CN115197855A - 白僵菌、白僵菌与草地贪夜蛾的卵寄生蜂联合及其应用 - Google Patents
白僵菌、白僵菌与草地贪夜蛾的卵寄生蜂联合及其应用 Download PDFInfo
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Abstract
本发明涉及白僵菌、白僵菌与草地贪夜蛾的卵寄生蜂联合及其应用。发明人提供“以菌治虫”和“以虫治虫”的生物防治手段,将球孢白僵菌直接用于防治草地贪夜蛾,或者与夜蛾黑卵蜂或玉米螟赤眼蜂联合,进行草地贪夜蛾的生物防治。本申请提供了球孢白僵菌、含有球孢白僵菌的杀虫剂、及防治草地贪夜蛾的方法,为防控草地贪夜蛾提供了重要参考。
Description
技术领域
本发明属于害虫防治领域,具体涉及白僵菌、白僵菌与草地贪夜蛾的卵寄生蜂联合及其在防治草地贪夜蛾中的应用。
背景技术
草地贪夜蛾又称秋黏虫(fall armyworm),属于鳞翅目(Lepidoptera)夜蛾科(Noctuidae),源产于美洲热带和亚热带地区,是新入侵我国的重大农业迁飞性害虫,对我国玉米的产量和品质构成严重威胁。草地贪夜蛾入侵我国后,研究者通过大量的室内毒力测定和田间药效试验,筛选出了乙基多杀菌素、氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐等化学药剂用于其防治,但长期使用化学农药不仅会增加农业生产成本、危害环境和人畜健康,也易使昆虫产生抗药性,因此,草地贪夜蛾的防控要寻求一种生态友好、不易产生抗性的绿色、安全的防治措施。此外,在生产中,由于田间复杂环境对单一使用昆虫病原真菌侵染或者寄生蜂寄生效果的影响,单独应用防效有限。
发明内容
本申请的目的在提供一种“以菌治虫”和“以虫治虫”的生物防治手段,将球孢白僵菌直接或间接用于防治草地贪夜蛾。
本申请提供球孢白僵菌在防治草地贪夜蛾中的应用。
进一步,所述球孢白僵菌的浓度为1×107-108孢子/mL。
进一步,所述球孢白僵菌为Bb20091317、Bb1237、Bb201017;优选的,所述球孢白僵菌为Bb20091317。
本申请提供一种草地贪夜蛾杀虫剂,所述草地贪夜蛾杀虫剂含有上述的球孢白僵菌。
球孢白僵菌对寄主的侵染途径分为以下阶段:分生孢子的附着、萌发、穿透表皮、菌丝在血腔内生长、产生毒素、寄主死亡、菌丝入侵所有器官、菌丝穿出表皮、产生分生孢子和分生孢子扩散。
进一步,所述草地贪夜蛾杀虫剂剂型为球孢白僵菌分生孢子悬浮液或粉剂;
优选的,所述孢子悬浮液有效成分为球孢白僵菌为Bb20091317菌株的分生孢子,浓度为1×107-108孢子/mL。
本申请还提供球孢白僵菌与卵寄生蜂联合,间接作用于草地贪夜蛾,对其实现生物防治的作用。
一种草地贪夜蛾杀虫剂,所述草地贪夜蛾杀虫剂为将球孢白僵菌分生孢子粘附于草地贪夜蛾的卵寄生蜂寄主卵表面。
草地贪夜蛾的卵寄生蜂主要有夜蛾黑卵蜂Telenomus remus和赤眼蜂属Trichogramma sp.,有研究报道夜蛾黑卵蜂、短管赤眼蜂T.pretiosum、螟黄赤眼蜂T.chiloni对草地贪夜蛾卵的寄生率均在90%以上,具有很好的田间防控潜力。
