Skip to main content
SpringerLink
Log in

Identification and analysis of toxins in novel Bacillus thuringiensis strain Bt S3076-1 against Spodoptera frugiperda and Helicoverpa armigera (Lep.: Noctuidae)

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

Despite the successful application of toxins from Bacillus thuringiensis as biological control agents against pests, pests are showing resistance against an increasing number of Bacillus thuringiensis toxins due to evolution; thus, new toxins with higher toxicity and broad-spectrum activity against insects are being increasingly identified. To find new toxins, whole genome sequencing of the novel B. thuringiensis strain Bt S3076-1 was performed, and ten predicted toxic genes were identified in this study, including six cry genes, two tpp genes, one cyt gene and one vip gene, among which six were novel toxins. Subsequently, SDS‒PAGE analysis showed that the major proteins at the spore maturation stage were approximately 120 kDa, 70 kDa, 67 kDa, 60 kDa and 40 kDa, while active proteins after trypsin digestion (approximately 70 kDa and 40 kDa) exhibited LC50 values of 149.64 μg/g and 441.47 μg/g against Spodoptera frugiperda and Helicoverpa armigera larvae, respectively. Furthermore, pathological observation results showed that the peritrophic membrane of Spodoptera frugiperda and Helicoverpa armigera larvae was degraded. These findings will provide an experimental reference for further research on the insecticidal activity, toxicity spectrum and synergism of these toxins in Bt S3076-1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Availability of data and materials

Not applicable.

Abbreviations

Bt:

Bacillus thuringiensis

°C:

Degree centigrade

RH:

Relative humidity

pH:

Potential of hydrogen

rpm:

Revolution per minute

h:

Hour/hours

min:

Minutes

g:

Gram/grams

µg:

Microgram

ml:

Millilitres

µl:

Microlitres

M:

Molar

mM:

Millimolar

V:

Ultraviolet

Kb:

Kilobase

bp:

Base pair

kDa:

Kilo Dalton

References

  • Beltrao HDBM, Silvafilha MHNL (2007) Interaction of Bacillus thuringiensis var. israelensis Cry toxins with binding sites from Aedes aegypti (Diptera: Culicidae) larvae midgut. FEMS Microbiol Lett 266:163–169

    Article  CAS  Google Scholar 

  • Ben-dov E (2014) Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins. Toxins 6:1222–1243

    Article  PubMed  PubMed Central  Google Scholar 

  • Bergamasco VB, Mendes DR, Fernandes OA (2013) Bacillus thuringiensis Cry1Ia10 and Vip3Aa protein interactions and their toxicity in Spodoptera spp. (Lepidoptera). J Invertebr Pathol 112:152–158

    Article  CAS  PubMed  Google Scholar 

  • Berry C, O’Neil S, Ben-Dov E (2002) Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 68:5082–5095

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bi Y, Zhang Y, Shu C, Crickmore N, Wang Q, Du L, Song F, Zhang J (2015) Genomic sequencing identifies novel Bacillus thuringiensis Vip1/Vip2 binary and Cry8 toxins that have high toxicity to scarabaeoidea larvae. Appl Microbiol Biotechnol 99:753–760

    Article  CAS  PubMed  Google Scholar 

  • Boonserm P, Mo M, Angsuthanasombat C (2006) Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-angstrom resolution. J Bacteriol 188:3391–3401

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bravo A, Gómez I, Porta H, García-Gómez BI, Rodriguez-Almazan C, Pardo L, Soberón M (2013) Evolution of Bacillus thuringiensis cry toxins insecticidal activity. Microb Biotechnol 6:17–26

    Article  PubMed  PubMed Central  Google Scholar 

  • Cao B, Nie Y, Guan Z, Chen C, Wang N, Wang Z, Shu C, Zhang J, Zhang D (2022) The crystal structure of Cry78Aa from Bacillus thuringiensis provides insights into its insecticidal activity. Commun Biol 95:801–817

    Article  Google Scholar 

  • Cao B, Shu C, Geng L, Song F, Zhang J (2020) Cry78Ba1, one novel crystal protein from Bacillus thuringiensis with high insecticidal activity against rice planthopper. J Agric Food Chem 68:2539–2546

    Article  CAS  PubMed  Google Scholar 

  • Casida JE, Durkin KA (2017) Pesticide chemical research in toxicology: lessons from nature. Chem Res Toxicol 30:94–104

