指導教授：張雅君臺灣大學：植物病理與微生物學研究所王柏荃Wang, Po-ChuanPo-ChuanWang2014-11-292018-06-292014-11-292018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/263048植物病毒病害由於無法有效以化學農藥達到防治目的，造成防治上的困難。前人研究發現土壤微生物及其產生之次級代謝物具有誘導植物抗性對抗真菌與細菌病害之潛力，但其利用於防治病毒病害的研究仍待研究。根基於土壤微生物的多樣性，本研究擬利用前人建立之土壤微生物篩選技術，結合實驗室建立之檢測抗病毒效力策略，期望可以篩選出能簡易培養且可產生誘導植物抗性並且對抗病毒病害之土壤微生物。檢測抗病毒效力的初步篩選是用Tobacco mosaic virus (TMV) 接種指示植物Nicotiana glutinosa利用產生出的病斑數目作為指標來判斷病害嚴重程度。將土壤微生物培養後的上清濾液塗佈於指示植物之葉表，篩選出一株平均可減少90%病斑表現的潛力菌株，後續綜合實驗室前人篩選到的另一株潛力菌株進行測定。兩株菌培養後的上清濾液塗佈於指示植物之葉背，平均可以減少60% - 65%之病徵表現，此結果顯示此兩株菌可能具有系統性保護之功能。選用對TMV具有高感病性的菸草品系Nicotiana benthamiana，利用接種後之倒伏病徵建立病害標準 (Disease index) 。將此兩株菌的培養濾液塗佈於N. benthamiana植株之葉背，接種TMV於葉面後，可觀察到發病時程有一至兩天之延遲。若改以塗佈整片葉後於上位葉接種TMV，也可觀察到病程發展之延緩，證實有系統性保護之功效。塗佈過兩株菌之培養濾液後的N. benthamiana，於處理葉皆有偵測到過氧化氫 (H2O2) 以及水楊酸抗性路徑之標誌基因PR-1a上升的情形，證明有水楊酸相關之抗性反應參與。利用ITS序列 (internal transcribed spacer)、26S rDNA D1/D2序列及形態鑑定，推測此兩株菌分別較接近Trichosporon scarabaeorum與Scedosporium dehogii。本實驗發現土壤微生物之培養濾液多可誘導植物抗性來對抗病毒，實驗中篩選出兩株真菌，其培養濾液不管在抗病或感病植物皆能有效誘導植物抗性，初步研究認為此兩株潛力菌株之培養濾液皆能誘導與水楊酸相關之抗性反應，並探討其在應用在病毒病病害管理之可行性。Recent research indicated that the secondary metabolites of soil microorganisms can induce plant resistance against fungus and bacterial disease; however, whether the metabolites are effective to virus disease remain to be resolved. Thus, we selected Nicotiana glutinosa, a local lesion host of Tobacco mosaic virus (TMV), as the indicator plant for analysis. The local lesion numbers induced by TMV were counted on plants treated with culture filtrates derived from soil microorganisms to measure the resistance response. The culture filtrates of the microorganisms was sprayed on to adaxial surface of plants then inoculated with TMV on the adaxial leaf surface to screen the filtrate with potential to induce the antiviral activity. Filtrate derived from two microorganisms can reduced 90% lesions on treated N. glutinosa. When the culture filtrates was sprayed on the leaves abaxial surface and inoculated the TMV on the adaxial surface of plants. About 60% - 65% lesions reduction was observed. It indicated that these culture filtrates can induce systemic protection against virus infection. Besides the TMV susceptible cultivar Nicotiana benthamiana was also selected for analysis, and the disease index was set up for further analysis. The culture filtrates was sprayed on lower leaves and then inoculated the TMV on the upper leaves, the symptoms delay was observed. It indicate that the culture filtrates of these two fungi have the ability to induce plant systemic protection. The hydrogen superoxide (H2O2) accumulation and pathogen-related protein gene, PR-1a, expression were detected on N. benthamiana leaves pretreated with the culture filtrates. These two fungi were identified by 26S rDNA D1/D2 region, ITS sequence analysis and morphology, they are highly related to Trichosporon scarabaeorum and Scedosporium dehogii. Our studies suggested that most of filtrates derived from soil microorganisms can induce plant resistance against viruses. We isolated two fungi from soil, and the filtrates derived from the two fungi can induce prominent salicylic acid related plant resistance response both on susceptible and resistance plants, and the potential application of the isolated fungi on virus disease management is discussed.