优选的,球孢白僵菌为Bb20091317浓度为1×104-108孢子/mL。
进一步,将球孢白僵菌分生孢子粘附于夜蛾黑卵蜂或玉米螟赤眼蜂寄主卵表面;
优选的,所述寄主卵为柞蚕卵、米蛾卵、斜纹夜蛾卵或者草地贪夜蛾卵。
一种防治草地贪夜蛾的方法,所述方法为将球孢白僵菌分生孢子粘附于草地贪夜蛾的卵寄生蜂寄主卵表面,待卵寄生蜂羽化后,将球孢白僵菌孢子传递至草地贪夜蛾卵表面,将球孢白僵菌孢子传递至草地贪夜蛾卵表面。
可选的,所述草地贪夜蛾的卵寄生蜂包括夜蛾黑卵蜂、玉米螟赤眼蜂、短管赤眼蜂、螟黄赤眼蜂;
进一步,所述球孢白僵菌与草地贪夜蛾的卵寄生蜂联合是指将球孢白僵菌分生孢子粘附于寄生玉米螟赤眼蜂的柞蚕卵表面,待玉米螟赤眼蜂羽化后,将球孢白僵菌孢子传递至草地贪夜蛾卵表面;
进一步,所述球孢白僵菌与草地贪夜蛾的卵寄生蜂联合是指将球孢白僵菌分生孢子粘附于寄生夜蛾黑卵蜂的斜纹夜蛾卵或者草地贪夜蛾卵表面,待夜蛾黑卵蜂羽化后,将球孢白僵菌孢子传递至草地贪夜蛾卵表面。
优选的,所述寄主卵为柞蚕卵、米蛾卵、斜纹夜蛾卵或者草地贪夜蛾卵,所述卵为寄生了夜蛾黑卵蜂或玉米螟赤眼蜂的卵。
进一步,所述球孢白僵菌为Bb20091317,优选的,球孢白僵菌为Bb20091317浓度为1×104-108孢子/mL。
一种防治草地贪夜蛾的方法:
获取草地贪夜蛾的卵寄生蜂寄主卵;
配置助剂溶液均匀喷施于夜蛾黑卵蜂或玉米螟赤眼蜂寄生10天左右的寄主卵表面;
将寄主卵置于球孢白僵菌Bb20091317分生孢子粉剂,球孢白僵菌Bb20091317粘附于柞蚕卵表面;
待寄主卵里面的夜蛾黑卵蜂或玉米螟赤眼蜂羽化后释放,所述夜蛾黑卵蜂或玉米螟赤眼蜂为载球孢白僵菌的夜蛾黑卵蜂或玉米螟赤眼蜂;
载球孢白僵菌的夜蛾黑卵蜂或玉米螟赤眼蜂将球孢白僵菌孢子传递至草地贪夜蛾卵表面,进而将球孢白僵菌孢子传递至草地贪夜蛾卵表面。
可选的,所述助剂溶液为淀粉溶液0.1%(W/V)、葡萄糖溶液0.1%(W/V)、吐温-80溶液0.1%(V/V)或无菌水。
一种防治草地贪夜蛾的方法:
获取寄生了夜蛾黑卵蜂的斜纹夜蛾卵或者草地贪夜蛾卵;
配置助剂溶液均匀喷施于夜蛾黑卵蜂寄生10天左右的斜纹夜蛾卵或者草地贪夜蛾卵表面;
将斜纹夜蛾卵或者草地贪夜蛾卵置于球孢白僵菌Bb20091317分生孢子粉剂,球孢白僵菌Bb20091317粘附于斜纹夜蛾卵或者草地贪夜蛾卵表面;
待斜纹夜蛾卵或者草地贪夜蛾卵里面的夜蛾黑卵蜂羽化后释放至温室或大田,所述夜蛾黑卵蜂为载球孢白僵菌的夜蛾黑卵蜂;
载球孢白僵菌的夜蛾黑卵蜂将球孢白僵菌孢子传递至草地贪夜蛾卵表面,进而将球孢白僵菌孢子传递至草地贪夜蛾卵表面。
一种防治草地贪夜蛾的方法:
获取寄生了玉米螟赤眼蜂的柞蚕卵;
配置助剂溶液均匀喷施于玉米螟赤眼蜂寄生10天左右的柞蚕卵表面;
将柞蚕卵置于球孢白僵菌Bb20091317分生孢子粉剂,球孢白僵菌Bb20091317粘附于柞蚕卵表面;
待柞蚕卵里面的玉米螟赤眼蜂羽化后释放至温室或大田,所述玉米螟赤眼蜂为载球孢白僵菌的玉米螟赤眼蜂;
载球孢白僵菌的玉米螟赤眼蜂将球孢白僵菌孢子传递至草地贪夜蛾卵表面,进而将球孢白僵菌孢子传递至草地贪夜蛾卵表面。
本发明的技术优势:
