Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30988
標題: 番茄內生性細菌 Bacillus cereus 之特性分析與其對青枯病之影響
Characterization of tomato endophytic Bacillus cereus and its effects on bacterial wilt disease
作者: 張郁靈
Jhang, Yu-Ling
關鍵字: endophytic bacteria
內生性細菌
Bacillus cereus
quorum sensing
quorum quenching enzymes
Bacillus cereus
集約反應
quorum quenching enzymes
出版社: 植物病理學系所
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摘要: 內生性細菌 (endophytic bacteria) 廣泛存在於不同種類的植物中,且可分布於植物的許多部位。由前人研究發現,部分內生性細菌具有獨特的代謝活性可改變寄主植物的生理狀態,有些則可產生抗生物質或具有分解性質的酵素,而能保護植物免於微生物之感染。因此,本研究自三個不同品種之番茄無菌培養苗分離得到 14 株內生菌,以測試植物內生性細菌是否具有保護番茄植株使其免於受到青枯病菌 (Ralstonia solanacearum) 感染之潛力。分離而得之菌株均經過脂肪酸圖譜 (fatty acid methyl ester profile) 以及 16S rRNA 基因序列之鑑定,其中一株菌為 Bacillus cereus (Bce1),經 PCR 檢測發現其帶有 aiiA 基因。AiiA 為一類 quorum quenching 酵素,具有 AHL (acyl homoserine lactone) lactonase 活性,可分解 quorum sensing (QS) 訊號傳遞分子 AHL。利用剪胚軸接種法將 Bce1 接種至番茄後,可知 Bce1 能於番茄維管束中生長,並且於接種後 30 天之莖部片段維持約 104 CFU/g 之菌量。此外,接種 Bce1 30 天後的番茄地上部與地下部之重量均較對照組高出 1 倍,顯示 Bce1 可促進番茄幼苗的生長。由平板對峙培養之結果顯示,Bce1 可減緩青枯病菌 R. solanacearum PS64 菌株之生長。於溫室試驗中,將接種內生菌 Bce1 30 天後的番茄以澆灌方式接種青枯病菌後,相較於無處理內生菌 Bce1 之對照組,青枯病之發病率降低 75%。由於 R. solanacearum 之毒力因子的表現受到 QS 調控,因此,本研究利用基因選殖方式增加 Bce1之 aiiA 表現量,以測試於植物中分解青枯病菌的 QS 訊號傳遞分子是否能使番茄青枯病之罹病度更為下降。然而,具有較高之 AiiA 表現量的 Bce1 經平板對峙培養測試發現,相較於野生型菌株,Bce1 轉形菌株無法降低 R. solanacearum PS64 之生長,也無法促進番茄生長。此外,將此轉形菌株接種於番茄幼苗 30 天後再進行青枯病菌之接種,亦無法降低青枯病之發病率,顯示 Bce1 中具有促進植物生長之活性物質可能直接或間接受到 AHL 自體誘導物 (autoinducer) 之調控。因 R. solanacearum 毒力因子胞外多醣體 (extracellular polysaccharide, EPS) 之生合成受到另一種 QS 訊號分子3-hydroxy palmitic acid methyl ester (3-OH PAME) 所調控,因此,未來可嘗試於具有促進植物生長能力之 Bce1 中表現 3-OH PAME 之分解酵素,以釐清基因選殖後之內生性細菌應用於番茄青枯病之管理的可能性。
Diverse endophytic bacteria that grow in the internal tissues of plants are found in a wide variety of plant species. Previous studies showed that some endophytic bacteria have unique metabolic activities that can alter the physiological conditions of the host plants, and some produce antibiotic substances and degradative enzymes that protect host plants from microbial infection. To test if endophytic bacteria have the potential in protecting tomato plants against Ralstonia solanacearum, fourteen endophytic bacteria were isolated from different cultivars of disinfested tomato seedlings in this study. All isolates were identified by FAME and 16S rRNA gene sequences, and one strain was identified as Bacillus cereus (Bce1) that harbors aiiA coding for the quorum quenching enzyme AHL (acyl homoserine lactone) lactonase. The endophytic growth of Bce1 in tomato seedlings was measured by hypocotyl cutting method to show that the bacterium can grow in the vascular tissues to reach a population density of 104 CFU/g at 30 days post inoculation (dpi). In comparison with the untreated plants, tomato seedlings inoculated with Bce1 showed at least one fold of increase in the total weight at 30 dpi, indicating that Bce1 can promote tomato growth. Bce1 had an inhibitory effect on the growth of R. solanacearum strain PS64 that can be readily observed by a dual culture method. When tomato seedlings were inoculated with Bce1 for 30 days and subsequently challenged with R. solanacearum strain PS152 by soil drench, the disease incidence was reduced by 75% in comparing with the 100% wilting symptom of the untreated plants. Knowing the expression of virulence factors in many plant pathogenic bacteria, including R. solanacearum, depends on the quorum sensing (QS) signaling pathways, Bce1 was genetically modified to increase the expression of the AHL-degrading enzyme AiiA to test if the disease severity of tomato bacterial wilt can be further reduced by the removal of QS signaling molecules in planta. Surprisingly, Bce1 with elevated AiiA lost the inhibitory capability on the in vitro growth of R. solanacearum PS64, the beneficial effect on tomato growth, and the protection against tomato bacterial wilt disease, suggesting the bioactive compounds of Bce1 that are involved in promoting plant growth and health may be directly or indirectly regulated by the AHL autoinducers. Genetic studies demonstrated that the extracellular polysaccharide (EPS) of R. solanacearum is an essential virulence factor whose synthesis depends on the other QS signal known as 3-hydroxy palmitic acid methyl ester (3-OH PAME). The feasibility of using genetically engineered endophytic bacteria in the management of tomato bacterial wilt disease will be tested again by expressing the 3-OH PAME degradative enzymes in the endophytic and growth-promoting bacterium Bce1.
URI: http://hdl.handle.net/11455/30988
其他識別: U0005-1808201114085000
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