鎖反應 (polymerase chain reaction; PCR) 多形性分析 ........23
viii
2. 增幅片段長度多形性 (amplified fragment length polymorphism; AFLP)
分析 ..........................................................................................................24
II. A1 和A2 配對型特性之探討 ...........................................................................25
菌株的配對型鑑定 ...........................................................................................25
生長形態特性 ...................................................................................................25
孢囊大小與形狀的觀察和在 28℃ 環境中的產孢量 ...................................26
兩種配對型的病原型評估 ...............................................................................27
III. 生體外評估番椒疫病菌對殺菌劑滅達樂之感受性 ......................................27
結果 ............................................................................................................................28
菌株種類的鑑定 ..................................................................................................28
I. 病原型 (pathotype) 特性之探討 ......................................................................28
病原型的鑑定 ...................................................................................................28
不同病原型之培養特性比較 ...........................................................................28
1. 在 PDA 及 V8 培養基平板之菌落形態與生長速度 ..........................28
2. 孢囊大小與形狀之比較 ...........................................................................29
3. 溫度對游走孢子產量之影響 ...................................................................29
利用分子檢測技術分析不同病原原型 P. capsici 之核酸多形性 ................29
1 聚合酶連鎖反應 (polymerase chain reaction; PCR) 多形性分析 ..........29
2 增幅片段長度多形性 (amplified fragment length polymorphism; AFLP)分
析 .................................................................................................................30
II. A1 和A2 配對型特性之探討 ...........................................................................30
菌株的配對型鑑定與所屬病原型 ................................................................30
生長形態特性..................................................................................................30
孢囊大小及形狀的觀察和在 28℃ 環境中的產孢量 ................................31
III. 生體外評估番椒疫病菌對殺菌劑滅達樂之感受性 ......................................31
討論 ...........................................................................................................................33
引用文獻 ...................................................................................................................37
表格說明 ...................................................................................................................43
圖示說明 ...................................................................................................................54
第三章：利用寄主抗病性防治疫病 .......................................................................64
中文摘要 ...................................................................................................................64
英文摘要 ...................................................................................................................65
前言 ...........................................................................................................................66
材料和方法 ...............................................................................................................69
自野生的番椒中篩選抗病種原 ............................................................................69
供試菌株 ..........................................................................................................69
供試植株 ..........................................................................................................69
游走孢子接種源的製備與接種 ......................................................................69
病害嚴重度的調查與抗病百分率之計算 ......................................................70
亞蔬高級番椒品系的抗病性評估 ........................................................................70
供試菌株 ..........................................................................................................70
供試植株 ..........................................................................................................70
游走孢子接種源的製備與接種 ......................................................................71
病害嚴重度的調查與抗病百分率之計算 ......................................................71
結果 ...........................................................................................................................72
自野生的番椒中篩選抗病種原 ......................................................................72
亞蔬高級番椒品系的抗病性評估....................................................................72
討論 ...........................................................................................................................74
引用文獻 ...................................................................................................................76
表格說明 ...................................................................................................................79
圖示說明 ...................................................................................................................83
第四章：結論 ...........................................................................................................86
附錄一 .......................................................................................................................8
