CN106993607B - 腐殖土作为载体在贮存昆虫病原线虫中的应用 - Google Patents
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Abstract
本发明公开了腐殖土作为载体在贮存昆虫病原线虫中的应用。本发明对腐殖土作为载体贮存线虫进行贮存效果测定，实验结果表明：腐殖土对S.carpocapsae All、S.feltiae JY‑90、H.bacteriophora H06、H.indica LN2线虫均有很好的贮存效果，是一种良好的昆虫病原线虫贮存载体。本发明用腐殖土作为载体贮存昆虫病原线虫，延长了线虫有效货架时间，实现了线虫的长距离运输和最佳时间的田间应用，为线虫产业化生产提供了技术支持；同时发掘了腐殖土的新用途，开拓了其应用领域。制得的昆虫病原线虫剂型可以直接撒施到田间用于防治田间害虫，同时也可以作为肥料提高田地肥力。
Description
技术领域：
本发明属于贮存技术领域，具体涉及腐殖土作为载体在贮存昆虫病原线虫中的应用。
背景技术：
昆虫病原线虫(Entomopathogenic nematodes，EPNs)是一类专门寄生昆虫的有益线虫，主要包括异小杆线虫(Heterorhabditis)和斯氏线虫(Steinernema)两个属。这类线虫具有易于大量生产，杀虫能力强，杀虫谱广，能主动搜索寄主，对人畜、环境安全等优点，对地下害虫特效(Kaya and Gaugler 1993；Grewal et al.，2005；Georgis et al.，2006；Yan et al.，2012)，应用前景广阔。在昆虫病原线虫的商品化过程中，延长线虫有效货架时间，以便于长距离运输和最佳时间的田间应用，是线虫产业化生产的关键技术之一。昆虫病原线虫的贮存效果与载体的种类、大小(Grewal,2002；Hussein and Abdel-Aty,2012；Shapiro-Ilan et al.,2012；Yan,2012；Vemmer and Patel,2013；Kim and Jaffuel,2015)、贮存温度(Grewal,2000b；Strauch et al.,2000；Peters,2016)等因素有关。
发明内容：
本发明的目的在于提供腐殖土作为载体在贮存昆虫病原线虫中的应用。
本实验室以硅藻土、蛭石、碎海绵作为比较，筛选到腐殖土对已商业化生产的四种线虫Steinernema carpocapsae All、S.feltiae JY-90、Heterorhabditis bacteriophoraH06以及H.indica LN2线虫具有很好的贮存效果，可作为线虫贮存一种有效的载体。
本发明的第一个目的是提供腐殖土作为载体在贮存昆虫病原线虫中的应用。
所述的昆虫病原线虫优选为Steinernema carpocapsae All、Steinernemafeltiae JY-90、Heterorhabditis bacteriophora H06或Heterorhabditis indica LN2。
本发明的第二个目的是提供腐殖土作为载体在制备昆虫病原线虫剂型中的应用。
所述的昆虫病原线虫优选为Steinernema carpocapsae All、Steinernemafeltiae JY-90、Heterorhabditis bacteriophora H06或Heterorhabditis indica LN2。
本发明的第三个目的是提供一种昆虫病原线虫剂型，包括线虫泥和腐殖土，所述的线虫泥和腐殖土的质量比为1:1，所述的线虫泥由昆虫病原线虫感染期线虫和水组成。
所述的线虫泥中线虫密度优选为70～210万条/g。
所述的昆虫病原线虫优选为Steinernema carpocapsae All、Steinernemafeltiae JY-90、Heterorhabditis bacteriophora H06或Heterorhabditis indica LN2。
所述的腐殖土的粒径优选为24目。
本发明对腐殖土作为载体贮存线虫进行贮存效果测定，实验结果表明：腐殖土对S.carpocapsae All、S.feltiae JY-90、H.bacteriophora H06、H.indica LN2线虫均有很好的贮存效果，是一种良好的昆虫病原线虫贮存载体。
本发明用腐殖土作为载体贮存昆虫病原线虫，延长了线虫有效货架时间，实现了线虫的长距离运输和最佳时间的田间应用，为线虫产业化生产提供了技术支持；同时发掘了腐殖土的新用途，开拓了其应用领域。制得的昆虫病原线虫剂型可以直接撒施到田间用于防治田间害虫，同时也可以作为肥料提高田地肥力。
附图说明：
图1是昆虫病原线虫Steinernema carpocapsae All、S.feltiae JY-90、Heterorhabditis bacteriophora H06以及H.indica LN2分别与硅藻土、蛭石和腐殖土按照质量比1:1或与碎海绵按照质量比3:1混配在5℃或15℃条件下的贮存效果；其中，*表明同一取样时间不同处理线虫存活率之间显著差异(P<0.05，Tukey’s test)；S.carpocapsae代表Steinernema carpocapsae All，S.feltiae代表S.feltiae JY-90，H.bacteriophora代表Heterorhabditis bacteriophora H06，H.indica代表H.indica LN2。
