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Biological characters of Bipolaris spp. causing brown spot on rice and water bamboo

By Shu-Mei Chen and 陳淑媚

Abstract

引起水稻 (Oryza sativa) 與茭白筍 (Zizania latifolia) 胡麻葉枯病之病原菌為Bipolaris oryzae與B. zizaniae,兩種病原菌之形態學差異在分生孢子基部臍的構造。前人依據形態觀察、病原性測定及有性世代配對結果,建議兩者應為同種,然他們所觀察之菌株數目有限,無法釐清兩者之關係。為釐清引起水稻與茭白筍胡麻葉枯病兩種Bipolaris菌株間之相互關係,本研究目的為調查田間水稻與茭白筍胡麻葉枯病病徵、觀察兩者分生孢子形態、親和性配對、病原性測定及分子特性分析。田間病徵調查結果得知,由Bipolaris sp.引起的茭白筍胡麻葉枯病,除具典型病徵外,亦有長條形病徵。形態觀察結果顯示,分離自水稻之112株或茭白筍之47株的Bipolaris屬分生孢子形態相似,且孢子基部以直角狀臍構造為主。孢子長度、寬度及隔膜數量測量與主成份分析顯示,分離自水稻或茭白筍之菌株無法以形態區分。利用前人發表可區別配對型之引子對,增幅所有供試菌株,結果顯示水稻與茭白筍菌株各有28.6 %與31.9 %為第二型配對型。親和性試驗結果證實,光照利於有性世代生成,且某些水稻與茭白筍菌株可配對產生有性世代;然配對結果與利用可鑑定配對型菌株之結果,並未完全符合。於接種試驗得知,分離自水稻與茭白筍之菌株均可感染兩種植株切離葉,引起典型胡麻葉枯病病徵,且少數菌株可引起葉枯或長形病斑。分析ITS (internal transcribed spacer) 與GPDH (glycerol-3-phosphate dehydrogenase) 序列區間之分子親緣性,結果指出分離自水稻與茭白筍之菌株於ITS序列分析形成同一分子群;而分析GPDH序列顯示分離自水稻之菌株分為兩群,其中的一群與茭白筍菌株形成同一分子群。將ITS與GPDH序列合併分析,所得結果與GPDH序列分析相似,由核苷酸序列與分子親緣性得知水稻菌株的分子多樣性高於茭白筍菌株。根據分生孢子形態觀察、有性世代配對、病原性測定與分子親緣性分析結果,顯示引起水稻與茭白筍胡麻葉枯病之病原菌生理特性相似。Bipolaris oryzae and B. zizaniae are the pathogens to cause brown spot on rice (Oryza sativa) and water bamboo (Zizania latifolia). Previous studies indicated that B. oryzae could be distinguished from B. zizaniae based on the hilum structure of conidia basal part. Several researchers suggested that B. zinaniae should be a synonym of B. oryzae due to similar morphology, pathogenicity and cross fertility test. However, previous studies showed the number limitation of observed isolates and the relationship between the two pathogens were still obscure. For carrying out the relationship of Bipolaris spp. causing brown spot on water bamboo and rice, the symptom was investigated in field and morphology, cross fertility, pathogenicity and molecular characters of Bipolaris spp. were be analyzed. Field investigation revealed that Bipolaris sp. could cause typical spot and long lesion on water bamboo. The conidia morphology indicated that 112 and 47 Bipolaris sp. isolates from rice and water bamboo, respectively, showed similar morphology and rectangular hilum was the major type. The conidial length, width and number of distoseptate could not diversify all isolates from rice or water bamboo based on principal component analysis. The MAT-2 gene amplification showed that 28.6 % from rice and 31.9 % from water bamboo were MAT-2 isolates. The cross fertility test confirmed that light could improve sexual reproduction. Certain isolates from rice or water bamboo mated successfully each isolate and produce ascospores. However, the result of cross fertility test did not company with the result of mating type identification completely. The virulence test demonstrated that the isolates from rice and water bamboo could infect both detached leaves of rice and water bamboo, resulting in typical brown spot, leaf blight or long lesion. Molecular phylogenetic analyses showed that Bipolaris isolates from rice and water bamboo formed a molecular group based on ITS (internal transcribed spacer) region. The other side, the analysis of GPDH (glycerol-3-phosphate dehydrogenase) sequence showed that isolates from rice could be separated into two molecular groups and one group isolates formed the same molecular group with isolates from water bamboo. ITS sequence combined with GPDH sequence showed similar result with GPDH sequence. Thus, the molecular diversity of isolates from rice is higher than the isolates from water bamboo. This study reveals that the Bipolaris spp. causing brown spot on rice or water bamboo have similar biological characters according to the morphology of conidia, affinity test, pathogenicity test and molecular analysis,.中文摘要 .................................................. i 英文摘要 ................................................ iii 目次 ..................................................... v 表目次 .................................................. vii 圖目次 ................................................. viii 前言 ..................................................... 1 材料與方法 ................................................ 4 一、罹病植株之收集 .......................................... 4 二、胡麻葉枯病菌之分離純化 ................................... 4 三、分生孢子形態觀察 ........................................ 4 四、供試菌株之DNA萃取 ...................................... 5 (一) 傳統萃取方法 .......................................... 5 (二) Plant DNA/RNA Extraction Kit法 ...................... 5 五、MAT-2配對型基因座的增幅 .................................. 6 六、有性世代配對 ............................................ 7 七、病原性測定 ............................................. 7 (一) 水稻切離葉接種 ......................................... 7 (二) 茭白筍切離葉接種 ....................................... 7 (三) 罹病度計算 ............................................ 8 八、核苷酸序列增幅 .......................................... 8 (一) ITS片段增幅 .......................................... 8 (二) GPDH片段增幅 ......................................... 9 (三) 菌株之分子親緣性分析 .................................... 9 結果 .................................................... 11 一、罹病葉與菌株之蒐集 ..................................... 11 二、分生孢子形態觀察 ....................................... 11 三、分生孢子之測量與主成份分析 ............................... 11 四、分生孢子基部之觀察 ...................................... 12 五、MAT-2配對型基因座之增幅 ................................. 13 六、有性世代配對 ........................................... 13 七、病原性測定 ............................................ 14 八、核苷酸序列增幅與親緣性分析 ............................... 15 (一) ITS基因序列解序與親緣性分析 ............................ 15 (二) GPDH序列解序與親緣性分析 .............................. 16 (三) ITS與GPDH序列合併分析 ................................ 17 討論 .................................................... 18 參考文獻 ................................................. 2

Topics: Bipolaris oryzae, B. zizaniae, 胡麻葉枯病, 分子多樣性, Bipolaris oryzae, B. zizaniae, brown spot, molecular diversity
Year: 2014
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/86036
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