Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (6): 1112-1121.doi: 10.3864/j.issn.0578-1752.2015.06.08

• PLANT PROTECTION • Previous Articles     Next Articles

Insecticidal Protein Genes of Bacillus thuringiensis Strain CPB012 and Its Effects in Controlling Different Insect Pests

LU Hui-hui, LIN Zhi-qiang, TAN Wan-zhong, LUO Hua-dong, XIAN Fei, BI Chao-wei, YU Yang, YANG Yu-heng   

  1. Research Institute of Alien Invasive Species, College of Plant Protection, Southwest University, Chongqing 400716
  • Received:2014-09-27 Online:2015-03-16 Published:2015-03-16

Abstract: 【Objective】The objective of this study is to identify the functional genes, with their expressed proteins, and insecticidal spectrum of Bacillus thuringiensis strain CPB012. This will be important for effective application of CPB012 in pest control in the future.【Method】CPB012 was cultured at 30℃ on LB plates for two to three days and the bacterial cells were stained with phenol magenta for microscopy observation of the protein crystals. cry1-cry10 were identified with the polymerase chain reaction-restriction frame length polymorphism (PCR-RFLP) technique, namely, a universal primer pair for a group of genes was designed and used to amplify the genes with PCR. The amplified genes were then catalyzed with restriction enzymes and thus the polymorphism of the insecticidal genes were decided. On the other hand, specific primers were designed for identifying sub-group or genes (such as cry1A, cry2Aa. etc.) in groups cry1-cry10 via PCR amplification and then these genes were sequenced and compared. cry1-cry40 and vip groups were identified with PCR through the design of primers. The crystal proteins were extracted, respectively, from the bacterial cells cultured on LB plates for 24, 47, 72 and 96 hours and SDS-PAGE chromatographic fingerprint analysis was applied to demonstrate different insecticidal proteins. The lethality of Bombyx mori (Lepidoptera), Leptinotarsa decemlineata (Cleoptera) and Bactrocera dorsalis (Diptera) by CPB012 was measured in the laboratory, the cooperative effects of CPB012 and MJ07 strains of Beauveria bassiana were tested under both laboratory and field conditions. 【Result】Continuous microscopic observations found that CPB012 produced spherical and cubic protein crystals in blastosporic cells. Gene type analysis showed that B. thuringiensis CPB012 contained cry1Aa, cry1Ia, cry2Aa, cry2Ab and vip3A genes. These genes were shown via SDS-PAGE chromatographic fingerprint to code crystal proteins of about 135, 80, 70, and 85-90 kD, respectively. Also, the bacterium was demonstrated to contain two new crystal proteins which were 30 and 50 kD. Laboratory tests showed that CPB012 infected B. mori, B. dorsalis and L. decemlineata caused more than 76% deaths of each pest. After a mixture of B. thuringiensis CPB012 and B. bassiana MJ07 (mixed at 0.5: 0.5) was applied in a laboratory test, the cumulative mortality rate of 2nd-instar L. decemlineata larvae was 96.5%, which was significantly higher than that of individual CPB012 (83.3%) and that of MJ07 (90.1%). The LC50 calculated from the multiple regression equation was 1.10×107 cfu/mL and the CTC was 254, which indicated the combined use of the two biocontrol agents increased pest control efficacy markedly. Under the local field condition, the mixture of CPB012+MJ07 treatment also improved the pest control effect markedly; the cumulative mortality of the 1st- and 2nd-instar larvae were 47.5% and 43.8%, respectively, which were significantly higher than those of separate CPB012, MJ07 and commercial Bt preparation.【Conclusion】The insecticidal function of B. thuringiensis CPB012 was decided by cry1Aa, cry1Ia, cry2Aa, cry2Ab and vip3A and these genes encoded insecticidal proteins of 135, 80, 70, and 85-90 kD. This bacterium had wide spectrum of insecticidal activity which was shown by high death rates of B. mori, B. dorsalis and L. decemlineata induced by CPB012 treatment. The insecticidal efficacy was significantly improved by combined application of CPB012 of B. thuringiensis and MJ07 of B. bassiana. Therefore, CPB012 is thought of as a wide-spectrum biocontrol agent and will be of potential significance and great value in developing effective bio-insecticide.

