Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31086
標題: 臺灣水稻稻熱病菌 (Pyricularia oryzae) 對黑色素生合成抑制劑與史托比類藥劑之感受性與族群多樣性研究
Studies on melanin biosynthesis inhibitors (MBIs) and strobilurins (QoIs) sensitivity and population diversity of Pyricularia oryzae in Taiwan
作者: 謝千祥
Hsieh, Chien-Hsiang
關鍵字: Pyricularia oryzae;Pyricularia oryzae;fungicides sensitivity;population diversity;methods for promoting conidial production;藥劑感受性研究;族群多樣性;促進產孢方法
出版社: 植物病理學系所
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摘要: 
本研究檢測 103 株從台灣各地所分離之稻熱病菌 (Pyricularia oryzae) 對黑色素生合成抑制劑 (melanin biosynthesis inhibitors, MBIs) 中的 75% 三賽唑 (tricyclazole) 可濕性粉劑與 15% 加普胺 (carpropamid) 水懸劑,和抑制粒線體呼吸作用電子傳遞之史托比類 (strobilurins) 藥劑中的 10% 亞托敏 (azoxystrobin) 水懸劑與 44.2% 克收欣 (kresoxim-methyl) 水懸劑的感受性,結果得知三賽唑對所有供試菌株之半數有效抑制濃度 (effective concentration for 50 % inhibition; EC50) 值介於 14.63 ± 15.40 μg/ml 至 179.34 ± 16.08 μg/ml 之間;而加普胺對所供試稻熱病菌株的 EC50 值介於 52.47 ± 5.33 μg/ml 至 463.71 ± 202.72 μg/ml 之間或 > 500 μg/ml。上述測試結果顯示,本研究所蒐集之稻熱病菌對加普胺已有低感受性菌株出現。另亞托敏對供試菌株之 EC50 值為 < 1 μg/ml,或介於 1.60 ± 0.61 μg/ml 至 88.12 ± 4.46 μg/ml 之間或 > 500 μg/ml,而克收欣對稻熱病菌株之 EC50 值,則介於 1.65 ± 0.49 μg/ml 至 483.39 ± 7.18 μg/ml 之間或 > 500 μg/ml,其中只有 10 株菌株對克收欣表現較敏感,其餘菌株對克收欣的感受性偏低。進一步分析對加普胺或克收欣表現低感受性 (EC50 > 500 μg/ml) 菌株於 scytalone dehydratase (SDH) 基因與粒線體細胞色素 (cytochrome b, cyt b) 基因之差異性,結果指出對加普胺表現低感受性菌株之 SDH 基因第 75 密碼子處無點突變的現象;而對克收欣表現低感受性的菌株,於 cyt b 基因第 129 或 143 密碼子處亦無點突變現象。本研究利用稻熱病菌核醣體 DNA (ribosomal DNA , rDNA) 中的非轉錄基因間隔區 (intergenic spacer, IGS) 結合簡單重複序列 (simple sequence repeat, SSR) 以 PCR 技術分析台灣稻熱病菌族群之多樣性,結果顯示 103 株菌株可分成 9 個 DNA 群組 (A-I),其中以 A 群組之菌株占的比例最高,B 群組次之,各有 72 與 15 株菌株。其餘 C-I 群組各有8、2、2、1、1、1、1 株稻熱病菌株。此外,分析結果指出 A 群組之菌株與 B 群組之菌株的親緣性最高,顯示 A 與 B 群組的菌株為田間主要優勢
ii
菌株。然此 9 個群組與菌株之採集區域及病原性強弱無明顯關聯性,反之對加普胺與亞托敏感受性較低 (EC50 > 500μg/ml) 的菌株皆屬於 A 群組,推測原因為 A 群組之菌株分布較廣,對藥劑低感受性的菌株出現機率較高。另對克收欣感受性表現較低的菌株,則因大多數稻熱病菌株皆屬低感受性,而帄均散布在各群組內。本實驗亦測試不同促使稻熱病菌產孢的方法,結果顯示刮除稻熱病菌菌落表面之氣生菌絲後,以近紫外光照射,並無促進產孢效果。另挑選 10 株稻熱病菌株培養於不同培養基上,添加或不添加不同濃度的過氧化氫 (H2O2) 於菌落,嘗試促進稻熱病菌在人工培養基上產生孢子,然此方法對促進菌株產孢的效果亦不佳。由於生理小種的判別需透過接種不同水稻品種,為了解台灣田間稻熱病菌株之致病力,尋找其他更有效促進稻熱病菌株在人工培養基穩定產孢的方法,為未來之重要課題。

A total of 103 isolates of Pyricularia oryzae from different rice production area in Taiwan were examined their sensitivities to tricyclazole, carpropamid, azoxystrobin and kresoxim-methyl. Results showed that the EC50 of tricyclazole to all isolates are 14.63 ± 14.40 ~ 179.34 ± 16.08 μg/ml and EC50 of carpropamid to all isolates are 52.47 ± 5.33 ~ 463.71 ± 202.72 μg/ml or > 500 μg/ml. Moreover, the EC50 of azoxystrobin to all isolates are <1 μg/ml, 1.60 ± 4.49 ~ 483.39 ± 7.18 μg/ml or > 500 μg/ml, and the EC50 of kresoxim-methyl to all isolates are 1.65 ± 