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dc.contributorJenn-Wen Huangen_US
dc.contributor.authorKuan-Lin Chenen_US
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dc.description.abstract西元2012年,在苗栗獅潭、大湖等栽培區,發現草莓植株出現矮化、新葉偏上生長、黃化及葉片萎凋乾枯等症狀;切開病株莖基部則可見到維管束褐變的病徵。由田間取回罹病植株,進行莖基部組織分離可疑病原菌,依柯霍氏法則測試後,確定各分離株的致病毒性後,選取毒力較強之Fofb 01-2與Fofb 4-13兩菌株進行草莓萎凋病菌之鑑定及其生物防治試驗。本研究主要目的在於 (1) 鑑定台灣草莓萎凋的病原菌;(2) 研發草莓萎凋病的生物防治方法。首先將Fofb 01-2與Fofb 4-13菌株分別培養於半醣Potato Dextrose Agar (PDA) 平板上,菌落顏色呈白色至灰色,中央有紫色色素沉積。病原菌有三種形態的孢子,小孢子呈現卵圓狀或臘腸狀;大孢子呈直至彎曲鐮刀狀,具有3-5個隔膜;厚膜孢子呈圓形。利用Lin等人 (2010) 針對Fusarium oxysporum Schl.所設計的專一性引子對FnSc-1 (5’-TACCACTTGTTGCCTCGGCGGATCAG-3’)/FnSc-2 (5’-TTGAGGAACGCGAATTAACGCGAGTC-3’)進行PCR (polymerase chain reaction),確定Fofb 01-2與Fofb 4-13兩菌株歸屬於F. oxysporum。進一步將病原菌株接種於胡瓜、西瓜、萵苣、白菜、番茄及草莓等不同寄主植物,結果僅有草莓植株受感染罹病,隨後以Suga等人 (2013) 針對尖鐮孢菌草莓分化種 (Fusarium oxysporum Schl. f. sp. fragariae Winks & Williams) 所設計的專一性引子對FofraF (5’-CAGACTGGGGTGCTTAAAGTT-3’)/FofraR (5’-AACCGCTAGGGTCGTAACAAA-3’) 進行Fofb 01-2與Fofb 4-13的分子生物學分析,並與NCBI資料庫進行比對,確認兩菌株的學名為F. oxysporum Schl. f. sp. fragariae Winks & Williams。Fofb 01-2與Fofb 4-13菌株的菌絲最適生長溫度及分生孢子與厚膜孢子最適發芽溫度皆為28 ℃。分生孢子最適發芽pH值則介於4-8,發芽率皆可達80 %以上。利用不同碳氮素源培養本病原菌,發現兩菌株最適生長於麥芽糖 (Maltose) 及山梨糖醇 (Sorbitol)、天門冬醯胺 (Asparagine)、纈氨酸 (Valine) 及硝酸鈉 (NaNO3)。在溫室測試蕈狀芽胞桿菌Bacillus mycoides BM103、BM104及BM105菌株防治草莓萎凋病之效果,結果發現施用三菌株均具有促進草莓植株根部增長2-4公分之功效。將處理過B. mycoides三菌株的草莓植株 (來自於組織培養苗) 分別種植於病菌土 (10^3 cfu/g) 中,結果顯示BM103菌株防治草莓萎凋病的功效最佳,可降低66%的罹病度。施用BM103菌株於草莓植株經過8天後,可發現植物根部與莖基部被纏據率達100%。進一步,利用B. mycoidesBM103之黃豆粉醱酵液搭配1% (w/v)幾丁聚醣澆灌於草莓植株後,可有效降低草莓萎凋病之罹病度達52.78 %以上。分析BM103的PGPR (Plant growth-promoting rhizobacteria) 特性,結果顯示BM103可生合成IAA約5.92 μg/ml,並釋放出氨。zh_TW
dc.description.abstractSince 2012, a new disease of strawberry has been found in Da-Hu, Shih-Tan, and other cultivation areas in Taiwan. The symptoms consisted of stunting, wilting of foliage, necrotic crowns and roots, discoloration of the internal vascular tissue, and eventually plant death. The isolates of the causal agents were obtained from diseased plants. Among them, two isolates named Fofb 01-2 and Fofb 4-13 showed highly virulent to strawberry plants and were selected for the following experiments. The purposes of the study were (1) to identify the causal agent of strawberry Fusarial wilt from Taiwan; (2) to develop the biocontrol measures for controlling the disease. Both isolates were subcultured on 1/2 PDA and formed gray-to-purple colonies. The isolates could produce microconidia, macroconidia, and chlamydospores. Microconidia were oval to ellipsoid. Macroconidia were straight to sickle, 3 to 5 septate. Chlamydospores were round. A 327 bp DNA fragment was successfully amplified from both fungal isolates by PCR (polymerase chain reaction) using two primers, FnSc-1 (5’-TACCACTTGTTGCCTCGGCGGATCAG-3’)/FnSc-2 (5’-TTGAGGAACGCGAATTAACGCGAGTC-3’) specific for Fusarium oxysporum. Virulence assays revealed that the isolates could only infect strawberry showing similar symptoms observed in the diseased plants in the field. No symptoms were observed after they were inoculated onto cucumber, watermelon, lettuce, Pak choi, and