CN113796460A - 蛔甙在制备促进H.indica LN2感染期线虫恢复发育制剂中的应用 - Google Patents
蛔甙在制备促进H.indica LN2感染期线虫恢复发育制剂中的应用 Download PDFInfo
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Abstract
本发明公开了蛔甙在制备促进H.indica LN2感染期线虫恢复发育制剂中的应用。H.indica LN2线虫在韭菜迟眼覃蚊Bradysia odoriphaga(简称韭蛆)绿色防控中发挥重要作用。本发明发现,相比于作为对照的共生菌菌液上清液,蛔甙能够显著地促进H.indica LN2感染期线虫恢复发育,可以使用蛔甙诱导H.indica LN2感染期线虫恢复发育。因此,在线虫大量培养过程中,将蛔甙添加到业已加入含相应共生细菌的H.indica LN2感染期线虫的培养基中,诱导感染期线虫恢复发育和生长。本发明为商品化生产具有成本优势的H.indica LN2线虫提供了核心技术。
Description
技术领域
本发明属于生物防治领域,具体涉及蛔甙在制备促进Heterorhabditis indicaLN2感染期线虫恢复发育制剂中的应用。
背景技术
昆虫病原斯氏属Steinernema和异小杆属Heterorhabditis线虫以3龄感染期虫态主动搜索寄主昆虫,对非靶标生物和环境安全,可规模化生产,是具有巨大应用潜力的天敌类生物制剂,已广泛用于防治农林、牧草、花卉和卫生等领域的害虫。昆虫病原线虫以感染期虫态随寄主食物或从昆虫的自然开口(如肛门、气门)、节间膜进入昆虫体内,随后释放肠腔中携带的Xenorhabdus属(与斯氏线虫Steinernema共生)或Photorhabdus属(与异小杆线虫Heterorhabditis共生)共生细菌。昆虫病原线虫与其体内携带的共生细菌共同作用致死寄主昆虫,然后利用寄主体内的营养物质繁殖后代。H.indica LN2线虫在韭菜迟眼覃蚊Bradysia odoriphaga(简称韭蛆)绿色防控中发挥重要作用。
当感染期线虫感染合适的寄主昆虫时或在线虫体外培养系统中加入合适的共生细菌时,感染期线虫将开始进入取食状态,并恢复发育,此过程称为发育恢复(Developmentrecovery)。昆虫血淋巴中含有的信号物质具有诱导感染期线虫恢复的作用。在人工培养基中培养的共生细菌也能产生此类信息物质诱导感染期幼虫恢复。这种诱导感染期线虫恢复的信号物质被命名为食物信号。在离体培养过程中,主要步骤是于线虫培养基中接入共生细菌,然后加入无污染感染期线虫种。感染期幼虫种的恢复比例影响线虫培养的工艺流程和成本。因此如何提高接种感染期线虫恢复比例是昆虫病原线虫商业化培养的核心技术。
一种共生菌不能使所有非特异共生的感染期线虫恢复,这表明不同种类的共生细菌所产生的食物信号不同。异丙基二苯乙烯Isopropylstilbene被认为是从Photorhabdus细菌中诱导感染期线虫发育的信号物质。但未见其它化合物诱导昆虫病原感染期线虫恢复发育的报道。线形动物普遍存在的蛔甙类Ascarosides信息物质在调节线虫的聚集、趋避、交配、多尔态(Dauer)形成及扩散传播等行为中发挥了重要作用。但这些蛔甙可否诱导昆虫病原感染期线虫恢复发育仍未见报道。
发明内容
本发明的第一个目的是提供蛔甙在制备促进H.indica LN2感染期线虫恢复发育制剂中的应用。
本发明通过实验发现,蛔甙能够诱导H.indica LN2感染期线虫恢复发育。因此,本发明提供蛔甙在制备促进H.indica LN2感染期线虫恢复发育制剂中的应用,所述的蛔甙,其结构式如式1中ascr#1、ascr#2、ascr#3、ascr#5、ascr#6、ascr#7、ascr#8、ascr#9、ascr#10、ascr#11ascr#12所示任一种。
优选的,所述的蛔甙是式1中ascr#1、ascr#2、ascr#3、ascr#5、ascr#6、ascr#7、ascr#8、ascr#9或ascr#10。
本发明的第二个目的是提供一种诱导H.indica LN2感染期线虫恢复发育的方法,是将蛔甙添加到业已加入含有相应共生细菌的H.indica LN2感染期线虫的培养基中,诱导感染期线虫恢复发育和生长。
所述蛔甙的结构式如式1中ascr#1、ascr#2、ascr#3、ascr#5、ascr#6、ascr#7、ascr#8、ascr#9、ascr#10、ascr#11ascr#12任一所示。
优选的,所述蛔甙是式1中ascr#1、ascr#2、ascr#3、ascr#5、ascr#6、ascr#7、ascr#8、ascr#9或ascr#10。
优选的,所述蛔甙在培养基中的浓度是0.04pM~0.04μM。
