Abstract In vitro culture of the symbiotic bacterium, Xenorhabdus indica, isolated from the entomopathogenic nematodes, Steinernema abbasi Taiwan isolate, caused ca. 95% mortality of Galleria mellonella mature larvae at 72 h after culturing, and remained ca. 93% at 96 h, indicating that this bacterium secreted insecticidal substances in its culture medium. After injection of mature larvae with different condensations of the bacterial cultured filtrates, the larval mortality of G. mellonella was ca. 85% with the 25-fold condensed filtrates and reached 100% with the 50-fold ones, whereas that of Spodoptera litura was ca. 75% with the 300-fold filtrates. The cultured filtrates could also inhibit some human pathogens tested, i.e., Staphylococcus aureus CCRC12652, Escherichia coli JM109, Klebsiella pneumonia CCRC10694, and Vibrio parahaemolyticus CCRC10806; a grass bacillus, Bacillus subtilis; and plant pathogenic fungi, i.e., Peronophythora litchi, Botrytis cinerea, Rhizoctonia solani (from cabbage), and Collectotrichum sp. (from strawberry). The cultured filtrates screened through 10 or 100 kDa molecular sieves could inhibit the growth of B. subtilis and B. cinerea while those through 3-kDa sieve could inhibit B. subtilis only. The filtrates sieved through a 10-kDa sieve caused 85.63 and 98.93% of necrotic rates, respectively, in an insect cell line, SF21, at 12 and 24 h post-treatment, whereas those through a 3 kDa sieve similarly caused 80.20 and 92.77% necrotic rates, respectively. However, only the filtrates through 10-kDa sieve resulted in 23.33, 75.00, and 96.67% mortality of G. mellonella larvae, respectively, at 6, 12, and 24 h after injection of G. mellonella larvae. It is thus indicated that both insecticidal and antimicrobial substances are present in the 10-kDa sieved filtrates. Proteins in the cultured filtrates were analyzed using SDS-PAGE electrophoresis. A protein band with 85 kDa of molecular weight was detected in the 100-kDa sieved filtrates while two bands with 22 and 25 kDa were found in the 10-kDa sieved filtrates. However, none were detected in the 3-kDa sieved one. On the basis of coloration tests, most of the separated molecules showed an amino acid structure. Furthermore, both exo- and endo-chitinases in the filtrates through 10-50 kDa sieves could be detected after reacting with different substrates, emitting fluorescence under the UV microscope. The concentration of lipopolysaccharide (LPS) isolated from X. indica was ca. 3 × 105 EU/mL, causing ca. 23, 43, and 93% mortality of G. mellonella larvae at 12, 24, and 36 h after injection, respectively. The LPS from X. indica resulted in ca. 7.67 mm of inhibition zone against a bacterium, B. subtilis and a fungus, B. cinerea, whereas that from Xenorhabdus nematophila caused ca. 8.00 and 5.33 mm of inhibition zone, respectively. In contrast, LPS from E. coli which is also an intestinal bacterium produced only ca. 1.33 mm of inhibition zone against B. subtilis. Therefore, the LPS from X. indica could inhibit both bacterial and fungal growth.
摘要 台灣產蟲生線蟲 (Steinernema abbasi) 之共生菌 (Xenorhabdus indica) 在體外培養72 h後，可造成大蠟蛾 (Galleria mellonella) 幼蟲95% 的死亡率，至96 h仍可維持93.33% 死亡率，顯示共生菌在此生長期可產生殺蟲物質。將培養濾液濃縮至25倍時能造成大蠟蛾幼蟲85% 死亡率，而50倍能造成100% 死亡率，但對斜紋夜蛾幼蟲濃縮300倍時，其死亡率僅為75%。濾液中所含之抗菌物質，對四種人體病原：金黃葡萄球菌 (Staphylococcus aureus) CCRC12652品系、大腸桿菌 (Escherichia coli) JM109品系、克雷伯氏菌 (Klebsiella pneumonia) CCRC10694、腸炎弧菌 (Vibrio parahaemolyticus) CCRC10806及枯草桿菌 (Bacillus subtilis) 等細菌及植物病原真菌，即Peronophythora litchi、Botrytis cinerea、Rhizoctonia solani (Cabbage) 和Collectotrichum sp. (Strawberry) 等菌株具抑制性。濾液經不同孔徑分子篩萃取後，對B. subtilis及B. cinerea具有抗生活性之物質，主要分佈在100及10 kDa分子篩中，但3 kDa分子篩萃取所得之物質只對B. subtilis有抑制效果；而對昆蟲SF21細胞株，主要在10及3 kDa。以孔徑10 kDa者所處理之細胞，經12及24 h分別引起85.63及98.93% 之細胞壞死率；3 kDa者亦可造成80.20及92.77% 細胞壞死率，但統計分析兩部份之壞死率則無顯著性。殺蟲物質之生物檢定結果，僅10 kDa者具殺蟲性，於注入血腔後6、12及24 h對大蠟蛾之幼蟲死亡率分別為23.33、75及96.67%，顯示10 kDa所篩之物質兼具抑菌及殺蟲性。濾液萃取物之蛋白質電泳分析，發現100 kDa分子篩物質在native gel上有一大分子，經SDS-PAGE分析結果，可見一濃縮物之條帶，分子量約為85 kDa；而10 kDa分子篩之萃取物，在電泳膠片上則有兩個條帶，其分子量約為22及25 kDa；但3 kDa萃取物在膠體則無條帶。經蛋白質之呈色反應結果，不同分子篩之篩出物均屬於蛋白質或胜肽之結構。在不同孔徑分子篩中萃取之濾液，50～10 kDa範圍中可測得exo及endo兩型幾丁質分解酵素。由共生菌所純化之脂多醣體 (LPS) 濃度約3 × 105 EU/mL，於12、24及36 h後造成大蠟蛾幼蟲死亡率分別約為23、43及93%。經測試結果發現X. indica之LPS對B. subtilis及B. cinerea均可引起約7.67 mm之抑制圈；而X. nematophila約為8.00與5.33 mm，但同為腸內菌科之E. coli，其LPS對B. subtilis之抑制圈僅約1.33 mm，顯示X. indica之LPS對細菌生長及真菌孢子發芽具有抑制作用。
| Key words: Steinernema abbasi, Xenorhabdus indica, insecticidal and antimicrobial substance, protein, lipopolysaccharide | | 關鍵詞: 蟲生線蟲、共生菌、殺蟲與抑菌物質、蛋白質、脂多醣體。 |
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