1本申请筛选了草地贪夜蛾高致病力菌株球孢白僵菌为Bb20091317,在1×108孢子/mL分生孢子浓度下,菌株Bb20091317对草地贪夜蛾3龄幼虫的累计校正致死率高达95.59%,3龄幼虫在1×107和1×108孢子/mL的Bb20091317浓度下的累计校正死亡率分别为62.50%和83.33%;
2本申请进一步提供了球孢白僵菌为Bb20091317对草地贪夜蛾的间接生物防治方法,并对联合防治的风险进行了评估,将夜蛾黑卵蜂和玉米螟赤眼蜂暴露于Bb20091317的1×104-108孢子/mL悬浮液的滤纸上1h后,两种蜂的死亡率与对照相比无显著差异,根据国际生物防治组织的规定,Bb20091317和夜蛾黑卵蜂和玉米螟赤眼蜂联合防治的风险属于无或者低风险,即菌株与夜蛾黑卵蜂和玉米螟赤眼蜂是相容的,可用于联合生物防治。
附图说明
图1是Bb20091317和MrCDTLJ1侵染后草地贪夜蛾3龄幼虫的生存曲线;
图2是不同浓度Bb20091317和MrCDTLJ1对草地贪夜蛾3龄幼虫的致病力;A、Bb20091317对草地贪夜蛾3龄幼虫的累计校正死亡率;B、MrCDTLJ1对草地贪夜蛾3龄幼虫的累计校正死亡率;
图3是不同浓度Bb20091317和MrCDTLJ1处理24h后对夜蛾黑卵蜂和玉米螟赤眼蜂成蜂的死亡率;A.Bb20091317对夜蛾黑卵蜂的致死率;B.Bb20091317对玉米螟赤眼蜂的致死率;C.MrCDTLJ1对夜蛾黑卵蜂的致死率;D.MrCDTLJ1对玉米螟赤眼蜂的致死率。
具体实施方式
下面结合具体实施例,进一步阐述本发明,仅用于解释本发明,而不能理解为对本发明的限制。本领域的普通技术人员可以理解:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。
实施例1高致病力菌株筛选
1.1材料
草地贪夜蛾是本实验饲养种群,采集于云南省德宏州芒市(24°26′1.64″N,98°35′17.34″E)玉米田,饲养到F3代用于实验,饲养温度(26±1)℃,光周期14L:10D,相对湿度为(60±5)%。利用夜蛾科通用人工饲料饲养至3龄幼虫进行试验;球孢白僵菌Bb20091317购买自苏州三亩良铺农业科技有限公司、Bb1237、Bb201017和金龟子绿僵菌Ma189由中国农业科学院植物保护研究所杂草鼠害与草地害虫组提供,莱氏绿僵菌MrCDTLJ1由广西农业科学院植物保护研究所生物防治研究室害虫生物防治研究团队提供;玉米品种为郑单958。
1.2实验方法
1.2.1高致病力菌株筛选
采用浸叶法(于健等,2020)筛选高致病力菌株。用0.05%吐温-80无菌溶液将供试菌株分生孢子配制成1×108孢子/mL孢子悬浮液备用。将V3时期玉米叶片剪成1cm2正方形,然后放入不同菌株的菌液中浸泡60s,取出后置于无菌滤纸上晾干,待其晾干后放入12孔组织培养皿中,每个培养皿分别接入1头饥饿4h的草地贪夜蛾3龄幼虫,待带菌叶片取食尽后再用未处理叶片饲喂。以浸渍0.05%吐温-80无菌溶液处理为对照。每组24头3龄幼虫,重复3次。处理后置于温度(26±1)℃、相对湿度(60±5)%、光周期14L:10D的人工气候箱中饲养。
接虫后每天观察记录草地贪夜蛾3龄幼虫的死亡数,处理组以幼虫变黑(僵虫)或体表长出菌丝的幼虫视为致死昆虫,连续记录10d,计算幼虫的累计死亡率和致死中时LT50。
