Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31686
標題: 產氰綠膿桿菌對蔬菜作物生長及番茄青枯病發生之影響
Effects of cyanogenic Pseudomonas aeruginosa on the growth of vegetable crops and tomato bacterial wilt caused by Ralstonia solanacearum
作者: 林宜賢
Lin, Yi-Hsien
關鍵字: Pseudomonas aeruginosa
綠膿桿菌
hydrogen cyanide
survival
vegetable crops
inhibitory effect
tomato bacterial wilt
phospholipase C
pathogenecity
氰酸
存活
蔬菜作物
抑制作用
番茄青枯病
磷脂分解酵素C
致病性
出版社: 植物病理學系
摘要: 綠膿桿菌(Pseudomonas aeruginosa)為自然環境常見的細菌。由植物葉表分離之綠膿桿菌大部分菌株皆具有產生氰酸之能力。本研究中發現葉表分離之綠膿桿菌在泥炭土中,以較低溫度下(24℃)之存活能力較佳,然綠膿桿菌氰酸產生能力之強弱與其在泥炭土中的存活能力無關。而綠膿桿菌氰酸的產生在不同溫度下,亦不影響其他螢光假單胞菌菌株於土壤中之存活。本研究利用密閉培養皿及密閉圓柱盒分別測試綠膿桿菌產氰菌株對萵苣發芽與生長的影響,結果顯示對萵苣種子的發芽的抑制作用與綠膿桿菌菌株之產氰能力有關。將綠膿桿菌WFP11r菌株中之hcnABC基因,應用聚合酵素連鎖反應(polymerase chain reaction, PCR)的方式增幅並予以選殖後,轉形至P. putida YLFP44菌株中表現,得到可產生大量氰酸之衍生菌株Y44H1。進一步以產氰衍生菌株Y44H1於密閉培養皿及密閉圓柱盒中分別測試其對萵苣、甘藍、白菜及番茄等蔬菜作物在種子發芽與發芽後幼苗生長之影響,顯示產氰衍生菌株Y44H1對所有蔬菜作物種子之發芽及幼苗之生長有明顯之抑制作用,且對番茄之抑制作用最強。利用Y44H1於密閉圓柱盒中測試其對不同蔬菜作物種子發芽率之影響,顯示茄科作物對氰酸的感受性高,並可造成種子之死亡。為了進一步了解植物對氰酸耐性的生化特性,分別測試萵苣、甘藍、白菜與番茄之種子及幼苗中代謝氰酸關鍵酵素b-cyanoalanine synthase (b-CAS)之活性,顯示在種子或幼苗上b-CAS之活性又以番茄為最低。由此證明綠膿桿菌及螢光假單胞菌氰酸的產生可抑制蔬菜種子發芽與幼苗生長,且不同蔬菜作物對氰酸之感受能力可能與植物中b-CAS之活性有關。此外,葉表分離之產氰綠膿桿菌菌株於密閉培養皿系統中可抑制許多不同植物病原細菌在培養基上之生長,且綠膿桿菌之產氰能力與抑菌程度具正相關。Y44H1衍生菌株對青枯病菌不僅抑制其生長,處理五天後也有致死作用,並可造成菌落及細胞形態之改變。然在溫室試驗中將此些產氰之綠膿桿菌與青枯病菌混合後共同接種於番茄根圈土壤中卻可促進番茄青枯病的發生。以Y44H1與青枯病菌混合後共同接種於番茄根圈土壤亦可促進番茄青枯病的發生,證明綠膿桿菌或螢光假單胞菌氰酸的產生可促進番茄青枯病的發生,推測綠膿桿菌菌株或產氰衍生菌株Y44H1氰酸的產生可傷害番茄之根系,進而增加番茄植株對青枯病菌之感病性。綠膿桿菌除可產生氰酸外,另可生成溶血性磷脂質分解酵素C (hemolytic phospholipase C, PLC-H,係由plcS基因轉譯所得)。在本研究中,應用PCR技術偵測台灣作物葉表分離之螢光假單胞菌菌株中plcS基因之存在情形,發現除供試之綠膿桿菌菌株可測得plcS基因外,其中一個P. fluorescens菌株(YLFP8)亦可測得plcS基因之存在,並具有溶血能力,為植物來源P. fluorescens菌株具有plcS基因及溶血能力之首次報導。由於接種綠膿桿菌WFP11r菌株與P. fluorescens菌株YLFP8可在大蒜蒜瓣上造成水浸狀褐化之病徵,為探討綠膿桿菌之PLC-H在植物致病上之角色,應用PCR技術選殖綠膿桿菌WFP11r菌株之plcSR基因(含構造基因與調控基因),並轉形至P. putida YLFP44菌株中,得到具有PLC-H活性之衍生菌株Y44P1,而此衍生菌株亦可在大蒜蒜瓣上引起水浸狀褐化之病徵,證明PLC-H為綠膿桿菌引起大蒜蒜瓣水浸狀褐化病徵之致病因子。
Pseudomonas aeruginosa is a gram-negative fluorescent bacterium that can be isolated from various habitats. Many P. aeruginosa strains isolated from leaf surface were cyanogenic, although cyanide did not appear to play an important role in their survival in our test conditions. Cyanide is known to inhibit aerobic respiration in many organisms. However, the viability of other pseudomonads in soil was not affected by the presence of cyanogenic P. aeruginosa. The cyanide toxicity on seed germination and seedling growth of lettuce was tested in this research, and the results showed the inhibition of seed germination was correlated with the cyanide produced by