对草地贪夜蛾高毒力的苏云金芽胞杆菌菌株筛选与杀虫活性研究

doi:10.16409/j.cnki.2095-039x.2021.03.008

摘要/Abstract

摘要： 草地贪夜蛾是一种世界性重大农业害虫，给玉米等多种主要粮食和经济作物造成严重危害。为实现对草地贪夜蛾的高效、绿色、持续防控，亟需筛选高毒力苏云金芽胞杆菌菌株资源。本研究以前期实验室储备的对斜纹夜蛾、小地老虎具有杀虫活性的363株野生菌株库为候选菌株，基于菌株杀虫基因差异及进化关系，去除冗余菌株后获得172株候选菌株。通过培养获取这些菌株胞晶混合物，进行杀虫蛋白定量后，测定杀虫活性，获得27株对草地贪夜蛾初孵幼虫具有较高杀虫活性的菌株，其中菌株B14-D2的杀虫活性最高，LC₅₀为0.155 μg/g。克隆了该菌株中的cry1Ea3基因并在HD73^-无晶体突变株中表达，Cry1Ea3蛋白对草地贪夜蛾初孵幼虫LC₅₀为1.789 μg/g。本研究为新的Bt产品和杀虫基因的开发利用提供了丰富资源。

关键词: 草地贪夜蛾, 苏云金芽胞杆菌, 生物防治, Cry1Ea蛋白

Abstract: Spodoptera frugiperda (J. E. Smith), a major agricultural pest in the world, causes serious damage to many major food and economic crops, such as corn. It is urgent to screen highly virulent Bacillus thuringiensis (Bt) strain resources to achieve high-efficiency, green and sustained control of S. frugiperda. Based on the difference of insecticidal genes and the evolutionary relationships among strains, 172 candidate strains were obtained from the 363 wild strains by eliminating repetition strains, which were stored in the laboratory and showed insecticidal activity against Spodoptera litura and Agrotis ypsilon. The spores and crystal mixtures of these strains were obtained by culture. Their virulence against S. frugiperda was tested after quantification of insecticidal proteins, and 27 Bt strains were obtained for their high virulence. The most virulent strain was B14-D2, with LC₅₀ of 0.155 μg/g. The cry1Ea3 gene in this strain was cloned and expressed in crystal-free mutant HD73^-. The LC₅₀ of Cry1Ea3 protein against neonates of S. frugiperda was 1.789 μg/g. This research adds to the Bt strain resources for the development of new Bt products.

Key words: Spodoptera frugiperda, Bacillus thuringiensis, biological control, Cry1Ea protein

中图分类号:

S476.1

王建, 杨小雪, 王丹丹, 张杰, 束长龙, 高继国, 耿丽丽. 对草地贪夜蛾高毒力的苏云金芽胞杆菌菌株筛选与杀虫活性研究[J]. 中国生物防治学报, 2021, 37(4): 660-670.

WANG Jian, YANG Xiaoxue, WANG Dandan, ZAHNG Jie, SHU Changlong, GAO Jiguo, GENG Lili. Screening and Insecticidal Activity of Bacillus thuringiensis Strains with High Toxicity against Spodoptera frugiperda[J]. Chinese Journal of Biological Control, 2021, 37(4): 660-670.

