Application of microbial agent Bacillus subtilis TKS1-1 on the control of rice bacterial blight and its putative control mechanisms

水稻白葉枯病為水稻上重要的細菌性病害，白葉枯病在田間通常於分蘗盛期發生，病徵開始在葉緣離葉尖五到六公分處出現暗綠色、水浸狀小斑點，然後逐漸延長而變成黃或枯黃色。目前常見推薦應用於水稻白葉枯病的防治措施主要有適當的田間操作模式與清園、肥培管理、施用化學藥劑防治、栽種抗病品種或是施用生物製劑等。桿菌屬 (Bacillus spp.) 已知被用於防治多種植物病害，考量到其對環境之友善性及可能具備多種機制以抑制病害發生，施用桿菌屬作為生物防治是一個深具潛力的防治方法。本研究旨在探討本土拮抗性枯草桿菌菌株TKS1-1於水稻白葉枯病之防治應用及其防治機制。在拮抗試驗中證實了枯草桿菌TKS1-1對於水稻白葉枯病菌WF6具有優異的抑制效果。在溫室試驗中，在接種白葉枯病菌菌株WF6前7天、2天或同時以剪葉浸漬法接種枯草桿菌TKS1-1，結果顯示不論哪一種處理都可以有效的抑制水稻白葉枯病的病徵發展，另外於水稻台農67號移植後每週澆灌一次枯草桿菌TKS1-1 100倍稀釋菌液，共澆灌三週後接種水稻白葉枯病，其病斑長度也較水處理組少了一公分。除此之外，澆灌枯草桿菌TKS1-1稀釋100倍菌液16小時後，結果顯示與處理水之對照組相比，處理枯草桿菌TKS1-1處理組之水稻台農67號之PR1a、PAL、NPR1及WRKY45基因也明顯被誘導。此外，在預先澆灌枯草桿菌TKS1-1再接種白葉枯病菌WF6之處理組中可以觀察到phenylalanine ammonia lyase、peroxidase以及polyphenol oxidase之活性皆較僅接種白葉枯病菌WF6、僅澆灌枯草桿菌TKS1-1稀釋100倍菌液及未處理組要高。為瞭解白葉枯病菌WF6在水稻葉片中之菌量是否受到影響，先以剪葉法接種經稀釋100倍之枯草桿菌TKS1-1培養液後再接種白葉枯病菌WF6，接種後0、3、6及9天採傷口下一公分葉片磨碎後以序列稀釋方式計算菌量，結果顯示在接種後第3天時TKS1-1之處理組之白葉枯病菌菌量明顯較水處理組低，此差距到接種後第9天依然存在，推測應為枯草桿菌TKS1-1產生拮抗水稻白葉枯病原菌WF6之物質而抑制其生長所致。此外，於水稻植株台農67號移植後開始處理第一次，之後每週一次共三次，分別以稀釋100倍、500倍及1000倍之枯草桿菌TKS1-1菌液澆灌處理水稻台農67號，結果顯示與水對照組相比三種不同稀釋濃度皆有明顯生長促進的效果。為鑑定出對水稻白葉枯病菌WF6具有抑制活性的物質，本研究中利用了Sep-pakⓇ C18管柱與Hi-trap Q陰離子交換樹脂進一步純化，收集對水稻白葉枯病菌WF6具有抑制活性之流出液後以等體積丁醇進行液液萃取，再利用液相層析串聯式質譜儀進行正、負離子全掃描分析，獲得之圖譜可以得知活性區段(滯留時間9.971分鐘與10.492分鐘) 與iturin A之滯留時間類似，與surfactin則不相似；但由質譜圖的數據中可以觀察到質荷比訊號大致落在1029.9至1187.7，此結果顯示由枯草桿菌TKS1-1產生對水稻白葉枯病菌WF6具有抑制活性之抗生物質與iturin A為不同之化合物。除此之外，0.0001 M之iturin A對水稻白葉枯病菌WF6僅有些微之抑制活性，而surfactin則完全沒有抑制活性。綜合上述特性顯示預先施用枯草桿菌TKS1-1可有效抑制水稻白葉枯病並促進水稻植株的生長，且其良好的防治效果可歸功於產生對水稻白葉枯病菌WF6具有抑制活性之抗生物質與誘導水稻植株抗病相關基因、蛋白質之表現。Xanthomonas oryzae pv. oryzae causes leaf blight of rice, which is one of the devastating diseases of rice plant. The use of chemical pesticides and resistant rice cultivars is the most common cultural practice for the control of rice bacterial blight. However, over-doses of chemical pesticides are detrimental to the ecological environment and breeding cultivars for resistant to pests may take long time. Additionally, with the emergence of drug-resistant strains, the efficacy of these control measures is hindered. Application of beneficial microorganisms may serve as an alternative strategy to solve this problem. Bacillus species are well known for their potential in disease suppression and plant promotion. Thus, they are considered as one of predominant genera of plant growth promotion rhizobacteria (PGPR). PGPR could be use as biological control agents by exhibiting several mechanisms including antagonism, competition for nutrient and induction of plant defense system. The main objectives of the study are to investigate the potential of Bacillus subtilis TKS1-1 for the control of rice bacterial blight and the possible mechanisms involved in disease control. By dual culture assay, B. subtilis TKS1-1 showed superior antagonistic activity against X. oryzae WF6. Application of B. subtilis strain TKS1-1 at the time of or