Taxonomic study of peronosporaceae (downy mildews) based on morphological and molecular characteristics = 형태적 및 분자적 특징에 의한 노균병균과 균류의 분류학적 연구

국문 초록 (Abstract)

노균병균과(Peronosporaceae)에 속하는 균류는 모두 식물병원균으로서 주요 작물에 심각한 피해를 일으키는 중요한 분류군이다. 본 연구는 1988년부터 2007년까지 한국에서 채집하여 고려대학교 생명과학대학 표본실(KUS-F)에 보존된 시료를 대상으로 한국에 존재하는 노균병균과 균류를 최초로 정리하였다. 총 78종의 기주식물로부터 9속, 35종의 노균병균을 동정하였고, 각각의 종에 대하여 상세한 병징과 함께 균학적 특징을 기재 묘사하였다. 또한, 경제적으로 상당한 피해를 일으키지만, 분류학적으로 많은 문제점을 가지는 분류군에 대하여 형태적 및 분자적 분석을 수행하였다.
노균병균과의 종의 개념(species concept)에 대한 연구를 위하여 경제적, 분류학적으로 가장 중요한 세 복합체(complex)인, Peronospora farinosa, Bremia lactucae, Plasmopara halstedii를 대상으로 형태적 및 분자적 특징과 기주 범위 또는 특이성에 관하여 분석하였다. 이 세 복합체는 지금까지 명아주과(Chenopodiaceae), 국화과 (Asteraceae)의 상추아과(Cichorioideae)와 국화아과(Asteroideae)의 많은 식물을 각각 감염하는 유일한 균들로 동정되었으나, 이 광의적 종개념(broad species concept)은 현재의 계통학적 분석의 결과와 일치하지 않았다. 다수유전자염기서열(multigene sequences)은 이 종들이 사실상 여러 종으로 구분되어야 함을 보였으며, 이것은 결과적으로 한 종의 노균병균이 특정 기주 속 또는 종만을 감염한다는 협의적 종개념(narrow species concept)이 광의적 종개념보다 노균병균의 분류체계에 더 적합하다는 것을 증명하였다. 그럼에도 불구하고, 여전히 이 한 종개념만으로 해결할 수 없는 여러 문제점들이 존재한다. Pseudoperonospora cubensis와 Ps. humuli에서의 연구에서처럼, 일부 종은 넓은 기주 범위를 가지는 것으로 보고되었다. 또한, 기주 식물을 모를 경우 이 종개념의 적용은 제한적이며, 만약 기주 식물을 알더라도 두 종 이상의 노균병균이 한 기주 식물을 감염하는 경우가 있으므로 적용하는데 한계가 있다. 게다가 계통학적 결과만을 토대로 종을 구분할 경우 그 유전적 다양성이 종간(interspecific) 또는 종내(infraspecific)의 유전적 차이인지 명확하지 않아 수 많은 신종(new species)의 도입을 피할 수 없다. 결과적으로 형태적 특징, 계통학적 분석, 그리고 기주 범위나 특이성에 기초한 종합적인 분석이 노균병균의 동정과 분류, 그리고 나아가 그들의 진화와 다양성을 이해하는데 필요하다.
본 연구는 전세계에서 가장 큰 경제적 피해를 주는 노균병균의 분류학적 문제점들을 해결하였다. 오랫동안 시금치와 사탕무에서 발생하는 노균병의 원인균으로 간주되었던 P. farinosa f.sp. spinaciae와 P. farinosa f.sp. betae에 대해 이전에 동물이명(synonym)으로 처리되었던 P. effusa와 P. schachtii을 각각 재도입하였다. 또한, 최근에 주요 곡물로서 널리 재배되기 시작한 퀴노아(quinoa)에서 발생하는 노균병의 원인균이 기존에 알려졌던 P. farinosa가 아닌, P. variabilis임을 밝혔다. 경제적으로 중요한 박과작물의 노균병균인 Ps. cubensis와 호프 노균병균인 Ps. humuli에 대한 형태적 및 분자적 결과를 통해 Ps. humuli를 Ps. cubensis의 동물이명으로 처리하였다. 상추를 비롯한 여러 작물을 감염하는 Bremia속에 대해, 오랫동안 한 종으로 분류되었던 B. microspora, B. ovata, B. saussureae, B. sonchicola, B. lactucae이 형태적, 분자적 특징에 근거하여 서로 다른 종임을 새롭게 밝혔다. 추가적으로 본 연구를 통해 새롭게 제시된 학명은 다음과 같다. 신종(new species): Plasmopara ambrosiae on Ambrosia artemisiifolia; Peronospora oblatispora on Potentilla chrysantha, P. paradoxa, P. supina, Aphanes microcarpa. 신조합(new combination): Plasmopara domingensis on Parthenium hysterophorus. 학명의 재도입(re-instatement): P. chenopodii, P. variabilis, P. boni-henrici, P. chenopodii-polyspermi on specific species of Chenopodium.