1.2.2致死中浓度测定
将筛选出的高致病力菌株配制成1×104-108孢子/mL 5个浓度梯度的分生孢子悬浮液备用。致病力测定和调查方法同1.2.1。每24头3龄幼虫为一组,重复3次,计算幼虫的累计死亡率和致死中浓度LC50。
1.3数据统计与分析
实验原始数据用Excel进行整理,用SPSS 19.0软件进行处理分析,统计各处理试虫的累计死亡率和累计校正死亡率。利用单因素ANOVA检验对实验数据进行差异显著性分析。采用Probit方法计算致死中时LT50以及求回归方程和计算致死中浓度LC50。用GraphpadPrism 8.0.2.263软件对浓度-时间生物测定进行生测分析。
结果与分析
草地贪夜蛾3龄幼虫高致病力菌株的筛选
毒力测定结果显示,在1×108孢子/mL分生孢子浓度下,菌株Bb20091317和MrCDTLJ1对草地贪夜蛾3龄幼虫的累计校正致死率较高,分别为95.59%和97.06%,而Bb1237、Bb201017和Ma189对草地贪夜蛾3龄幼虫的致死率低,最高仅为13.89%(表1)。Bb20091317和MrCDTLJ1对草地贪夜蛾3龄幼虫的LT50值分别为3.42d和5.72d(表1)。生存曲线结果表明,在第3d时,Bb20091317处理下的草地贪夜蛾3龄幼虫存活率为38.89%,在第8d时仅有4.17%的幼虫存活。MrCDTLJ1处理下的幼虫在第5d才开始死亡,第5d存活率为55.56%,第8d的存活率为2.78%(图1)。
表1不同菌株对草地贪夜蛾3龄幼虫的致病力
Bb20091317和MrCDTLJ1对草地贪夜蛾3龄幼虫的致死中浓度
图2结果显示,随着孢子液浓度的增加,Bb20091317和MrCDTLJ1对草地贪夜蛾3龄幼虫的累计校正死亡率逐渐升高。Bb20091317和MrCDTLJ1在不同浓度下对3龄幼虫的致病力差异显著(Bb20091317,F5,12=63.292,P<0.05;MrCDTLJ1,F5,12=75.861,P<0.05)。3龄幼虫在1×107和1×108孢子/mL的Bb20091317浓度下的累计校正死亡率分别为62.50%和83.33%,在1×106、1×107和1×108孢子/mL的MrCDTLJ1浓度下的累计校正死亡率分别为80.56%、93.06%和95.85%。通过Probit方法,得到Bb20091317对3龄幼虫的LC50为7.05×106孢子/mL,95%置信区间为3.68×106-1.45×107孢子/mL,P<0.001,MrCDTLJ1对3龄幼虫的LC50为2.25×105孢子/mL,95%置信区间为8.51×104-5.32×105孢子/mL,P<0.001(表2)。
表2 Bb20091317和MrCDTLJ1对草地贪夜蛾3龄幼虫的致死中浓度
实施例2与卵寄生蜂的相容性研究
2.1材料
夜蛾黑卵蜂Telenomus remus由安徽科技学院赠予,玉米螟赤眼蜂Trichogrammaostriniae是本实验饲养种群,分别用实验室饲养的草地贪夜蛾卵块和玉米螟卵块饲养,成蜂用10%的蜂蜜水饲喂。饲养温度为(27±1)℃,光周期16L:8D,相对湿度为(60±5)%;
