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造成芝麻、洛神花及長梗紫麻病害的植物菌質體之診斷鑑定及特性分析

By 曾意雯 and Yi-Wen Tseng

Abstract

植物菌質體 (phytoplasmas) 為一群絕對寄生於植物韌皮部、不具細胞壁且細胞具有多型性的革蘭氏陽性細菌。在自然界中,植物菌質體可藉由取食韌皮部的昆蟲進行傳播,例如: 葉蟬、飛蝨及木蝨等。植物菌質體可在寄主上造成多種病徵,例如: 葉部黃化、花器綠化、花器葉化及簇葉等。今年六月、九月及十月,分別發現了疑似植物菌質體感染的芝麻、洛神及長梗紫麻病株。在彰化埤頭鄉採集而得之芝麻植株出現葉部黃化、莖部不正常彎曲及花器葉化等病徵,在台中潭子鄉採集而得之洛神病株,其病徵為花器葉化及捲葉,而在苗栗南庄鄉採集的長梗紫麻病株則出現了葉部黃化及簇葉的病徵。分別抽取芝麻、洛神及長梗紫麻之病株及健株的植物總量DNA、進行巢氏PCR並將所得之片段進行解序後結果顯示,分別由芝麻曲莖及花葉植物菌質體 (sesame abnormal stem curling and phyllody phytoplasma)、洛神捲葉及花葉植物菌質體 (roselle wrinkled leaves and phyllody phytoplasma) 及長梗紫麻簇葉植物菌質體 (purple woodnettle witches’-broom phytoplasma) 所感染。進行RFLP及親緣關係的分析,其結果顯示芝麻曲莖及花葉植物菌質體歸屬於16SrII-A群。利用RFLP分析結果顯示,洛神捲葉及花葉植物菌質體及長梗紫麻簇葉植物菌質體則屬於16SrI群內的新亞群。利用親緣關係分析結果顯示長梗紫麻簇葉植物菌質體屬於16SrI群內的新亞群,而洛神捲葉及花葉植物菌質體則與16SrI-L內的菌株親緣關係相近。切取芝麻、洛神及長梗紫麻植物之莖部分別進行穿透式及掃描式電子顯微鏡的鏡檢,可看到大小約200 – 800 nm的多形性細胞。 這是台灣第一次報導芝麻被16SrII-A之植物菌質體所感染、洛神及長梗紫麻受16SrI之植物菌質體所感染,而長梗紫麻同時也為植物菌質體的新寄主。另外,在芝麻種子種皮、胚乳及幼苗的檢測結果顯示,植物菌質體僅能存在於芝麻種子種皮但卻無法在一個月及兩個月大之幼苗上檢測到植物菌質體,初步結果顯示芝麻曲莖與花葉植物菌質體無法藉由種子傳播,而植物菌質體在田間危害芝麻、洛神及長梗紫麻為台灣的新興病害,其病害傳播模式與對作物產量與品質的影響則有待進一步的調查與研究。Phytoplasmas, Gram-positive bacteria, are cell-wall free with pleomorphic cell bodies and phloem-limited in situ. In nature, phytoplasma is transmitted by phloem-feeding insects, e.g. leafhoppers, planthoppers and psyllids. Phytoplasmas can cause various symptoms such as leaf yellowing, virescence, phyllody, and witches’-broom. In June, September and October 2013, both symptomatic and asymptomatic plants of sesame, roselle and purple woodnettle were collected from the fields in Taiwan, respectively. Symptomatic sesame plants exhibiting leaf yellowing, abnormal stem curling and phyllody were observed in the field in Pitou Township, Changhua County, roselle showing phyllody and wrinkled leaves in Tantsu Township, Taichung County and purple woodnettle with leaf yellowing and witches''-broom (WB) symptoms in Nanchuang Township, Miaoli County. The extracted total DNA from plant tissues were amplified by a nested PCR followed by sequencing the putative sequences from symptomatic plants of sesame, roselle and purple woodnettle, separately. The results showed that the symptomatic plants of sesame, roselle, and purple woodnettle were strongly associated with various phytoplasmas. The 16S rDNA restriction fragment length polymorphism (RFLP) pattern and phylogenetic analysis revealed that the sesame abnormal stem curling and phyllody phytoplasma in Taiwan belongs to 16SrII-A group / subgroup. The RFLP analysis revealed that roselle wrinkled leaves and phyllody phytoplasma and purple woodnettle witches’-broom phytoplasma belong to a new unnamed subgroup of 16SrI group. The phylogenetic analysis showed that purple woodnettle witches’-broom phytoplasma belongs to a new subgroup of 16SrI group and roselle wrinkled leaves and phyllody phytoplasma is closely related to 16SrI-L subgroup. Based on the observation results under scanning electron microscopy and transmission electron microscopy, 200 – 800 nm pleomorphic bodies were only found in the phloem sieve cells of symptomatic sesame, roselle, and purple woodnettle tissues. This represent the first report of a 16SrII-A subgroup phytoplasma infecting sesame in Taiwan, 16SrI group phytoplasmas