不同饲喂下棉铃虫肠道内生菌的分离鉴定及脱毒效果研究

Isolation, identification and detoxification of intestinal endophytes of Helicoverpa armigera under different feeding

摘要:

【目的】本研究对比饲喂正常饲料和棉粕饲料下棉铃虫（Helicoverpa armigera）肠道中可培养微生物的异同，并对筛选菌株进行棉酚耐受及降解作用的研究，为棉铃虫肠道微生物在棉源饲料中的脱毒作用提供理论和实验支持。【方法】通过饲喂不同饲料、醋酸棉酚单一碳源微生物选择性培养，分离饲喂正常饲料和棉粕饲料下棉铃虫肠道可培养棉酚耐受性内生菌，并进行16S rRNA基因序列分析鉴定；在一定浓度的醋酸棉酚液体中培养，对菌株进行脱毒作用分析，复合配比后，研究棉铃虫肠道复合菌在棉酚降解中的效果。【结果】共计分离得到的32株菌株，其中17株细菌可能是新分类单元，其中14株为潜在新种，3株为可能的新属。饲喂正常饲料组棉铃虫肠道中分离出17株分属于3个门、10个属，饲喂棉粕饲料组棉酚虫肠道中分离出15株属于2个门、8个属。随着选择性培养基醋酸棉酚浓度的升高，饲喂棉粕饲料组肠道菌耐受100、300、500、1 000 mg/kg醋酸棉酚的菌株数均比饲喂正常饲料组高。在所有的耐受菌株中棉酚降解率达到50%以上的有15株，对棉酚最高降解率达90.83%。在肠道菌复合降解棉酚实验中，棉铃虫肠道内生菌实验组对棉酚的降解率显著高于对照组（P<0.05），棉酚降解率最高为87.04%。【结论】本研究证明了棉铃虫肠道内生细菌中存在对棉酚具有较高降解能力的菌株，丰富了棉酚降解菌的菌种资源，并对复合菌在棉源饲料开发应用的潜力进行了初步研究，为棉源饲料中棉酚的生物降解提供了一条新途径。

关键词:棉酚;棉铃虫;肠道内生菌;分离鉴定;脱毒

Abstract:

[Objective] This study compared the differentiation of endophytes in the intestines of Helicoverpa armigera under feeding normal diet and cottonseed meal diet, and studied gossypol tolerance and degradation effects of the screened strains, which provided theoretical and experimental support for further research on the detoxification of Helicoverpa armigera intestinal microorganisms in cotton-derived feed.[Methods] The gossypol tolerant endophytic bacteria were isolated in the intestinal tract of Helicoverpa armigera under different feeding conditions, selective culture of gossypol acetate single carbon source microorganism, and 16S rRNA gene sequence were analyzed and identified. The detoxification effect of the strains was analyzed by cultivated in a certain concentration of gossypol acetate liquid, and the compound ratio was used to study the effect of the compound bacteria on gossypol degradation.[Results] A total of 32 strains were isolated from Helicoverpa armigera, of which 17 strains were potential new taxon, and 14 were potential new species, and 3 were possible potential new genera. 17 strains were isolated from the intestinal tract of Helicoverpa armigera under feeding normal diet, belonging to 10 genera in 3 phyla of bacteria, 15 strains were isolated from the intestinal tract of Helicoverpa armigera under feeding cottonseed meal diet, belonging to 8 genera in 2 phyla of bacteria. With the increase of the concentration of gossypol acetate in the selective medium, the number of intestinal bacteria resistant to 100, 300, 500, 1 000 mg/kg gossypol acetate in the cottonseed meal feed groups were higher than those in the normal feed groups. Among all the tolerant strains, 15 strains had a degradation rate of more than 50%, and the highest degradation rate of gossypol was 90.83%. In the experiment of compound degradation of gossypol by intestinal bacteria, the degradation rate of gossypol in the experimental group were significantly higher than that in the control group (P<0.05), and the highest degradation rate of gossypol was 87.04%.[Conclusion] It was proved that there were some higher ability of degrading gossypol strains in the intestinal endophytic bacteria of Helicoverpa armigera, which enriches the resource of the strains degrading gossypol, and preliminary research on the potential of compound bacteria in the development and application of cotton-derived feed was conducted, which provided a new way for the biodegradation of gossypol in cotton-derived feed.

Key words:gossypol;Helicoverpa armigera;intestinal endophytic bacteria;isolation and identification;detoxification