Abstract：Lepidopteran pests cause huge losses to agricultural production every year, and long-term application of chemical pesticides cause serious damage to the ecological environment. Bacillus thuringiensis(Bt) is widely used as microbial insecticide because of its high insecticidal activity and environmental friendliness. The aim of this study was to isolate Bt strains from the soil of Liaoning province and to characterize them on the basis of the morphology of insecticidal crystal proteins (ICPs), the cry gene, the ICPs profiles and the insecticidal activities against lepidopteran pests. The temperature screening method was applied to isolate the wild Bt strains. The cry genotypes were analyzed by restriction fragment length polymorphism PCR (PCR-RFLP). The ICPs profiles were analyzed by SDS-PAGE, and the isolates were tested for their insecticidal activities against 5 species of Lepidopteran pests. A total of 524 soil samples were collected from different areas of Liaoning province, and 66 wild Bt strains were isolated, with a prevalence rate of 12.6%. The ICPs of the strains exhibited varied shapes such as rhombus, sphere, short biconical and irregular shape. The isolated strains expressed ICPs, including 130, 90 and 60 kD proteins. The insecticidal toxicity test showed that Bt strains DD229, DD214-2, YK524 and YK540 had high activities against the tested Lepidoptera pests. The results showed that Bt was abundant in the soils of Liaoning province. And 4 strains that showed high insecticidal activities against Lepidopteran pests which would have good research and application value.
倪鹤嘉, 程雪, 王静, 高泽平, 魏盼盼, 谭佳丽, 孙敬, 李海涛, 高继国. 辽宁省部分地区对鳞翅目害虫高毒力的苏云金芽胞杆菌菌株的分离与筛选[J]. 农业生物技术学报, 2019, 27(8): 1513-1520.
NI He-Jia, CHENG Xue, WANG Jing, GAO Ze-Ping, WEI Pan-Pan, TAN Jia-Li, SUN Jing, LI Hai-Tao, GAO Ji-Guo. Isolation and Screening of High Virulent Bacillus thuringiensis Strains Against Lepidopteran Pests in Some Areas of Liaoning Province. 农业生物技术学报, 2019, 27(8): 1513-1520.
[1] 雷梦英. 2015. 苏云金芽胞杆菌cry5Ba3基因的表达及对松材线虫的毒杀效果[D]. 硕士学位论文, 浙江农林大学, 导师: 张立钦; 王勇军. pp. 11-20. (Lei M Y.2015. the Expression of Bacillus thuringiensis cry5Ba3 and its lethal effect on pine wood nematode[D]. Thesis for M.S., Zhejiang Agriculture and Forestry University, Supervisor: Zhang L Q; Wang Y J, pp. 11-20.) [2] 李帅, 张茜, 高剑, 等. 2016. 牡丹江火山口原始森林苏云金芽胞杆菌的分离和cry基因鉴定[J]. 牡丹江师范学院学报(自然科学版), 97(4): 47-49. (Li S, Zhang Q, Gao J, et al.2016. Reserch on the distribution and cry genetic diversity of Bacillus thuringiensis from the primeval forests of volcanic vent in Mudanjiang[J]. Journal of Mudanjiang Normal University, 97(4): 47-49.) [3] 刘东明. 2011. 苏云金芽胞杆菌新型cry1基因克隆、表达及活性分析[D]. 硕士学位论文, 东北农业大学, 导师: 高继国. pp. 29-33. (Liu D M.2011. Cloning, expression and activity analysis of a novel cry1 gene from Bacillus thuringiensis[D]. Thesis for M.S., Northeast Agriculture University, Supervisor: Gao J G, pp. 29-33.) [4] 刘琴, 马谈斌, 祁建杭, 等. 2011. 苏云金芽胞杆菌毒素蛋白和粘虫颗粒体病毒对甜菜夜蛾中肠围食膜的破坏作用[J]. 中国生物防治学报, 27(2): 182-187. (Liu Q, Ma T B, QI J H, et al.2011. Damaging effects of PuGV-Ps and Bacillus thuringiensis on peritrophic membrane of Spodoptera exigua[J]. Chinese Journal of Biological Control, 27(2): 182-187.) [5] 茅洁瑜. 2011. 