- 學位論文
分離自水稻種子之病原菌 Monographella albescens 之研究
Studies of Pathogen Monographella albescens Isolated from Rice Seeds
摘要
臺灣地處亞熱帶，為高溫多濕的海島型氣候，很適合各種稻作病原菌之繁殖及為害，每年因稻作病害引起的經濟損失約6%。取自台東縣池上鄉新興育苗場之高雄139號水稻品種種子，經分離培養到3個真菌菌株，分別為Ma-1027、Bo-1027和Tl-1101，以光學顯微鏡觀察Ma-1027，其孢子透明橢圓、紡綞形狀，有1-2隔膜，孢子大小約為11-21 × 4-8 μm；Bo-1027孢子呈長圓筒形至長紡綞形狀，6-11個隔膜，孢子大小約為72-128 × 19-29 μm；Tl-1101分生孢子呈黃綠色近圓形或蛙卵形狀；並於水稻葉片分離到一個細菌菌株定名為Bs-1101。再以分子生物技術鑑定，分別利用18S-F / 28S-R和A1-16S-23S-F / A1-16S-23S-R引子對，進行Ma-1027、Bo-1027、Tl-1101和Bs-1101 genomic DNA之聚合酶連鎖反應（PCR）增幅、水平電泳膠體分析。 Ma-1027、Bo-1027、Tl-1101之PCR核酸產物均約為650 bp，Bs-1101約480 bp。PCR產物之核苷酸序列經譯讀與比較，結果Ma-1027與NCBI中的Monographella albescens isolate CIAT 11（AJ132506.1）核苷酸序列相同度達100％；Bo-1027與NCBI中的Bipolaris oryzae isolate San Pablo-Monopolar(SM)（DQ300207.1）核苷酸序列相同度達99％；Tl-1101與NCBI中的Trichoderma longibrachiatum strain CIB T29（EU280095.1）核苷酸序列相同度達99％；Bs-1101與NCBI中的Bacillus subtilis strain Tpb55 （DQ672263.1）核苷酸序列相同度達98％。Ma-1027及Bo-1027菌株，可以回接到水稻高雄139號葉面上，產生相同之病徵。經上述分析試驗結果Ma-1027為Monographella albescens，此為台灣首次發現之水稻病原菌。Ma-1027菌株之生理特性調查，在20℃溫度，果糖（fructose）碳素源，硝酸鈉氮素源，pH 6等條件對菌絲的生長最佳。39個水稻品種感病性測定，對Ma-1027全部不具抗病性。藥劑室內篩選以25%撲克拉乳劑（Prochloraz）1000倍、34%殺紋滅達樂溶液（Hymerxazol+Metalaxy）1000倍、40%亞賜圃乳劑（Isoprothiolane）1000倍、10﹪菲克利乳劑（Hexaconazole）1500倍，對Ma-1027菌絲之抑制性較強。拮抗菌Tl-1101與Bs-1101，亦可以有效抑制Ma-1027菌絲生長。
並列摘要
Taiwan is located at tropical and subtropical areas belonging to island weather with high temperature and humility which is suitable for pathogens propagation and infection to rice crop. Every year the economic loss caused by rice diseases is about 6%. Three fungal isolates, named as Ma-1027, Bo-1027and Tl-1101, were isolated and cultured from seeds of Kaohsiung 139 rice variety at Hsing-sing rice seedling production Station in Chihshang of Taitung county. Under microscope of Ma-1027 were observed as rhombus-shaped with 1-2 septa and with size of 11-21×4-8 μm; conidia of Bo-1027 were observed as ellipse-shaped with 6-11 septa and with size of 72-128×19-29 μm; conidia of Tl-1101 were observed at ground-shaped. One bacterial isolate was also obtained from rice leaf and named as Bs-1101. Four isolates were further identified and analyzed by PCR amplification with 18S-F/28SR primer for Ma-1027, Bo-1027 and Tl-1101, and AI-16S-23S-F/Al-16S-23S-R primer for Bs-1101. Sizes of PCR DNA products were estimated about 650 bp for Ma-1027, Bo-1027and Tl-1101, and 480 bp for Bs-1101, respectively. The results of comparison of nucleotide sequences of 4 PCR DNA products indicated that Ma-1027 was close to Monographella albescens isolate CIAT 11(AJ132506.1) in NCBI with identity of 100% and first report in Taiwan. Bo-1027 was close to Bipolaris oryzae isolate San Pablo-Monopolar (SM)(DQ300207.1) in NCBI with identity of 99%. Tl-1101 was close to Trichoderma longibrachiatum strain CIB T29（EU280095.1）in NCBI with identity of 99%. Bs-1101 was close to Bacillus subtilis strain Tpb55（DQ672263.1）in NCBI with identity of 98%. Infectivity of Ma-1027 and Bo-1027 were confirmed by inoculation on Kaohsiung 139 rice variety. The optimal physical conditions for mycelial growth of Ma-1027 were determined at 20℃ temperature, and medium with fructose carbon source, NaNO3 nitrogen source and pH 6 value. The susceptibility of 39 rice varieties determined by Ma-1027 inoculation showed that no resistant variety was found. The result of agrochemicals screening in laboratory indicated that 25% prochlorage, 34% Hymerxazol＋Metalaxy, and 40% Isoprothiolane with 1,000 time dilution, 10% Hexaaconazole with 1,500 time dilution and antigonism agents of Tl-1101 and Bs-1101 had good effect on mycelial growth of Ma-1027.