参考文献

[1] Sparks A N. A review of the biology of the fall armyworm[J]. The Florida Entomologist, 1979, 62(2):82-87.
[2] 郭井菲, 赵建周, 何康来, 等. 警惕危险性害虫草地贪夜蛾入侵中国[J]. 植物保护, 2018, 44(6):1-10.
[3] Montezano D G, Specht A, Sosa-Gómez D R, et al. Host plants of Spodoptera frugiperda (Lepidoptera:Noctuidae) in the Americas[J]. African Entomology, 2018, 26(2):286-300.
[4] FAO. Integrated management of the fall armyworm on maize[R]. Rome:Food and Agriculture Organization of the United Nations, 2017.
[5] Goergen G, Kumar P L, Sankung S B, et al. First report of outbreaks of the fall armyworm Spodoptera frugiperda (J E Smith) (Lepidoptera, Noctuidae), a new alien invasive pest in west and central Africa[J]. PLoS ONE, 2016, 11(10):e0165632.
[6] FAO. Fall armyworm likely to spread from India to other parts of Asia with South East Asia and South China most at risk[R]. Rome:Food and Agriculture Organization of the United Nations, 2018.
[7] Li Y H, Wang Z Y, Romeis J. Managing the invasive fall armyworm through biotech crops:a Chinese perspective[J]. Trends in Biotechnology, 2020, 39(2):105-107.
[8] 吴孔明. 中国草地贪夜蛾的防控策略[J]. 植物保护, 2020, 46(2):1-5.
[9] Young J R. Fall armyworm:control with insecticides[J]. Florida Entomologist, 1979, 62(2):130-133.
[10] 农业农村部.农业农村部办公厅关于做好草地贪夜蛾应急防治用药有关工作的通知[EB/OL].[2019-06-03]. http://www.moa.gov.cn/gk/tzgg_1/tfw/201906/t20190605_6316201.htm.
[11] Rasko D A, Altherr M R, Han C S, et al. Genomics of the Bacillus cereus group of organisms[J]. FEMS Microbiology Reviews, 2005, 29(2):303-329.
[12] Schenpf E, Crickmore N, Rie V J, et al. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiology and Molecular Biology Reviews, 1998, 62(3):775-806.
[13] Sanahuja G, Banakar R, Twyman R M, et al. Bacillus thuringiensis:a century of research, development and commercial applications[J]. Plant Biotechnology Journal, 2015, 9(3):283-300.
[14] Palma L, Muñoz D, Berry C, et al. Bacillus thuringiensis toxins:an overview of their biocidal activity[J]. Toxins, 2014, 6(12):3296-3325.
[15] Andrade R, Rodriguez C, Oehlschlager A C. Optimization of a pheromone lure for Spodoptera frugiperda (Smith) in central America[J]. Journal of the Brazilian Chemical Society, 2000, 11(6):609-613.
[16] 李红梅, 万敏, 顾蕊, 等. 基于文献计量学的重大入侵害虫草地贪夜蛾的研究动态分析[J]. 植物保护, 2019, 45(4):34-42.
[17] EPA. Details for DIPEL^® DF biological insecticide dry flowable[EB/OL].[2018-12-10]. https://iaspub.epa.gov/apex/pesticides/f?p=PPLS:8:::::P8_PUID,P8_RINUM:15274,73049-39.
[18] Figueiredo S C, Lemes A R N, Sebastião I, et al. Synergism of the Bacillus thuringiensis Cry1, Cry2, and Vip3 proteins in Spodoptera frugiperda control[J]. Applied Biochemistry and Biotechnology, 2019, 188:798-809.
[19] Ingber D A, Mason C E, Flexner L. Cry1 Bt susceptibilities of fall armyworm (Lepidoptera:Noctuidae) host strains[J]. Journal of Economic Entomology, 2018, 111(1):361-368.
[20] Grossi-De-Sa M F, Magalhaes M Q D, Silva M S, et al. Susceptibility of Anthonomus grandis (cotton boll weevil) and Spodoptera frugiperda (fall armyworm) to a Cry1Ia-type toxin from a Brazilian Bacillus thuringiensis strain[J]. Journal of Biochemistry and Molecular Biology, 2007, 40(5):773-782.
[21] Chakroun M, Banyuls N, Bel Y, et al. Bacterial vegetative insecticidal proteins (Vip) from entomopathogenic bacteria[J]. Microbiology and Molecular Biology Reviews, 2016, 80(2):329-350.