0.49 ~ 483.39 ± 7.18 μg/ml or > 500 μg/ml. According the examinations, low sensitive isolates of P. oryzae to carpropamid, azoxystrobin and kresoxim-methyl were identified. For analyzing the mechanism of resistance to different fungicides, two genes of scytalone dehydratase (SDH) and cytochrome b (cyt b) gene were sequenced and analyzed. The previous reported demonstrated that the point mutation in SDH gene can produce resistance to carpropamid and point mutation in cyt b gene can produce resistance to strobilurins. The SDH gene of P. oryzae isolates showed low sensitivity to carpropamid were amplified and sequenced, and the results indicated that low sensitive isolates did not have point mutation at codon 75 in SDH gene. Meanwhile, the isolates of P. oryzae showed low sensitivity to azoxystrobin and kresoxim-methyl also did not have mutation in codon 129 or 143 in cyt b gene. The results revealed that resistant mechanism of isolates showed low sensitivity to carpropamid or showed low sensitivity to azoxystrobin and kresoxim-methyl did not correspond with mutation in SDH or cyt b genes, respectively. For realizing the genetic diversity of population, 103 isolates of P. oryzae were analyzed by PCR-base method. The rDNA of intergenic spacer (IGS) and simple sequence repeat (SSR) were amplified by CNS1 and (CAG)5, and the PCR results were analyzed by
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FreeTree, MEGA 4 and VisionWorks LS software. According to the results, 103 isolates of P. oryzae from different area could be separated into 9 groups, A~I. Among these groups, group A included 72 isolates, group B included 15 isolates and groups C~I included 8, 2, 2, 1, 1, 1, 1 isolates, respectively. Thus, isolates of groups A and B are most dominant population among P. oryzae in Taiwan and have highly molecular relationship with each group. However, these groups did not correspond with collected area or rice cultivar in Taiwan. The other side, the isolates showed low sensitivity to carpropamid and azoxystrobin are located into group A and the isolates showed low sensitivity to kresoxim-methyl are scattered into each group. In this study, several methods were tested for increasing the sporulation of P. oryzae in article media, including hydrogen peroxide (H2O2) added, blue light incubated, colony wounded etc. However, there no significant sporulation on P. oryzae colony after treated with different methods. It is necessary to improve the sporulation of P. oryzae in future.
URI: http://hdl.handle.net/11455/31086
其他識別: U0005-2608201100241300
Appears in Collections:植物病理學系

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