CN104073441B - 一株蝗虫绿僵菌及其应用 - Google Patents
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Abstract
本发明涉及一种蝗虫绿僵菌的制备方法及应用,尤其对防治蝗虫类害虫有效。本发明提供的蝗虫绿僵菌Metarhizium acridum TRCC21601,其在中国微生物菌种保藏管理委员会普通微生物中心的登记入册编号为CGMCC No.7992。该菌株产孢能力强,对菱蝗3龄跳蝻杀虫活性强,LT50(致死中时)为5.59天。利用该菌株能有效控制菱蝗的种群数量。另外,蝗虫绿僵菌对其他害虫松毛虫、玉米螟、蛴螬等也具有很好的防效,值得推广应用。
Description
技术领域
一株蝗虫绿僵菌的制备方法及应用
背景技术
蝗虫是蝗科,直翅目昆虫。全世界有超过10,000种。分布于全世界的热带、温带的草地和沙漠地区。口器坚硬,前翅狭窄而坚韧,后翅宽大而柔软,善于飞行,后肢很发达,善于跳跃。蝗虫是农、林、牧业生态系统的重要组成部分,不少有害蝗种对农、林、牧业可造成不同程度危害。全世界的蝗虫已有1万种以上,其中对农、林、牧业可造成危害的蝗虫约300种左右,全球除南极洲、欧亚大陆北纬550以北地区外均可发生蝗虫。
多年来草原蝗虫一直采用化学农药防治,对迅速控制蝗害的发生、扩散起到了积极作用。但化学农药治蝗存在成本高、用量大、安全性差等弊端且大量使用易造成人畜中毒、污染草原同时杀灭蝗虫天敌危害草原尘态环境破坏生态平衡。因此生物防治技术是目前草原灾害综合治理的积极有效的措施。白僵菌是一种半知菌类的虫生真菌,具有营养器官--菌丝和繁殖器官-分生孢子,菌丝有横隔有分枝。白僵菌的分布范围很广,从海拔几米至2000多米的高山均发现过白僵菌的存在,白僵菌可以侵入6个目15科200多种昆虫、螨类的虫体内大量繁殖,同时不断产尘白僵素(大环脂类毒素)和草酸钙结晶,这些物质可引起昆虫中毒,使体液发现机能发生变化,打乱新陈代谢以致死亡。所谓白僵虫就是白僵菌的分生孢子落在昆虫体上,在合适温湿条件下,即可发芽直接侵入昆虫体内,以昆虫体内的血细胞及其他组织细胞作为营养,大量增殖,以后菌丝穿出体表,产生白粉状分生孢子,从而使害虫呈白色僵死状,称为白僵虫。白僵菌制剂对人、畜无毒,对作物安全,无残留、无污染,但能感染家蚕幼虫,形成僵蚕病。
发明内容
本发明要解决的技术问题是提供了一株蝗虫绿僵菌。
本发明所提供的蝗虫绿僵菌Metarhizium acridum TRCC21601,其在中国微生物菌种保藏管理委员会普通微生物中心的登记入册编号为CGMCCNo.7992。该菌株已于2013年08月13日把藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)。
本发明所要解决的另一个技术问题是提供一种杀虫菌剂。
本发明提供的杀虫菌剂,它的活性成分是上述蝗虫绿僵菌Metarhizium acridumTRCC21601。
所述杀虫菌剂用于防治以下害虫的几种或一种:松毛虫、玉米螟、蛴螬、蝗虫、马铃薯甲虫、松褐天牛、白蚁、茶小绿叶蝉、桃小食心虫等。
本发明的蝗虫绿僵菌Metarhizium acridum TRCC21601对3龄蝗蝻具有高致病力。
菌种名称:蝗虫绿僵菌
拉丁名:Metarhizium acridum
菌种编号:TRCC21601.
