【作者】 温发园；

【导师】 尹新明； 张永安；

【作者基本信息】 河南农业大学 ， 农业昆虫与害虫防治， 2005， 硕士

【摘要】 松墨天牛(Monochamus alternatus)是我国重要的林业害虫,除取食危害马尾松(Pinus massoniana Lamb)、油松(Pinus tabulae formis Carr)等松树外,还传播松材线虫病(Bursaphelenchus xyphduslo),已对我国松树造成严重危害。微孢子虫是专性细胞内寄生的单细胞原生动物,是一类重要的昆虫病原微生物,它主要寄生在昆虫体内,通过水平传播和垂直传播引起昆虫疾病的发生与流行,是自然界中制约昆虫种群密度的重要因素之一。作为一种生物杀虫剂,它能持续控制害虫并对环境安全。但国内外尚无运用微孢子虫防治天牛的报道。白僵菌是一类重要的生防真菌,在生产上应用广泛,防治松墨天牛在生产上也早有应用,目前多利用其孢子粉剂或液剂,但存在施用方法不便和防治效果容易受外界环境的影响等局限性,在应用中受到很大限制。 本研究主要包括两个方面:1) 以从松墨天牛体内分离到的一种微粒子虫为研究对象,观察其形态特征,超微结构、明确其分类地位,调查了其野外流行情况,以期为松墨天牛的生物防治提供理论与实践依据。2) 从白僵菌中寻找对松墨天牛有毒杀作用的蛋白,该研究可从生物化学和分子生物学的角度揭示虫生真菌的致病机理,同时为培育抗天牛苗木奠定基础。 通过对分离出的微孢子虫的形态和超微结构观察认为其应归于微粒子属,命名为松墨天牛微粒子虫(Nosema alternatus Zhang);野外调查证明不同地点松褐天牛均有不同程度的微粒子虫感染,说明其广泛存在于自然界,是昆虫的制约因子之一。 选取12株有代表性的白僵菌菌株,通过室内接种松褐天牛筛选出对松褐天牛高毒力的两个菌株,Bb6和Bb8,两个菌株在25℃下对松褐天牛的致死中浓度分别是3.34×10~7孢子/ml和5.76×10~7孢子/ml;比较了菌株Bb6接种松褐天牛后天牛体内酯酶同工酶谱变化,松墨天牛感染Bb6后其酯酶谱明显增加1条E5酶带,E1、E2、E3、E4酶带比健虫的相应酶带颜色深,说明Bb6可明显引起松墨天牛幼虫的身体病变,而致其酯酶同工酶发生了变化;用硫酸铵沉淀浓缩、透析的方法提取了Bb6胞外全蛋白提取液,再通过制备电泳和洗脱凝胶的方法从胞外全蛋白提取液中分离纯化出了5种单一蛋白。 通过对5种单一蛋白的生测,筛选出1种高毒性蛋白,松墨天牛幼虫经毒蛋白处理后,前期多表现为拒食,而后行动迟缓、不爱活动,在饲料中的钻蛀活动明显减少,多数时间停卧在饲料中不动,死亡前多从饲料中钻出,在饲料表面痉挛、抽搐,不久死亡。刚死亡的幼虫体色正常无明显症状,死亡后1～2天内虫体软化、变褐、变黑。有些试虫经毒蛋白处理后虽然未死亡但不能正常化蛹,化蛹为畸形,不能羽化,羽化出的成虫不正常。 毒性最强的蛋白Bb6W-D经SDS-PAGE电泳后解离为2个亚基,分子量较大的一个为大亚基,较小的一个为小亚基。经SDS-PAGE电泳测定,大亚基的分子量约为24kD,小亚基的分子量约为17kD;经等电聚焦电泳测定,大亚基的pI为9.59,小亚基的pI为9.40。该蛋白N-末端起始的10个氨基酸序列为:E→P→M→G→I→P→G→E→P→T更多还原

【Abstract】 Monochamus alternatus is a serious forest pest which not only attacks pine trees but also drills in the host tree stems and branches, and it is also a major vector of pine wood nematode (Bursaphelenchus xylophelus).It has already endangered our country’s pines seriously. Microsporida is a kind of unicellular protozoa which parasitizes in the living cell specially and it is a kind of important entomopathogen.It’s efficient to control the insect populations persistently and it’s safe for the enviroment as a bio-pestcide. It can cause insect’s disease to prevail through propagate or transmit vertically and levelly.So it is one of the important factors which restricts insect’s populations in the nature. But there is no report using Microsporida to control the longhorn beetles both at home and abroad. Beauveria sp is a kind of important biological control agent which has been widely applied to control insect pest. It is for some years to control M. alternatus by Beauveria sp. But it is limited to control pest because only spore powder or liquid can be used.This research includes two parts mainly: 1)Microsporida was extracted from Monochamus alternatus and its characteristic and ultrastructure were observed in order to define its categorised position. And its epidemic situation was investigated in the field in order to provide the theory and practice basis for biological control of the Monochamus alternatus. 2) Extracting toxic proteins to M. alternatus from Beauveria sp will reveal the mechanism how the fungi infect their host from the view of biochemistry and molecular biology and it is also the foundation for cultivating anti-longhorn beetles trees .Through observing its exterior characteristic and ultrastructure we think this kind of Microsporida is belong to Nosema and name it Microsporida of Monochamus alternatus (Nosema alternatus Zhang ) Field investigation prove there are Microsporida infection to the Monochamus alternatus in different places. And Microsporida which exist in the nature extensively is one of the restriction factors of the insects .Twelve representative Beauveria sp strains were choosed from the collections in our laboratory. The result of bioassy for 12 strains shows that Bb6 and Bb8 have high toxic to Monochamus alternatus under 25℃, Bb6 and Bb8’s LC50 is3.34×10~7spores/ml and 5.76×10~7 spores/ml; There are significant difference in zymograms of esterase isozymes between the host larvae infected with Bb6 and the control. The zymograms of the treated increase band E5, and the color of the band E1、 band E2、 band E3、 bandE4 is heavier than the更多还原