Key words: Bacillus thuringiensis CPB012, functional genes, insecticidal crystal proteins, Leptinotarsa decemlineata, Beauveria bassiana MJ07, biological control

[1]    罗华东, 严加林, 余洋, 谭万忠. 马铃薯甲虫病原细菌分离及生防菌的筛选与鉴定. 中国农业科学, 2012, 45(18): 3744-3754.
Luo H D, Yan J L, Yu Y, Tan W Z. Isolation, screening and identification of bacterial agents for biological control of Colorado potato beetle (Leptinotarsa decemlineata). Scientia Agricultura Sinica, 2012, 45(18): 3744-3754. (in Chinese)
[2]    Grafius E. Economic impact of insecticide resistance in the Colorado potato beetle (Coleoptera: Chrysomelidae) on the Michigan potato industry. Journal of Economic Entomology, 1997, 90(5): 1144-1151.
[3]    Jiang W H, Guo W C, Lu W P, Shi X Q, Xiong M H, Wang Z T, Li G Q. Target site insensitivity mutations in the AChE and LdVssc1 confer resistance to pyrethroids and carbamates in Leptinotarsa decemlineata in northern Xinjiang Uygur autonomous region. Pesticide Biochemistry and Physiology, 2011, 100: 74-81.
[4]    李源, 赵珮, 尹春艳, 刘小侠, 张青文. 多种植物挥发物及马铃薯甲虫聚集素对马铃薯甲虫的引诱作用. 昆虫学报, 2010, 53(7): 734-740.
Li Y, Zhao P, Yin C Y, Liu X X, Zhang Q W. Attraction of Leptinotarsa decanlineata (Say) (Coleoptera Chrysomelidae) by several plant volatiles and aggregation pheromone. Acta Entomologica Sinica, 2010, 53(7): 734-740. (in Chinese)
[5]    Bravo A, Likitvivatanaavong S, Gill S S, Soberón M. Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochemistry and Molecular Biology, 2011, 41: 423-431.
[6]    Raymond B, Johnston P R, Nielsen-LeRoux C, Lereclus D, Crickmore N. Bacillus thuringiensis: an impotent pathogen? Trends in Microbiology, 2010, 18(5): 189-194.
[7]    Crickmore N, Zeigler D R, Bravo A, Schnepf E, Lereclus D, Baum J, Narva K, Van Rie J, Sampson K, Sun M. Bacillus thuringiensis toxin nomenclature. 2014, Bt/index. html.
[8]    Bravo A, Sarabia S, Lopez L, Ontiveros H, Abarca C, Ortíz A, Ortíz M, Lina L, Villalobos F J, Peña G, Nuñez-Valdez M E, Soberón M, Quintero R. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Applied and Environmental Microbiology, 1998, 64(12): 4965-4972.
[9]    Bradley D, Harkey M A, Kim M K, Biever K D, Bauer L S. The insecticidal CryIB crystal protein of Bacillus thuringiensis ssp. thuringiensis has dual speci?city to Coleopteran and Lepidopteran larvae. Journal of Invertebrate Pathology, 1995, 65: 162-173.
[10]   Grossi-de-Sa M F, Quezado de Magalhaes M, Silva M S, Silva S M, Dias S C, Nakasu E Y, Brunetta P S, Oliveira G R, Neto O B, Sampaio de Oliveira R, Soares L H, Ayub M A, Siqueira H A, Figueira E L. Susceptibility of Anthonomus grandis (cotton boll weevil) and Spodoptera frugiperda (fall armyworm) to a Cry1Ia-type toxin from a Brazilian Bacillus thuringiensis strain. Journal of Biochemistry and Molecular Biology, 2007, 40 (5): 773-782.
