Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89320
標題: 以 Streptomyces sp. C6 菌株防治番茄青枯病之研究
Studies on Streptomyces sp. C6 to control tomato bacterial wilt caused by Ralstonia solanacearum
作者: Hoi-Weng Choi
蔡凱榮
關鍵字: 生物防治
鏈黴菌
二次代謝物
抗生物質
青枯病菌
青枯病
醱酵培養基
溫室試驗
碳素源
氮素源
Biocontrol
Streptomyces spp.
secondary metabolites
antibiotic
Ralstonia solanacearum
bacterial wilt
fermentation medium
carbon source
nitrogen source
greenhouse trials
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摘要: 番茄青枯病 (bacterial wilt of tomato) 為臺灣番茄作物上重要的細菌性病害之一, 其病原菌為 Ralstonia solanacearum。此病原菌主要棲息於土壤,田間,經傷口入侵,並以風雨等方式傳播﹔可以在田間殘株上長期存活。本研究從三個堆肥材料及一個根圈土壤總共分離出 56 株放射線菌,經測試後,共有 7 株對青枯病菌有拮抗能力,其中菌株 C4、C6 對峙青枯病菌可產生最大的抑制圈,再測試其產生五種分解酵素的能力,發現 C6 產生幾丁質,蛋白質,纖維素,澱粉及 β-1,3-葡聚糖酶 (β-1,3-glucanase) 這五種分解酵素的能力之總和最佳,故之後乃以 C6 菌株進行 10 種天然素材培養液及最佳培養碳、氮素源的篩選。發現 C6 於馬鈴薯葡萄糖培養液 (PDB) 中培養所得的醱酵濾液,具有最佳的拮抗能力,且此能力表現的時間可達第 7 天,C6 在馬鈴薯葡萄糖培養液培養第三天醱酵液拮抗青枯病菌的能力逹到高峰 (抑制圈直徑為 33.33 mm)。經碳、氮源之篩選,顯示澱粉與硝酸鈣可促進 C6 菌株生長,碳源為木糖時則可促進其產生抗生物質。溫室防治試驗係將 C6 及 C4 分別培養於在馬鈴薯葡萄糖培養液中,在 150 rpm,30℃條件下培養三天後,澆灌至 28 天大的番茄幼苗,7 天後再接種青枯病菌。第三次溫室試驗結果顯示,對照組之病害嚴重度 (Disease severity) 在接種後第 7 天為 40%,而 C6 及 C4 處理組未發病;第 18 天時,對照組病害嚴重程度達 80%,C4 處理組為 69%,而 C6 處理組為 20%;至 43 天時,C4 與對照組之病害嚴重度皆為 80%,C6 為 40%的病害嚴重度。將培養 C6 菌株之馬鈴薯葡萄糖培養液的濾液經兩種理化測試,C6 菌株產生的抗生物質為非蛋白質類,不耐高溫 (以 100℃處理 45 分鐘即失去活性) 將 C6 與 6 種病原細菌在營養洋菜培養基 (Nutrient Agar) 對峙培養,C6 菌株尚可抑制 6 種病原細菌。
Bacterial wilt of tomato caused by Ralstonia solanacearum is an important bacterial disease of tomato crops in Taiwan. This pathogen inhabited in soil, dispersed by wind or rain, infected host plants through wounds, survived on plant debris for a long time. In this study, 56 actinomycetes isolates were obtained from the rhizosphere soil form R. solanacearum infected tomato and the composts. Seven isolates showed antagonism against R. solanacearum. Among them, the C6 and C4 isolates produced the biggest inhibition zone when dual-cultured with R. solanacearum. The C6 isolate also had the better ability of producing chitinase, protease, cellulase, amylase and β-1, 3-glucanase than other isolates, and got 15 points on the total value. The C6 isolate was cultured in 10 different organic media and tested for its antagonistic ability to R. solanacearum. Eight sources of carbon or nitrogen substituted on Czapek's media were also tested. The results showed that C6 isolate had the best antagonism against R. solanacearum when cultured in PDB medium, the inhibition ability reached the peak on the third day, and could last for seven days. C6 isolate had the maximum growth rate when incubated in starch-Czapek's medium or nitrate-calcium-Czapek's medium, and the xylose-Czapek's medium could promote the production of antibiotics of C6 isolate. In the greenhouse experiment, C6 or C4 was grown in PDB at 30℃, 150 rpm on shaker for three days, then drenched onto the 28-days-old tomato seedlings. After seven days, the seedlings were inoculated with R. solanacearum. The results of the 3rd greenhouse experiment showed that the C6 treatment were able to inhibit disease development until the 7th day. On the 18th day, C6 and C4 had 20% and 69% disease severity, respectively wile the blank control already had 80%. To study the physical and chemical properties of the antibiotic produced by the C6 isolate, the cultural broth were treated with Proteinase K or heat and proceeded to test the antagonistic ability to R. solanacearum. The results showed that the antibiotic was not proteins and intolerant of high temperature. Further research needs to be done to clarify the properties of this substance. In order to evaluate the C6 strain for field application, six plant pathogenic bacteria were dual-cultured. The results showed that C6 could inhibit the growth of all 6 pathogens indicating that the C6 isolate could be applied for other plant bacterial disease management.
URI: http://hdl.handle.net/11455/89320
文章公開時間: 2018-07-15
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