臺灣博碩士論文加值系統

百香果種苗及其果實在台灣及國際貿易上具高經濟價值，而由Fusarium solani所造成的百香果頸腐病為一百香果重要病害，並會造成其嚴重苗株死亡及產量損失。本研究目的為開發以Bacillus 屬菌株做為防治百香果頸腐病之生物防治資材，篩選具潛力可防治頸腐病的化學藥劑，並評估以Bacillus 屬菌株及化學藥劑共同防治潛力，另進一步探討Bacillus 屬具植物生長促進及生物防治潛力之相關機制。由本研究前期試驗結果顯示B. subtilis YBC及151B1 對許多種真菌病原具拮抗性，其中也包括百香果病原菌Colletotrichum karstii 及 Fusarium solani PF7。而本研究中發現B. subtilis YBC及151B1可抑制F. solani PF7菌絲生長及孢子發芽，其抑制率分別為41.8 %及39.5%。而施用B. subtilis YBC and 151B1 培養液可以降低Fusarium於葉片感染之罹病度並提升帶病土中百香果扦插苗的存活率。另試驗結果亦顯示施用B. subtilis YBC 及 151B1 培養液，具促進百香果種苗生長效果，可提升黃百香果種苗植株高度、增加葉片大小及數量與植株鮮重及乾重。另經測試十三種延伸使用於百香果病害防治及其他作物上子囊菌引起之病害防治用化學殺菌劑對F. solani PF7之生長抑制及引起病害之防治效果，結果顯示得克利具有最佳菌絲生長抑制能力並可以顯著提升百香果扦插苗於病土中之存活率。且亦發現供試測試藥劑皆不會影響拮抗菌B. subtilis YBC 及151B1生長，顯示所測試藥劑與B. subtilis YBC 及 151B1具有親和性。另探討B. subtilis YBC及151B1於百香果Fusarium病害防治機制，結果顯示經B. subtilis YBC 及B. subtilis 151B1 培養濾液處理之F. solani PF7，其發芽管畸形膨大且會喪失細胞膜完整性，且會造成病原真菌粒線體膜潛勢降低及干擾其能量代謝。另亦發現經B. subtilis YBC及151B1培養濾液處理後會導致F. solani PF7細胞之激活態氧累積及造成膜磷脂絲胺酸外顯性、染色質凝聚及DNA 片段化等細胞程序性凋亡之特徵性生理生化表現，因此證實B. subtilis YBC及151B1可以誘導病原之細胞程序性凋亡。另本研究中亦證實B. subtilis YBC及151B1兩株菌株具產生吲哚乙酸及溶磷能力等植物生長促進性狀。綜合上述實驗結果顯示，B. subtilis YBC 及151B1具有防治由F. solani所造成百香果病害之潛力，而其病害防治機制可能因其可誘導病原菌細胞程序性凋亡，並降低粒線體膜潛勢及干擾能量代謝。另B. subtilis YBC 及151B1可透過產生吲哚乙酸及具溶磷能力而促進植物生長。另研究中亦發現化學藥劑-得克利、B. subtilis YBC 及151B1單獨使用或是得克利與B. subtilis 混合應用皆具可防治由F. solani PF7所造成的病害之潛力，因此未來可以用於田間進行綜合防治由F. solani所造成百香果頸腐病。

The seedlings and the fresh fruits of passion fruits are of high value in local and global trade. Fusarium solani (Teleomorph: Neocosmospora solani (Martius) is one of the main disease causal agents affecting passion fruits. The objectives of this study are to develop Bacillus-based biocontrol agents for the management of fusarium disease on passion fruits; to assess the potential fungicides to control fusarium disease on passion fruits; to evaluate of the compatibility of potential Bacillus species strains with chemical fungicides; and to develop integrated strategy for controlling Fusarium disease on passion fruits; to investigate the putative plant protection mechanisms by Bacillus species. Our previous studies indicated that Bacillus subtilis YBC and 151B1 native in Taiwan showed antagonistic activity to several fungal pathogens on passion fruits including Colletotrichum karstii and F. solani PF7. They also inhibited mycelial growth and conidial germination of F. solani PF7. Application of B. subtilis 151B1 and YBC culture broth caused a reduction in the disease severity of fusarium wilt on leaves of passion fruits and enhanced the survival rates of passion fruit seedlings with challenge of F. solani. Application of B. subtilis 151B1 and YBC culture broth promoted the growth of plant height, increased the size and numbers of leaves, and fresh and dry weights of passion fruit seedlings. Among thirteen fungicides commosnly used for the control of Ascomycetes tested, the results showed that tebuconazole showed the best effecacy on the inhibition of hyphal growth of F. solani PF7 and the best survival of the passion fruit cuttings in the infested soil. We also found that the growth of B. subtilis 151B1 and YBC was not affected by the above tested fungicides and the efficacy in enhancing survival rates of passion fruit cuttings in the Fusarium-infested soil remained, suggesting the compatibility between B. subtilis and tested fungicides. To investigate the putative mechanisms of disease control, the results indicated that the treatments of culture filtrates from B. subtilis 151B1 and YBC both caused aberrant conidial morphology and loss of cell membrane integrity. Additionally, the treatments caused reduction in mitochondrial membrane potential and interfered with energy metabolism of Fusarium solani PF7. The treatments also enhanced the reactive oxygen species accumulation, and resulted in externalization of phosphatidylserine, chromatin condensation, and DNA fragmentation, suggesting the functions of culture filtrates in triggering apoptotic-like cell death. To investigate the mechanisms of plant growth promotion by B. subtilis 151B1 and YBC, our results indicated that both Bacillus strains exhibited activities of producing indole-3-acetic acid (IAA) and solubilizing phosphate. In conclusions, B. subtilis 151B1 and YBC are potential biocontrol agents for passion fruit disease caused by F. solani. The control efficacy may be contributed to their ability in triggering apoptotic-like cell death, reducing the mitochondrial membrane potential and interfering with energy metabolism of the pathogen. B. subtilis 151B1 and YBC also exhibited growth promotion traits by producing IAA and solubilizing phosphates. Chemical fungicide, tebuconazole, B. subtilis 151B1 and YBC, or combination of tebuconazole and B. subtilis could be used to prevent the passion fruits from the infection by F. solani PF7. The integrated control strategies by combination use of Bacillus species and chemical fungicides could be further implemented to control the collar rot disease of passion fruits caused by F. solani.

