CN107006430A - 一种规模化生产小卷蛾斯氏线虫新方法 - Google Patents
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Abstract
本发明提供了一种利用嗜线虫杆菌与线虫共生的原理，通过非离体培养与离体培养相互结合，稳定生产小卷蛾斯氏线虫F3代的新方法。选取健康的大蜡螟末龄幼虫，接种小卷蛾斯氏线虫，得到的线虫为F0代。再将F0代线虫接种到培养了2天嗜线虫杆菌的营养琼脂培养皿中，得到F1代。最后将F1代接种于扩大海绵培养基中，培养14天得到大量的F3代作为商品线虫。本方法所生产的商品线虫都是F3代线虫，生产周期短，品质稳定。
Description
技术领域
本发明属于生物防治技术，具体涉及一种利用嗜线虫杆菌与线虫共生的原理，通过非离体培养与离体培养相互结合，稳定生产小卷蛾斯氏线虫F3代的新方法。
背景技术
小卷蛾斯氏线虫是一种以昆虫为寄主的昆虫病原线虫。处于侵入期的昆虫病原线虫能自主寻找寄主，侵入寄主的线虫从肠道中释放昆虫致病杆菌，杆菌在寄主中大量繁殖导致寄主迅速死亡。国际上常用于防治害虫的昆虫病原线虫主要属于斯氏线虫属和异小杆线虫属。斯氏线虫有10个种，异小杆线虫有3个种。在工业化国家生物农药市场上，市场份额仅次于苏云金芽孢杆菌，占第二位。这类线虫对土栖性害虫和钻蛀性害虫有较强的杀灭作用。昆虫病原线虫可规模化培养，且使用方便，对人畜安全，不污染环境，在美国是免注册产品。我国已开发了不少杀虫线虫品种，目前处于中试推广应用阶段，均无注册成产品。
产业化昆虫病原线虫，需要高效，稳定的病原线虫培养方法。在美国、德国等生物农药发达的国家一般采用液体法培养昆虫病原线虫。而我国昆虫病原线虫产业化起步较晚，从事昆虫病原线虫产业化的企业采用都是固体培养和非离体培养。以上两种方法各有其局限性，使用非离体培养法，经过多代培养后培养基（虫子尸体）易发霉，并且易染蝇蛆。采用离体培养法，经多代培养后线虫质量退化，加上培养周期过长原生嗜线虫杆菌易转变成次生杆菌。以上等等原因严重限制了昆虫病原线虫的的规模化培养和影响了线虫的品质。
发明内容
为了克服原来技术上培养周期28天导致嗜线虫杆菌转变为次生杆菌这一技术难题，本发明提供了一种利用嗜线虫杆菌与线虫共生的原理，通过非离体培养与离体培养相互结合，稳定生产小卷蛾斯氏线虫F3代的新方法。选取健康的大蜡螟末龄幼虫，接种小卷蛾斯氏线虫，一天后待大蜡螟死亡，挑选出来集中于通气、恒温、恒湿环境下培养10天，得到的线虫为F0代。再将F0代线虫接种到培养了2天嗜线虫杆菌的营养琼脂培养皿中，培养10天得到繁殖后的线虫作为F1代。最后将F1代接种于扩大海绵培养基中，培养14天得到大量的F3代作为商品线虫。本方法所生产的商品线虫都是F3代线虫，生产周期短，保证嗜线虫杆菌不转变成次生杆菌，从而保证品质稳定。
具体实施方式：
下面列举了实例，对本发明进行进一步说明：
实施例1选取健康的大蜡螟末龄幼虫1000条，将带小卷蛾斯氏线虫的海绵放入去离子水中，搅拌多次，将海绵挑出，显微镜检测线虫密度100万尾/ml，对1000条大蜡螟体表进行接种。一天后待大蜡螟死亡，挑选一天死亡的大蜡螟，在恒温25摄氏度下、90%湿度下恒湿环境下培养10天，得到的线虫为F0代。将F0代从大蜡螟尸体内用无菌去离子水浸出线虫，再将线虫用无菌生理盐水清洗3-5次，离心分离。将去杂菌的F0代50万尾线虫接种到培养了2天嗜线虫杆菌的15mm营养琼脂培养皿中，培养皿营养蛋白胨10%，酵母膏5%，琼脂2%，将培养皿用锡箔纸封严，培养10天得到繁殖后的线虫作为F1代250万尾。选取无杂菌的F1代作种用线虫，含杂菌的培养皿直接去除。最后将F1代接种于扩大海绵培养基中，海绵培养基中营养为虫粉60%，速溶豆粉40%，蔗糖5%，猪油5%。培养14天繁殖2代，得到大量的F3代作为商品线虫2.5亿尾。
附图1生产小卷蛾斯氏线虫流程图对照图。
Claims (10)
1.本发明为一种生产小卷蛾斯氏线虫新方法，其特征在于利用大蜡螟将小卷蛾斯氏线虫复壮，再用固体培养将线虫放大和扩大化生产，稳定产出商品化的F3代。
2.根据权利要求1利用大蜡螟将小卷蛾斯氏线虫复壮，其特征在于选用活的末期大蜡螟接种线虫进行线虫的复壮，作为F0代。
