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標題: The improvement on liquid culture of the entomopathogenic nematodes, Steinernema abbasi
本土產蟲生線蟲 (Steinernema abbasi) 液體培養之改進
作者: 賴億彰
Yi-Chang Lai
關鍵字: Steinernema abbasi;液體培養;斜紋夜蛾;Steinernema abbasi;liquid culture;Spodoptera litura;in vivo;in vitro;IJs
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為探討本土產蟲生線蟲Steinernema abbasi之液體培養方式及培養基成分之改進,將S. abbasi以搖瓶之方式於14天的培養過程中產生兩個成蟲高峰期,於兩高峰期將培養轉速調整為70、90 rpm並培養14天,侵染期幼蟲(infective juveniles, IJs)回收之總產量皆低於調整前之產量,但不具顯著性差異。添加市售OAK特級全脂、低脂、脫脂奶粉於培養基中,所得產量依序為8×104、2.6×104、4.5×103 IJs/ml,以添加全脂奶粉所得IJs產量最高;將全脂奶粉濃度由1.65%(w/w)調高至6.6%(w/w),IJs產量由1.7×105下降至125 IJs/ml;1.65%全脂奶粉培養17天,於第16天可達最高IJs產量;添加1.65%、3.3%(w/w)全脂奶粉後之IJs產量皆高於未添加前之產量且具顯著性差異。於培養基中加入1.5、3、4.5 ml共生菌液並培養48小時後接種線蟲,所得IJs產量依序為6.7×104、2.2×104、1.8×104 IJs/ml,隨著共生菌液添加量增加產量逐漸下降但不具顯著性差異;於培養基中加入1.5、3、4.5 ml共生菌液並同時接種線蟲,所得IJs產量皆低於接種量。經一對一生物檢定測試,體內培養與體外液體培養之線蟲對斜紋夜蛾(Spodoptera litura)第五齡幼蟲之致死率經72小時皆達23.3%,且於各時間點間不具顯著性差異。以10、20、30隻IJs對斜紋夜蛾五齡幼蟲之致病力測試,隨著濃度增加LT50逐漸減短,不論體內培養或體外液體培養,接種10 IJs之LT50皆與接種20、30 IJs之LT50具顯著性差異;接種20 IJs與30IJs之LT50則不具顯著性差異;體內培養與體外液體培養之線蟲於各濃度間之LT50皆不具顯著性差異,經72小時皆可達到近100%之死亡率。本試驗於培養基中添加全脂奶粉可有效提升S. abbasi之產量,且具有與體內培養線蟲相同之致病效果。

This study was conducted to culture the entomopathogenic nematode, Steinernema abbasi, on liquid media containing different components. There were two peaks of nematode adult production in the shaker flasks at 14 days after culturing. When changed the rotation speed at 70 and 90 rpm in these cultures, the yields of infective juveniles (IJs) were lower than those of the unchanged one. However, there was no significant difference between the changed and unchanged groups. When the OAK whole milk powder, low-fat milk powder, and non-fat milk powder were added into the liquid media, the IJ yields were 8×104, 2.6×104, and 4.5×103 IJs/ml, respectively. Among them, the IJ yield of the whole milk powder based medium was the highest one; however, as increased its concentration from 1.65% to 6.6%, the nematode yields decreased from 1.7×105 to 125 IJs/ml. When inoculated with 500 IJs/ml and cultured for 17 days in the 1.65% whole milk powder medium, the maximum yields reached at 16th day after culturing. The IJ yields of 1.65% and 3.3%whole milk powder media were higher than those without adding milk powder. There were statistically significant differences among these media. When incubated 1.5, 3.0, or 4.5 ml of symbiotic bacteria in the culture medium for 48 h before inoculating nematodes, the IJ yields were 6.7×104, 2.2×104, and 1.8×104 IJs/ml, respectively. The yields between these three treatments were not significantly different. However, the yields by adding 1.5, 3.0, and 4.5 ml of symbiotic bacteria together with nematodes were less than those of the amount of their initial IJ inocula. Using one-on-one bioassay, the mortalities of 5th instar larvae of Spodoptera litura treated with in vivo- and in vitro-cultured S. abbasi were 23.3% at 72 h after inoculation. There were no significant differences among the assayed time course. When applied 10, 20, and 30 IJs to each 5th instar larva of S. litura, the LT50 values of 10 IJs was significantly different with those of 20 and 30 IJs. However, the LT50 value of 20 IJs was not significantly different with that of 30 IJs. The LT50 values at the same inoculated dosage in both in vivo and in vitro cultures were not significantly different. The mortalities of 5th instar larvae of S. litura in all treatments were near 100% after incubating for 72 h. In conclusion, our results showed that addition of whole milk powder to the liquid media could raise the yield of IJs and that the nematodes cultured in the liquid medium exhibited similar pathogenicity to S. litura as those cultured in vivo.
其他識別: U0005-0610201414101400
Rights: 同意授權瀏覽/列印電子全文服務,2017-12-30起公開。
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