臺灣博碩士論文加值系統

馬鈴薯瘡痂病為馬鈴薯種植期間常見的病害，主要病原為馬鈴薯瘡痂病菌 (Streptomyces scabies)，可造成薯塊壞疽病斑或木栓化病徵而使馬鈴薯商品價值降低，臺灣目前無推薦防治藥劑或微生物資材，僅能以種植健康種薯、水稻輪作與調節土壤酸鹼值來降低危害，然防治效果有限。本實驗室先前研究發現液化澱粉芽孢桿菌(Bacillus amyloliquefaciens) Ba01能有效防治田間瘡痂病，於雲林斗南田間試驗中，以Ba01發酵液 (2×107 CFU/mL) 澆灌，罹病度可從14.4 ± 2.9% (無Ba01發酵液處理) 降至5.6 ± 1.1% (P＜0.05; Tukey’s test)。為更進一步了解Ba01抑菌機制，以質譜儀分析Ba01分泌之二次代謝物，推測表面素 (surfactin) 為主要抑制瘡痂病原菌的物質之一，故本研究藉由剔除Ba01 srf基因簇 (gene cluster) 突變株證明表面素為主要的抑菌物質。實驗利用電穿孔方式將選殖成功的質體轉入Ba01進行in-frame deletion，而後以不同溫度條件篩選srf 基因簇突變株，最後藉由專一性引子及質譜儀分析確認FRY1、FRY3為srf基因簇缺失突變株。srf基因簇突變株FRY1及FRY3於濾紙片擴散試驗中抑制馬鈴薯瘡痂病菌之能力降低 (P < 0.001；t-test)，且突變株表面移行能力亦顯著下降 (P < 0.001；t-test)，另外添加表面素予srf基因簇突變株FRY1則會恢復其表面移行能力。根據文獻報導表面移行能力與生物膜生成有關，故測試srf基因簇是否與生物膜相關，本研究亦發現Ba01能形成皺褶狀生物膜，而srf基因簇突變株則無法形成。生理生化測試發現srf基因簇突變株之澱粉、蛋白水解酶及溶磷能力皆明顯下降，且srf基因簇突變株無法抑制馬鈴薯薯片瘡痂病菌PS07的生長及產孢。由以上結果推測Ba01產生之表面素除了能抑制馬鈴薯瘡痂病菌的生長，施用於田間可能具保護馬鈴薯根部並增加纏聚能力以抵抗病害。未來希望運用此基因剔除技術探討液化澱粉芽孢桿菌更多基因功能、益菌及促進植物生長之機制。

Potato common scab mainly caused by Streptomyces scabies widely occurs during the potato planting fields. It causes necrotic lesions or cork symptoms on potato tubers and decreases the economic value of potato. At present, there is no recommended chemical or biological pesticides from combating potato common scab in Taiwan. We could only reduce its occurrence by planting healthy seed potato, rotation with rice and regulating soil pH, but they are not efficacious. Previous study in our laboratory found that Bacillus amyloliquefaciens Ba01 can effectively control potato common scab in the field. In the field trial in Dounan, Yunlin, the disease severity of potato common scab was reduced from 14.4 ± 2.9% (without Ba01) to 5.6 ± 1.1% after Ba01 treatment (P＜0.05; Tukey’s test). To further investigate the antibacterial mechanism of Ba01, we analyzed the second metabolites secreted by Ba01 by imaging mass spectrometry, and suggested that surfactin is one of major compounds inhibiting S. scabies. Therefore, this study will test if surfactin is the main antibacterial substance by knocking out srf gene cluster in Ba01. The cloning plasmid pRY1 was transformed to Ba01 by electroporation for in-frame deletion, and the srf gene cluster mutants were screened at different temperatures. Finally, these srf gene cluster mutants were confirmed by specific primers and mass spectrometry. We further found that the ability of srf gene cluster mutants FRY1 and FRY3 to inhibit S. scabies was decreased (P < 0.001; t-test) in disc diffusion asssay, and the swarming ability of these mutants was also decreased (P < 0.001; t- test). Interestingly, the swarming ability of Δsrf mutant FRY1 was restored by the addition of surfactin. According to literatures, the swarming ability was linked to biofilm formation. We found that Ba01 formed wrinkled biofilm in MSgg liquid medium, but the mutants did not. In physiological and biochemical tests, the α-amylase, protease, and phosphate-solubilizing ability of Δsrf mutants were significantly decreased, and the mutant could not effectively inhibit the growth and sporulation of S. scabies PS07 on potato tuber slices. As a consequence, we suggest that surfactins produced by Ba01 may not only inhibit the growth of S. scabies but also protect the potato roots and increase the colonization to combat potato common scab. In the future, we plan to use this gene knockout technique to investigate gene functions of B. amyloliquefaciens, mechanisms of beneficial bacteria and plant growth promoting ability.

口試委員會審定書 i
致謝 ii
中文摘要 iv
英文摘要 vi
目錄 viii
表目錄 xi
圖目錄 xii
1. 前言 1
1.1馬鈴薯之經濟重要性 1
1.2馬鈴薯重要病害及臺灣種薯驗證制度 2
1.3馬鈴薯瘡痂病危害概況及防治策略 4
1.4液化澱粉芽孢桿菌 5
1.5液化澱粉芽孢桿菌突變株相關研究 7
1.6實驗室前人研究及研究目的 8
2. 材料與方法 10
2.1試驗菌株培養條件及培養基 10
2.2 細菌染色體DNA純化 10
2.3 srf基因簇突變株質體的建立 11
2.3.1 建立Ba01 srf 基因簇 (gene cluster) 突變株cassette 11
2.3.2 選殖srf基因簇突變株質體 12
2.4 Ba01轉型與突變株的建立 13
2.4.1電穿孔方式進行Ba01的轉型 13
2.4.2 以In-frame deletion建立突變株 14
2.5 Ba01菌株生長試驗 14
2.6 Ba01菌株內生孢子形成率試驗 15
2.7 濾紙片擴散之拮抗試驗 15
2.8以MALDI-TOF質譜儀分析Ba01菌株之表面素分泌量 15
2.9 Ba01菌株表面移行能力的測試 16
2.10 生物膜形成試驗 16
2.11 Ba01菌株胞外水解酶及溶磷能力測試 17
2.12馬鈴薯薯片試驗 (Tuber slice assays) 18
3. 結果 19
3.1 Ba01 srf基因簇突變株之獲得 19
3.2 srf基因簇突變株生長下降 19
3.3 srf基因簇突變株產孢能力下降 19
3.4 srf基因簇突變株抑制瘡痂病菌能力明顯下降 20
3.5 srf基因簇突變株無法生成表面素 20
3.6 srf基因簇突變株表面移行能力明顯降低 20
3.7 添加表面素能夠回復srf基因簇突變株表面移行能力 21
3.8 srf基因簇突變株無法形成正常生物膜及菌落結構 21
3.9 srf基因簇突變株之澱粉酶、蛋白酶以及溶磷能力降低 22
3.10 srf基因簇突變株在薯片上無法抑制瘡痂病菌的生長及產孢 22
4. 討論 23
5. 參考文獻 26
6. 圖表 34
7. 附錄 50
7.1附錄圖表 50
7.2防治馬鈴薯瘡痂病之化學藥劑及生物製劑應用 51

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