枯草桿菌Bacillus subtilis BS-99-H之iturin A抗菌物質對蓮霧果腐病原菌Pestalotiopsis eugeniae之生物活性

Bioactivity of Antifungal Substance Iturin A Produced by Bacillus Subtilis Strain BS-99-H Against Pestalotiopsis Eugeniae, a Causal Pathogen of Wax Apple Fruit Rot

林秀芬(H. F. Lin) ； 陳滄海(T. H. Chen) ； 劉顯達(S. D. Liu)

《植物病理學會刊》 19卷3期 (2010/09) Pp. 225-233

https://doi.org/10.6649/PPB.201009_19(3).0006

摘要

從玻璃紙抗生法的研究顯示，令枯草桿菌(Bacillus subtilis) BS-99-H之瓊脂塊放在玻璃紙上，再置於培養基平板上培養1-4天，去除含有枯草桿菌玻璃紙之培養基經90℃加熱10分鐘及未經加熱處理，皆對蓮霧果腐病原菌（Pestalotiopsis eugeniae）的菌絲生長有相同的抑制效果，至培養第5天之後，對P. eugeniae菌絲生長的抑制率達100%。由上述具有拮抗作用之培養基中萃取可能的抗菌物質，經HPLC及串聯式質譜儀（MS/MS）分析得知其為iturin A抗生物質。另將部分純化iturin A與P. eugeniae菌絲混合處理後，發現菌體細胞導電度於8小時後顯著增加3.4倍，至28小時達到高峰，為5.3倍；以顯微鏡觀察P. eugeniae菌絲呈現異常膨脹與扭曲。BS-99-H所產生的iturin A抑制P. eugeniae孢子發芽之IC50為0.51 mg/ml，當iturin A濃度為≥ 0.84 mg/ml則完全抑制P. eugeniae孢子發芽與菌絲生長。從本研究結果顯示出BS-99-H產生的iturin A是抑制P. eugeniae很重要的因子，在發展對P. eugeniae之生物防治上將可扮演重要的角色，因此本研究所得的枯草桿菌BS-99-H未來具有潛力開發作為蓮霧果腐病害之生物性殺菌劑。

關鍵字

枯草桿菌BS-99-H ； 蓮霧果腐病原菌 ； iturin A

並列摘要

Using the cellophane paper antibiosis method, an agar disc containing Bacillus subtilis BS-99-H was placed on a cellophane paper which was overlaid on the surface of an agar medium plate. After incubation, the cellophane paper was removed, and the medium that was heated at 90℃ for 10 min or non-heated was assayed for the inhibitory activity against mycelial growth of Pestalotiopsis eugeniae. Inhibition of mycelial growth of P. eugeniae was observed on both heated and non-heated media one day after incubation, and a complete inhibition can be observed at 5 days post incubation. The extracts from the heated and non-heated media were analyzed by the HPLC and tandem mass spectrometry (MS/ MS) methods, and iturin A was detected from both media. The partially purified iturin A was found to cause electrolyte leakage of P. eugeniae mycelia. The leakage was increased 3.4 folds at 8 hours post treatment and reached maximum increase of 5.3 folds at 28 hours post treatment. Treatment with the BS-99-H iturin A also caused swelling and malformed shape of the hyphae of P. eugeniae. The IC50 value of iturin A from BS-99-H for inhibition of P. eugeniae conidial germination was 0.51 mg/ml. The conidial germination and mycelia growth were both completely inhibited by iturin A at ≥ 0.84 mg/ml. The above results indicate that antibiotic iturin A produced by BS-99-H played an important role in antagonism against P. eugeniae. Therefore, B. subtilis BS-99-H could be a useful organism for development of a biofungicide agent for biocontrol of wax apple fruit rot.

並列關鍵字

Bacillus subtilis BS-99-H ； Pestalotiopsis eugeniae ； iturin A