柞蚕空胴病病原拮抗菌的分离、鉴定及防治效果研究

Screening, identification, and biocontrol effect of bacteria against empty-gut disease of Antheraea pernyi

摘要:

【目的】本研究从辽宁省蚕业科学研究所柞园土壤中分离筛选对柞蚕空胴病病原菌有显著拮抗作用的细菌，为该病的生物防治奠定研究基础。【方法】采用稀释涂布平板法分离柞园土壤中细菌，利用抑菌圈法筛选拮抗效果显著的菌株；根据形态学、生理生化及分子生物学对拮抗菌进行鉴定；利用自然转化法对拮抗菌进行荧光蛋白标记，测定其在柞树叶片和柞蚕肠道内的定殖规律，并对其室内和野外防效进行测定。【结果】从柞园土壤中分离获得87株细菌，其中BF-49对柞蚕肠球菌的拮抗效果显著(P<0.001)；鉴定结果显示该BF-49与贝莱斯芽孢杆菌(Bacillus velezensis)的亲缘关系最近，故将该菌株鉴定为Bacillus velezensis，命名为B. velezensis BF-49。荧光蛋白标记菌株BF-49-GFP在柞蚕肠道中能定殖5 d，在柞树叶片上接种20 d后浓度仍达 1.25×10⁴ CFU/g。BF-49发酵液的10倍稀释液对柞蚕空胴病的室内防效为78.25%，野外防效为74.42%，均显著高于对照药剂。【结论】筛选获得的B. velezensis BF-49对柞蚕空胴病防效显著，可作为开发柞蚕空胴病生防制剂的候选菌株。

Abstract:

[Objective] To isolate and screen bacteria against Enterococcus pernyi (the pathogen of Antheraea pernyi empty-gut disease) from the oak garden soil of Sericultural Research Institute of Liaoning Province and thus to lay a foundation for the biocontrol of the disease. [Methods] Bacteria were isolated from soil by spread plate technique and the antagonistic strain was screened out with the inhibition zone method. The strain was identified according to morphological features, physiological and biochemical characteristics, and molecular biology. The antagonistic strain was labeled with green fluorescent protein with natural transformation method, and the colonization of the antagonistic strain in oak leaves and gut of A. pernyi was determined. The control of empty-gut disease of A. pernyi was identified by indoor and field experiments. [Results] A total of 87 bacterial strains were isolated from soil. Among them, BF-49 was found to have significant antagonistic activity against E. pernyi. The identification results revealed that BF-49 had the closest genetic relationship with Bacillus velezensis. Therefore, it was identified as B. velezensis and named B. velezensis BF-49. The fluorescence-labeled strain BF-49-GFP could colonize in the gut of A. pernyi for 5 days, and the count of BF-49-GFP reached 1.25×10⁴ CFU/g 20 days after inoculation on the oak leaves. The indoor and field control of empty-gut disease by the 10-fold diluted BF-49 fermentation broth was 78.25% and 74.42%, respectively, which were significantly higher than those of the control agents. [Conclusion] The B. velezensis BF-49 has significant control effect on empty-gut disease of A. pernyi, being a candidate strain for developing the biological control agent against empty-gut disease.