篩選台灣土壤細菌應用於茄科青枯病之防治

Abstract

由 Ralstonia solanacearum 所引起的青枯病，又稱細菌性萎凋病 (bacterial wilt)，是植物重要的細菌性病害之一，目前尚無有效的防治方式。在本研究中，我們從自然環境中分離了77 株細菌菌株，並利用特殊培養基測試其螯鐵蛋白的合成及拮抗青枯病菌能力。將其中14 株具有上述兩種抗菌能力的細菌株，接種在兩種番茄品種上觀察其生物防治效力。實驗結果顯示，WF02, WF03 與 DH25 這三株細菌株具有明顯降低青枯病在中等抗病品種番茄 (Micro-Tom) 的死亡率、病情指數及病情發展曲線下面積 (area under disease progress curve, AUDPC) 之效能，其中 WF02 菌株在易感病品種番茄 (L390) 上亦有防治效果。經比對三株細菌株的16S rDNA 和 gyrB 基因序列以及利用 GEN III MicroPlate 進行菌種鑑定，顯示三株細菌株均屬於液化澱粉芽孢桿菌 (Bacillus amyloliquefaciens)。為進一步探討其可能之抑病機制，我們以 MPN-PCR 檢測土壤中的病原菌數。在預先處理 WF02 菌株的土壤樣本中的病原菌數量明顯低於僅處理病原菌的對照組。我們同時也利用 real-time PCR 偵測植株體內與 salicylic acid (SA)、jasmonic acid (JA) 和 ethylene (ET) 等信息傳導路徑相關的植物防禦基因的表現量，發現若在病原菌感染之前預先處理WF02的植株，在兩種番茄品種的葉片中，與 SA 及 JA 相關之基因表現量皆明顯上昇，而預先處理WF03 及 DH25的植株，只在 Micro-Tom 中觀察到植物抗病相關基因表現之提升。因此我們推測 B. amyloliquefaciens WF02 菌株可能是利用拮抗作用或活化植物防禦反應等機制達到抑制青枯病的效果。

Bacterial wilt caused by a soil-borne pathogen, Ralstonia solanacearum is a serious plant disease and there is no effective method to control presently. In this study, 77 bacterial strains were screened by in vitro medium test for siderophore production ability and antagonist against R. solanacearum. Fourteen bacteria that have both abilities were assessed their in vivo biocontrol actives against R. solanacearum by pot experiments with two tomato cultivars. We found that three potential isolates (WF02, WF03 and DH25) could reduce the mortality, disease incidence and area under disease pathogenesis curve (AUDPC) in the resistance cultivar Micro-Tom. WF02 could also reduce the disease severity in the susceptive cultivar L390. Analysis of 16S rRNA and gyrB gene sequences and GEN III MicroPlate revealed that these isolates all belong to Bacillus amyloliquefaciens. We determined the pathogen population in rhizosphere soils by MPN-PCR, and found that the number of pathogen population was significantly reduced by WF02 pretreated soil as compared with that of control. We used real-time PCR to analyze the expressions of the plant defense genes that are related to salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. Treating with the WF02 isolate could obviously induce the expressions of SA and JA related genes in both tomato cultivars, but WF03 and DH25 only induced the plant resistance in tomato cv. Micro-Tom. Taken together, we deduced that the B. amyloliquefaciens WF02 isolate can use various mechanisms, such as antagonistic effects or activation of plant defense reactions to suppress tomato bacterial wilt disease.