Mikrobiol. Z. 2022; 84(4):9-29.
doi: https://doi.org/10.15407/microbiolj84.04.009

Insecticidal and Potato Growth Stimulation Activity of Bacillus thuringiensis kurstaki HD-1

S.A. López-Pazos1, F.M. Chavarrio Cañas1, A.C. Rojas Arias2

1Universidad Antonio Nariño
Carrera 3 Este 47A-15, Bogotá D.C., Colombia

2Fundación Universitaria Agraria de Colombia — UNIAGRARIA
Calle 170 54A-10, Bogotá D.C., Colombia

Bacillus thuringiensis (Bt) produces Cry toxins against pest insects. Cry proteins are conformed by domains related to pore formation and recognition of protein receptors. Plant-induced systemic resistance (ISR) is triggered due to pest attack, it could be activated by Bacillus sp. Tecia solanivora (Ts) is a potato pest, susceptible to Cry1Ac and Cry1B proteins. This paper indicates the endorsement of Bt kurstaki HD-1 (BtkHD1) in relation to Ts control (Cry1Ac and Cry1B proteins), potato growth promotion, and plant ISR due to pests related to the BtkHD1-potato system. To ensure that ongoing quality control of BtkHD1 was maintained, crystal synthesis (microscopy), cry1 genes presence, and Cry protein production were checked. Bioassays Ts larvae and potato plantlets and an in silico analysis of the hybrid Cry1Ac-Cry1Ba protein and potato ISR related to the BtkHD1 infl uence were performed. Bioassay on Ts larvae shows an LC50 of 536 ng/cm2 of diet. A potato growth promotion assay revealed the effect of BtkHD1 on the length and dry weight of stems. The prospective analysis took into account relevant factors affecting the biological function of the hybrid protein focused on domain II. In silico identification of 15 BtkHD1 proteins and 68 potato proteins related to plant ISR due to pests was completed. This project serves to validation of toxicity on Ts larvae and potato growth effect based on BtkHD1, including a forward analysis of the hybrid Cry1Ac1-Cry1Ba1, and proteins associated with this strain and potato for eliciting plant ISR due to pests.

Keywords: Bacillus thuringiensis strain ABTS-351, Cry protein, Tecia solanivora, Solanum tuberosum development promotion, induced systemic resistance.