다국어 초록 (Multilingual Abstract)

The Peronosporaceae (downy mildews) have caused spectacular and catastrophic epidemics on several crops in the past, and some of them continue to cause epidemics and severe losses to the present. In this study, downy mildew fungi in Korea have first been compiled. Many materials of host plants infected with downy mildew fungi were collected from 1988 to 2007, and deposited in the mycological herbarium KUS-F. Through the monographic study, it was described and illustrated a total of 35 species belonging to nine genera of Peronosporaceae, and arranged in alphabetical order. Their synonymy, morphological characteristics of taxonomic values, host plants, specimens examined, geographical distribution, taxonomic notes, and line drawings were given for each species. Two genera of the family, Basidiophora and Plasmoverna, were recorded new to Korea, and 10 species of three genera were firstly reported and illustrated: Basidophora entospora, Peronospora agrestis, Peronospora conferta, Peronospora chrysosplenii, Peronospora galii, Peronospora swinglei, Peronospora viciae, Plasmopara pileae, Plasmopara wilsonii, and Plasmoverna pygmaea. Sixteen species of 12 genera were added as new host plants in Korea; Anemone raddeana, Anemone reflexa, Cerastium holosteoides var. hallaisanense, Chrysosplenium flagelliferum, Chrysosplenium japonicum, Conyza canadensis, Galium spurium, Impatiens noli-tangere, Pilea hamaoi, Pilea mongolica, Plantago major var. japonica, Potentilla paradoxa, Salvia plebeia, Veronica didyma var. lilacina, Veronica persica, and Vicia angustifolia var. segetilis.
The present studies are dealing with the morphological and molecular characteristics, and host range or specificity for Peronospora farinosa, Bremia lactucae, and Plasmopara halstedii within the Peronosporaceae. The three species were usually regarded as sole parasitic fungus causing downy mildew disease on many plants covering the family Chenopodiaceae and two subfamilies, the Cichorioideae and the Asteroideae, of the family Asteraceae, respectively. This view, however, has been challenged with the present extensive results. Phylogenetic analysis of the multigene sequences indicates that they are not monophyletic, but falls into several different branches according to their host plants. Therefore, it indicated that the narrow species concept is largely adequate for species delimitation of the downy mildews. However, as in Ps. humuli and Ps. cubensis, a few downy mildew may have a host range beyond the family of the host plant. Also, if we did not know the host plants the fungus infects, the species concept could be not applied, and even if we kwno, more than two distinct species parasitic on the same host genus or even the same host species have been often reported in the Peronosporaceae. Besides, phylogenetic analysis alone without regarding of morphological characteristics would also inevitably lead to the creation of many more species. Therefore, the narrow species concept alone is insufficient to define all species within the Peronosporaceae. Before this study, the splitting of species based on morphological features has been criticized due to the lack of clear-cut difference, but the present results revealed that many traditional morphological species well correspond to units defined by host specialization and phylogenetic analysis. Therefore, a combining investigation of the morphological characteristics, molecular phylogenetic reconstructions, and host range or specialization, will certainly yield important results which contribute to define species and further to the understanding of the evolution and diversification of downy mildews.