[11]   Naimov S, Weemen-Hendriks M, Dukiandjiev S, de Maagd R A. Bacillus thuringiensis delta-endotoxin Cry1 hybrid proteins with increased activity against the Colorado potato beetle. Applied and Environmental Microbiology, 2001, 67(11): 5328-5330.
[12]   Arrieta G, Hernández A, Espinoza A M. Diversity of Bacillus thuringiensis strains isolated from coffee plantations infested with the coffee berry borer Hypothenemus hampei. Revista de Biologia Tropical, 2004, 52(3): 757-764.
[13]   Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamraev A, Baizhanov M, Glupov V, Margalith Y. Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Applied and Environmental Microbiology, 1999, 65(8): 3714-3716.
[14]   Ben-Dov E, Manasherob R, Zaritsky A, Barak Z, Margalith Y. PCR analysis of cry7 genes in Bacillus thuringiensis by the ?ve conserved blocks of toxins. Current Microbiology, 2001, 42: 96-99.
[15]   Yamaguchi T, Sahara K, Bando H, Asano S. Discovery of a novel Bacillus thuringiensis Cry8D protein and the unique toxicity of the Cry8D-class proteins against scarab beetles. Journal of Invertebrate Pathology, 2008, 99(3): 57-62.
[16]   Zhang J, Hodgman T C, Krieger L, Schnetter W, Schairer H U. Cloning and analysis of the ?rst cry gene from Bacillus popilliae. Journal of Bacteriology, 1997, 179(13): 4336-4341.
[17]   Nazarian A, Jahangiri R, Jouzani G S, Seifinejad A, Soheilivand S, Bagher O, Keshavarzi M, Alamisaeid K. Coleopteran-speci?c and putative novel cry genes in Iranian native Bacillus thuringiensis collection. Journal of Invertebrate Pathology, 2009, 102: 101-109.
[18]   Kuo W S, Chak K F. Identi?cation of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-ampli?ed DNA. Applied and Environmental Microbiology, 1996, 62(4): 1369-1377.
[19]   宋福平, 张杰, 黄大昉, 谢天健, 杨自文, 戴莲韵, 李国勋. 苏云金芽孢杆菌cry基因PCR-RFLP鉴定体系的建立. 中国农业科学, 1998, 31(3): 1-4.
Song F P, Zhang J, Huang D F, Xie T J, Yang Z W, Dai L Y, Li G X. Establishment of PCR-RFLP identification system of cry genes from Bacillus thuringiensis. Scientia Agricultura Sinica,1998, 31(3): 1-4. (in Chinese)
[20]   Zimmermann G. Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii. Biocontrol Science and Technology, 2007, 17(5/6): 553-596.
[21]   Shapiro-Ilan D I, Jackson M, Reilly C C, Hotchkiss M W. Effects of combining an entomopathogenic fungi or bacterium with entomopathogenic nematodes on mortality of Curculio caryae (Coleoptera: Curculionidae). Biological Control, 2004, 30: 119-126.
[22]   罗华东, 王利君, 谭万忠, 周军, 杜喜翠. 马铃薯甲虫虫生真菌分离及强致病菌株筛选. 南京农业大学学报, 2011, 34(1): 61-67.
Luo H D, Wang L J, Tan W Z, Zhou J, Du X C. Fungal isolates from Colorado potato beetles and their pathogenicity to silkworm larvae. Journal of Nanjing Agricultural University, 2011, 34(1): 61-67. (in Chinese)
[23]   Ejiofor A O, Johnson T. Physiological and molecular detection of crystalliferous Bacillus thuringiensis strains from habitats in the South Central United States. Journal of Industrial Microbiology & Biotechnology, 2002, 28: 284-290.