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、实验材料与方法
1.昆虫病原线虫的培养：S.carpocapsae All、S.feltiae JY-90、H.bacteriophora H06和H.indica LN2线虫感染期线虫(IJs，Infective juveniles)，按照韩日畴(1996)的固体培养方法进行培养。收获线虫将线虫装于纱网袋中置于洗衣机中按照常规程序清洗，收集洗衣机排水液于沉降槽(2.0m×1.0m×0.5m)中，加水清洗3次后收集线虫沉淀于棉布袋中收集线虫泥。
2.线虫泥密度统计：取线虫泥0.1g分别溶于1000mL水中，用玻璃棒搅拌均匀，取1mL于计数板中。每种线虫泥与每一线虫悬浮液中均重复取样3个，于显微镜下计数，取平均值计算收获的S.carpocapsae All、S.feltiae JY-90、H.bacteriophora H06和H.indicaLN2线虫泥中的线虫密度分别为130、70、150、210万条/g。
3.线虫与不同载体按比例配制剂型：将S.carpocapsae All、S.feltiae JY-90、H.bacteriophora H06和H.indica LN2线虫泥分别与硅藻土(150目)按照质量比1:1混合均匀，分别得到S.carpocapsae All线虫硅藻土剂型、S.feltiae JY-90线虫硅藻土剂型、H.bacteriophora H06线虫硅藻土剂型和H.indica LN2线虫硅藻土剂型；将S.carpocapsaeAll、S.feltiae JY-90、H.bacteriophora H06和H.indica LN2线虫泥分别与蛭石(200目)按照质量比1:1混合均匀，分别得到S.carpocapsae All线虫蛭石剂型、S.feltiae JY-90线虫蛭石剂型、H.bacteriophora H06线虫蛭石剂型和H.indica LN2线虫蛭石剂型；将S.carpocapsae All、S.feltiae JY-90、H.bacteriophora H06和H.indica LN2线虫泥分别与腐殖土(24目)按照质量比1:1混合均匀，分别得到S.carpocapsae All线虫腐殖土剂型、S.feltiae JY-90线虫腐殖土剂型、H.bacteriophora H06线虫腐殖土剂型和H.indica LN2线虫腐殖土剂型；将S.carpocapsae All、S.feltiae JY-90、H.bacteriophora H06和H.indica LN2线虫泥分别与碎海绵按照质量比3:1混合均匀，分别得到S.carpocapsae All线虫碎海绵剂型、S.feltiae JY-90线虫碎海绵剂型、H.bacteriophora H06线虫碎海绵剂型和H.indica LN2线虫碎海绵剂型。
4.线虫剂型分装：将混合均匀后的线虫剂型按照40g/瓶(硅藻土与蛭石剂型)、23g/瓶(腐殖土剂型)、13g/瓶(碎海绵剂型)装入塑料杯(pp材质，200mL，上口径为4.5cm，底部直径为7cm，高度为7.5cm)中，所有剂型在塑料杯中的高度均为5.5±0.5cm。将塑料杯用透气封口膜(透气封口膜包括pp膜和设置在pp膜中间的一透气膜，透气膜的直径为1cm，透气膜的膜孔径为5μm)进行热封。
5.线虫剂型的贮存温度设置：将S.carpocapsae All和S.feltiae JY-90线虫不同载体不同比例的剂型处理分别置于5℃贮存室中进行贮存，H.bacteriophora H06和H.indica LN2线虫各种载体不同比例的剂型处理分别置于15℃贮存室中进行贮存，每一处理为30个重复(30个塑料杯)。
6.线虫贮存效果测定：每隔一定时间对线虫的存活率进行统计，取样时间为S.carpocapsae All、S.feltiae JY-90线虫0、20、40、60、80、100、120、140、160、180d；H.bacteriophora H06和H.indica LN2线虫0、10、20、30、40、50、60、70、80、90d。每次每个处理取样3个重复，每一重复取3个样品计数。取样方法为：将每个处理每一杯子内所有线虫全部溶于水中，搅拌均匀后取样10mL于灭菌培养皿中，置于25℃条件下恢复24h后计数，统计线虫的存活数量及死亡数量，计算线虫的存活率。
7.数据分析：线虫死亡率的百分数值均经反正弦arcsin(X1/2)转换后，以SPSS软件(版本为SPSS 16.0for Windows)进行方差分析，多重比较采用Tukey检验各处理之间的差异显著性，显著水平P<0.05(SPSS 16.0，SPSS Inc.，Chicago，IL，USA)。
二、实验结果
各线虫与不同载体混用的贮存效果详见图1。各线虫腐殖土剂型的存活率与蛭石、碎海绵剂型的存活率之间无显著差异，但均显著高于硅藻土剂型(F≥6.448；df＝3，8；P≤0.016)。