Taxonomic problems with downy mildew pathogens parasitic on economically important crops have been also resolved in this study. The names Peronospora effusa and P. schachtii have been reinstated for the downy mildew fungi parasitic on spinach and beet, respectively, and the causal agent of the disease on an economically important quinoa (Chenopodium quinoa) has been identified as Peronospora variabilis, and not P. farinosa as claimed by most studies. For two economically important downy mildews, Pseudoperonospora humuli and Ps. cubensis, the former species has been to the taxonomic synonyms of the latter one. The taxonomic study of five Bremia species demonstrated that B. microspora, B. ovata, B. saussureae, and B. sonchicola should be regarded as independent taxa from B. lactucae, parasitic on lettuce. There were also introduced following taxonomic novelties and revisions: Plasmopara ambrosiae sp. nov. on Ambrosia artemisiifolia, Peronospora oblatispora sp. nov., parasitic on Potentilla chrysantha, P. paradoxa, P. supina and Aphanes microcarpa, P. domingensis comb. nov. on Parthenium hysterophorus, and re-instatement of P. boni-henrici, P. chenopodii, P. chenopodii-polyspermi, and P. variabilis infecting specific species of Chenopodium.

목차 (Table of Contents)

• CONTENTS
• ABSTRACT .................................................................. i
• KOREAN ABSTRACT .....................................................iii
• CONTENTS .................................................................v
• LIST OF FIGURES .........................................................x
• LIST OF TABLES ........................................................xvi
• CHAPTERS
• I. General introduction
• 1.1. The family Peronosporaceae ................................2
• 1.2. Taxonomy of downy mildews ..............................3
• 1.3. Species concept of downy mildews ......................5
• 1.4. Objectives of the study ........................................6
• II. Peronosporaceae of Korea
• 1. Introduction ..............................................................10
• 2. Morphological study of downy mildews .......................10
• 3. Downy mildews in Korea ...........................................11
• Taxonomy ...................................................................15
• Additional list of Peronosporaceae from Korea ................128
• III. Phylogenetic studies of Peronosporaceae
• 1. Re-consideration of Peronospora farinosa infecting Spinacia oleracea as distinct species, Peronospora effusa
• 1.1. Abstract ...............................................................132
• 1.2. Introduction ..........................................................132
• 1.3. Materials and methods ..........................................134
• 1.3.1. Fungal specimens ..............................................134
• 1.3.2. DNA extraction, PCR amplification, and sequencing ................................................................134
• 1.3.3. Sequence alignment and phylogenetic analysis .....134
• 1.4. Results ................................................................139
• 1.4.1. Molecular analysis .............................................139
• 1.4.2. Morphological analysis .......................................140
• 1.5. Discussion ...........................................................141
• Taxonomy .................................................................150
• 2. Morphological and molecular analyses support the existence of host-specific Peronospora species infecting Chenopodium
• 2.1. Abstract ...............................................................154
• 2.2. Introduction ..........................................................154
• 2.3. Materials and methods ..........................................155
• 2.3.1. Fungal specimens ..............................................155
• 2.3.2. Morphological analysis .......................................155
• 2.3.3. DNA extraction, PCR amplification, and sequencing ................................................................155
• 2.3.4. Sequence alignment and phylogenetic analysis .....158
• 2.4. Results and discussion .........................................158
• Taxonomy ..............................................................161
• 2.5. Conclusion ..........................................................168
• 3. Multigene phylogenetic analyses support recognition of the downy mildew complex (Peronospora schachtii-effusa-rumicis) as separate species
• 3.1. Abstract ...............................................................170
• 3.2. Introduction ..........................................................170
• 3.3. Materials and methods ..........................................172
• 3.3.1. Fungal specimens ..............................................172
• 3.3.2. DNA extraction, PCR amplification, and sequencing ................................................................172
• 3.3.3. Sequence alignment and phylogenetic analysis .....175
• 3.4. Results ................................................................176
• 3.4.1. Sequence alignment ...........................................176
• 3.4.2. Phylogenetic analysis of ITS rDNA .......................176
• 3.4.3. Phylogenetic analysis of COX2 and ND1 mtDNA .....177
• 3.4.4. Phylogenetic analysis of a combined dataset ........177
• 3.5. Discussion ...........................................................186
• 4. Morphological and molecular identification of the causal agent of downy mildew disease on quinoa (Chenopodium quinoa)
• 4.1. Abstract ...............................................................192
• 4.2. Introduction ..........................................................192
• 4.3. Materials and methods ..........................................193
• 4.3.1. Fungal specimens ..............................................193
