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-  2016 

Screening and identification of cry genes from Bacillus thuringiensis isolates highly toxic to the larvae of Holotrichia oblita

DOI: 10.13802/j.cnki.zwbhxb.2016.03.018

Keywords: 苏云金芽胞杆菌 多样性分析 大黑鳃金龟甲 cry8基因 组织病理
Bacillus thuringiensis diversity analysis Holotrichia oblita cry8 histopathology

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为获得对大黑鳃金龟甲幼虫具有较高杀虫活性的菌株,利用拌土法测定了本实验室分离的500株苏云金芽胞杆菌(Bacillus thuringiensis,Bt)对大黑鳃金龟甲的杀虫活性,建立Bt菌株比较鉴别技术来进行多样性分析,并对菌株的晶体形态及幼虫感染Bt后的中肠组织切片进行观察。结果显示,从500株Bt菌株中筛选到42株对大黑鳃金龟甲幼虫具有不同程度活性的菌株,分属于14个菌株类型;其中有10株校正死亡率大于60%,261-1菌株杀虫活性最高,7 d校正死亡率达100%。从14个菌株类型中各选取1个代表菌株进行基因鉴定,仅P65-1、1126-1、FCD114和78-2菌株分别含有cry8Ma、cry8Ca、cry8Ab、cry8Ga、cry8Ea共5个cry8类新基因,其它10个菌株均不含cry3、cry8、cry18、cry23、cry37、cry43等对鞘翅目害虫有效的杀虫基因。14个菌株中,78-3、127-2和1198-1菌株能产生双锥体型晶体,261-1、FCD114、P65-1、FTL84、78-2和1126-1菌株能产生球形晶体。幼虫感染261-1、1198-1、FCD114、1126-1、QDL40-2菌株后,中肠组织发生明显的病理变化:2 d时肠壁细胞明显出现空洞化,排列疏松,4 d时受到严重破坏并脱离底膜。表明筛选到的Bt菌株具有防治大黑鳃金龟甲幼虫的潜力。
In order to obtain Bacillus thuringiensis (Bt) isolates with higher insecticidal activity for the larvae of Holotrichia oblita, 500 Bt isolates in the laboratory were investigated; the diversity analysis method was developed, and the crystal shapes were observed in Bt isolates. Furthermore, the histopathological changes were observed in the midgut of H. oblita larvae infected by a mixture of crystals and spores produced by tested isolates by using histopathological slice. The results showed that 42 isolates were found toxic to larvae of this pest, and these isolates were classified into 14 different types, of which ten isolates had a corrected mortality of over 60%, and the isolate 261-1 had the highest toxicity reaching 100% in seven days after infection. One isolate was selected from each type of isolates to identify cry genes. The results indicated that four isolates, P65-1, 1126-1, FCD114 and 78-2, contained five novel cry8 genes, i.e. cry8Ma, cry8Ca, cry8Ab, cry8Ga and cry8Ea, and the other ten isolates, including the isolate 261-1, did not contain cry genes toxic to H. oblita larvae, such as cry3, cry8, cry18, cry23, cry37and cry43. Three isolates, 78-3, 127-2 and 1198-1, contained double pyramid-shaped crystals, and spherical crystals were observed in six isolates, 261-1, FCD114, P65-1, FTL84, 78-2 and 1126-1. Isolates 261-1, 1198-1, FCD114, 1126-1 and QDL40-2 could cause obvious histopathological changes in larval midgut. The midgut cells became loose arrangement and appeared with obvious vacuolization, and were seriously damaged and even deviated from the membrane. The Bt isolates screened in this study may have applications in biological control of H. oblita larvae.


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