vuzbiochemiИзвестия вузов. Прикладная химия и биотехнологияProceedings of Universities. Applied Chemistry and Biotechnology2227-29252500-1558ИРНИТУ10.21285/achb.941AQLWEOvuzbiochemi-1344Research ArticleФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯPHYSICOCHEMICAL BIOLOGYЧастота встречаемости cry-подобных генов в штаммах Bacillus thuringiensis Крымской коллекции микроорганизмовFrequency of cry-like genes in Bacillus thuringiensis strains of the Crimean microorganism collectionhttps://orcid.org/0000-0001-5401-0579КрыжкоА. В.KryzhkoA. V.

Крыжко Анастасия Владимировна, к.с-х.н., ведущий научный сотрудник

295453, г. Симферополь, ул. Киевская, 150

Anastasiia V. Kryzhko, Cand. Sci. (Agriculture), Leading Researcher

150, Kievskaya St., Simferopol, 295453

nk_lib@mail.ruНаучно-исследовательский институт сельского хозяйства КрымаРоссияResearch Institute of Agriculture of CrimeaRussian Federation202413012025144567577Copyright © Крыжко А.В., 20242024Крыжко А.В.Kryzhko A.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vuzbiochemi.elpub.ru/jour/article/view/1344

Энтомопатогенные штаммы Bacillus thuringiensis используются для разработки биопрепаратов нового поколения против листогрызущих вредителей. Целью проведенного исследования было изучение частоты встречаемости cry-подобных генов в штаммах и выбор перспективного для создания на его основе энтомопатогенного биопрепарата. Материалом для исследования послужили энтомопатогенные штаммы Bacillus thuringiensis, полученные из Крымской коллекции микроорганизмов Научно-исследовательского института сельского хозяйства Крыма. Энтомопатогенное действие перспективных штаммов изучали в ходе лабораторных опытов на личинках представителей отрядов Coleoptera, Lepidoptera. В качестве наиболее перспективных, содержащих не менее четырех генов токсинообразования, отобраны штаммы Bacillus thuringiensis 708 (cry1, thuE, cry7-8, cry11), 942 (cry1, thuE, cry11, vip), 949 (cry1, thuE, cry4, cry7-8), 989 (cry1, thuE, cry11, vip), 0162 (cry1, thuE, cry11, vip), 0307 (cry1, thuE, cry4, cry7-8), 0308 (cry1, thuE, cry4, cry7-8), 0363 (cry1, thuE, cry5, cry11) и 0371 (cry1, thuE, cry9, cry11). Установлено, что выделенные штаммы Bacillus thuringiensis 0162, 0307, 0363 и 0371 оказывают высокое энтомопатогенное действие против личинок колорадского жука, ильмового листоеда (88,3–100%), гусениц плодовой моли, капустной совки, златогузки и американской бабочки (92,3–100%). Показано, что штамм Bacillus thuringiensis 0371 проходит все классические фазы развития и в течение 45–48 ч демонстрирует полный выход кристаллов и спор из спорангия. Таким образом, штамм 0371 перспективен для разработки регламента производства биопрепарата для защиты сельскохозяйственных растений.

