誘導圓葉菸草系統性抗病之細菌通透酶相關外泌因子鑑定

Identification of bacterial permease-related secreted factors involved in the induction of systemic disease resistance in Nicotiana benthamiana

蔡瑋祥(Wei-Hsiang Tsai)

指導教授 ： 陳昭瑩

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201704381

摘要

施用化學農藥是常見的高效植物病害管理策略之一，但過度施用化學農藥可能造成環境污染以及抗藥性病原菌的出現，因此，研發生物性農藥及其活性成份作為替代方案顯得特別重要。一類常用的生物性農藥來自植物促生根圈細菌，它擁有群聚於植物根圈的能力並有利於植物生長。部份植物促生根圈細菌亦可增強植物全體抵抗病原微生物的危害，此現象稱為誘導性系統抗病。前人研究顯示由臺灣花蓮太魯閣國家公園的臺灣百合根圈分離出之植物促生根圈細菌臘狀芽孢桿菌 (Bacillus cereus) 菌株 C1L, 被證實有降低百合灰黴病及南方玉米葉枯病罹病嚴重度的功效。本研究自臘狀芽孢桿菌C1L跳躍子突變庫，篩選誘導圓葉菸草對灰黴病菌 (Botrytis cinerea) 系統性抗病能力減弱之突變株，結果顯示突變株 M177經由定序與南方雜合分析確認跳躍子之單插入位置為巨環內酯通透酶基因 (macrolide permease)，且經反轉錄聚合酶連鎖反應確定無法表現巨環內酯通透酶之訊息核酸。隨後藉由液相層析串連質譜儀分析臘狀芽孢桿菌野生株C1L與突變株M177的外泌液差異性累積物質，得知臘狀芽孢桿菌 C1L 與誘導系統性抗病能力相關之細菌產物可能有flagellin、hemolysin 與 phospholipase C等，而這些候選因子的外泌與巨環內酯通透酶的關係，以及其對菸草誘導抗病性的促進作用尚待進一步研究。綜合上述，本研究增進對臘狀芽孢桿菌 C1L 介導系統性抗病之瞭解，且提供進一步探視的相關線索。

關鍵字

植物促生根圈細菌 ； 誘導系統性抗病 ； 臘狀芽孢桿菌 ； 圓葉菸草 ； 巨環內脂通透酶

並列摘要

Applying chemical pesticides is one of the common and efficient plant disease management measures. However, overuse of chemical pesticides causes environmental pollution and emergence of antibiotic-resistant strains of pathogens. Therefore, developing active ingredients of biopesticides has become an important issue. Several kinds of the popular biopesticides are developed based on plant growth-promoting rhizobacteria (PGPR) which are capable of colonizing plant rhizosphere and beneficial for plant growth. Some PGPR also increase whole-plant resistance against biotic and abiotic stresses, a phenomenon of induced systemic resistance (ISR). A PGPR strain named Bacillus cereus C1L, isolated from the rhizosphere of Lilium formosanum in Taroko National Park, Hualien County, Taiwan, was proved able to reduce disease severity of lily gray mold and southern corn leaf blight after soil-drench application. The aim of this study was to identify the bacterial genes involved in the systemic disease resistance induced by B. cereus C1L. To find the related genes, a transposon insertion library of B. cereus C1L was used and screened by the phenotype of decreased ISR on Nicotiana benthamiana against fungal pathogen Botrytis cinerea. In the mutant strain M177 of B. cereua C1L, a single transposon insertion was located in the gene encoding macrolide permease as confirmed by sequencing and Southern blot hybridization. The failure to express macrolide permease gene was demonstrated by reverse transcription-polymerase chain reaction. Presumably, a lack of macrolide permease function in M177 would retard the secretion of products required for ISR elicitation. LC-MS/MS analysis of the compounds differentially present in the secretions of B. cereus C1L wild-type strain and mutant M177 showed that flagellin, hemolysin and phospholipase C might be involved in the ISR elicitation of B. cereus C1L in N. benthamiana. The implication of permease involving in the secretion of candidate factors for eliciting effect of candidate factors on systemic resistance needs to be further confirmed. In this study, the mechanisms of B. cereus C1L-mediated ISR are better understood and give some clues for further investigations.

並列關鍵字

Plant growth-promoting rhizobacteria ； induced systemic resistance ； Bacillus cereus ； Nicotiana benthamiana ； macrolide permease

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