2.2实验方法
Bb20091317和MrCDTLJ1对夜蛾黑卵蜂和玉米螟赤眼蜂成蜂存活率的影响:参考Rannback et al.(2015)的方法,稍作修改。采用残膜生物测定法测试Bb20091317和MrCDTLJ1对初孵的夜蛾黑卵蜂和玉米螟赤眼蜂的直接毒性。分别将浓度为1×104-108孢子/mL的Bb20091317和MrCDTLJ1分生孢子悬浮液均匀喷洒在滤纸上,然后将滤纸覆盖在55.6cm2玻璃管中,以0.05%吐温-80溶液为对照。每个管中引入20头成蜂(<24小时日龄),每组20头,3次重复,使其在管中暴露1h后,记录死蜂数量。然后将活蜂引入新的管中,并饲喂10%的蜂蜜水,24h后检查并记录管中存活和死亡的成蜂数量(日光照射下轻敲试管不动即为死亡蜂)。
Bb20091317与玉米螟赤眼蜂联合对草地贪夜蛾的毒力测定:在无菌操作台中用接菌针将Bb20091317菌株的分生孢子挑至90mm一次性培养皿中,用小镊子夹走分生孢子粉中较大的菌丝块及杂质,只留下细小的孢子粉,薄薄的铺满培养皿底部,用电动吸虫器吸取刚刚羽化出来的玉米螟赤眼蜂成虫,移入培养皿中,盖上培养皿盖子,待玉米螟赤眼蜂在铺满孢子粉的培养皿中爬行数十秒后,挑取触角和足部粘有孢子,翅膀不沾菌的玉米螟赤眼蜂,放置一个新的培养皿中,用蜂蜜水(蜂蜜:无菌水=1:9)进行饲养,置于24±1℃,光周期为L:D=16:8的人工气候箱中。每日固定时间来记录草地贪夜蛾死亡数。用不携菌的刚羽化的玉米螟赤眼蜂作为对照。
Bb20091317与夜蛾黑卵蜂联合对草地贪夜蛾的毒力测定:在无菌操作台中用接菌针将Bb20091317菌株的分生孢子挑至90mm一次性培养皿中,用小镊子夹走分生孢子粉中较大的菌丝块及杂质,只留下细小的孢子粉,薄薄的铺满培养皿底部,用电动吸虫器吸取刚刚羽化出来的夜蛾黑卵蜂成虫,移入培养皿中,盖上培养皿盖子,待夜蛾黑卵蜂在铺满孢子粉的培养皿中爬行数十秒后,挑取触角和足部粘有孢子,翅膀不沾菌的夜蛾黑卵蜂,放置一个新的培养皿中,用蜂蜜水(蜂蜜:无菌水=1:9)进行饲养,置于24±1℃,光周期为L:D=16:8的人工气候箱中。每日固定时间来记录草地贪夜蛾死亡数。用不携菌的刚羽化的夜蛾黑卵蜂作为对照。
结果与分析
Bb20091317和MrCDTLJ1对夜蛾黑卵蜂和玉米螟赤眼蜂成虫存活率的影响:成蜂存活率的测定结果显示,当将夜蛾黑卵蜂和玉米螟赤眼蜂分别暴露于Bb20091317、MrCDTLJ1的1×104-108孢子/mL悬浮液的滤纸上1h后,两种蜂的死亡率与对照相比无显著差异,处理组与对照组蜂的存活率均在100%。暴露24h后,夜蛾黑卵蜂和玉米螟赤眼蜂在1×104-106孢子/mL的Bb20091317处理下和在1×104孢子/mL的MrCDTLJ1处理下的死亡率与对照相比均无显著差异(图3)。两种蜂在Bb20091317处理下的最高的死亡率分别为15.00%和18.33%(图3A和图3B),在MrCDTLJ1处理下的最高的死亡率分别为18.33%和20.00%(图3C和图3D)。相对于不携带菌的对照组,Bb20091317与夜蛾黑卵蜂、玉米螟赤眼蜂的联合对草地贪夜蛾均有很好的防治效果。
Claims (10)
