臘狀芽孢桿菌誘導玉米系統性抗葉枯病之機制研究

Abstract

玉米為單子葉禾本科植物，為目前世界重要的糧食及飼料作物，具高度經濟價值；在臺灣，玉米常見的病害有紋枯病、銹病、露菌病，以及南方玉米葉枯病。南方玉米葉枯病由病原真菌Cochliobolus heterostrophus所引起，其孢子可藉空氣傳播，玉米受到感染後，病斑可快速擴展相連致使葉片枯死，對產量有顯著影響，目前以抗病品種或施用錳乃浦藥劑為主要防治方式。本研究室使用能誘導百合抗灰黴病之臘狀芽孢桿菌Bacillus cereus C1L防治玉米葉枯病，已於田間試驗指出，定期於玉米根圈澆灌C1L菌液對葉枯病有良好的防治效果。本研究在基因表現與顯微層次探討C1L菌株誘導玉米系統性抗葉枯病之機制。C1L菌株或其培養後產生之氣體分子二甲基二硫醚處理組之玉米葉片的抗壞血酸過氧化物酶基因與酚類化合物合成相關之苯丙胺酸氨解酶基因皆於葉枯病菌接種後，表現量有提前上升的情形。由組織化學染色結果得知，玉米根圈處理C1L菌株後接種葉枯病菌，葉表細胞癒傷葡聚糖及酚類化合物之累積增加；而過氧化氫累積量較對照組低，顯示具較優之抗氧化力。其次，探討植物荷爾蒙誘導物及荷爾蒙生合成抑制劑對玉米葉枯病罹病程度的影響，發現外施乙烯生合成抑制劑艾微激素能降低感染葉枯病之病斑大小；並以玉米已知之荷爾蒙誘導性標誌基因作為偵測目標的反轉錄聚合酶連鎖反應結果指出，C1L菌株處理致使葉片EIN2基因表現量呈下降趨勢，故C1L菌株可能藉由抑制乙烯路徑增加植物抗病性。另一方面，以基因微陣列分析及酵素活性測試指出，C1L菌株處理可誘發玉米罹病葉片提高一系列穀胱甘肽轉移酶相關基因及活性表現，暗示C1L菌株可透過增強植物解毒能力，提升其對葉枯病之抗性。

Corn is one of the most important crops in the world since it is a staple food of many people and animals. Southern corn leaf blight (SCLB) is caused by the air-borne fungal pathogen Cochliobolus heterostrophus. Infected lesions expand and merge rapidly, cauing corn leaves shrivel and ultimately end up with low yield. Foliar spray with fungicide Maneb is a usual measure of disease control. Previous field trials had shown Bacillus cereus C1L application in corn rhizosphere suppressed SCLB disease severity. This research attempts to demonstrate possible defense mechanisms involved in C1L-induced systemic resistance to SCLB by gene expression analysis and microscopical examination. Gene expressions of cytosolic ascorbate peroxidase (cAPX) and phenylalanine ammonia lyase (PAL) increased earlier and to higher level in the C1L- and dimethyl disulfide (DMDS)-treated corn leaves after pathogen inoculation as compared with the control. In addition, through histochemical staining, lower H2O2, more phenolic compounds, and increased callose deposition were observed in the C1L- and DMDS-treated corn leave than the control after inoculation with C. heterostrophus. In addition, since lesion sizes were significantly reduced by application of ethylene biosynthesis inhibitor Retain® and the ethylene-inducible EIN2 gene were down regulated in the leaves by C1L treatment, it appears that strain C1L can prevent the disease by suppressing ethylene pathway. On the other hand, increased expression of glutathione transferase genes and activities in C. heterostrophus-inoculated leaves were observed in microarray analysis and GST activity assay, suggesting that strain C1L could build disease resistance capacity through enhancing detoxification in maize.