P. aeruginosa. The hcnABC genes coding for amino acid dehydrogenase and oxidase were cloned from cyanogenic P. aeruginosa strain WFP11r and transformed into a non-cyanogenic P. putida YLFP44 by electroporation. The P. putida derivative Y44H1, produced high quantities of cyanide could strongly inhibit seed germination and seedling growth of lettuce, cabbage, Chinese cabbage, and tomato. Among these, tomato was the most sensitive vegetable to cyanide, which might be due to its low activity of b-cyanoalanine synthase (b-CAS) that is responsible for cyanide detoxification. We also found the growth of many plant pathogenic bacteria was inhibited by cyanogenic P. aeruginosa strains in the paired-plate system. The more cyanide P. aeruginosa produced, or the longer it incubated with other organisms, the fewer viable cells were recovered from the paired-plates. For example, prolonged incubation (5 days) with Y44H1 led to the death of R. solanacearum and altered the colony and cell morphology of the bacterium. However, when tomato plants were co-inoculated with R. solanacearum and cyanogenic P. aeruginosa or Y44H1, the disease severity was greater than that of plants treated with R. solanacearum only. It was likely the effect of cyanide on tomato plants was greater than that on bacterial wilt pathogen R. solanacearum in the experimental conditions; the question as to how cyanide increases bacterial wilt disease remains elusive. In addition to cyanide, hemolytic phospholipase C (PLC-H), which is encoded by plcS gene, also plays an important role in the virulence of P. aeruginosa on animals and plants. We applied PCR technique and found plcS gene in all strains of P. aeruginosa and one strain of P. fluorescens (YLFP8) in Taiwan. Moreover, all the plcS-harboring pseudomonads were hemolytic. Both P. aeruginosa WFP11r and P. fluorescens YLFP8 caused brownish water-soaking symptoms on garlic, but the other non-hemolytic pseudomonads did not. To elucidate the role of plcSR genes in bacterial pathogenicity on plants, plcSR genes from P. aeruginosa WFP11r were cloned and transformed into non-hemolytic P. putida YLFP44. The resulting transformant, Y44P1, showed hemolytic activity and caused brownish water-soaking symptoms on garlic, which was comparable to the parental strain YLFP8. The results presented in this research indicate that cyanide and PLC-H were important virulence factors in P. aeruginosa pathogenecity on plants.
URI: http://hdl.handle.net/11455/31686
Appears in Collections:植物病理學系

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