3.根据权利要求2利用大蜡螟将小卷蛾斯氏线虫复壮，其特征在于选用接种后1-2天死亡的没有变色的大蜡螟作为线虫培养对象。
4.根据权利要求2利用大蜡螟将小卷蛾斯氏线虫复壮，其特征在于接种死亡的大蜡螟，集中于通气装备中、在恒温25摄氏度下、90%湿度下恒湿环境下培养10天。
5.根据权利要求2利用大蜡螟中复壮线虫作F0代，其特征在于将F0代从大蜡螟尸体内用无菌去离子水浸出线虫，再将线虫用无菌生理盐水清洗3-5次。
6.根据权利要求1固体培养将线虫放大生产，其特征在于将去杂菌的F0代线虫接种到培养了2天嗜线虫杆菌的15mm营养琼脂培养皿中，培养10天得到繁殖1代后的线虫作为F1代。
7.根据权利要求6中营养琼脂培养，其特征在于营养配方蛋白质浓度高于20%，脂类大于5%。
8.根据权利要求1固体培养将线虫扩大化生产，其特征在于将培养皿中移取整皿含线虫的琼脂直接倒入扩大化培养基中。
9.根据权利要求8固体培养将线虫扩大化生产，其特征在于扩大培养基营养配方为虫粉60%，速溶豆粉40%，蔗糖5%，猪油5%。
10.根据权利要求8固体培养将线虫扩大化生产，其特征在于扩大培养天数为14天，繁殖2代。
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| CN109496989A (zh) * | 2018-11-01 | 2019-03-22 | 李小龙 | 一种昆虫病原线虫活体培养方法 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102972446A (zh) * | 2012-11-22 | 2013-03-20 | 北京市西山试验林场 | 小卷蛾斯氏线虫的制备方法 |
| CN104222023A (zh) * | 2014-08-30 | 2014-12-24 | 北京安和亿泰生物工程技术有限公司 | 一种斯氏线虫大批量繁殖培育方法 |
| CN106035250A (zh) * | 2016-08-18 | 2016-10-26 | 浙江绿神天敌生物技术有限公司 | 一种昆虫病原线虫的培养工艺 |
| CN106035238A (zh) * | 2016-05-30 | 2016-10-26 | 中国科学院东北地理与农业生态研究所 | 一种线虫培养装置及供试昆虫病原线虫的简易繁殖方法 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102972446A (zh) * | 2012-11-22 | 2013-03-20 | 北京市西山试验林场 | 小卷蛾斯氏线虫的制备方法 |
| CN104222023A (zh) * | 2014-08-30 | 2014-12-24 | 北京安和亿泰生物工程技术有限公司 | 一种斯氏线虫大批量繁殖培育方法 |
| CN106035238A (zh) * | 2016-05-30 | 2016-10-26 | 中国科学院东北地理与农业生态研究所 | 一种线虫培养装置及供试昆虫病原线虫的简易繁殖方法 |
| CN106035250A (zh) * | 2016-08-18 | 2016-10-26 | 浙江绿神天敌生物技术有限公司 | 一种昆虫病原线虫的培养工艺 |
Non-Patent Citations (3)
| Title |
|---|
| 徐洁莲等: "连续传代培养对病原斯氏线虫的带菌率和种群结构的影响", 《昆虫天敌》 * |
| 李春杰: "昆虫病原线虫大量繁殖技术研究", 《中国优秀硕士学位论文全文数据库》 * |
| 王颖等: "斯氏线虫繁殖技术及其防治杨干象的初步研究", 《林业科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109496989A (zh) * | 2018-11-01 | 2019-03-22 | 李小龙 | 一种昆虫病原线虫活体培养方法 |
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