CN116267891B - 一种昆虫病原线虫保护剂及其应用 - Google Patents
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Abstract
本发明涉及生物杀虫剂技术领域,具体涉及一种昆虫病原线虫保护剂及其应用,昆虫病原线虫保护剂包括依克多因和纳他霉素,依克多因和纳他霉素的质量比为200~20000:1~20,应用于保存昆虫病原线虫,利用依克多因能缓解高渗、高温、冻融、干燥和辐射对蛋白、核酸、生物膜及整个细胞的毒害作用和纳他霉素抑制真菌生长的能力,可以提高昆虫病原线虫在低温、高温、干燥及辐射等逆境下的存活率,降低储存过程中的真菌污染率,并维持昆虫病原线虫高的浸染力,提高昆虫病原线虫的生物防治效果。
Description
技术领域
本发明涉及昆虫病原线虫保存技术领域,具体涉及一种昆虫病原线虫保护剂及其应用。
背景技术
昆虫病原线虫是一类专性寄生昆虫的致病性线虫,包括斯氏线虫科[Steinernematidae carpocapsae(Sc-2)]和异小杆线虫科[Herorhabditidaebacteriophora(Hb-1)],其消化道内携带有病原菌,当它从昆虫消化道或体壁侵入昆虫寄主体内后,共生病原菌从线虫体内释放出来,在昆虫血液内增殖,最终使寄主昆虫感染病原菌患败血症而死亡,能有效防治农林、园艺植物的土栖和钻蛀性害虫,如韭蛆、蛴螬、桃小食心虫、黄曲跳甲等,具有寄主广泛、致病力强且对人畜无害的特点,是一种新型的生物杀虫剂。
昆虫病原线虫从生产到应用需经过繁殖、收获、清洗、存储及施用等过程,存储是其中的重要步骤,目的是尽可能长时间维持昆虫病原线虫高存活率和高侵染力,对昆虫病原线虫的大规模应用和应用效果至关重要。目前昆虫病原线虫大批量贮存技术主要是通过一定的方式脱水使线虫进入低湿休眠(anhydrobiosis)状态,或者采用低温储存(4℃左右),以降低线虫代谢,从而延长线虫贮存时间。
昆虫病原线虫在存储和施用过程中会经历低温、高温、干燥、紫外线及真菌污染等胁迫,导致昆虫病原线虫死亡、侵染力下降,防治害虫效果不佳。
发明内容
针对昆虫病原线虫在存储和施用过程中存活率不高、侵染力低的问题,本发明提供一种昆虫病原线虫保护剂及其应用。
第一方面,本发明提供一种昆虫病原线虫保护剂,包括依克多因和纳他霉素,依克多因和纳他霉素的质量比为200~20000:1~20。
依克多因是耐盐微生物为维持渗透压平衡而在细胞内产生的一种相容性溶质,属于亲水性、兼性离子的生物小分子,能缓解高渗、高温、冻融、干燥和辐射对生物大分子及整个细胞的毒害作用;纳他霉素是天然抗真菌化合物,可以广泛有效的抑制各种霉菌、酵母菌的生长,二者结合能有效提高昆虫病原线虫保存过程中的存活率,延长昆虫病原线虫储存时间;维持昆虫病原线虫的侵染力,提高对虫害的防治效果,有助于昆虫病原线虫的大规模推广应用。
进一步的,依克多因和纳他霉素的质量比为4998:2。
第二方面,本发明提供上述昆虫病原线虫保护剂在保存昆虫病原线虫方面的应用。
进一步的,保存方法包括以下步骤:
(1)将昆虫病原线虫保护剂和昆虫病原线虫悬浊液按照质量比2~200:800~998混合均匀,得混合液;
(2)将混合液用海绵吸附,密封包装后低温保存。
进一步的,步骤(1)中,昆虫病原线虫保护剂和昆虫病原线虫悬浊液的质量比为50:950。
进一步的,步骤(2)中,混合液与海绵的质量比为5~9:1~5。
进一步的,混合液与海绵的质量比为7:3。
进一步的,步骤(2)中,低温保存的温度为4℃。
本发明的有益效果在于:
1、本发明提供的昆虫病原线虫保护剂,添加天然、安全的微生物发酵产物依克多因和纳他霉素,和昆虫病原线虫生物杀虫剂的定位相得益彰,安全绿色。