Full text

  1. Melo AL, Soccol VT, Soccol CR. Bacillus thuringiensis: mechanism of action, resistance, and new applications: a review. Crit Rev Biotechnol. 2016; 36(2):317‒326. https://doi.org/10.3109/07388551.2014.960793
  2. van Frankenhuyzen K. Insecticidal activity of Bacillus thuringiensis crystal proteins. J Invertebr Pathol. 2009; 101(1):1‒16. https://doi.org/10.1016/j.jip.2009.02.009
  3. Pigott CR, Ellar DJ. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol Mol Biol Rev. 2007; 71(2):255‒281. https://doi.org/10.1128/MMBR.00034-06
  4. de Maagd RA, Weemen-Hendriks M, Stiekema W, Bosch D. Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specificity determinant for Spodoptera exigua in different, but not all, Cry1-Cry1C hybrids. Appl Environ Microbiol. 2000; 66(4):1559‒1563. https://doi.org/10.1128/AEM.66.4.1559-1563.2000
  5. Crickmore N, Berry C, Panneerselvam S, Mishra R, Connor TR, Bonning BC. A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins. J Invertebr Pathol. 2020; 107438. https://doi.org/10.1016/j.jip.2020.107438
  6. Pitre L, Hernández-Fernández J, Bernal Villegas J. Toxicidad de delta-endotoxinas recombinantes de Bacillus thuringiensis sobre larvas de la polilla guatemalteca (Tecia solanivora) (Lepidóptera: Gelechiidae). Rev Colomb Biotecnol. 2008; 10:85‒96.
  7. Valderrama AM, Veásquez N, Rodríguez E, et al. Resistance to Tecia solanivora (Lepidoptera: Gelechiidae) in three transgenic Andean varieties of potato expressing Bacillus thuringiensis CrylAc protein. J Econ Entomol. 2007; 100(1):172‒179. https://doi.org/10.1603/0022-0493(2007)100[172:RTTSLG]2.0.CO;2
  8. Frost CJ, Mescher MC, Carlson JE, De Moraes CM. Plant defense priming against herbivores: getting ready for a different battle. Plant Physiol. 2008; 146(3):818‒824. https://doi.org/10.1104/pp.107.113027
  9. Azizoglu U. Bacillus thuringiensis as a Biofertilizer and Biostimulator: a Mini-Review of the Little-Known Plant Growth-Promoting Properties of Bt. Curr Microbiol. 2019; 76(11):1379‒1385. https://doi.org/10.1007/s00284-019-01705-9
  10. Takahashi H, Nakaho K, Ishihara T, et al. Transcriptional profile of tomato roots exhibiting Bacillus thuringiensis induced resistance to Ralstonia solanacearum. Plant Cell Rep. 2014; 33(1):99‒110. https://doi.org/10.1007/s00299-013-1515-1
  11. Deng Q, Wang R, Sun D, et al. Complete Genome of Bacillus velezensis CMT-6 and Comparative Genome Analysis Reveals Lipopeptide Diversity. Biochem Genet. 2020; 58(1):1‒15. https://doi.org/10.1007/s10528-019-09927-z
  12. Harun-Or-Rashid M, Kim HJ, Yeom SI, et al. Bacillus velezensis YC7010 Enhances Plant Defenses Against Brown Planthopper Th rough Transcriptomic and Metabolic Changes in Rice. Front Plant Sci. 2018; 9:1904. https://doi.org/10.3389/fpls.2018.01904
  13. Valenzuela-Soto JH, Estrada-Hernández MG, Ibarra-Laclette E, Délano-Frier JP. Inoculation of tomato plants (Solanum lycopersicum) with growth-promoting Bacillus subtilis retards whitefly Bemisia tabaci development. Planta. 2010; 231(2):397‒410. https://doi.org/10.1007/s00425-009-1061-9
  14. He M, Wilde A, Kaderbhai MA. A simple single-step procedure for small-scale preparation of Escherichia coli plasmids. Nucleic Acids Res. 1990; 18(6):1660. https://doi.org/10.1093/nar/18.6.1660
  15. Cobb BD, Clarkson JM. A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methods. Nucleic Acids Res. 1994; 22(18):3801‒3805. https://doi.org/10.1093/nar/22.18.3801
  16. Bravo A, Sarabia S, Lopez L, et al. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl Environ Microbiol. 1998; 64(12):4965‒4972. https://doi.org/10.1128/AEM.64.12.4965-4972.1998
  17. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248‒254. https://doi.org/10.1016/0003-2697(76)90527-3
  18. Sievers F, Wilm A, Dineen D, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011; 7:539. https://doi.org/10.1038/msb.2011.75
  19. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins M, Appel R. Protein identification and analysis tools on the Ex-Pasy server, In: Walker JM, editor. The proteomics protocols handbook. New York, USA: Human Press; 2005. p. 571‒660. https://doi.org/10.1385/1-59259-890-0:571
  20. Hadzipasic O, Wrabl JO, Hilser VJ. A horizontal alignment tool for numerical trend discovery in sequence data: application to protein hydropathy. PLoS Comput Biol. 2013; 9(10):e1003247. https://doi.org/10.1371/journal.pcbi.1003247
  21. Bjellqvist B, Hughes GJ, Pasquali C, et al. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis. 1993; 14(10):1023‒1031. https://doi.org/10.1002/elps.11501401163
  22. Kolaskar AS, Tongaonkar PC. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett. 1990; 276(1‒2):172‒174. https://doi.org/10.1016/0014-5793(90)80535-Q
  23. Parker JM, Guo D, Hodges RS. New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X-ray-derived accessible sites. Biochemistry. 1986; 25(19):5425‒5432. https://doi.org/10.1021/bi00367a013
  24. Waterhouse A, Bertoni M, Bienert S, et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018; 46(W1):W296‒W303. https://doi.org/10.1093/nar/gky427