    Article  CAS  PubMed  Google Scholar 

  • Chakroun M, Banyuls N, Bel Y (2016) Bacterial vegetative insecticidal proteins (Vip) from entomopathogenic bacteria. Microbiol Mol Biol Rev 80:329–350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen YJ, Liu JX (2021) Insecticidal activities of Salvia hispanica L.essential oil and combinations of their main compounds against the beet armyworm Spodoptera exigua. Ind Crops Prod 162:310–324

    Article  Google Scholar 

  • Ehler LE (2004) An evaluation of some natural enemies of Spodoptera exigua on sugar beet in northern California. Biocontrol 49:121–135

    Article  Google Scholar 

  • Elleuch J, Jaoua S, Darriet F (2015) Cry4Ba and Cyt1Aa proteins from Bacillus thuringiensis israelensis: interactions and toxicity mechanism against Aedes aegypti. Toxicon 104:83–90

    Article  CAS  PubMed  Google Scholar 

  • Fatima N, Rehman A, Bukhari DA (2022) Biotoxicity assessment of cloned cry 11 protein gene from Bacillus thuringiensis 9NF. Saudi J Biol Sci 29:103–163

    Article  Google Scholar 

  • Fatoretto JC, Michel AP, Silva FMC (2017) Adaptive potential of fall armyworm (Lepidoptera: Noctuidae) limits Bt trait durability in Brazil. J Integrated Pest Manag 8:1–10

    Article  Google Scholar 

  • Fayad N, Kambris Z, El Chamy L, Mahillon J, Kallassy AM (2020) A novel anti-dipteran Bacillus thuringiensis strain: unusual cry toxin genes in a highly dynamic plasmid environment. Appl Environ Microbiol 11:85–87

    Google Scholar 

  • Fu BW, Xu L, Zheng MX, Chen QX, Shi Y, Zhu YJ (2022) Stability is essential for insecticidal activity of Vip3Aa toxin against Spodoptera exigua. AMB Express 12:88–92

    Article  Google Scholar 

  • Fu X, Feng H, Liu Z, Wu K (2017) Trans-regional migration of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), in North-East Asia. PLoS ONE 25:8–12

    Google Scholar 

  • Grossi-De-Sa MF, Magalhaes MQD, Silva MS (2007) Susceptibility of Anthonomus grandis (cotton boll weevil) and Spodoptera frugiperda (fall armyworm) to a Cry1Ia-type toxin from a Brazilian Bacillus thuringiensis strain. J Biochem Mol Biol 40:773–782

    CAS  PubMed  Google Scholar 

  • Guan P, Dai X, Zhu J, Li Q, Li S, Wang S, Li P, Zheng A (2014) Bacillus thuringiensis subsp. sichuansis strain MC28 produces a novel crystal protein with activity against Culex quinquefasciatus larvae. World J Microbiol Biotechnol 30:1417–1421

    Article  CAS  PubMed  Google Scholar 

  • Hayakawa T, Yoneda N, Okada K (2016) Bacillus thuringiensis Cry11Ba works synergistically with Cry4Aa but not with Cry11Aa for toxicity against mosquito Culex pipiens (Diptera: Culicidae) larvae. Appl Entomol Zool 52:1–8

    Google Scholar 

  • Hernández-Rodríguez CS, Boets A, Van Rie J, Ferré J (2009) Screening and identification of vip genes in Bacillus thuringiensis strains. J Appl Microbiol 107:219–225

    Article  PubMed  Google Scholar 

  • Hernández-Soto A, Cristina M, Rincón-Castro D, Espinoza AM, Ibarra JE (2009) Parasporal body formation via overexpression of the Cry10Aa toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa synergism. Appl Environ Microbiol 75:4661–4667

    Article  PubMed  PubMed Central  Google Scholar 

  • Huang SF, Zhu YJ, Liu B, Ruan CQ, Lin J (2006) Morphological characteristics of parasporal crystals of Bacillus thuringiensis LSZ9408. Wuyi Science 40:37–40

    Google Scholar 

  • Ingber DA, Mason CE, Flexner L (2018) Cry1 Bt susceptibilities of fall armyworm (Lepidoptera: Noctuidae) host strains. J Econ Entomol 111:361–368

    Article  CAS  PubMed  Google Scholar 

  • Jakka SR, Knight VR, Jurat-Fuentes JL (2014) Spodoptera frugiperda (J.E. Smith) with field-evolved resistance to Bt maize are susceptible to Bt pesticides. J Invertebr Pathol 122:52–54