S.carpocapsae All线虫在5℃腐殖土、蛭石、碎海绵中贮存180天时存活率仍大于90％，而在硅藻土中贮存超过100天时存活率降低至90％以下，超过120天时存活率降低至80％以下；S.feltiae JY-90线虫在5℃腐殖土、蛭石、碎海绵中贮存140天时存活率仍大于90％，而在硅藻土中贮存超过80天时存活率降低至90％以下，超过100天时存活率降低至80％以下。H.bacteriophora H06线虫在15℃硅藻土中贮存仅10天线虫存活率降低至75％，而在腐殖土、蛭石、碎海绵中贮存40天时线虫的存活率仍大于80％。而H.indica LN2线虫在15℃硅藻土中贮存仅10天线虫存活率降低至50％，而在腐殖土、蛭石、碎海绵中贮存20天时线虫的存活率仍大于80％。
实验结果表明，腐殖土是一种良好的昆虫病原线虫贮存载体，对昆虫病原线虫的贮存效果与蛭石、碎海绵无差异，显著高于硅藻土的贮存效果。
但是由于海绵比较难操作，而腐殖土可以作为肥料使用，因此将线虫与腐殖土混合，即可以很好的贮存好线虫，又可以作为肥料使用，一举两得，且腐殖土价格便宜，比蛭石价格便宜一半，因此最优剂型还是腐殖土剂型的。
Claims (5)
1.腐殖土作为载体在贮存昆虫病原线虫中的应用，所述的昆虫病原线虫为Steinernema carpocapsae All或Steinernema feltiae JY-90。
2.腐殖土作为载体在制备昆虫病原线虫剂型中的应用，所述的昆虫病原线虫为Steinernema carpocapsae All或Steinernema feltiae JY-90。
3. 一种昆虫病原线虫剂型，其特征在于，包括线虫泥和腐殖土，所述的线虫泥和腐殖土的质量比为1:1，所述的线虫泥由昆虫病原线虫感染期线虫和水组成，所述的昆虫病原线虫为Steinernema carpocapsae All或Steinernema feltiae JY-90。
4.根据权利要求3所述的昆虫病原线虫剂型，其特征在于，所述的线虫泥中线虫密度为70~210万条/g。
5.根据权利要求3或4所述的昆虫病原线虫剂型，其特征在于，所述的腐殖土的粒径为24目。
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| CN1088046A (zh) * | 1992-09-10 | 1994-06-22 | 联邦科学和工业研究组织 | 昆虫致病性线虫的储存方法 |
| EP0909532A1 (en) * | 1997-10-16 | 1999-04-21 | Development Center For Biotechnology | Environmentally compatible porous material comprising beneficial nematodes and its preparation |
| EP2806740A1 (en) * | 2012-01-25 | 2014-12-03 | Bayer Intellectual Property GmbH | Active compounds combination containing fluopyram bacillus and biologically control agent |
| WO2016176764A1 (en) * | 2015-05-04 | 2016-11-10 | Green Advantage Technology Inc. | Methods and formulations for storing entomopathogenic nematodes |
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| CN1088046A (zh) * | 1992-09-10 | 1994-06-22 | 联邦科学和工业研究组织 | 昆虫致病性线虫的储存方法 |
| EP0668718A4 (en) * | 1992-09-10 | 1997-07-23 | Commw Scient Ind Res Org | METHOD FOR STORING ENTOMOPATHOGENIC NEMATODES. |
| EP0909532A1 (en) * | 1997-10-16 | 1999-04-21 | Development Center For Biotechnology | Environmentally compatible porous material comprising beneficial nematodes and its preparation |
| EP2806740A1 (en) * | 2012-01-25 | 2014-12-03 | Bayer Intellectual Property GmbH | Active compounds combination containing fluopyram bacillus and biologically control agent |
| CN104507314A (zh) * | 2012-01-25 | 2015-04-08 | 拜耳知识产权有限责任公司 | 含有氟吡菌酰胺芽孢杆菌和生物防治剂的活性化合物结合物 |
| WO2016176764A1 (en) * | 2015-05-04 | 2016-11-10 | Green Advantage Technology Inc. | Methods and formulations for storing entomopathogenic nematodes |
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