| Bacillus thuringiensis (Bt) is a bio-pesticide that has been successfully used in the field of pest control for many years. About the mechanism of Bt, several recent reviews provided the explanations of the mechanism or mode of action of Cry toxins from different attitude, and it has been lasted for several decades from1970s. In recent years, some experts have proposed that the insect gut bacteria can inhibit the growth of pathogens or reduce the insect mortality rates caused by pathogen. The existing of intestinal microflora also has influence on the death caused by Bacillus thuringiensis.In this study, we examined the diversity and the structure of the intestinal microbiota community of diamondback moth, Plutella xylostella (L.), from two different treatments, one is the gut microbiota of normal healthy diamondback moth larvae, and the other is the gut microbiota of diamondback moth treated with Bacillus thuringiensis Cry1Ac toxin. Furthermore, we isolated and identified the cultured intestinal bacteria from P. xylostella. We also did some research on the relationship between the insecticidal activity of Bt and part of the cultured intestinal bacteria. This work provide a basis to illustrate the roles of gut microflora and the insecticidal mechanism of Bt, and also offer new ideas to improve the insecticidal activity of Bt for pest control.1. Using PCR-DGGE molecular methods to clarify if there are some differences between the intestinal microbiota community of P. xylostella which is under normal growth conditions and treated with Cry toxins. To explore the differences of the gut bacteria in diamondback moth, two16S rDNA gene clone libraries were constructed. With the comparision of microbial community richness and composition among the two clone libraries, the results showed that the gut microbial species of diamondback moth is abundant, mainly consist of a-Proteobacteria, y-Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. In all the clones, y-Proteobacteria and Firmicutes are the dominant species in the gut, accounting for about94.24%to the total library clones. Enterobacteriaceae, which belonged to y-Proteobacteria, was the most prominent group that was composed of Enterobacter, Pantoea, Klebsiella, Serratia and Citrobacter. There are significant differences between the gut bacteria community structure of two16S rDNA gene clone libraries after analyzing the characteristics, community structure and neighbor-joining phylogennetics tree. Compared to the normal healthy diamondback moth larvae, the number of clones in the libraries of the Bt toxins-treatment which belonged to Serratia, Pantoea, Pseudomonas, Bacillus and Pectobacterium was increased; and the number of Klebsiella and Carnbacterium was decreased. There are only three clones belonged to Curtobacterium and Flavobacterium have been detected.2.80cultured bacteria were obtained from the gut of normal healthy diamondback moth larvae, and then were classified into11representative strains with bacterial16S rDNA gene sequences combined with the results of conventional identification methods characteristic, colony morphology, physiological and biochemical characteristic. Our results revealed that the name of11representative strains were Sphingomonas paucimobilis, Chryseobacterium indologenes, Stenotrophomonas maltophilia, Pseudomonas luteola, Enterobacter cancerogenus, Serratia plymuthica, Pantoea spp, Klebsiella pneumoniae, Enterobacter cloacea, Citrobacter freundii, Enterobacter aerogenes.3. By adding different concentration of gut cultural bacteria to Bacillus thuringiensis CrylAc toxins, we can explore the role of six kinds of gut cultural bacteria to the insecticidal activity of Bt with the changes of mortality. The presented results suggests that Serratia plymuthica and Pantoea spp potentially protected diamondback moth larvae from Bt CrylAc toxins. With the increasing of bacterial concentration, the mortality of diamondback moth larvae decreased. |
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