中国血吸虫病防治杂志 ›› 2021, Vol. 33 ›› Issue (3): 248-.

• 论著 • 上一篇    下一篇

贝莱斯芽孢杆菌Y6杀螺活性及其机制的初步研究

杨成1,乔云龙2,刘辉1,潘中武1*   

  1. 1皖南医学院微生物学与免疫学教研室(芜湖 241002);2 皖南医学院药学院
  • 出版日期:2021-07-09 发布日期:2021-07-09
  • 作者简介:杨成,男,硕士,副教授。研究方向:微生物活性物质
  • 基金资助:
    2020年活性生物大分子研究安徽省重点实验室自主课题项目(LAB202002);安徽省大学生创新创业训练项目(S202010368061)

Molluscicidal activity of the Bacillus velezensisis Y6 strain against Oncomelania hupensis and its preliminary mechanisms of molluscicidal actions

YANG Cheng1, QIAO Yun⁃Long2, LIU Hui1, PAN Zhong⁃Wu1*   

  1. 1 Department of Microbiology and Immunology, Wannan Medical College, Wuhu 241002, China; 2 School of Pharmacy, Wannan Medical College, China
  • Online:2021-07-09 Published:2021-07-09

摘要: 目的 评价芽孢杆菌Y6实验室内杀螺活性,初步探讨其杀螺作用机制。方法 应用形态学、生理生化特性及16S rDNA 基因序列同源性分析,对菌株Y6进行生物学鉴定。配制浓度分别为0.005、0.010、0.015 g/mL的芽孢杆菌Y6悬液,采用浸泡法评价其室内杀螺效果。检测芽孢杆菌Y6悬液浸泡后钉螺软体组织内Y6菌体含量和糖原含量变化,初步探讨其杀螺机制。结果 Y6菌落呈乳白色、不透明,在营养琼脂培养基表面有菌膜形成;革兰氏染色呈阳性,杆状;芽孢位于菌体中间,呈无色透明折光小体,被菌体包裹;卵磷脂酶试验呈阳性,Y6菌体16S rDNA基因序列与多株贝莱斯芽孢杆菌(Bacillus velezensisis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和枯草芽孢杆菌(Bacillus subtilis)同源性均为100%,将菌株Y6鉴定为贝莱斯芽孢杆菌。0.005、0.010、0.015 g/mL 芽孢杆菌Y6悬液浸泡24、48、72 h后,钉螺死亡率分别为28.3%、31.7%和81.6%,43.3%、58.3%和93.3%及63.3%、78.3%和98.3%;随着悬液浓度增加、浸泡时间延长,菌株Y6悬液杀螺效果提高。采用染色镜检法和平板菌落计数法观察发现,菌株Y6悬液浸泡后的死螺体内Y6菌体含量最高、活螺体内最低;经芽孢杆菌Y6悬液浸泡后,死螺、濒死螺和活螺软体组织内平均糖原含量分别为(0.68 ± 0.06)、(1.09 ± 0.16) μg/mg和(2.56 ± 0.32) μg/mg(F = 59.519,P < 0.05),活螺软体组织内平均糖原含量显著高于死螺(t = 14.073,P < 0.05)和濒死螺(t = 10.027,P < 0.05),而濒死螺软体组织内平均糖原含量显著高于死螺(t = 5.983,P < 0.05)。结论 贝莱斯芽孢杆菌Y6对钉螺具有较好杀灭效果,其杀螺机制可能是菌体侵染致螺体内糖原代谢紊乱而引起钉螺死亡。  

关键词: 钉螺, 贝莱斯芽孢杆菌, 杀螺效果, 糖原

Abstract: Objective To assess the molluscicidal activity of the of Bacillus Y6 strain against Oncomelania hupensis in laboratory, and to preliminarily investigate its mechanisms of molluscicidal actions. Methods Biological identification of the Y6 strain was performed based on analysis of its morphological and physiochemical features and homology analysis of the 16S rDNA gene sequence. Bacillus Y6 suspensions were formulated at concentrations of 0.005, 0.010 g/mL and 0.015 g/mL, and the molluscicidal activity of Bacillus Y6 suspensions against O. hupensis was tested in laboratory using the immersion method. In addition, the Bacillus Y6 content and glycogen content were detected in O. hupensis following exposure to Bacillus Y6 suspensions to preliminarily explore the molluscicidal mechanism of the Bacillus Y6 strain against O. hupensis. Results The colony of the Bacillus Y6 strain appeared non⁃transparent milky white, and mycoderma was produced on the surface of the nutrient agar liquid medium. The Y6 stain was Gram positive and rod⁃shaped, and the endospore was located at the center of the Bacillus Y6 strain and appeared an achromatic, transparent and refractive body, which was encapsulated by the Y6 strain. The Y6 strain was positive for the lecithinase test, and the 16S rDNA gene sequence showed a 100% homology with those of multiple B. velezensisis strains, B. amyloliquefaciens and B. subtilis. The Y6 strain was therefore identified as B. velezensisis. Following immersion in the Bacillus Y6 suspensions at concentrations of 0.005, 0.010 g/mL and 0.015 g/mL for 24, 48 h and 72 h, the mortality rates of Oncomelania snails were 28.3%, 31.7% and 81.6%, 43.3%, 58.3% and 93.3%, and 63.3%, 78.3% and 98.3%, respectively. The molluscicidal activity of the Bacillus Y6 suspensions increased with the suspension concentration and duration of immersion. Microscopy and colony counting revealed the highest Y6 content in dead snails and the lowest in living snails following immersion in Bacillus Y6 suspensions, and the mean glycogen contents were (0.68 ± 0.06), (1.09 ± 0.16) μg/mg and (2.56 ± 0.32) μg/mg in the soft tissues of dead, dying and living snails following immersion in Bacillus Y6 suspensions (F = 59.519, P < 0.05), and the mean glycogen content was significantly higher in living snails than in dead (t = 14.073, P < 0.05) and dying snails (t = 10.027, P < 0.05), while the mean glycogen content was significantly higher in dying snails than in dead snails (t = 5.983, P < 0.05). Conclusion The B. velezensisis Y6 strain shows a high molluscicidal activity against O. hupensis snails, and its invasion may cause glycogen metabolism disorders, leading to snail death.

Key words: Oncomelania hupensis, Bacillus velezensisis, Molluscicidal activity, Glycogen

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