• 4.3.2. Morphological analysis .......................................193
• 4.3.3. Molecular analysis .............................................193
• 4.4. Results ................................................................196
• 4.4.1. Morphological analysis .......................................196
• 4.4.2. Phylogenetic analysis .........................................198
• 4.5. Discussion ...........................................................200
• 5. A new downy-mildew of the Rosaceae: Peronospora oblatispora sp. nov. (Chromista, Peronosporales)
• 5.1. Abstract ...............................................................202
• 5.2. Introduction ..........................................................202
• 5.3. Materials and methods ..........................................202
• 5.3.1. Fungal specimens ..............................................203
• 5.3.2. Morphological analysis .......................................203
• 5.3.3. DNA extraction, PCR amplification, and sequencing ................................................................203
• 5.3.4. Sequencing alignment and phylogenetic analysis ..203
• 5.4. Results ................................................................205
• 5.5. Discussion ...........................................................208
• Taxonomy .................................................................209
• 6. Revision of Pseudoperonospora cubensis and Ps. humuli based on molecular and morphological data
• 6.1. Abstract ...............................................................214
• 6.2. Introduction ..........................................................214
• 6.3. Materials and methods ..........................................216
• 6.3.1. Fungal isolates ...................................................216
• 6.3.2. Light microscopy ................................................216
• 6.3.3. DNA extraction, PCR amplification, and sequencing ................................................................216
• 6.3.4. Sequence alignment and phylogenetic analysis .....219
• 6.4. Results ................................................................220
• 6.4.1. Morphological analysis .......................................220
• 6.4.2. Molecular analysis .............................................222
• 6.5. Discussion ...........................................................222
• 7. Extreme size and sequence variation in the ITS rDNA of Bremia lactucae
• 7.1. Abstract ...............................................................230
• 7.2. Introduction ..........................................................230
• 7.3. Materials and methods ..........................................231
• 7.4. Results and discussion .........................................233
• 8. Taxonomy of five Bremia species infecting the Cichorioideae of Asteraceae based on morphological characteristics and molecular analysis
• 8.1. Abstract ...............................................................240
• 8.2. Introduction ..........................................................240
• 8.3. Materials and methods ..........................................242
• 8.3.1. Fungal materials .................................................242
• 8.3.2. Nomenclature of host plants ................................242
• 8.3.3. Light microscopy ...............................................242
• 8.3.4. DNA extraction, PCR, and sequencing ..................244
• 8.3.5. Sequence alignment and phylogenetic analysis .....244
• 8.4. Results ................................................................245
• 8.4.1. Phylogenetic analysis .........................................245
• 8.4.2. Morphological analysis .......................................250
• 8.5. Discussion ...........................................................253
• 9. Plasmopara ambrosiae sp. nov., a new species causing downy mildew on Ambrosia, with notes on Plasmopara halstedii, based on morphological and multiple gene phylogenies
• 9.1. Abstract ...............................................................260
• 9.2. Introduction ..........................................................262
• 9.3. Materials and methods ..........................................262
• 9.3.1. Downy mildew specimens ..................................262
• 9.3.2. Morphology studies ............................................262
• 9.3.3. DNA extraction and PCR amplification and sequencing of the D1/D2 28S rDNA, COX2, and ND1 regions ..............262
• 9.3.4. Phylogenetic analyses ........................................264
• 9.4. Results ................................................................265
• 9.4.1. Morphological analysis .......................................265
• 9.4.2. Sequence alignment ...........................................265
• 9.4.3. Phylogenetic analysis D1/D2 region of 28S rDNA ....270
• 9.4.4. Phylogenetic analysis of COX2 mtDNA ..................270
• 9.4.5. Phylogenetic analysis of ND1 mtDNA ....................271
• 9.5. Discussion ...........................................................271
• Taxonomy .................................................................271
• 10. Reclassification of Bremia domingensis to the genus Plasmopara as Plasmopara domingensis comb. nov.
• 10.1. Abstract ..............................................................280
• 10.2. Introduction .........................................................280
• 10.3. Materials and methods .........................................280
• 10.4. Results ...............................................................280
• 10.5. Discussion .........................................................281
• New combination proposed .......................................283
• VI. General discussion ................................................285
• References .................................................................291