[22] Bergamasco V B, Mendes D R, Fernandes O A, et al. Bacillus thuringiensis Cry1Ia10 and Vip3Aa protein interactions and their toxicity in Spodoptera spp. (Lepidoptera)[J]. Journal of Invertebrate Pathology, 2013, 112(2):152-158.
[23] ISAAA. Global status of commercialized biotech/GM crops:2018[J]. ISAAA Briefs, 2018, 54:143.
[24] Siebert M W, Nolting S P, Hendrix W, et al. Evaluation of corn hybrids expressing Cry1F, Cry1A.105, Cry2Ab2, Cry34Ab1/Cry35Ab1, and Cry3Bb1 against Southern United States insect pests[J]. Journal of Economic Entomology, 2012, 105(5):1825-1834.
[25] 张丹丹, 吴孔明. 国产Bt-Cry1Ab和Bt-(Cry1Ab+Vip3Aa)玉米对草地贪夜蛾的抗性测定[J]. 植物保护, 2019, 45(4):54-60.
[26] Fatoretto J C, Michel A P, Silva F M C, et al. Adaptive potential of fall armyworm (Lepidoptera:Noctuidae) limits Bt trait durability in Brazil[J]. Journal of Integrated Pest Management, 2017, 8(1):1-10.
[27] Wang X Q, Shu C L, Jiang J, et al. Screening and identification of cry genes from Bacillus thuringiensis isolates highly toxic to the larvae of Holotrichia oblita[J]. Journal of Plant Protection, 2016, 43(3):483-492.
[28] Calabrese D M, Nickerson K W, Lane L C. A comparison of protein crystal subunit sizes in Bacillus thuringiensis[J]. Canadian Journal of Microbiology, 1980, 26(8):1006-1010.
[29] Song F P, Zhang J, Gu A X, et al. Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene[J]. Applied and Environmental Microbiology, 2003, 69(9):5207-5211.
[30] 单月明. 苏云金芽胞杆菌新型杀虫基因的发掘与活性分析[D]. 哈尔滨:东北农业大学, 2019.
[31] 萨姆布鲁克J, 拉塞尔D W. 黄培堂, 译. 2002分子克隆实验指南(第3版)[M]. 北京:科学出版社, 2008.
[32] Wang G J, Zhang J, Song F P, et al. Engineered Bacillus thuringiensis G033A with broad insecticidal activity against Lepidopteran and Coleopteran pests[J]. Applied Microbiology and Biotechnology, 2006, 72(5):924-930.
[33] Sambrook J, Fritsch E F, Maniatis T. Molecular cloning[M]. New York:Cold Spring Harbor Laboratory Press, 1989.
[34] Lereclus D, Arantès O, Chaufaux J, et al. Transformation and expression of a cloned δ-endotoxin gene in Bacillus thuringiensis[J]. FEMS Microbiology Letters, 1989, 60(2):211-217.
[35] Zhou Z S, Yang S J, Shu C L, et al. Comparison and optimization of the method for Cry1Ac protoxin preparation in HD73 strain[J]. Journal of Integrative Agriculture, 2015, 14(8):1598-1603.
[36] 农药市场信息. 我国批准登记第3个草地贪夜蛾防治药剂[EB/OL].[2020-10-10]. http://jsppa.com.cn/news/yanfa/3547.html.
[37] Monnerat R G, Batista A C, Medeiros P T, et al. Screening of Brazilian Bacillus thuringiensis isolates active against Spodoptera frugiperda, Plutella xylostella and Anticarsia gemmatalis[J]. Biological Control, 2007, 41(3):291-295.
[38] 刘华梅, 胡虓, 王应龙, 等. 对草地贪夜蛾高毒力的苏云金杆菌菌株筛选[J]. 中国生物防治学报, 2019, 35(5):721-728.
[39] Frankenhuyzen K V. Insecticidal activity of Bacillus thuringiensis crystal proteins[J]. Journal of Invertebrate Pathology, 2009, 101(1):1-16.
[40] Chang J H, Roh J Y, Je Y H, et al. Isolation of a strain of Bacillus thuringiensis ssp. kurstaki HD-1 encoding δ-endotoxin Cry1E[J]. Letters in Applied Microbiology, 1998, 26(5):387-390.
[41] IRAC. Susceptibility test method 020, version 3.2[EB/OL].[2011-5]. https://irac-online.org/methods/spodoptera-helicoverpa-heliothis-larvae.
[42] 李国平, 姬婷婕, 孙小旭, 等. 入侵云南草地贪夜蛾种群对5种常用Bt蛋白的敏感性评价[J]. 植物保护, 2019, 45(3):15-20.
[43] Gouffon C, Vliet A V, Rie J V, et al. Binding sites for Bacillus thuringiensis Cry2Ae toxin on Heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin[J]. Applied and Environmental Microbiology, 2011, 77(10):3182-3188.