2、本发明提供的昆虫病原线虫保护剂及其应用,利用依克多因能缓解高渗、高温、冻融、干燥和辐射对蛋白、核酸、生物膜及整个细胞的毒害作用和纳他霉素抑制真菌生长的能力,可以提高昆虫病原线虫在低温、高温、干燥及辐射等逆境下的存活率,降低储存过程中的真菌污染率,并维持昆虫病原线虫高的浸染力,提高昆虫病原线虫的生物防治效果。
具体实施方式
为了使本技术领域的人员更好地理解本发明中的技术方案,下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂、材料、器具,如无特殊说明,均为现有技术,可从商业途径获得。
下述实施例中,所用依克多因纯度大于98%,内毒素含量低于0.1EU/mg;纳他霉素纯度大于95%。昆虫病原线虫悬液是通过采用固体培养的方法培养昆虫病原线虫Hb-1,培养完成后经水洗浓缩得到。
下述实施例和对比例所使用依克多因、纳他霉素和昆虫病原线虫悬液均为同一批,品质相同。
实施例1
取49.98g依克多因和0.02g纳他霉素混合均匀,加入950g昆虫病原线虫悬液中混合均匀,用428.5g碎海绵块吸附,均匀分成5份密封后放于4℃冰箱保存。
实施例2
取99.98g依克多因和0.02g纳他霉素混合均匀,加入900g昆虫病原线虫悬液中混合均匀,用428.5g碎海绵块吸附,均匀分成5份密封后放于4℃冰箱保存。
实施例3
取49.95g依克多因和0.05g纳他霉素混合均匀,加入950g昆虫病原线虫悬液中混合均匀,用428.5g碎海绵块吸附,均匀分成5份密封后放于4℃冰箱保存。
实施例4
取19.90g依克多因和0.1g纳他霉素混合均匀,加入980g昆虫病原线虫悬液中混合均匀,用428.5g碎海绵块吸附,均匀分成5份密封后放于4℃冰箱保存。
对比例1
取49.98g蒸馏水和0.02g纳他霉素混合均匀,加入950g昆虫病原线虫悬液中混合均匀,用428.5g碎海绵块吸附,均匀分成5份密封后放于4℃冰箱保存。
对比例2
取49.98g依克多因和0.02g蒸馏水混合均匀,加入950g昆虫病原线虫悬液中混合均匀,用428.5g碎海绵块吸附,均匀分成5份密封后放于4℃冰箱保存。
对比例3
取50g蒸馏水加入950g昆虫病原线虫悬液中混合均匀,用428.5g碎海绵块吸附,均匀分成5份密封后放于4℃冰箱保存。
试验例1昆虫病原线虫存活率测定
将实施例1和对比例1-3得到的昆虫病原线虫产品于4℃的冰箱中保存30天后取出,置于室温2小时,解剖镜下观察昆虫病原线虫死亡情况,判断标准为:昆虫病原线虫经针头机械刺激无反应活动,则认为个体已经死亡。记录并计算昆虫病原线虫的存活率(公式如下所示),结果如表1所示。
表1昆虫病原线虫保护剂对昆虫病原线虫存活率的影响
组别 | 实施例1 | 对比例1 | 对比例2 | 对比例3 |
存活率(%) | 96.5 | 90.1 | 83.7 | 72.9 |
由表1可知,实施例1保存昆虫病原线虫存活率最高,对比例1、对比例2、对比例3保存昆虫病原线虫的存活率依次递减,说明单独使用依克多因或纳他霉素可以提高昆虫病原线虫的存活率,而本发明复配使用依可多因和纳他霉素提高昆虫病原线虫的存活率效果更显著,可以达到96.5%。
试验例2昆虫病原线虫侵染力的测定
根据试验例1计算的昆虫病原线虫存活率,将于4℃的冰箱中保存30天的实施例1和对比例1-3昆虫病原线虫产品分别稀释至成活昆虫病原线虫浓度为500条/mL,然后取2mL稀释液加到铺有双层滤纸的培养皿中,分别作为处理组1-4,并设置不加稀释液的铺有双层滤纸的培养皿为对照组,处理组和对照组各3个重复。处理组1-4和对照组的每个培养皿放置10条大蜡螟,在25℃黑暗条件下培养,24小时后,每隔12小时调查每个培养皿中大蜡螟的死亡数量,计算大蜡螟校正死亡率(公式如下所示),结果如表2所示。
