Development of Simple Detection Methods of Plant Pathogenic Title Oomycetes( 本文(Fulltext) ) Author(s) FENG, WENZHUO Report No.(Doctoral Degree) 博士(農学) 甲第708号 Issue Date 2019-03-13 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/77944 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。 Development of Simple Detection Methods of Plant Pathogenic Oomycetes （植物病原性卵菌類の簡易検出法の開発） 2018 The United Graduate School of Agriculture Science, Gifu University Science of Biological Environment (Gifu University) FENG WENZHUO Development of Simple Detection Methods of Plant Pathogenic Oomycetes （植物病原性卵菌類の簡易検出法の開発） FENG WENZHUO INDEX PREFACE .......................................................................................................... 1 CHAPTER 1 Establishment of simple LAMP procedures with detection of Pythium irregulare in field samples ................................................................... 12 MATERIALS AND METHODS ..................................................................................... 13 RESULTS ................................................................................................................... 17 1. Primer design and specificity ............................................................................... 17 2. Sensitivity of LAMP ............................................................................................. 18 3. Practical detection of P. irregulare in field samples .............................................. 18 DISCUSSION ............................................................................................................. 28 CHAPTER 2 Use of LAMP detection to identify potential contamination sources of plant- pathogenic Pythium species in hydroponic culture systems of tomato and eustoma............................................................................................... 29 MATERIALS AND METHODS ..................................................................................... 40 RESULTS ................................................................................................................... 34 1. Detection of P. aphanidermatum in tomato cultivation.......................................... 34 2. Detection of P. irregulare in eustoma cultivation .................................................. 35 DISCUSSION ............................................................................................................. 44 CHAPTER 3 LAMP detection of four plant pathogenic oomycetes and its application in lettuce fields ........................................................................................ 49 MATERIALS AND METHODS ..................................................................................... 50 RESULTS ................................................................................................................... 55 1. Specificity of the LAMP assay ............................................................................. 55 2. Sensitivity of the LAMP assay ............................................................................. 55 3. Optimization of the procedures for LAMP detection in field samples .................... 56 4. Infection sites in lettuce plants ............................................................................. 57 5. Detection of pathogens in soil samples from the field .......................................... 57 DISCUSSION ............................................................................................................. 74 CHAPTER 4 A simple LAMP detection of Phytophthora colocasiae in infected taro fields ........................................................................................................... 78 MATERIALS AND METHODS ..................................................................................... 79 RESULTS ................................................................................................................... 83 1. Specificity and sensitivity of the LAMP primers .................................................... 83 2. Identification of isolates maintained on agar media ............................................. 83 3. Development of the Plant-LAMP assay ............................................................... 84 4. Detection of the pathogen in field samples .......................................................... 84 DISCUSSION ............................................................................................................. 95 OVERALL DISCUSSION ................................................................................. 99 SUMMARY ..................................................................................................... 103 ACKNOWLEDGMENTS ................................................................................ 107 REFERENCES ............................................................................................... 109 PREFACE Plant is the very precious part of our earth for it maintaining the atmosphere, supplying food and energy, cycling water and nurturing soils and so on. However, almost all species of wild and cultivated plants are subject to disease. The diseases are recognized by many symptoms, such as chlorosis, interveinal chlorosis, stunting, and purpling (McCauley et al. 2009), and can happen in a forest, a field, and even a greenhouse. The occurrence and prevalence of plant diseases depends on the presence of pathogens, environmental conditions, weather and crops and varieties grown. Pathogen is a major class of disease- causing agents that include viruses, bacteria, fungi, nematodes, parasitic plants, and especially, oomycetes. Plant pathogenic oomycetes once classified as fungi, because of their filamentous growth, nutrition by absorption, and reproduction via spores, are now classified as a distinct group based on a number of unique characteristics (Rossman and Palm 2006). It has several hundred organisms that include some of the most devastating plant pathogens, particularly within the genera of Pythium and Phytophthora, and have colonized almost all ecosystems all over the world. This group causes devastating diseases of crop, ornamentals, and native plants including damping-off, seedling blight, root rot, foliar blight and downy mildew, and are thought to be not only the most important group of pathogens of dicotyledonous plants (Erwin et al. 1996), but also the source of yield reduction in cereal crop (Cook et al. 1987; Paulitz and Adams 2003; Harvey and Lawrence 2008). Oomycete grows best in running surface water, so it also known as “water molds”. One of its most distinguishing characteristics is the production of zoospores produced in sporangia that lead to the disease spreading rapidly (Fry and Grünwald 2010). Zoospores can swim in water films on leaf surfaces, in soil water, in 1 hydroponic media and in natural water. When zoospores are attracted to exudates and extracts from roots, they begin to settle on the baits and in the vicinity of the baits, to round up, and to begin to germinate. Moreover, “Oomycota” means “egg fungus”, and refers to the large round structures containing the female gametes, the oogonia (Waggoner and Speer 1994). Usually, each individual produces antheridia and oogonia. Almost all Pythium and some Phytophthora species are homothallic. In these species, sexual reproduction occurs in a single culture. The other Phytophthora species, in which two distinct mating types occur, and sexual reproduction requires the presence of both mating types, are heterothallic. The fertilized oogonia develop into sexual spores called oospores, which have a thick-walled structure shown to be able to survive for years in soil (Fry and Grünwald 2010). The genus Pythium includes more than 140 species with more than 40 new species having been described since 2000 (Kageyama 2014); Most of the Pythium members live in soil or aquatic environments and widely distributed all over the world. Many species cause seed, stem and root rot, and seedling damping-off in various crops, inflicting serious economic. While others are more restricted in the host and geographic range, or affect plants only under special environmental conditions (Hendrix and Campbell 1973). Pythium irregulare Buisman is one of the most important Pythium species with regard to agriculture as it has a complex and extensive host range, and typically exhibits high virulence (van der Plaats-Niterink 1981; Chen et al. 1992; Barr et al. 1997; Garzón et al. 2005, 2007). It distinguished from other Pythium species on the basis of oogonium morphology, which has an irregular number (0-5) of projections, and spherical sporangia is highly pathogenic to a wide range hosts, and has been identified on over 200 host species, such as eustoma, pineapple, grasses, tobacco, pecan trees, cucumber, onion, carrot, pepper and a number of floricultural crops. Matsumoto et al. (2000) classified Py. 2 irregulare isolates into four DNA groups based on randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), PCR-restriction fragment length polymorphism analyses, and phylogenetic analysis of the ribosomal DNA internal transcribed spacer (rDNA ITS) regions. They demonstrated that among these groups I to IV, groups III and IV were not important as plant pathogens and phylogenetically distinct from groups I and II. Groups I and II are generally regarded as a single species (Spies et al. 2011a), although Garzón et al. (2007) proposed group II as a new species, Py. cryptoirregulare. In this study, we treat group