致謝 i 中文摘要 ii Abstract iv Contents vi 1 INTRODUCTION 1 2 MATERIALS AND METHODS 5 2.1 Preparation of selective medium 5 2.2 Isolation of soil microorganisms by vegetable mixture 5 2.3 Preparation of culture filtrates derived from the isolated soil microorganisms 6 2.4 Plant materials 7 2.5 In vitro transcription and inoculum preparation 7 2.6 Antiviral activity assays 8 2.7 Plants treatment and sampling 8 2.8 RNA extraction 9 2.9 DNase treatment 9 2.10 Semi-quantitative PCR 10 2.11 Hydrogen peroxide staining 11 2.12 Fungi identification 11 2.13 Phylogenic analysis 12 3 RESULTS 13 4 DISCUSSION 21 REFERENCES 25 Tables and Figures 30 Table 1. Primer sequence used in the study 31 Table 2. Infection rate of Tobacco mosaic virus (TMV) on Nicotiana benthamiana inoculated with different concentrations of TMV inoculum 32 Figure 1. Lesion numbers induced by Tobacco mosaic virus (TMV) inoculum prepared from TMV infected freeze-dried leaves on Nicotiana glutinosa 33 Figure 2. Lesion numbers and necrosis induced by Tobacco mosaic virus (TMV) on Nicotiana glutinosa leaves pretreated with salicylic acid (SA) 34 Figure 3. The ratio of lesion numbers induced by Tobacco mosaic virus (TMV) on treated Nicotiana glutinosa 35 Figure 4. Lesion numbers induced by Tobacco mosaic virus (TMV) on Nicotiana glutinosa leaves pretreated with culture filtrates derived from collected fungi 36 Figure 5. Lesion numbers induced by Tobacco mosaic virus (TMV) on Nicotiana glutinosa leaves adaxial surface pretreated with culture filtrates derived from selected fungi 37 Figure 6. Lesion numbers induced by Tobacco mosaic virus (TMV) on Nicotiana glutinosa leaves abaxial surface pretreated with culture filtrates derived from selected fungi 38 Figure 7. Lesion numbers induced by Tobacco mosaic virus (TMV) on Nicotiana glutinosa leaves pretreated with culture filtrates derived from two different broths 39 Figure 8. Symptoms induced by Tobacco mosaic virus (TMV) on Nicotiana benthamiana inoculated with optimized TMV inoculum 40 Figure 9. Disease index of Nicotiana benthamiana inoculated with optimized Tobacco mosaic virus(TMV)inoculum 41 Figure 10. Severity of symptoms induced by Tobacco mosaic virus (TMV) on Nicotiana benthamiana 42 Figure 11. Severity of symptoms induced by systemic Tobacco mosaic virus (TMV) inoculation on Nicotiana benthamiana within two days after treatment 43 Figure 12. Severity of symptoms induced by systemic inoculation of Tobacco mosaic virus (TMV) on Nicotiana benthamiana within five days after treatment 44 Figure 13. Semi-quantification of PR1a and PDF1.2 genes on Nicotiana benthamiana 46 Figure 14. The detection of hydrogen peroxide on Nicotiana benthamiana treated leaves 47 Figure 15. Lesion numbers induced by Tobacco mosaic virus (TMV) on Nicotiana glutinosa leaves adaxial surface pretreated with sterilized filtrates 48 Figure 16. Morphology of PHF2 on culture mediums 49 Figure 17. Morphology of PHF14 on culture mediums 50 Figure 18. Morphology of NTU1F8 on cultured mediums 51 Figure 19. Phylogenetic tree of PHF2 and PHF14 52 Figure 20. Phylogenetic tree of NTU1F8 53 Figure 21. Colony morphology of PHF2 on cultured medium 54 Figure 22. Colony morphology of NTU1F8 on cultured medium 55 Appendix 56 Appendix Table 1. Detailed properties of soil samples collected in this study 57 Appendix Figure 1. Three classes of screened soil microorganisms isolated from the soil sample 58 Appendix Figure 2. Lesion numbers induced by Tobacco mosaic virus (TMV) on Nicotiana glutinosa leaves abaxial surface pretreated with crude filtrates or sterilized filtrates 59 Appendix Figure 3. Colony morphology of PHF14 on cultured medium 603227489 bytesapplication/pdf論文公開時間：2017/08/21論文使用權限：同意有償授權(權利金給回饋學校)土壤微生物菸草嵌紋病毒誘導抗性次級代謝物thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/263048/1/ntu-103-R01633018-1.pdf