[24]   Jouzani G S, Abad A P, Seifinejad A, Marzban R, Kariman K, Maleki B. Distribution and diversity of Dipteran-specific cry and cyt genes in native Bacillus thuringiensis strains obtained from different ecosystems of Iran. Journal of Industrial Microbiology & Biotechnology, 2008, 35: 83-94.
[25]   Seifinejad A, Salehi Jouzani G R, Hosseinzadeh A, Abdmishani C. Characterization of Lepidoptera-active cry and vip genes in Iranian Bacillus thuringiensis strain collection. Biological Control, 2008, 44: 216-226.
[26]   Chan Y H. Biostatistics 201: linear regression analysis. Singapore Medicine Journal, 2004, 45(2): 55-61.
[27]   Krupke C H, Hunt G J, Eitzer B D, Andino G, Given K. Multiple routes of pesticide exposure for honey bees living near agricultural fields. PLoS ONE, 2012, 7(1): e29268.
[28]   Sun Y P, Sun Y Q.Insect resistance to insecticides and dynamics of insect toxicology. Entomologia Sinica, 1994, 1(3): 217-241.
[29]   Konecka E, Baranek J, Hrycak A, Kaznowski A. Insecticidal activity of Bacillus thuringiensis strains isolated from soil and water. The Scienti?c World Journal, 2012, 2012: Article ID 710501.
[30]   He J, Wang J P, Yin W, Shao X H, Zheng H J, Li M S, Zhao Y W, Sun M, Wang S Y, Yu Z N. Complete genome sequence of Bacillus thuringiensis subsp. chinensis strain CT-43. Journal of Bacteriology, 2011, 193(13): 3407-3408.
[31]   Sedlak M, Walter T, Aronson A. Regulation by overlapping promoters of the rate of synthesis and deposition into crystalline inclusions of Bacillus thuringiensis δ-endotoxins. Journal of Bacteriology, 2000, 182(3): 734-741. 
[32]   Wirth M C, Georghiou G P, Federict B A. CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94: 10536-10540.
[33]   Tailor R, Tippett J, Gibb G, Pells S, Pike D, Jordan L, Ely S. Identification and characterization of a novel Bacillus thuringiensis δ-endotoxin entomocidal to Coleopteran and Lepidopteran larvae. Molecular Microbiology, 1992, 6(9): 1211-1217.
[34]   Wraight S P, Ramos M E. Synergistic interaction between Beauveria bassiana- and Bacillus thuringiensis tenebrionis-based biopesticides applied against field populations of Colorado potato beetle larvae. Journal of Invertebrate Pathology, 2005, 90: 139-150.
[1] SHA YueXia, HUANG ZeYang, MA Rui. Control Efficacy of Pseudomonas alcaliphila Strain Ej2 Against Rice Blast and Its Effect on Endogenous Hormones in Rice [J]. Scientia Agricultura Sinica, 2022, 55(2): 320-328.
[2] CHEN Yang,ZHAO HongYi,YAN JunJie,HUANG Jian,GAO YuLin. Chemical Synthesis View on Sex Pheromones of Potato Tuberworm (Phthorimaea operculella) [J]. Scientia Agricultura Sinica, 2021, 54(3): 556-572.
[3] CAO YuHan,LI ZiTeng,ZHANG JingYi,ZHANG JingNa,HU TongLe,WANG ShuTong,WANG YaNan,CAO KeQiang. Analysis of dsRNA Carried by Alternaria alternata f. sp. mali in China and Identification of a dsRNA Virus [J]. Scientia Agricultura Sinica, 2021, 54(22): 4787-4799.
[4] HU ChangXiong,FAN Wei,ZHANG Qian,CHEN GuoHua,YIN HongHui,XU TianYang,YANG JinBo,YANG Hang,WU DaoHui,ZHANG XiaoMing. Control Effect of Orius similis on Frankliniella occidentalis Based on the Two-Sex Life Table and the Age-Stage-Specific Predation Rate [J]. Scientia Agricultura Sinica, 2021, 54(13): 2769-2780.