    Article  CAS  PubMed  Google Scholar 

  • Jouzani GS, Valijanian E, Sharafi R (2017) Bacillus thuringiensis: a successful insecticide with new environmental features and tidings. Appl Microbiol Biotechnol 101:2691–2711

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, Chandra A, Pandey KC (2008) Bacillus thuringiensis (Bt) transgenic crop: an environment friendly insect-pest management strategy. J Environ Biol 29:641–653

    CAS  PubMed  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Machado EP, Dos S, Rodrigues Junior GL, Führ FM, Zago SL, Marques LH, Santos AC, Nowatzki T, Dahmer ML, Omoto C, Bernardi O (2020) Cross-crop resistance of Spodoptera frugiperda selected on Bt maize to genetically-modified soybean expressing Cry1Ac and Cry1F proteins in Brazil. Sci Rep 22:10–14

    Google Scholar 

  • Maeda M, Mizuki E, Nakamura Y, Hatano T, Ohba M (2000) Recovery of Bacillus thuringiensis from marine sediments of Japan. Curr Microbiol 40:418–422

    Article  CAS  PubMed  Google Scholar 

  • Mehrkhou F, Talebi AA, Moharramipour S, Naveh VH (2012) Demographic parameters of Spodoptera exigua (Lepidoptera: Noctuidae) on different soybean cultivars. Environ Entomol 41:326–332

    Article  PubMed  Google Scholar 

  • Meissle M, Romeis J, Bigler F (2011) Bt maize and integrated pest management—a European perspective. Pest Manag Sci 67:1049–1058

    CAS  PubMed  Google Scholar 

  • Montezano DG, Specht A, Sosa-Gómez DR (2018) Host plants of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas. Afr Entomol 26:286–300

    Article  Google Scholar 

  • Navon A, Ascher KRS (2000) Bioassys of entomopathogenic microbes and nematodes. CABI Publ Wallingford 10:1–49

    Google Scholar 

  • Panwar BS, Kaur J, Kumar P, Kaur S (2018) A novel cry52Ca1 gene from an Indian Bacillus thuringiensis isolate is toxic to Helicoverpa armigera (cotton boll worm). J Invertebr Pathol 159:137–140

    Article  CAS  PubMed  Google Scholar 

  • Peña-Cardeña A, Grande R, Sánchez J, Tabashnik BE, Bravo A, Soberón M, Gómez I (2018) The C-terminal protoxin region of Bacillus thuringiensis Cry1Ab toxin has a functional role in binding to GPI-anchored receptors in the insect midgut. J Biol Chem 293:20263–20272

    Article  PubMed  PubMed Central  Google Scholar 

  • Qiu L, Zhang B, Liu L, Ma W, Wang X, Lei C, Chen L (2017) Proteomic analysis of Cry2Aa-binding proteins and their receptor function in Spodoptera exigua. Sci Rep 9:402–422

    Google Scholar 

  • Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, Swerdlow H, Turner DJ (2008) A large genome center’s improvements to the Illumina sequencing system. Nat Methods 5:1005–1010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Revina LP, Kostina LI, Ganushkina LA (2004) Reconstruction of Bacillus thuringiensis ssp. israelensis Cry11a endotoxin from fragments correspondingto its N- and c-moieties restores its original biological activity. Biochemistry 69:181–187

    CAS  PubMed  Google Scholar 

  • Sam BJ, Russell DW, Huang PT (2008) Molecular cloning: a laboratory manual, 3d edn. Science Press, Beijing, pp 20–25

    Google Scholar 

  • Siebert MW, Nolting SP, Hendrix W (2012) Evaluation of corn hybrids expressing Cry1F, Cry1A15, Cry2Ab2, Cry34Ab1/Cry35Ab1, and Cry3Bb1 against Southern United States insect pests. J Econ Entomol 105:1825–1834

    Article  CAS  PubMed  Google Scholar 

  • Soaresdasilva J, Pinheiro VCS, Litaiffabreu E (2015) Isolation of Bacillus thuringiensis from the state of Amazonas, in Brazil, and screening against Aedes aegypti (Diptera, Culicidae). Revista Brasileira De Entomologia 59:1–6

    Article  Google Scholar 

  • Sparks AN (1979) A review of the biology of the fall armyworm. Fla Entomol 62:82–87

    Article  Google Scholar 

  • Thiery I, Delécluse A, Tamayo MC (1997) Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative B. thuringiensis strain. Appl Environ Microbiol 63:468–473