  25. Bhattacharya D, Nowotny J, Cao R, Cheng J. 3Drefine: an interactive web server for efficient protein structure refinement. Nucleic Acids Res. 2016; 44(W1):W406‒W409. https://doi.org/10.1093/nar/gkw336
  26. Dulmage HT, Boening OP, Rehnborg CS, Hansen GD. A proposed standardized bioassay for formulations of Bacillus thuringiensis based on the international unit. J Invertebr Pathol. 1971; 18(2):240‒245. https://doi.org/10.1016/0022-2011(71)90151-0
  27. Porcar M, Juárez-Pérez V. PCR-based identification of Bacillus thuringiensis pesticidal crystal genes. FEMS Microbiol Rev. 2003; 26(5):419‒432. https://doi.org/10.1111/j.1574-6976.2003.tb00624.x
  28. Barboza-Corona JE, Delgadillo-Ángeles JL, Castañeda-Ramírez JC, et al. Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74Δsp chitinase inclusions, Cry crystals and spores for applied use. Microb Cell Fact. 2014; 13:15. https://doi.org/10.1186/1475-2859-13-15
  29. Navon A. Bioassays of Bacillus thuringiensis products used against agricultural pests, In: Navon A, editor. Bioassays of entomopathogenic Microbes and Nematodes. Wallingford, UK: CABI Publishing; 2000. p. 1‒24. https://doi.org/10.1079/9780851994222.0001
  30. Jouzani GS, Valijanian E, Sharafi R. Bacillus thuringiensis: a successful insecticide with new environmental features and tidings. Appl Microbiol Biotechnol. 2017; 101(7):2691‒2711. https://doi.org/10.1007/s00253-017-8175-y
  31. Elazouni I, Abdel-Aziz S, Rabea A. Microbial efficacy as biological agents for potato enrichment as well as biocontrols against wilt disease caused by Ralstonia solanacearum. World J Microbiol Biotechnol. 2019; 35(2):30. https://doi.org/10.1007/s11274-019-2596-y
  32. Palma L, Muñoz D, Berry C, Murillo J, Caballero P. Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins (Basel). 2014; 6(12):3296‒3325. https://doi.org/10.3390/toxins6123296
  33. Berry C, Crickmore N. Structural classification of insecticidal proteins ‒ Towards an in silico characterisation of novel toxins. J Invertebr Pathol. 2017; 142:16‒22. https://doi.org/10.1016/j.jip.2016.07.015
  34. Karlova R, Weemen-Hendriks M, Naimov S, Ceron J, Dukiandjiev S, de Maagd RA. Bacillus thuringiensis deltaendotoxin Cry1Ac domain III enhances activity against Heliothis virescens in some, but not all Cry1-Cry1Ac hybrids. J Invertebr Pathol. 2005; 88(2):169‒172. https://doi.org/10.1016/j.jip.2004.11.004
  35. Atsumi S, Mizuno E, Hara H, et al. Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin. Appl Environ Microbiol. 2005; 71(7):3966‒3977. https://doi.org/10.1128/AEM.71.7.3966-3977.2005
  36. Tien MZ, Meyer AG, Sydykova DK, Spielman SJ, Wilke CO. Maximum allowed solvent accessibilites of residues in proteins. PLoS One. 2013; 8(11):e80635. https://doi.org/10.1371/journal.pone.0080635
  37. Karim S, Dean DH. Pesticidal and receptor binding properties of Bacillus thuringiensis Cry1Ab and Cry1Ac delta-endotoxin mutants to Pectinophora gossypiella and Helicoverpa zea. Curr Microbiol. 2000; 41(6):430‒440. https://doi.org/10.1007/s002840010163
  38. Wu SJ, Koller CN, Miller DL, Bauer LS, Dean DH. Enhanced toxicity of Bacillus thuringiensis Cry3A delta-endotoxin in coleopterans by mutagenesis in a receptor binding loop. FEBS Lett. 2000; 473(2):227‒232. https://doi.org/10.1016/S0014-5793(00)01505-2
  39. Creighton TE. Proteins: structures and molecular properties. 2nd Edition. New York, USA: WH Freeman and Company, 1993.
  40. León I, Alonso ER, Mata S, Cabezas C, Alonso JL. Unveiling the Neutral Forms of Glutamine. Angew Chem Int Ed Engl. 2019; 58(45):16002‒16007. https://doi.org/10.1002/anie.201907222
  41. Habka S, Sohn WY, Vaquero-Vara V, et al. On the turn-inducing properties of asparagine: the structuring role of the amide side chain, from isolated model peptides to crystallized proteins. Phys Chem Chem Phys. 2018; 20(5):3411‒3423. https://doi.org/10.1039/C7CP07605C
  42. Oi C, Treado JD, Levine ZA, et al. A threonine zipper that mediates protein-protein interactions: Structure and prediction. Protein Sci. 2018; 27(11):1969‒1977. https://doi.org/10.1002/pro.3505
  43. Biedermannova L, E Riley K, Berka K, Hobza P, Vondrasek J. Another role of proline: stabilization interactions in proteins and protein complexes concerning proline and tryptophane. Phys Chem Chem Phys. 2008; 10(42):6350‒6359. https://doi.org/10.1039/b805087b
  44. Renfrew PD, Butterfoss GL, Kuhlman B. Using quantum mechanics to improve estimates of amino acid side chain rotamer energies. Proteins. 2008; 71(4):1637‒1646. https://doi.org/10.1002/prot.21845
  45. Naimov S, Weemen-Hendriks M, Dukiandjiev S, de Maagd RA. Bacillus thuringiensis delta-endotoxin Cry1 hybrid proteins with increased activity against the Colorado potato beetle. Appl Environ Microbiol. 2001; 67(11):5328‒5330. https://doi.org/10.1128/AEM.67.11.5328-5330.2001
  46. Choudhary DK, Prakash A, Johri BN. Induced systemic resistance (ISR) in plants: mechanism of action. Indian J Microbiol. 2007; 47(4):289‒297. https://doi.org/10.1007/s12088-007-0054-2
  47. Ongena M, Jacques P. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol. 2008; 16(3):115‒125. https://doi.org/10.1016/j.tim.2007.12.009