infecting roselle and purple woodnettle, and purple woodnettle is considered as a new host of phytoplasmas. Based on the results of PCR detection of seeds and one-month- and two-month-old seedlings germinated from symptomatic and asymptomatic sesames for the presence of phytoplasmas, it showed that phytoplasmas can only be detected in seed coats but not in the endosperm and the one-month- and two-month-old seedlings thus far. They will be monitored further for up to 6 months after germination. The occurrence of phytoplasmas on sesame, roselle, and purple woodnettle are newly identified in Taiwan, their transmission in the fields and impacts on crop yields and quality will require further investigation.中文摘要………………………………………………………………….……..……iii 英文摘要…………………………………………………….…….……………….....v 目錄………….…………………………………………………………...……..……vii 表目次……….……………………………………………………………………......ix 圖目次……….………………………………………………………………………..x 中文前言…….……………………………………………………………………..…1 英文前言…….…………………………………………………………………..……4 前人研究 植物菌質體的發現與命名……………….………………………………..….…..7 植物菌質體的特性、傳播特性與防治方法………………………..……………7 植物菌質體的分類方法……………….…………………………..………..…...10 植物菌質體的基因體特性……………….……………………………….……..12 芝麻 (sesame)、洛神 (roselle) 及長梗紫 (苧) 麻 (purple woodnettle) 的病害 研究……………….…………………………………………………..….………12 Materials and Methods Plant source and symptoms of the infected plants……………………...………15 Extraction of plant total DNA and performance of a nested polymerase chain reaction (nested PCR)…………………………...……….…………..………….15 Amplification of the F2nR2 region of 16S rRNA by universal primers..............17 Sequencing and the sequence analyses of the PCR products…….……..………18 (1) Cloning the putative PCR products and analyses the putative transformants……………………………………………………………..18 (2)Sequencing of the cloned PCR products……………………..…..……….18 (3)Identification and classification in 16S rDNA……………….……………19 (4)Phylogenetic tree constructed of the 16S rRNA gene sequences……..…..19 Transmission electron microscopy (TEM)…………………………….……..…20 Detection of phytoplasmas in sesame seeds and seedlings…...…………….......21 Result Plant source and symptoms of the infected plants…………………...…………22 Extraction of plant total DNA and performance of a nested polymerase chain reaction (nested PCR)…………………...……………….…………..………….22 Amplification of the F2nR2 region of 16S rRNA by universal primers......…....22 Sequencing and the sequence analyses of the PCR products……………...……23 (1) Cloning the putative PCR products and analyses the putative transformants……………………………………………………………..23 (2)Sequencing of the cloned PCR products………………………….………23 (3)Identification and classification in 16S rDNA………………..…………...24 (4)Phylogenetic tree constructed of the 16S rRNA gene sequences…………26 Transmission electron microscopy (TEM)…………………………...……....…27 Detection of phytoplasmas in sesame seeds and seedling……………………....27 Discussion..………………………………………….…………….……………....…29 Reference……………………………………………………………………………35 Tables………………………………………………………………………...………45 Figures…………………………………………………………..…………….……..5

Topics: 植物菌質體, 芝麻, 洛神, 長梗紫麻, 花器葉化, 簇葉, 葉部黃化, Phytoplasma, sesame, roselle, purple woodnettle, phyllody, witches’-broom, leaf yellowing
Year: 10000
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/86004
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