对鞘翅目害虫具有活性的新型cry基因的研究[D]. 硕士学位论文, 华侨大学, 导师: 林毅; 张杰. pp.19-27. (Mao J Y.2011. Investigation of novel cry genes specific to coleopteran pests[D]. Thesis for M.S., Huaqiao University, Supervisor: Lin Y; Zhang J, pp. 19-27.) [6] 曲慧东, 孙明, 谷祖敏,等. 2005. 辽宁土壤中苏云金芽孢杆菌分布调查[J]. 植物保护, 31(3): 71-74. (Qu H D,Sun M, Gu Z M, et al.2005. A preliminary investigation on the distribution of Bacillus thuringiensis in soil in Liaoning Province[J]. Plant Protection, 31(3): 71-74.) [7] 宋福平, 张杰, 谢天健, 等. 1998. 苏云金芽胞杆菌cry基因PCR-RFLP鉴定体系的建立[J]. 中国农业科学, 31(3): 13-18. (Song F P, Zhang J, Xie T J, et al.1998. Establishment of PCR-RFLP identification system for Bacillus thuringiensis cry gene[J]. Scientia Agricultura Sinica,31(3): 13-18.) [8] 孙棋棋. 2018. 侵蚀环境中土壤微生物群落. 变化特征[D]. 博士学位论文, 中国科学院大学(中国科学院教育部水土保持与生态环境研究中心), 导师: 郭胜利, pp. 11-15. (Sun Q Q.2018. Characteristics of soil microbial community change in erosive environment[D]. Thesis for Ph.D., University of Chinese Academy of Sciences (Institute of Soil and Water Conservation), Supervisor: Guo S L, pp. 11-15.) [9] 赵银娟, 魏炜, 李荣鹏, 等. 2008. 一株海洋来源的高活性Bt菌的生物学特性研究[J]. 微生物学杂志, (04): 43-46. (Zhao Y J, Wei W, Li R P, et al. 2008. (Biological characteristics of highly active Bt strain of marine origin[J]. Journal of Microbiology, (04): 43-46.) [10] Bravo A, Sarabia S, Lopez L, et al.1998. Characterization of cry genes in Mexican Bacillus thuringiensis strain collection[J]. Applied and Environmental Microbiology, 64(12): 4965-4972. [11] Bukhari D A,Fatima N,Rehman A.2018. Molecular characterization of Cry11 crystal protein gene from Bacillus thuringiensis isolated from different soil samples[J]. Pakistan Journal of Zoology, 50(6): 2351-2356. [12] Crialesi-Legori P C B, Davolos C C, Lemes A R N, et al.2014. Interaction of Cry1 and Vip3A proteins of Bacillus thuringiensis for the control of lepidopteran insect pests[J]. Pesquisa Agropecuária Brasileira, 49(2): 79-87. [13] Georgina S, Raviraj B, Twyman M, et al.2011. Bacillus thuringiensis: A century of research, development and commercial applications[J]. Plant Biotechnology Journal, 9(3): 283-300. [14] Jara S, Maduell P, Orduz S.2006. Diversity of Bacillus thuringiensis strains in the maize and bean phylloplane and their respective soils in Colombia[J]. Journal of Applied Microbiology, 101(1): 117-24. [15] Kotik M, Kočanová M, Marešová H, et al.2004. High-level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer[J]. Protein Expression and Purification, 36(1): 61-69. [16] Kuo W S, Chak K F.1996. Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA[J]. Applied & Environmental Microbiology, 62(4): 1369-1377. [17] Li Z, Jin D Y.2017. Isolation and biological determination of Bacillus thuringiensis with high toxicity in soil of Changbai Mountain Area[J]. Agricultural Science & Technology, 18(3): 506-508; 513. [18] Maeda M, Mizuki E, Nakamura Y, et al.2000. Recovery of Bacillus thuringiensis from marine sediments of Japan[J]. Current Microbiology, 40(6): 418-422. [19] Martínez C, Ibarra J, Caballero P.2005. Association analysis between serotype, cry gene content, and toxicity to Helicoverpa armigera larvae among Bacillus thuringiensis