中文摘要 i
Abstract iii
目錄 v
圖目次 ix
前言 1
材料與方法 8
一、供試藥品來源 8
二、供試菌種來源與病原性測試 8
（一）供試百香果病原菌來源與保存 8
（二）供試拮抗性枯草桿菌群菌株之來源與保存 9
（三）百香果病原菌病原性測試 9
三、百香果頸腐病原PF7之形態及分子生物學鑑定 9
（一）形態鑑定 9
（二）分子生物學鑑定 10
四、B. subtilis YBC、YBX及151B1對F. solani PF7之拮抗作用
11
五、具拮抗性之芽孢桿菌屬菌株YBC、YBX及151B1之生理生化特性
及１6S rRNA 序列分析 11
（一）Biolog 鑑定及生理生化分析 11
（二）B. subtilis YBC及151B1之碳、氮素源利用分析 13
（三）16S rRNA 序列分析 14
六、具拮抗性之B. subtilis YBC及151B1培養液的製備 15
七、B. subtilis YBC及151B1對百香果防治潛力測試 15
（一）施用微生物培養液對百香果離葉之防治測試 15
（二）施用微生物培養液對於扦插苗存活性測試 16
八、B. subtilis YBC及151B1對百香果實生苗生長之影響測試 16
九、化學藥劑對百香果病原菌PF7之菌絲生長抑制測試及孢子發芽抑制測
試 17
十、化學藥劑與B. subtilis YBC及151B1親和性及共同防治試驗 18
十一、B. subtilis YBC及151B1培養濾液對F. solani PF7孢子發芽及於百香
果葉片上侵入感染之影響 19
十二、B. subtilis YBC及151B1培養濾液是否會導致百香果頸腐病菌PF7之
細胞凋亡之檢測 20
（一）細胞膜通透性測試 20
（二）激活態氧(Reactive oxygen species, ROS)累積 20
（三）膜磷脂絲胺酸(phosphatidylserine, PS )外顯性 21
（四）染色質凝聚 22
（五）DNA 片段化 22
十三、 B. subtilis YBC and 151B1 培養濾液對百香果頸腐病原菌PF7粒線體
膜潛勢及呼吸作用之影響 22
十四、B. subtilis YBC及151B1之植物生長促進特性分析 23
十五、統計分析 25
結果 26
一、百香果果實壞疽病原菌之分離及病原性測定 26
二、百香果頸腐病原PF7之形態及分子生物學鑑定 26
（一）形態鑑定 26
（二）分子生物學鑑定 27
三、根圈土壤分離之芽孢桿菌屬菌株對百香果病原菌之拮抗作用 28
四、以生理生化特性及分子生物學鑑定具拮抗性芽孢桿菌屬菌株 28
（一）Biolog 鑑定及生理生化分析 28
（二）B. subtilis YBC及151B1之碳、氮素源利用分析 29
（三）16S rRNA 序列分析 29
五、B. subtilis YBC及151B1對百香果頸腐病害防治潛力測試 29
六、B. subtilis YBC及151B1對百香果實生苗生長之影響測試 30
七、化學藥劑對百香果病原菌PF7之菌絲生長抑制測試及孢子發芽抑
制測試 31
八、化學藥劑與B. subtilis YBC及151B1親和性及共同防治試驗 31
九、B. subtilis YBC及151B1培養濾液對F. solani PF7孢子發芽及於百香果
葉片上侵入感染之影響 32
十、B. subtilis YBC及151B1 培養濾液是否會導致百香果頸腐病菌PF7之
細胞凋亡之檢測 33
十一、B. subtilis YBC and 151B1 培養濾液對百香果頸腐病原菌PF7粒線體
膜潛勢及呼吸作用之影響 35
十二、B. subtilis YBC及151B1之植物生長促進特性分析 36
討論 38
參考文獻 43
圖說明 53
附錄 83

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