表2大蜡螟校正死亡率
由表2中昆虫病原线虫Hb-1浸染力测定结果显示,培养24h后,实施例1保存的昆虫病原线虫使大蜡螟校正死亡率最高,对比例2次之,之后依次是对比例1和对比例3;培养36h,后实施例1保存的昆虫病原线虫使大蜡螟校正死亡率最高,对比例2次之,之后依次是对比例1和对比例3,且实施例1大蜡螟校正死亡率明显高于对比例;培养48h后,各保存方法所保存的昆虫病原线虫使大蜡螟校正死亡率均达到90%以上,本发明实施例1保存的昆虫病原线虫使大蜡螟校正死亡率可达到100%。由上述可见,显相较于其他保存方法,使用本发明的昆虫病原线虫保护剂及方法保存的昆虫病原线虫的侵染力更强,更高效。
试验例3阳光照射处理后昆虫病原线虫浸染力的测定
根据试验例1计算的昆虫病原线虫存活率,将于4℃的冰箱中保存30天的实施例1和对比例1-3昆虫病原线虫产品分别稀释至成活昆虫病原线虫浓度为500条/mL,放于太阳光下照射2小时,然后取2mL稀释液加到铺有双层滤纸的培养皿中,分别作为处理组1-4,并设置不加稀释液的铺有双层滤纸的培养皿为对照组,处理组和对照组各3个重复。处理组1-4和对照组的每个培养皿放置10条大蜡螟,在25℃黑暗条件下培养,24小时后,每隔12小时调查每个培养皿中大蜡螟的死亡数量,计算大蜡螟校正死亡率(公式如下所示),结果如表3所示。
表3阳光照射处理后大蜡螟校正死亡率
由表3对昆虫病原线虫Hb-1浸染力测定结果显示,培养24h后,实施例1保存的昆虫病原线虫使大蜡螟校正死亡率最高,对比例2次之,之后依次是对比例1和对比例3;培养36h后,实施例1保存的昆虫病原线虫使大蜡螟校正死亡率最高,对比例2次之,之后依次是对比例1和对比例3,且实施例1和对比例2大蜡螟校正死亡率明显高于对比例1和对比例3;培养60h,各保存方法所保存的昆虫病原线虫使大蜡螟校正死亡率均达到70%以上,本发明实施例1保存的昆虫病原线虫使大蜡螟校正死亡率可达到100%。由上述可见,经阳光照射后,相比于其他保存方法,使用本发明的昆虫病原线虫保护剂及方法保存的昆虫病原线虫的侵染力更强,更高效。
综上所述,本发明提供的一种含有依克多因的昆虫病原线虫保护剂能有效提高昆虫病原线虫在保存过程中的存活率,维持较高的侵染能力,有助于昆虫病原线虫的推广应用。
尽管通过优选实施例的方式对本发明进行了详细描述,但本发明并不限于此。在不脱离本发明的精神和实质的前提下,本领域普通技术人员可以对本发明的实施例进行各种等效的修改或替换,而这些修改或替换都应在本发明的涵盖范围内/任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。
Claims (6)
1.一种昆虫病原线虫保护剂在保存昆虫病原线虫方面的应用,其特征在于,昆虫病原线虫保护剂由依克多因和纳他霉素组成,依克多因和纳他霉素的质量比为200~20000:1~20;
保存方法包括以下步骤:
(1)将昆虫病原线虫保护剂和昆虫病原线虫悬浊液按照质量比2~200:800~998混合均匀,得混合液;
(2)将混合液用海绵吸附,密封包装后低温保存。
2.如权利要求1所述的应用,其特征在于,依克多因和纳他霉素的质量比为4998:2。
3.如权利要求1所述的应用,其特征在于,步骤(1)中,昆虫病原线虫保护剂和昆虫病原线虫悬浊液的质量比为50:950。
4.如权利要求1所述的应用,其特征在于,步骤(2)中,混合液与海绵的质量比为5~9:1~5。
5.如权利要求4所述的应用,其特征在于,混合液与海绵的质量比为7:3。
6.如权利要求1所述的应用,其特征在于,步骤(2)中,低温保存的温度为4℃。
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