isolates native to Spain[J]. Journal of Invertebrate Pathology, 90(2): 91-97. [20] Patel K D, Bhanshali F C, Chaudhary A V, et al.2013. A new enrichment method for isolation of Bacillus thuringiensis from diverse sample types[J]. Applied Biochemistry and Biotechnology, 170(1): 58-66. [21] Pigott C R, King M S, Ellar D J, et al.2008. Investigating the properties of Bacillus thuringiensis Cry proteins with novel loop replace-ments created using combinatorial molecular biology[J]. Applied and Environmental Microbiology, 74: 3497-3511. [22] Sauka D H, Benintende G B, 2017. Diversity and distribution of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Argentina[J]. Revista Argentina de Microbiología, 49(3): 273-281. [23] Saadaoui I, Al-Thani R, Al-Saadi F, et al.2010. Characterization of Tunisian Bacillus thuringiensis strains with abundance of kurstaki subspecies harbouring insecticidal activities against the lepidopteran insect Ephestia kuehniella[J]. Current Microbiology, 61(6): 541-548. [24] Salama H S, Abd El-Ghany N M, Saker M M, 2015. Diversity of Bacillus thuringiensis isolates from Egyptian soils as shown by molecular characterization[J]. Journal of Genetic Engineering and Biotechnology, 13(2): 101-109. [25] Schnepf H E, Whiteley H R, 1981. Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli[J]. Proceedings of the National Academy of Sciences of the USA, 78(5): 2893-2897. [26] Schnepf E, Crickmore N, van Rie J, et al.1998. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiology and Molecular Biology Review, 62(3): 755-806. [27] Soares-da-Silvaa J, Pinheiro V C S, Litaiff-Abreu E, et al.2015. Isolation of Bacillus thuringiensis from the state of Amazonas, in Brazil, and screening against Aedes aegypti (Diptera, Culicidae)[J]. Revista Brasileira de Entomologia, 59(1): 1-6. [28] Song F P, Zhang J, Gu A X, et al.2003. Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene[J]. Applied & Environmental Microbiology, 69(9): 5207-5211. [29] Thamthiankul S, Moar W J, Miller M E, et al.2004. Improving the insecticidal activity of Bacillus thuringiensis subsp.aizawai against Spodoptera exigua by chromosomal expression of a chitinase gene[J]. Applied Microbiology and Biotechnology, 65(2): 183-192. [30] Wang J H, Boets A, Van Rie J, et al.2003. Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China[J]. Journal of Invertebrate Pathology, 82(1): 63-71. [31] Whiteley H R, Schnepf H E.1986. The molecular biology of parasporal crystal body formation in Bacillus thuringiensis[J]. Annual Review of Microbiology, 40: 549-576. [32] Zhang H Y, Yu Z N, Deng W X, 2000. Composition and ecological distribution of cry proteins and their genotypes of Bacillus thuringiensis isolates from warehouses in China[J]. Journal of Invertebrate Pathology, 76(3): 191-197 [33] Zhang W F, Neil C, Zenas G, et al.2012. Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensis-An alternative to Bti?[J]. Journal of Invertebrate Pathology, 109(2012): 217-222. [34] Zhang L L,Lin J,Luo L, et al.2010. A novel Bacillus thuringiensis strain LLB6, isolated from bryophytes,and its new cry2Ac-type gene[J]. Letters in Applied Microbiology, 44(3): 301-307.
