建立Bacillus amyloliquefaciens PMB05 之發酵液配方於植物免疫反應與防治番茄青枯病之探討

由Ralstonia solanacearum所引起之番茄青枯病為番茄生產之重要限制因子，目前仍無推薦藥劑可供防治，應用拮抗微生物之防治策略被視為一種具有潛力的方法，已有多篇研究證實Bacillus spp.對青枯病具有防治效果，這些微生物之防治機制均與拮抗能力及誘導抗病有關。在近來的報導中亦證實利用提升植物免疫之蛋白質對青枯病的防治具有效果，本研究擬篩選可提升植物免疫反應之菌株並分析其處理為青枯病之抗病性。首先，選殖R. solanacearum的PopW蛋白做為植物免疫路徑之誘導物，進一步利用9株Bacillus spp.菌株在PopW共同處理下，評估其於菸草過敏性之分析之影響。其中，僅PMB05菌株顯著提升過敏性反應的發生，在番茄青枯病防治試驗中顯示此菌株可顯著降低青枯病之發生。為了更深入地了解這種抗病性的可能機制，在阿拉伯芥植物中進行了免疫通道抑制劑與PopW誘導的激活化氧產生和癒傷葡聚糖累積之探討。於添加植物NADPH氧化酶抑制劑與鈣離子通道抑制劑結果顯示，PMB05所提升PopW誘導的過敏性反應隨著上述兩種抑制劑添加的濃度增加而下降，且在PMB05與PopW共同處理時，顯示ROS產生與callose累積皆顯著提升。隨後，進一步評估如何應用發酵技術來強化免疫反應訊號，於搖瓶條件下進行發酵配方調整，結果顯示PMBFL-1發酵培養基的配方條件所獲得之發酵液可顯著增強PMB05所提升的免疫反應，且經500倍稀釋後仍能有效減少番茄植株青枯病之發生。綜上所述，本論文證明，Bacillus amyloliquefaciens PMB05為一拮抗微生物可增強植物免疫反應並提升植物對抗青枯病的抗病性。另，本研究亦說明利用植物免疫反應篩選之微生物可應用於植物病害之防治。

關鍵字

番茄青枯病； Bacillus amyloliquefaciens PMB05 ；植物免疫； PopW ；發酵培養

並列摘要

Tomato bacterial wilt caused by Ralstonia solanacearum is an important limiting factor for tomato production, and there is currently no recommended pesticide for control. The use of antagonistic bacterium is seen as a potential approach. Among them, Bacillus spp. has been applied to control bacterial wilt of tomato. However, the possible mechanisms are related to the antagonistic effect and induced resistance. In recent reports, it has been confirmed that the use of proteins that enhance plant immunity has an effect on the control of bacterial wilt. In this study, we attend to screen plant immunity intensifying Bacillus spp strain for further control tomato bacterial wilt. First, the PopW from R. solanacearum was expressed as an elicitor of PAMP-triggered immunity, and 9 Bacillus spp. strains were used to evaluate the effect on tobacco hypersensitive reaction (HR) assay upon PopW treatment. Among them, only the PMB05 strain significantly increased the incidence of HR ratio. This strain was shown to significantly reduce the occurrence of bacterial wilt in the tomato bacterial wilt. To gain a deeper understanding of the possible mechanisms of this disease resistance, we demonstrated that PopW-induced responses intensified by PMB05 were associated with PTI actilation in Arabidopsis plants, including rapid ROS generation and callose deposition. Subsequently, further evaluation of how to adjust the fermentation formula to strengthen the immune response signal, the results show that the PMBFL-1fermentation medium can enhance the PTI-intensification of PMB05. The 500-fold dilution of fermentation liquid was still effective to reduce the occurrence of tomato bacterial wilt. We concluded that Bacillus amyloliquefaciens PMB05 is microorganism that enhances disease resistance against bacterial wilt through intensifying PTI. Moreover, this study indicated that the screening plant immunty intensifying microorganisms to control plant disease is a feasible strategy.