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
地址:北京市朝阳区北辰西路1号院3号
保藏日期:2013年08月13日
保藏中心登记入册编号:CGMCC No.7992
以下结合具体实施实例详细描述本发明,这些实例仅仅是用于理解本发明而非限制。
实施实例1
蝗虫绿僵菌的菌株鉴定
分子系统学鉴定
DNA提取方法:采用石英砂研磨CTAB提取法(Scott et al.,2000)。
遗传标记:选用翻译延伸因子(tefl-alpha)基因(tef),扩增引物:EF1T,EF2T(Bischoffet al.,2006)。
PCR扩增反应条件:先94摄氏度加热3分钟使模板DNA变性,然后进入下列温度循环:94摄氏度变性30秒,50摄氏度退火30秒,72摄氏度延伸45秒,共进行35个循环,最后在72摄氏度延伸5分钟(Wang,2012)。
PCR产物检测和测序:PCR产物与100bpDNAladder(MBIFermentas)用2.0%的琼脂糖凝胶(agarosegel)在0.5×TBE电泳缓冲液中于80V电压下电泳20分钟,然后置于0.5μg/ml的溴乙锭(EB,ehidium bromide)溶液中染色15分钟,在波长365和254nm的紫外光下检测为明显单一条带的产物由擎科生物技术有限公司纯化后用ABi3700(Applied Biosystems)进行双向直通测序。
数据处理和统计学分析:原始序列用生物软件Bioedit7.0.9(Hall,1999)编辑后得到准确无误的序列,与GenBank下载的模式和权威菌株序列组成序列数据矩阵。对于序列矩阵,在Bioedit7.0.9下做必要的人工编辑和调整得到的新矩阵。将该矩阵用MEGA5.0(Tamura et al.,2011)进行邻居连接法(neighbor-joining,NJ)分析,推断出系统发育树,并用自展法(bootstrap)进行1000次重复评估各分支的可靠性(Fig.1,见下页)。
菌株TRCC21601tef序列:
TRCC21601
5’-TCACATCAACGTGGTCGTTATCGTAAGTCACCTGCCTCTATTTCGAACCTTGTACGAGCTTTTATACTGACTTGGTCGTCGTAGGGGTATGTTTTGGAGCCTACACTCTTCGCCGTCTCGAATTTTGATAACTAACTGGTCCTGTCAGCCACGTCGACTCCGGCAAGTCTACCACCACTGGTCACTTGATCTACCAGTGCGGTGGTATCGACAAGCGTACCATTGAGAAGTTCGAGAAGGTAAGCCAAAATCCCGTTCAATGAATTGCTGTCGTTTGGCGATGAAAGTTATTGGGTTTCTCGCTGCCTGTCGGCCACTACCCCTCACTGTGACACGAAAATTTTCGCGGGGCCTTGTCTTGGACTTTGATGGGGCATCTATCCCCGCCAGCTGTCGAGAGTGTGGTCTGGCTGTTGAAACCACAATGTTTTCGATGCTTTTAGATACCAAAAAAAAACATGAAGCTAATCTGCATCGCTGTATAGGAAGCCGCCGAACTCGGTAAGGTCCTTCAAGTACGCATGGGTTCTTGACAAGCTCAAGGCCGAGCGTGAGCGTGGTATCACCATCGACATTGCCCTCTGGAAGTTCGAGACTCCCAAGTATTATGTCACCGTCATTGGTATGTCGATTGTCAAACTGACAGATACTTCTCTCCCGACTTGAATGCTAATGTTCCTCTCATAGACGCTCCCGGTCACCGTGATTTCATCAAGAACAT-3’
系统发育树
蝗虫绿僵菌的室内杀蝗特性测定
该实验设两个处理,分别是蝗虫绿僵菌Metarhizium acridum TRCC21601实验组和空白对照组。
实验处理方法如下:25摄氏度下,接种孢子量为1.0×108个/mL,处理笼放入菱蝗(pygmy grasshoppers)3龄蝗蝻,每个处理有3个重复,每组处理30头蝗虫,药剂用量为5ml/30头,利用小喷雾器直接喷雾于养虫笼内。计录死虫数,记录存活率。经SPSS17.0统计分析得到LT50(致死中时)。结果表明,蝗虫绿僵菌Metarhizium acridum TRCC21601对菱蝗(pygmy grasshoppers)3龄蝗蝻的LT50为5.59天(见表1)。对其他害虫的防治效果均大于90%(见表2)。
表1蝗虫绿僵菌CGMCC No.7992室内杀蝗活性测定
表2蝗虫绿僵菌CGMCC No.7992对害虫的防治效果
附图说明
图1蝗虫绿僵菌Metarhizium acridum TRCC21601CGMCC No.7992)的16S rDNA序列。
图2蝗虫绿僵菌Metarhizium acridum TRCC21601CGMCC No.7992)基于16sRNA序列构建的系统发育树。
Claims (7)
1.一株蝗虫绿僵菌(Metarhizium acridum),其保藏编号为CGMCC No.7992。
2.权利要求1所述的蝗虫绿僵菌在产生分生孢子或菌丝体中的应用。
3.一种杀虫菌剂,它的活性成分是权利要求1所述的蝗虫绿僵菌。
4.根据权利要求3所述的杀虫菌剂,其特征在于:所述的蝗虫绿僵菌为分生孢子或菌丝体。
5.根据权利要求3或4所述的杀虫菌剂,其特征在于:所述杀虫菌剂用于防治以下害虫的几种或一种:松毛虫、玉米螟、蛴螬、蝗虫、马铃薯甲虫、松褐天牛、白蚁、茶小绿叶蝉、桃小食心虫。
6.权利要求1所述的蝗虫绿僵菌在制备杀虫剂中的应用。
7.权利要求3或4所述的杀虫菌剂在制备杀虫剂中的应用。
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