本发明发现,相比于作为对照的共生菌菌液上清液,蛔甙能够显著地诱导H.indica LN2感染期线虫恢复发育,感染期线虫呈现良好的恢复率。因此,使用蛔甙诱导H.indica LN2感染期线虫恢复发育,将为商品化生产具有成本优势的H.indica LN2线虫提供核心技术并发挥支撑作用。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1
一、实验材料
1、蛔甙包括11种人工合成的蛔甙,代号分别为ascr#1、ascr#2、ascr#3、ascr#5、ascr#6、ascr#7、ascr#8、ascr#9、ascr#10、ascr#11、ascr#12,其结构式分列如下:
分别将上式中的11种蛔甙以无菌PBS溶解至0.04mM,然后稀释至一定的浓度,以一定的量加入共生细菌上清液或培养基中,使共生细菌上清液或培养基中的浓度分别为0.04μM、0.04nM、0.04pM。
2、共生细菌单菌培养的H.indica LN2:将常规固体培养基培养的线虫经清洗后,加入硫酸链霉素溶液(作用浓度:100ppm)25℃过夜处理,用无菌PBS缓冲清洗三次后,沉淀以无菌PBS配制成10μl含50感染期线虫(IJs)的浓度(5000IJs/mL),装入250ml三角瓶中(装50ml线虫悬液),置于9-13℃。最好7天内使用。
3、Photorhabdus luminescens LN2共生细菌的制备:以无菌营养肉汤(121℃灭菌30min)于25℃,120rpm摇床中培养H.indica LN2线虫的共生细菌(P.luminescens LN2)3天;12000rpm,4℃下离心10min;在超净工作台上将获得的上清液以细菌过滤器(0.22μm)过滤,用于IJ恢复(IJ recovery)测定。
4、硫酸链霉素:100mg/mL。
5、无菌1×PBS缓冲液。
6、无菌超纯水。
7、0.2μm过滤器。
二、实验方法
于一次性48孔细胞培养板中,分别加入0.1ml P.luminescens LN2共生细菌的上清液;每个孔中加入10μl硫酸链霉素(原液为100mg/ml,稀释10倍使用);以10μl的量加入约50条感染期线虫;再分别加入10μl不同浓度的蛔甙,使其在共生细菌上清液或培养基中的浓度分别为0.04μM、0.04nM、0.04pM。以等体积加入10mg/ml硫酸链霉素的上清液、无菌PBS、加入10mg/ml硫酸链霉素的无菌PBS、质量分数0.1%DMSO作为对照。每个处理设置4个孔的重复。
将孔板以封口膜封口后,装入无菌带盖的塑料盒内,放置25℃下黑暗培养。于第6天在超净工作台中利用解剖镜观察统计每个孔中感染期线虫的恢复数(即恢复发育的线虫数量),并计算恢复率,恢复率=恢复数/总数×100%。
结果如表1所示。
表1:蛔甙对H.indica LN2感染期线虫恢复率的影响(6天)
注:PBS代表无菌PBS中加入硫酸链霉素;PBS(无)代表未加入硫酸链霉素的无菌PBS。表中大写字母表示同一处理下不同浓度之间的显著性差异;小写字母表示同一浓度下不同处理之间的显著性差异。
从表1可以看出,所述11种蛔甙都具有促进H.indica LN2感染期线虫恢复发育的良好效果,特别是其中的ascr#1、ascr#2、ascr#3、ascr#5、ascr#6、ascr#7和ascr#8具有非常良好的促进感染期线虫恢复的效果。
Claims (5)
2.根据权利要求1所述的应用,其特征在于,所述的蛔甙是所述式中的ascr#1、ascr#2、ascr#3、ascr#5、ascr#6、ascr#7、ascr#8、ascr#9或ascr#10。
4.根据权利要求3所述的方法,其特征在于,所述蛔甙所述式中的ascr#1、ascr#2、ascr#3、ascr#5、ascr#6、ascr#7、ascr#8、ascr#9或ascr#10。
5.根据权利要求3或4所述的方法,其特征在于,所述的蛔甙在培养基中的浓度是0.04pM~0.04μM。
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US20200281213A1 (en) * | 2019-03-06 | 2020-09-10 | The United States Of America, As Represented By The Secretary Of Agriculture | Methods and compositions for increasing infectivity of entomopathogenic nematodes |
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