tomato. Further analysis using primers FofraF (5’-CAGACTGGGGTGCTTAAAGTT-3’)/FofraR (5’-AACCGCTAGGGTCGTAACAAA-3’) identified two isolates as F. oxysporum Schl. f. sp. fragariae Winks & Williams. The optimal temperature for mycelial growth, conidial and chlamydospore germination of isolates Fofb 01-2 and Fofb 4-13 was at 28℃. The pH values between 4-8 was optimal for both conidial germination more than 80%. Two isolates were more suitable for mycelial growth in media with carbon sources, maltose and sorbitol or nitrogen sources, asparagine, valine, and NaNO3. The strawberry seedlings were treated respectively with Bacillus mycoides isolates BM103, BM104, and BM105. The results indicated that the three biocontrol agents could significantly increase root growth about 2-4cm compared to the control. After three consecutive drenches, each of BM103, BM104, and BM105 suspension (10^8 cfu/ml) was applied to strawberry seedlings weekly, and the treated plants were transferred to grow in the infested soil (10^3 cfu/g). The results indicated that BM103 could reduce the disease severity more than 66% compared to the control. It was found that the roots and crown of strawberry seedlings could be completely colonized by B. mycoides BM103 eight days after drenching treatment. In order to improve efficacy of B. mycoides BM103 for controlling the disease, 1% (w/v) chitosan was mixed with soybean meal fermented broth of BM103. The results indicated that the disease severity was reduced more than 52.78 % compared to the control. In addition, analyzing PGPR characteristics showed that B. mycoides BM103 could produce IAA about 5.92 μg/ml and release ammonia.en_US
dc.description.tableofcontents中文摘要 i ABSTRACT iii 目錄 v 表次索引 viii 圖次索引 ix 前言 1 材料與方法 6 (一) 供試植株來源 6 (二) 供試菌株來源、培養與保存 6 (三) 病原菌鑑定 7 1. 病原菌形態觀察 7 2. 寄主圍範測試 7 3. Fofb 01-2與Fofb4-13菌株之分子生物鑑定 7 (四) 接種平台之建立 10 1. 病菌土製作 10 2. 病菌土濃度測定 10 3. 病害調查方式 11 4. 接種濃度對罹病度之影響 11 (五) 病原菌生理生化特性探討 11 1. 溫度對菌絲生長影響 11 2. 溫度對分生孢子發芽之影響 12 3. pH值對分子孢子發芽之影響 12 4. 溫度對厚膜孢子發芽之影響 12 5. 碳素源對菌落生長之影響 13 6. 氮素源對菌落生長之影響 13 (六) 拮抗微生物防治草莓萎凋病之效果評估 14 1. 拮抗微生物來源 14 2. 拮抗微生物對草莓植株生長之影響 14 3. 拮抗微生物防治草莓萎凋病之效果評估 14 4. 拮抗微生物於草莓植株根部纏據之分析 14 5. 拮抗微生物醱酵液製備 15 6. 拮抗微生物醱酵液對草莓植株生長之影響 15 7. 拮抗微生物醱酵液防治草莓萎凋病之效果評估 15 8. 拮抗微生物之PGPR特性分析 16 結果 18 (一) 病徵描述 18 (二) 病原菌鑑定 18 1. 病原菌形態觀察 18 2. 寄主圍範測試 19 3. Fofb 01-2與Fofb4-13之分子生物學鑑定 19 (三) 接種平台之建立 19 1. 接種菌量濃度與罹病度之關係 19 (四) 病原菌生理生化特性 20 1. 溫度對菌絲生長之影響 20 2. 溫度對分生孢子發芽之影響 20 3. pH值對分子孢子發芽之影響 20 4. 溫度對厚膜孢子發芽之影響 20 5. 碳素源對菌落生長之影響 21 6. 氮素源對菌落生長之影響 21 (五) 拮抗微生物防治草莓萎凋病之效果評估 21 1. 拮抗微生物對草莓植株生長之影響 21 2. 拮抗微生物防治草莓萎凋病之效果 22 3. 拮抗微生物於草莓植株根部纏據之分析 22 4. 拮抗微生物醱酵液對草莓植株生長之影響 22 5. 拮抗微生物醱酵液防治草莓萎凋病之效果 23 6. 拮抗微生物PGPR特性分析 23 討論 24 參考文獻 30 圖表 37 附錄 62zh_TW
dc.subjectBacillus mycoideszh_TW
dc.subjectFusarium oxysporum f. sp. fragariaeen_US
dc.subjectBacillus mycoidesen_US
dc.titleIdentification for the causal agent of strawberry Fusarial wilt from Taiwan and its biocontrol experimentsen_US
dc.typethesis and dissertationen_US
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item.openairetypethesis and dissertation-
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