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Torres-Quintero MC, Arenas-Sosa I, Zuñiga-Navarrete F, Hernández-Velázquez VM, Alvear-Garcia A-C (2022) Characterization of insecticidal Cry1Cb2 protein from Bacillus thuringiensis toxic to Myzus persicae (Sulzer). J Invertebr Pathol 189:107–731

    Article  Google Scholar 

  • Warren GW (1997) Vegetative insecticidal proteins: novel proteins for control of corn pests. Advances in insecet control: the role of transgenic plants. Taylor and Francis, London, pp 109–122

  • Wei H, Xincheng Z, Abid A (2021) Population dynamics and reproductive developmental analysis of Helicoverpa armigera (Lepidoptera: Noctuidae) trapped using food attractants in the field. J Econ Entomol 114:2–14

    Google Scholar 

  • Wu C, Yao G, Zou M (2007) N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner. Biochem Biophys Res Commun 357:360–365

    Article  CAS  PubMed  Google Scholar 

  • Wu ZQ, Zhou Y, Liu PP, Wei YJ, Zhang Y, Li YZ, Liu SK, Fang XJ (2016) Draft genome sequence of Bacillus thuringiensis strain S3076–1. Bt Research 1:1–7

    Google Scholar 

  • Yang F, Kerns DL, Head GP, Price P, Huang F (2017) Cross-resistance to purified Bt proteins, Bt corn and Bt cotton in a Cry2Ab2-corn resistant strain of Spodoptera frugiperda. Pest Manag Sci 73:2495–2503

    Article  CAS  PubMed  Google Scholar 

  • Zhang B, Liu H, Wang JJ, Zhou X (2008) Advance in the research on Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). Chin Agric Sci Bull 24:427–433 ((in Chinese))

    CAS  Google Scholar 

  • Zhou Y, Wu ZQ, Zhang J, Wan SR, Jin WJ, Li YZ, Fang XJ (2020) Cry80Aa1, a novel Bacillus thuringiensis toxin with mosquitocidal activity to Culex pipiens pallens. J Invertebrate Pathol 173:107386

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants Guangxi Minzu University Introduction of Talent Research Start-up Fund (2020KJQD23), Guangxi Natural Science Foundation (Grant No.2022GXNSFBA035446), The Basic Ability Enhancement Program for Young and Middle-aged Teachers of Guangxi (2022KY0159).

Funding

This study was funded by grants Guangxi Minzu University Introduction of Talent Research Start-up Fund (2020KJQD23), Guangxi Natural Science Foundation (Grant No.2022GXNSFBA035446), The Basic Ability Enhancement Program for Young and Middle-aged Teachers of Guangxi (2022KY0159).

Author information

Authors and Affiliations

  1. Guangxi Key Laboratory for Polysaccharide Materials and Modifications, School of Marine Sciences and Biotechnology, Guangxi Minzu University, Nanning, 530008, People’s Republic of China

    Tianbao Yang, Liuping Li, Mingguo Jiang & Yan Zhou

  2. Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, Hainan, People’s Republic of China

    Zhongqi Wu & Xuanjun Fang

  3. Institute of Life Science, Jiyang College of Zhejiang A&F University, Zhuji, 311800, Zhejiang, People’s Republic of China

    Zhongqi Wu & Xuanjun Fang

  4. Guangxi Lvyounong Biological Technology Co., Ltd, Nanning, 537100, People’s Republic of China

    Wenshan Huang

Authors
  1. Tianbao Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhongqi Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Liuping Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mingguo Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xuanjun Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wenshan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yan Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

TY completed the strain culture, and proteome identification and their analysis conducted the prokaryotic expression of the predicted proteins, performed the larvae bioassays experiments, and wrote and revised the whole manuscript. ZW and LL complete genome sequenced and made genome-wide analysis of strain Bt S3076-1. MJ and XF were involved in project design and manuscript modification. WH participated in the bioassay analysis. YZ is responsible for writing, revising, and finalizing the manuscript. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Yan Zhou.

Ethics declarations

Conflict of interest

Authors declare no competing interests.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Additional information

Communicated by Yusuf Akhter.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, T., Wu, Z., Li, L. et al. Identification and analysis of toxins in novel Bacillus thuringiensis strain Bt S3076-1 against Spodoptera frugiperda and Helicoverpa armigera (Lep.: Noctuidae). Arch Microbiol 205, 168 (2023). https://doi.org/10.1007/s00203-023-03490-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00203-023-03490-3

Keywords

Search

Navigation