Studies on the colonization of Bacillus cereus C1L in theoots of Lilium formosanum

百合灰黴病(Lily gray mold)為臺灣百合之真菌性病害，由灰黴病菌(Botrytis elliptica (Berk.) Cooke)感染造成葉片焦枯、花器畸形及花瓣萎凋，對臺灣百合之園藝栽作影響甚鉅。為利用生物防治法解決灰黴病菌危害的問題，乃自臺灣百合根圈篩選具有生物防治潛力的菌株，其中以臘狀芽孢桿菌C1L菌株之表現最佳。為便於剖析C1L菌株之群聚 (colonization) 特性，利用對立復黴素(rifampicin)具抗性的C1L菌株，處理於臺灣百合幼苗根圈後，回收根內及根表的細菌，計算族群密度，發現C1L菌株在根表及根內有良好的群聚能力。利用源自臘狀芽孢桿菌幾丁質分解酵素基因(chiCH)之啟動子及pHY300PLK建構可表現綠色螢光蛋白的載體系統，使C1L菌株產生自發性螢光。經綠色螢光標定之C1L菌株，其誘導植株系統性抗病能力(induced systemic resistance)、抑制真菌孢子發芽、菌絲生長的能力及泳動性(motility)皆與野生菌株之功效相近。利用共軛焦雷射顯微鏡觀察螢光標定之C1L菌株在臺灣百合幼苗根部群聚的情形，發現C1L菌株可附著根毛進入根部表層細胞間隙，並進入根部組織之深層細胞間隙。進一步利用轉位誘變之方式，獲得340個C1L菌株之突變株，針對突變株在玉米根內的群聚能力及泳動性進行篩選，經由單邊側翼序列比對方式，發現失去群聚能力之突變株被破壞之基因可能解碼nitric oxide dioxygenase cAMP-dependent protein kinase、N-acetylgalactosamine-6-phosphate deacetylase、glucose-specific IIABC component、replication protein等，而失去泳動性之突變株被破壞的基因可能解碼DNA binding protein、hypothetical protein以及possible collagen-like protein integrase。未來，期望進一步分析C1L菌株在植物根部群聚的模式。Gray mold disease, caused by Botrytis elliptica (Berk.) Cooke, is one of the most important fungal diseases in Lilium formosanum that causes severe losses of yield and quality of lilies. In general, field management by application of fungicides to prevent and decrease disease incidence frequently results in the evolvement of fungicide-resistant strains. Therefore, alternative control measures such as biological control are developed. A biocontrol strain of Bacillus cereus C1L had been screened from the rhizosphere of L. formosanum and it could suppress the disease severity of lily gray mold. In this study, fluorescent C1L strain was constructed by using a promoter driving the expression of chitinase CH of Bacillus cereus and a shuttle vector pHY300PLK to express green fluorescence protein (GFP) in both Escherichia coli and Bacillus cereus. The tests on the abilities of induced systemic resistance (ISR), inhibition of spore germination and hyphal growth, and the bacterial motility indicated that the GFP-labeled C1L strain was similar to the wild-type strain. Examination by confocal laser scanning microscopy showed the presence of GFP-labeled C1L bacterial cells inner the roots of L. formosanum seedlings. The C1L strain could adhere to the root hairs, enter the intercellular space of root surface layer, and present in the in-depth intercellular space of root tissues, indicating that this bacterium could be