1.球孢白僵菌在防治草地贪夜蛾中的应用。
2.根据权利要求1所述的应用,其特征在于,所述球孢白僵菌的浓度为1×107-108孢子/mL。
3.根据权利要求1所述的应用,其特征在于所述球孢白僵菌为Bb20091317。
4.一种草地贪夜蛾杀虫剂,其特征在于,所述草地贪夜蛾杀虫剂含有权利要求1-3任意一项所述的球孢白僵菌。
5.根据权利要求4所述的草地贪夜蛾杀虫剂,其特征在于,所述草地贪夜蛾杀虫剂剂型为球孢白僵菌分生孢子悬浮液或粉剂;优选的,所述孢子悬浮液有效成分为球孢白僵菌为Bb20091317菌株的分生孢子,浓度为1×107-108孢子/mL。
6.一种草地贪夜蛾杀虫剂,其特征在于,所述草地贪夜蛾杀虫剂为将权利要求1-3任意一项所述的球孢白僵菌分生孢子粘附于草地贪夜蛾的卵寄生蜂寄主卵表面。
7.根据权利要求6所述的草地贪夜蛾杀虫剂,其特征在于,将球孢白僵菌分生孢子粘附于夜蛾黑卵蜂或玉米螟赤眼蜂寄主卵表面;优选的,采用淀粉溶液、葡萄糖溶液或吐温-80溶液将球孢白僵菌分生孢子粘附于夜蛾黑卵蜂或玉米螟赤眼蜂寄主卵表面。
8.一种防治草地贪夜蛾的方法,其特征在于,所述方法为将球孢白僵菌分生孢子粘附于草地贪夜蛾的卵寄生蜂寄主卵表面,待卵寄生蜂羽化后,将球孢白僵菌孢子传递至草地贪夜蛾卵表面,将球孢白僵菌孢子传递至草地贪夜蛾卵表面。
9.根据权利要求8所述的应用,其特征在于,所述球孢白僵菌与草地贪夜蛾的卵寄生蜂联合是指将球孢白僵菌分生孢子粘附于夜蛾黑卵蜂或玉米螟赤眼蜂寄主卵表面,待夜蛾黑卵蜂或玉米螟赤眼蜂羽化后,将球孢白僵菌孢子传递至草地贪夜蛾卵表面;
优选的,所述防治草地贪夜蛾的方法为:
获取寄生了玉米螟赤眼蜂的柞蚕卵;
配置助剂溶液均匀喷施于玉米螟赤眼蜂寄生10天左右的柞蚕卵表面;
将柞蚕卵置于球孢白僵菌Bb20091317分生孢子粉剂,球孢白僵菌Bb20091317粘附于柞蚕卵表面;
待柞蚕卵里面的玉米螟赤眼蜂羽化后释放至温室或大田,所述玉米螟赤眼蜂为载球孢白僵菌的玉米螟赤眼蜂;
载球孢白僵菌的玉米螟赤眼蜂接触草地贪夜蛾卵表面,进而将球孢白僵菌孢子传递至草地贪夜蛾卵表面;
优选的,所述防治草地贪夜蛾的方法为:
获取寄生了夜蛾黑卵蜂的斜纹夜蛾卵或者草地贪夜蛾卵;
配置助剂溶液均匀喷施于夜蛾黑卵蜂寄生10天左右的斜纹夜蛾卵或者草地贪夜蛾卵表面;
将斜纹夜蛾卵或者草地贪夜蛾卵置于球孢白僵菌Bb20091317分生孢子粉剂,球孢白僵菌Bb20091317粘附于斜纹夜蛾卵或者草地贪夜蛾卵表面;
待斜纹夜蛾卵或者草地贪夜蛾卵里面的夜蛾黑卵蜂羽化后释放至温室或大田,所述夜蛾黑卵蜂为载球孢白僵菌的夜蛾黑卵蜂;
载球孢白僵菌的夜蛾黑卵蜂接触草地贪夜蛾卵表面,进而将球孢白僵菌孢子传递至草地贪夜蛾卵表面。
10.根据权利要求8或9所述的应用,其特征在于,所述寄主卵为柞蚕卵、米蛾卵、斜纹夜蛾卵或者草地贪夜蛾卵,可选的,所述助剂溶液为淀粉溶液0.1%、葡萄糖溶液0.1%、吐温-80溶液0.1%或无菌水;可选的,所述球孢白僵菌为Bb20091317,优选的,球孢白僵菌为Bb20091317浓度为1×104-108孢子/mL。
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