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標題: 影響Fusarium proliferatum產生fumonisin B1之因子探討
Factors Affecting Fumonisin B1 Production by Fusarium proliferatum
作者: 林秀橤
Lin, Shiou-Ruei
關鍵字: fumonisin B1
Fusarium proliferatum
Fusarium proliferatum
出版社: 植物病理學系所
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摘要: 鐮孢菌屬Liseola群的真菌會產生伏馬鐮孢毒素 (Fumonisin B1, FB1)。這種真菌毒素可引起馬的腦白質部軟化症、豬的肺水腫、大鼠肝病或腎病,干擾禽類的免疫系統及可能引發人類的食道癌等疾病。利用Nash-PCNB選擇性培養基分離水稻與玉米等穀粒上的真菌,共計獲得17株Liseola群的鐮孢菌。隨後以Fumonisin kit及HPLC檢測各菌株是否可產生FB1,結果發現ST-P01 (來自玉米飼料)、PR-14、PR-33、PR-44及PR-143 (來自稻穀)等五菌株均具有產生FB1的能力;其中以ST-P01菌株產生FB1的量最高,達581.52 ppm。利用孢子形態、大小、產孢方式及ITS序列比對等方法,將ST-P01、PR-14、PR-33、PR-44及PR-143等五菌株的學名鑑定為Fusarium proliferatum (Matsushima) Nirenberg。進一步,利用玉米液態培養基培養ST-P01菌株,評估溫度、培養日數、培養基質種類、培養基基質型式、培養方式、培養基酸鹼度、濕度及病原菌接種濃度等因子對其產生FB1的影響,結果發現靜置培養ST-P01五日後,才可測到FB1的產生,且在32℃的產毒量最高。以小麥、燕麥、裸麥、蕎麥、糙米、紫糯米、白米、小米及玉米等九種液態培養基質分別培養ST-P01菌株,結果發現其在紫糯米粉液態培養基產生FB1的量最高,達431 ppm,其次在玉米粉液態培養基上的產量則為351 ppm;至於以燕麥粉液態培養的產毒量最少,僅有75 ppm;此外,在靜置方式培養ST-P01五天後,固態培養基中FB1產量遠高於液態培養,達5000倍;但在液態培養中,震盪培養ST-P01所產生FB1之量顯著高於靜置培養者。以不同酸鹼值之玉米粉液態培養基培養ST-P01,發現在培養基pH值2時,其產毒量最多,達453 ppm;而在pH值6時產生FB1最少,僅有19 ppm。在不同相對濕度條件下,將ST-P01培養15天後,除在相對濕度(R.H.)97.5%時可產生FB1外,其餘在RH 7、20、34.5及75.5%時,均無法產生。以不同ST-P01孢子濃度(1*105、106及107 conidia/ml)分別接種於液態玉米培養基中,發現三者的最初接種濃度不會影響FB1的產量。比較ST-P01、PR-14、PR-44及PR-143培養條件,發現ST-P01適合於32℃在pH值2之培養基培養,而PR-44則適合於28℃在pH值6之培養基培養,兩者才可產生高量的FB1。至於其餘兩菌株產生FB1量均低於偵測範圍。調控玉米的儲存條件,發現於4℃下保存受汙染之穀物,經過15天其不會有菌絲長出,亦不會產生FB1。此外,篩選多種添加物可否降低伏馬鐮孢毒素B1的產生,結果發現茶胺酸可降低ST-P01產生FB1毒素達34%。
Fusarium species of the Section Liseola could produce fumonisins. This mycotoxin was associated with equine leukoencephalomacia, procine pulmonary edema nephrotoxicity, liver or kidney cancer in rats, immunosuppression in poultry, and high rate of human esophageal cancer. Seventeen Fusarium isolates in the Section Liseola were isolated from rice and corn by Nash-PCNB selective medium, and evaluated their ability of producing FB1 by Fumonisin kit and HPLC. Five isolates, ST-P01(from corn feed), PR-14, PR-33, PR-44, and PR-143(from rice), were able to produce FB1, especially isolate ST-P01 was able to produce the highest amount of FB1, up to 581.52 ppm. Isolates ST-P01, PR-14, PR-33, PR-44, and PR-143 were identified as Fusarium proliferatum (Matsushima) Nirenberg by conidia morphology, conidigenous cells, and ITS sequencing. Furthermore, the factors affecting FB1 production were evaluated including temperature, culture time, culture substrates, culture methods, pH value of culture medium, relative humidity, and inoculum density. The FB1 production by isolate ST-P01 could be detected five days after its static culturing, and FB1 yield was the highest at 32℃ compared to the other temperature treatment. Isolate ST-P01 produced the highest amount of FB1 up to 431 ppm in purple glutinous rice meal liquid media among nine substrates, including purple glutinous rice, unpolished rice, rice, millet, wheat, corn, oat, naked barley, and buckwheat. Isolate ST-P01 cultured in corn meal liquid media produced 351 ppm. The less one was 75 ppm when isolate ST-P01 cultured in oat meal liquid media. In addition, isolate ST-P01 cultured staticly in corn solid media produced 5000 times more FB1 than in liquid media. Isolate ST-P01 produced more amount of FB1 cultured in corn meal liquid with shaking compared to with static. Isolate ST-P01 cultured in corn meal liquit media with pH 2 produced the highest amount of FB1 at 453 ppm. On the contrary, it cultured in corn meal liquid media with pH 6 produced the lowest amount of FB1 at 19 ppm. Among five relative humidities including 7, 20, 34.5, 75.5 and 97.5%, isolate ST-P01 could produce FB1 only at RH 97.5%. Three initial inoculum density of isolate ST-P01, 105, 106, and 107 conidia/ml, inoculated into liquid corn meal media did not make difference on producing FB1. In our studies, among four isolates tested, ST-P01 was able to produce largest amount of FB1 when it was cultured at 32℃ in medium with pH 2. However, isolate PR-44 was suitable for FB1 production at 28℃ in medium with pH 6. By manipulalting the conditions for corn storage, it was found that the inoculated corn kernels stored at 4 ℃ for 15 days did not grow out the fungal mycelia and produce FB1. In addition, amendment of FB1-solution with teamine could reduce 34% of FB1.
其他識別: U0005-2008200921255900
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