臘狀芽孢桿菌C1L菌株誘導百合系統性抗灰黴病菌之研究

Studies on Bacillus cereus C1L-induced systemic resistance against Botrytis elliptica in Lilium

劉益宏(Yi-Hung Liu)

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/博士(2008年)

https://doi.org/10.6342/NTU.2008.10017

摘要

已知施用臘狀芽孢桿菌C1L菌株之菌液於葵百合及臺灣百合根圈，可有效地降低灰黴病之發病嚴重度，本論文研究C1L菌株誘導百合之抗病機制，並推測可能之訊息傳遞路徑。首先利用抑制性扣減雜交（suppression subtractive hybridization, SSH）技術，於臺灣百合上鑑定出預先處理C1L菌株而有差異性表現的基因LfGRP1、LfMT1、LfPsbR等。其中LfMT1為正調控表現，LfGRP1及LfPsbR呈負調控表現；在葵百合上，同源性基因之表現亦有有相同趨勢，推測C1L菌株由改變百合之基因表現，影響百合之生理功能，致使百合對灰黴病菌具有抵抗性，其作用機制與水楊酸誘導表現的抗病機制不同。其次，觀察到C1L菌株可促使葵百合抑制由灰黴病菌感染所造成之過氧化氫累積及百合細胞過度氧化及細胞死亡的情形。由活性氧(reactive oxygen species, ROS)抑制劑之作用及北方雜合分析之結果推測LfGRP1及LfPsbR基因的負調控表現與ROS代謝途徑有關。再者，利用乙烯及甲基茉莉酸處理葵百合時，LfGRP1及LfPsbR基因有增量表現，但預先處理離層酸時，此二基因之表現則會受到抑制，指出C1L菌株誘導百合對灰黴病產生抗病性，可能參與離層酸訊息傳遞路徑。另一方面，於葵百合葉片上單獨接種灰黴病菌24小時，與對照組比較之下，以Evan blue染色可以偵測到高含量的細胞死亡，而預先處理C1L菌株再接種灰黴病菌 24小時，可抑制百合葉片過氧化氫累積及細胞死亡。此外，以C1L菌株處理百合時，百合葉之過氧化酶活性，於處理後48小時達到最高，單一接種灰黴病菌 24小時，過氧化酶活性最強，但48小時即呈現負調控；預先處理C1L菌株24小時後再接種灰黴病菌時，過氧化酶活性並不會增加。另一方面，分析β-1,3-葡聚糖酶基因之表現，預先處理C1L菌株24小時再接種灰黴病菌，可提早於接種後48小時偵測到β-1,3-葡聚糖酶基因之表現，可能與百合葉抗灰黴病菌感染的能力有關。為進一步分析C1L菌株誘導百合抗灰黴病之作用機制，利用cDNA-AFLP技術在不同處理中共獲得葵百合之104個cDNA片段，以半定量反轉錄聚合酶鏈鎖反應偵測基因之表現，發現C1L菌株處理百合根系，可經由鈣離子的訊息傳遞，啟動抗病反應，而此過程會帶動與離層酸或似離層酸訊息路徑有關之清除活性氧物質的抗氧化反應。本研究並發現C1L菌株可誘導麩醯胺酸合成酵素基因之表現，其可能減輕灰黴病菌感染對百合細胞的毒化作用，因而降低灰黴病之嚴重度。

關鍵字

根圈細菌 ； 誘導系統性抗病 ； 過氧化氫 ； 細胞死亡 ； 差異性表現 ； 抗氧化作用 ； 離層酸

並列摘要

Application of Bacillus cereus C1L suspension in the rhizosphere of Lilium formosanum and lily cv. Star Gazer to reduce gray mold severity has been demonstrated. In this dissertation, the mechanism of induced systemic resistance (ISR) was approached to figure out the signaling pathway involved. Firstly, differentially expressed genes in strain C1L-treated L. formosanum were identified to be LfGRP1, LfMT1, and LfPsbR, by the use of suppression subtractive hybridization method. When L. formosanum was treated with strain C1L, LfMT1 was up-regulated. However, LfGRP1 and LfPsbR were down-regulated. The expression patterns of the 3 homologues in oriental lily cv. Star Gazer were identical to those of LfGRP1, LfMT1 and LfPsbR. When using ethylene and methyl jasmonate to treat lily cv. Star Gazer, LsGRP1 and LsPsbR were up-regulated. On the contrary, these genes were down-regulated by the treatment of abscissic acid (ABA), indicating that ABA signaling pathway is involved in the systemic resistance induced by strain C1L. When ‘Stra Gazer’ lily plants were treated with strain C1L 24 hours before inoculation with B. elliptica, accumulation of hydrogen peroxide and cell death in the leaves was suppressed as compared to those exhibited in the plant singly inoculated with B. elliptica. The peroxidase activity was not increased in the leaves inoculated with B. elliptica post strain C1L treatment, unlike that inoculated with B. elliptica singly. On the other hand, strain C1L induced early expression of LpGlu1 (β-1,3-glucanase) in response to B. elliptica infection post strain C1L treatment as compared to that inoculated with B. elliptica singly. In order to dissect the mechanism of ISR activated by strain C1L, cDNA-AFLP was analyzed and 104 cDNA clones representative of differentially expressed genes of lily were obtained. Expressions of selected genes were analyzed by semi-quantitative reverse-transcription polymerase chain reaction. The results showed that strain C1L could induce systemic resistance via calcium ion signaling pathway and ABA signaling pathway, followed by anti-oxidative response. Additionally, strain C1L induced expression of glutamate synthase of lily which might decrease cellular toxicity caused by B. elliptica and therefore reduced the disease severity.

並列關鍵字

Rhizobacteria ； induced systemic resistance ； hydrogen peroxide ； plant cell death ； antioxidation ； abscisic acid

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