[5] LI YangFan,SHAO MeiQi,LIU CHANG,GUO QingGang,WANG PeiPei,CHEN XiuYe,SU ZhenHe,MA Ping. Identification of the Antifungal Active Compounds from Bacillus amyloliquefaciens Strain HMB33604 and Its Control Efficacy Against Potato Black Scurf [J]. Scientia Agricultura Sinica, 2021, 54(12): 2559-2569.
[6] LI Shu,WANG Jie,HUANG NingXing,JIN ZhenYu,WANG Su,ZHANG Fan. Research Progress and Prospect on Banker Plant Systems of Predators for Biological Control [J]. Scientia Agricultura Sinica, 2020, 53(19): 3975-3987.
[7] DONG Cheng,CHEN ZhiYong,XIE YingXin,ZHANG YangYang,GOU PeiXin,YANG JiaHeng,MA DongYun,WANG ChenYang,GUO TianCai. Effects of Successive Biochar Addition to Soil on Nitrogen Functional Microorganisms and Nitrous Oxide Emission [J]. Scientia Agricultura Sinica, 2020, 53(19): 4024-4034.
[8] ZHANG Lei,JIA Qi,WU Wei,ZHAO LuPing,XUE Bing,LIU HuanHuan,SHANG Jing,YONG TaiWen,LI Qing,YANG WenYu. Species Identification and Virulence Determination of Beauveria bassiana Strain BEdy1 from Ergania doriae yunnanus [J]. Scientia Agricultura Sinica, 2020, 53(14): 2974-2982.
[9] SHA YueXia,SUI ShuTing,ZENG QingChao,SHEN RuiQing. Biocontrol Potential of Bacillus velezensis Strain E69 Against Rice Blast and Other Fungal Diseases [J]. Scientia Agricultura Sinica, 2019, 52(11): 1908-1917.
[10] LI YuJia, LI Qian, ZHANG ZhiXiang, LI ShiFang. Screening and identification of peach endophytic bacteria with antagonism against Agrobacterium tumefaciens [J]. Scientia Agricultura Sinica, 2017, 50(20): 3918-3929.
[11] ZHANG Fan, LI Shu, XIAO Da, ZHAO Jing, WANG Ran, GUO Xiao-jun, WANG Su. Progress in Pest Management by Natural Enemies in Greenhouse Vegetables in China [J]. Scientia Agricultura Sinica, 2015, 48(17): 3463-3476.
[12] LIU Lin-Zhou-1, DAI Peng-1, 吕Bing-1 , ZANG Lian-Sheng-1, DU Wen-Mei-1, WAN Fang-Hao-2. Interspecific Competition Between Encarsia sophia and E. formosa and Their Impacts on Suppression of Trialeurodes vaporariorum [J]. Scientia Agricultura Sinica, 2013, 46(22): 4837-4841.
[13] ZHANG Rong-Sheng, WANG Xiao-Yu, LUO Chu-Ping, LIU Yong-Feng, LIU You-Zhou, CHEN Zhi-Yi. Identification of the Lipopeptides from Bacillus amyloliquefaciens Lx-11 and Biocontrol Efficacy of Surfactin Against Bacterial Leaf Streak [J]. Scientia Agricultura Sinica, 2013, 46(10): 2014-2021.
[14] LUO Hua-Dong, YAN Jia-Lin, YU Yang, TAN Wan-Zhong. Isolation, Screening and Identification of Bacterial Agents for Biological Control of Colorado Potato Beetle (Leptinotarsa decemlineata) [J]. Scientia Agricultura Sinica, 2012, 45(18): 3744-3754.
[15] WANG Jing, HUANG Yun, YAO Jia, LIN Shan, LI Xiao-Lan, QIN Yun. Identification and Control Effects of Two Antagonistic Actinomycetes Against Clubroot [J]. Scientia Agricultura Sinica, 2011, 44(13): 2692-2700 .
Full text



No Suggested Reading articles found!