an epiphyte and endophyte in the plant roots. In order to identify the genes of B. cereus C1L involved in the root colonization, a transposon insertion mutant library including 340 mutant strains was constructed. These mutants were screened for motility and colonization ability within corn roots. The genes identified from mutants of losing colonization ability were predicted to encode nitric oxide dioxygenase cAMP-dependent protein kinase, N-acetylgalactosamine-6-phosphate deacetylase, glucose-specific IIABC component, replication protein…et al. The genes possibly involved in motility were predicted to encode DNA binding protein, hypothetical protein, and possible collagen-like protein integrase. A build-up of model for the colonization of strain C1L in the roots of L. formosanum is the future prospect.壹、 中文摘要 5、 英文摘要 6、 前 言 8、 前人研究 10、 百合與百合灰黴病 10、 根圈微生物 11、 根圈細菌之生物防治作用 12、 植物內生性細菌群聚植物之作用 13、 植物內生性細菌促進植物生長之應用 14、 植物內生性細菌防治病害之應用 15、 植物內生性細菌群聚作用之相關因子 16、 材料與方法 18、 供試菌株與載體 18、 觀察C1L菌株在臺灣百合根圈族群密度的消長 19. 溫室試驗植株之栽培 19. 篩選具有立復黴素抗性之臘狀芽孢桿菌 C1L突變株 19. C1L菌株處理臺灣百合根圈土壤及回收試驗 19、 建構綠色螢光蛋白標定之C1L菌株 20. 分子生物技術之利用 20. 建構表現GFP之穿梭載體系統 23. 確認C1L菌株電穿孔轉形株表現GFP之北方雜合分析 27. 標定GFP之C1L菌株電穿孔轉形株與野生株之比較 29、 利用螢光顯微鏡觀察標定GFP之C1L菌株 31、 觀察標定GFP之C1L菌株在臺灣百合根部組織群聚情形 32、 利用轉位子Tn917ac1建構突變庫及篩選失去根部群聚能力之C1L突變株 32. 誘變株篩選系統 32. Tn917ac1插入C1L菌株染色體位置分析 34. 臘狀芽孢桿菌C1L菌株及突變株之ISR作用 35、 結 果 36、 觀察C1L菌株在臺灣百合根圈的族群密度消長 36、 建構GFP標定之C1L電穿孔轉形株 36、 確認表現GFP之C1L電穿孔轉形株 37、 以螢光顯微鏡觀察標定GFP之C1L菌株 38、 GFP電穿孔轉形株特性 38. 生長曲線 38. 對峙培養 38. 抑制真菌孢子發芽試驗 39. 誘導植物抗病性之作用 39. 泳動性試驗 39、 觀察標定GFP之C1L電穿孔轉形株在臺灣百合根部組織群聚情形 40、 篩選失去群聚能力及泳動性之C1L突變株 40、 討 論 43、 總 結 48、 參考文獻 50拾、圖表集 59一、供試菌株 60二、供試載體 61三、引子 62四、臘狀芽孢桿菌C1L野生株及電穿孔轉形株CHG之抑制真菌生長能力比較 63五、臘狀芽孢桿菌C1L野生株及電穿孔轉形株CHG之ISR作用 64六、失去群聚能力之臘狀芽孢桿菌突變株Tn917ac1單邊側翼基因序列比對 65七、失去泳動能力之臘狀芽孢桿菌突變株Tn917ac1單邊側翼基因序列比對 66一、臘狀芽孢桿菌C1L菌株在臺灣百合根部表面的族群密度消長 67二、臘狀芽孢桿菌C1L菌株在臺灣百合根內組織的族群密度消長 68三、載體pHY300PLK穩定性之測試 69四、pGTZCH及pGTZCHG圖譜 70五、pPLKCH及pPLKCHG圖譜 71六、載體pPLKCHG之序列分析(1~1,900 bp) 72七、測定臘狀芽孢桿菌電穿孔轉形株CHG培養之自發綠色螢光強度 73八、北方雜合分析臘狀芽孢桿菌C1L野生株、電穿孔轉形株CH及CHG之GFP mRNA表現情形 74九、 利用螢光顯微鏡觀察臘狀芽孢桿菌C1L野生株及電穿孔轉形株CHG之自發綠色螢光 75十、臘狀芽孢桿菌C1L 野生株、電穿孔轉形株CH 及CHG之生長曲線 76十一、臘狀芽孢桿菌C1L野生株及電穿孔轉形株CHG對真菌之拮抗能力 77十二、 臘狀芽孢桿菌C1L野生株及電穿孔轉形株CHG上清濾液對真菌孢子發芽之抑制率 78十三、臘狀芽孢桿菌C1L野生株、電穿孔轉形株CH及CHG泳動能力之比較 79十四、 利用共軛焦雷射掃描顯微鏡觀察臘狀芽孢桿菌C1L菌株及電穿孔轉形株CHG在臺灣百合根部群聚情形 80十五、臘狀芽孢桿菌突變株之泳動能力等級 81十六、臘狀芽孢桿菌C1L菌株及突變株之ISR作用 82十七、臘狀芽孢桿菌C1L菌株於臺灣百合根部群聚之初步模式圖 83 錄 84一、pgroESp-GFP之載體圖譜 84二、pD917之載體圖譜 8