Objective: To evaluate the storage stability of metabolites from actinomycetes Streptomyces nigrogriseolus XD 2-7 and the mollcuscicidal activity against Oncomelania hupensis in the laboratory, and to preliminarily explore the mechanisms of the molluscicidal activity.
Methods: The fermentation supernatant of S. nigrogriseolus XD 2-7 was prepared and stored at -20, 4 °C and 28 °C without light for 10 d; then, the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The fermentation supernatant was boiled in a 100 °C water bath for 30 min and recovered to room temperature, and then the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The pH values of the fermentation supernatant were adjusted to 4.0, 6.0 and 9.0 with concentrated hydrochloric acid and sodium hydroxide, and the fermentation supernatant was stilled at room temperature for 12 h, with its pH adjusted to 7.0; then, the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The fermentation product of S. nigrogriseolus XD 2-7was isolated and purified four times with macroporous resin, silica gel and octadecylsilane bonded silica gel. The final products were prepared into solutions at concentrations of 10.00, 5.00, 2.50, 1.25 mg/L and 0.63 mg/L, and the molluscicidal effect of the final productswas tested against O. hupensis following immersion for 72 h, while dechlorination water served as blank controls, and 0.10 mg/L niclosamide served as positive control. The adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels were measured in in O. hupensis soft tissues using high performance liquid chromatography (HPLC) following exposure to the final purified fermentation products of S. nigrogriseolus XD 2-7.
Results: After the fermentation supernatant of S. nigrogriseolus XD 2-7 was placed at -20, 4 °C and 28 °C without light for 10 d, immersion in the stock solution and solutions at 10- and 50-fold dilutions for 72 h resulted in a 100% (30/30) O. hupensis mortality. Following boiling at 100 °C for 30 min, immersion in the stock solution and solutions at 10- and 50-fold dilutions for 72 h resulted in a 100.00% (30/30) O. hupensis mortality. Following storage at pH values of 4.0 and 6.0 for 12 h, immersion in the fermentation supernatant of S. nigrogriseolus XD 2-7 for 72 h resulted in a 100.00% (30/30) O. hupensis mortality, and following storage at a pH value of 9.0 for 12 h, immersion in the fermentation supernatant of S. nigrogriseolus XD 2-7 for 72 h resulted in a 33.33% (10/30) O. hupensis mortality (χ2 = 30.000, P < 0.05). The minimum concentration of the final purified fermentation products of S. nigrogriseolus XD 2-7 was 1.25 mg/L for achieving a 100% (30/30) O. hupensis mortality. The ATP level was significantly lower in O. hupensis soft tissues exposed to 0.10 mg/L and 1.00 mg/L of the final purified fermentation products of S. nigrogriseolus XD 2-7 than in controls (F = 7.274, P < 0.05), while no significant difference was detected in the ADP level between the treatment group and controls (F = 2.485, P > 0.05).
Conclusions: The active mollcuscicidal ingredients of the S. nigrogriseolus XD 2-7 metabolites are maintained stably at -20, 4 °C and 28 °C for 10 d, and are heat and acid resistant but not alkali resistant. The metabolites from S. nigrogriseolus XD 2-7 may cause energy metabolism disorders in O. hupensis, leading to O. hupensis death.
［摘要］ 目的 评价放线菌黑色浅灰链霉菌 (Streptomyces nigrogriseolus XD 2-7) 代谢产物储存稳定性及其分离纯化产物 实验室杀螺活性, 并初步探讨其杀螺作用机制。方法 制备黑色浅灰链霉菌发酵上清液, 将其于 −20、4 °C 和 28 °C 无光照 条件下放置 10 d 后, 测定其 72 h 浸杀灭螺效果; 将发酵上清液置于 100 °C 水浴中煮沸 30 min 后恢复至室温, 测定其 72 h 浸杀灭螺效果; 用浓盐酸和氢氧化钠调节发酵上清液 pH 值为 4.0、6.0 和 9.0, 室温下静置 12 h, 再调节发酵上清液 pH 至 7.0, 测定其 72 h 浸杀灭螺效果。用大孔树脂、硅胶和十八烷基硅烷键合硅胶依次对黑色浅灰链霉菌发酵产物进 行 4 次分离纯化, 将最终分离纯化产物配置成 10.00、5.00、2.50、1.25 mg/L 和 0.63 mg/L 浓度药液, 测定其 72 h 浸杀灭螺效 果。各实验设立空白对照组以脱氯水处理, 阳性对照组以 0.10 mg/L 氯硝柳胺处理。采用高效液相色谱法测定经纯化代 谢产物浸泡后钉螺软体组织中三磷酸腺苷 (adenosine triphosphate, ATP) 和二磷酸腺苷 (adenosine diphosphate, ADP) 含 量。结果 黑色浅灰链霉菌发酵上清液在 −20、4 °C 和 28 °C 无光照条件下放置 10 d 后, 原液及稀释 10、50 倍药液浸杀钉 螺 72 h, 钉螺死亡率均为 100.00% (30/30); 经 100 °C 煮沸 30 min 后, 原液及稀释 10、50 倍药液浸杀钉螺 72 h, 钉螺死亡率 均为 100.00% (30/30)。发酵上清液在pH值为 4.0、6.0 条件下存储 12 h 后浸杀钉螺 72 h, 钉螺死亡率均为 100.00% (30/30); 在 pH 值为 9.0 条件下存储 12 h 后浸杀钉螺72 h, 钉螺死亡率为 33.33% (10/30, χ2 = 30.000, P < 0.05)。发酵上清 液最终分离纯化产物100.00% (30/30) 杀死钉螺所需最低浓度为1.25 mg/L。以 0.10 mg/L 和 1.00 mg/L 浓度最终分离纯化 产物处理钉螺后, 钉螺软体组织中ATP水平较对照组显著降低 (F = 7.274, P < 0.05), ADP 水平与对照组差异无统计学意 义 (F = 2.485, P > 0.05)。结论 黑色浅灰链霉菌代谢产物中杀螺活性成分可在 −20、4 °C 和 28 °C 条件下稳定储存 10 d, 且耐热、耐酸而不耐碱, 其杀螺机制可能是使钉螺能量代谢发生紊乱而死亡。.
Keywords:
Molluscicidal effect;