拮抗微生物枯草桿菌生物防治作用機制之探討及其高產能發酵技術開發

The Mechanism of Biological Control with Antagonistic Microorganism Bacillus subtilis and Development of High Yeild Fermentation Technique

林秀芬(Hsiu-Fen Lin)

指導教授 ： 劉顯達 ； 陳滄海

國立屏東科技大學/農學院/生物資源研究所/博士(2011年)

https://doi.org/10.6346/NPUST.2011.00240

摘要

有鑑於施用化學農藥於植物病害防治對環境及人類等造成之負面影響，因此開發安全性之微生物農藥乃成為重要之課題。本論文研究目的期望能開發枯草桿菌微生物農藥之商品化，作為有效的病害生物防治藥劑。本研究取自本土性土壤之拮抗微生物細菌，經微生物學方法及分子技術鑑定為枯草桿菌Bacillus subtilis，進一步以溫度篩選出代號BS-99-H之耐高溫菌株。枯草桿菌BS-99-H具有產生內生孢子之特性，因而菌體不易有自溶現象產生；平板對峙培養結果得知，枯草桿菌 BS-99-H 對 12 屬 26 種的供試植物病原真菌皆具抑制其菌絲生長的作用，其中對蓮霧果腐病菌(Pestalotiopsis eugeniae)抑制效果為最佳。萃取枯草桿菌BS-99-H之DNA，利用ituD 及 lpa-14兩個專一性引子對進行PCR反應結果得知，枯草桿菌BS-99-H菌株分別可增幅出1203 bp及675 bp片段，顯示BS-99-H含有產生iturin之基因。自枯草桿菌BS-99-H發酵液萃取可能的抗菌物質，經高效能液相層析儀(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 濃度達 ≥ 0.84 mg/mL 則可完全抑制 P. eugeniae 孢子發芽與菌絲生長。由本研究結果顯示出 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。高產能SYM-d液態發酵基質配方C/N比率高，每公升以3 %接菌量、180 rpm、通氣量0.82 L/min培養枯草桿菌BS-99-H，於28℃經培養2天，其細菌濃度可達1010 cfu/mL以上；枯草桿菌BS-99-H粉劑成品，細菌濃度為5 × 109 cfu/g，稀釋500倍可有效地防治蓮霧病害及芒果炭疽病等。綜合以上試驗結果得知，本枯草桿菌BS-99-H製劑生產成本低，極具未來開發為植物病害生物性殺菌劑之市場競爭力。

關鍵字

枯草桿菌BS-99-H ； 生物防治 ； 抗生物質 ； 發酵技術

並列摘要

Due to adverse effect of chemical pesticides on the environment and human health, development of the environmentally friendly and safe microbial pesticides has become an important issue. The purpose of this study was to develop the microbial pesticide Bacillus subtilis, into a commercial product for effective control of plant diseases. The antagonistic bacterium used in this study was isolated from the local soil in Taiwan. The identification of bacterial isolate was performed both by microbiological methods using Gram stain and Biolog system, and by molecular methods. According to these data, the isolate was identified as Bacillus subtilis. The effective BS-99-H strain was obtained by screening from the original culture using temperature treatment. B. subtilis BS-99-H produced a large amount of endospores compared with stock isolate. The dual culture tests showed that B. subtilis BS-99-H was strongly inhibiting to the mycelium growth of 12 genera and 26 species of fungal plant pathogens, especially the wax apple fruit rot pathogen of Pestalotiopsis eugeniae. The DNA of BS-99-H was extracted for identification of antibiotic compound producing genes. In the PCR reaction test, the amplification products with expected length of 1203 bp for ituD and 675 bp for lpa were detected, In suggesting that the iturin coding genes are present in the region specific for the primers used. When the extract of the BS-99-H fermentation broth medium was analyzed with HPLC and tandem mass spectrometry (MS/MS), iturin A was detected.
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 conidial germination inhibition of P. eugeniae was 0.51 mg/mL. The conidial germination and mycelia growth were both completely inhibited by iturin A at ≥ 0.84 mg/mL. The results indicated that antibiotic iturin A produced by BS-99-H played an important role in the antagonism against P. eugeniae. A new liquid fermentation medium SYM-d was formulated to meet the purpose of high yield mass production. The substrate C/N ratio is high in SYM-d liquid medium. Under the shaking condition of 180 rpm, aeration rate of 0.82 liter/min and 3% bacterial inoculum, culture concentration of Bacillus subtilis BS-99-H reached 1010 cfu/mL or more at 28℃ in 2 days. The powder-formulated product of Bacillus subtilis BS-99-H had the bacterial concentration of 5 × 109 cfu/g. The product at 500X dilution effectively controlled wax apple and mango anthracnose and other diseases in the field tests. The method developed in this study greatly decreased the cost for mass production of BS-99-H and increased the performance of the product which is important in the commercialization of this product in the future.

並列關鍵字

Bacillus subtilis BS-99-H ； biocontrol ； antibiotics ； fermentation technique

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