prior to inoculation with X. oryzae WF6 was found to suppress rice bacterial blight. In addition, the lesion length of leaf blight was 1 cm less by the drenching application with 100-fold diluted endospores formulation of B. subtilis TKS1-1 compared to the water control, suggesting a contribution to induce systemic resistance. Spray-application of B. subtilis TKS1-1 endospores formulation (5 x 107 CFU/ ml) was shown to increase the expression of several plant defense-related genes inclulding PR1a, PAL, NPR1, and WRKY45 at 16 hours post application in comparsion with the water control. Furthermore, drenching application of strain TKS1-1 7 days prior to inoculation of the pathogen was found to increase the activity of phenylalanine ammonia lyase, peroxidase and polyphenol oxidase at 48 hrs post-inoculation in comparision with other treatments. Reduction in the cell population of X. oryzae WF6 in rice leaves was observed when B. subtilis TKS1-1 was introduced to the leaves, suggesting putative antibiotics may be produced by B. subtilis TKS1-1. Application of B. subtilis TKS1-1 formulation diluted 100-fold, 500-fold and 1000-fold on rice cultivar TNG67 all showed significant promotion of plant growth. To determine the putative compounds that inhibit growth of the leaf blight pathogen, cultural broth of B. subtilis TKS1-1 in SYM broth was subjected to solid phase extraction with Sep-pak® C18 cartridge, purification by ion-exchange chromatography using a Hi-trap Q column and then LC/MS/MS analysis. The chromatograms corresponding to LC/MS/MS analysis indicated that the antibiotics from B. subtilis TKS1-1 retained at 9.971 and 10.492 mins were similar to iturin A but distinct from surfactins. Results from mass spectra showed that the mass of charge ratio of antibiotics produced by B. subtilis TKS1-1 are around 1.029.9 to 1187.7 which are different from iturin A. Additionally, iturin A and surfactins at the concentration of 0.0001 M showed trivial or did not show antagonistic activity to X. oryzae WF6, respectively. The findings suggested that B. subtilis TKS1-1 exhibited superior efficacy in suppression of rice bacterial blight and in growth promotion. The biocontrol potential is partly attributed to the antibiotics produced and to the induction of plant defense response by B. subtilis TKS1-1.目錄 中文摘要 I 英文摘要 (Abstract) III 目錄 VI 圖目次 IX 壹、前言 10 一、水稻栽培現況 10 二、水稻白葉枯病的危害與重要性 11 三、施用於植物細菌性病害之生物防治方法 13 四、桿菌屬 (Bacillus spp.) 之特性與應用概況 15 貳、材料與方法 23 一、供試藥品試劑來源與培養基種類 23 二、供試水稻之種植 23 三、供試水稻白葉枯病菌菌株來源與保存 24 四、供試拮抗性枯草桿菌菌株TKS1-1之來源與保存 24 五、枯草桿菌TKS1-1菌液對於水稻白葉枯病防治應用性之探討 25 六、枯草桿菌TKS1-1對水稻白葉枯病菌WF6在水稻葉片上族群之影響 26 七、枯草桿菌TKS1-1抗生物質之萃取及分析 26 八、枯草桿菌TKS1-1對水稻防禦相關基因表現量之影響 29 九、枯草桿菌TKS1-1於水稻白葉枯病菌WF6接種下對水稻氧化酵素活性之影響 30 十、枯草桿菌TKS1-1對水稻植株生長影響評估 31 參、結果 32 一、水稻白葉枯病原之分離與鑑定 32 二、枯草桿菌TKS1-1於水稻白葉枯病之防治應用 32 三、剪切傷口浸漬處理枯草桿菌TKS1-1可顯著降低水稻白葉枯病菌WF6於水稻葉片上族群量 33 四、對白葉枯病菌WF6具拮抗活性之枯草桿菌TKS1-1抗生物質為iturin family之類似物 34 肆、討論 39 伍、參考文獻 45 陸、圖表說明 55 柒、附錄 7