The entomopathogenic strains of Bacillus thuringiensis are used in the development of new-generation biopreparations against leaf-eating insects. The present study was aimed at analyzing the frequency of cry-like genes in the strains and at identifying a promising strain for the development of an entomopathogenic biopreparation on its basis. The study materials included the entomopathogenic strains of Bacillus thuringiensis obtained from the Crimean microorganism collection of the Crimean Agricultural Research Institute. The entomopathogenic effect of promising strains was studied in laboratory experiments on Coleoptera and Lepidoptera larvae. The following strains of Bacillus thuringiensis were identified as the most promising, i.e., containing at least four toxin genes: 708 (cry1, thuE, cry7-8, cry11), 942 (cry1, thuE, cry11, vip), 949 (cry1, thuE, cry4, cry7-8), 989 (cry1, thuE, cry11, vip), 0162 (cry1, thuE, cry11, vip), 0307 (cry1, thuE, cry4, cry7-8), 0308 (cry1, thuE, cry4, cry7-8), 0363 (cry1, thuE, cry5, cry11) и 0371 (cry1, thuE, cry9, cry11). The isolated strains of Bacillus thuringiensis 0162, 0307, 0363, and 0371 were found to have a high entomopathogenic effect on the larvae of the Colorado potato beetle and elm-leaf beetle (88.3–100%), as well as the caterpillars of ermine moth, cabbage moth, brown-tail moth, and fall webworm (92.3–100%). It is shown that Bacillus thuringiensis strain 0371 goes through all traditional stages of development and exhibits complete release of crystals and spores from the sporangium within 45–48 h. Thus, strain 0371 can be used to develop specifications for manufacturing a plant protection biopreparation.

Bacillus thuringiensiscry-подобные генылистогрызущие вредителиColeopteraLepidopteraBacillus thuringiensiscry-like genesleaf-eating insectsColeopteraLepidopteraРабота выполнена в рамках государственного задания № FNZW-2022-0006.The work was financially supported by the state assignment no. FNZW-2022-0006.ReferencesArthurs S., Dara S.K. Microbial biopesticides for invertebrate pests and their markets in the United States // Journal of Invertebrate Pathology. 2019. Vol. 165. P. 13–21. DOI: 10.1016/j.jip.2018.01.008.Arthurs S., Dara S.K. Microbial biopesticides for invertebrate pests and their markets in the United States. Journal of Invertebrate Pathology. 2019;165:13-21. DOI: 10.1016/j.jip.2018.01.008.Jouzani G.S., Valijanian E., Sharafi R. Bacillus thuringiensis: a successful insecticide with new environmental features and tidings // Applied Microbiology and Biotechnology. 2017. Vol. 101. P. 2691–2711. DOI: 10.1007/s00253-017-8175-y.Jouzani G.S., Valijanian E., Sharafi R. Bacillus thuringiensis: a successful insecticide with new environmental features and tidings. Applied Microbiology and Biotechnology. 2017;101:2691-2711. DOI: 10.1007/s00253-017-8175-y.Duarte Neto J.M.W., Wanderley M.C.A., da Silva T.A.F., Marques D.A.V., da Silva G.R., Gurgel J.F., et al. Bacillus thuringiensis endotoxin production: a systematic review of the past 10 years // World Journal of Microbiology and Biotechnology. 2020. Vol. 36. P. 128. DOI: 10.1007/s11274-020-02904-4.Duarte Neto J.M.W., Wanderley M.C.A., da Silva T.A.F., Marques D.A.V., da Silva G.R., Gurgel J.F., et al. Bacillus thuringiensis endotoxin production: a systematic review of the past 10 years. World Journal of Microbiology and Biotechnology. 2020;36:128. DOI: 10.1007/s11274-020-02904-4.Jo H., Tagele S.B., Pham H.Q., Kim M.-C., Choi S.-D., Kim M.-J., et al. Response of soil bacterial community and pepper plant growth to application of Bacillus thuringiensis KNU-07 // Agronomy. 2020. Vol. 10, no. 4. P. 551. DOI: 10.3390/agronomy10040551.Jo H., Tagele S.B., Pham H.Q., Kim M.-C., Choi S.-D., Kim M.-J., et al. Response of soil bacterial community and pepper plant growth to application of Bacillus thuringiensis KNU-07. Agronomy. 2020;10(4):551. DOI: 10.3390/agronomy10040551.Perez M.P., Sauka D.H., Onco M.I., Berretta M.F., Benintende G.B. Selection of Bacillus thuringiensis strains toxic to cotton boll weevil (Anthonomus grandis, Coleoptera: Curculionidae) larvae // Revista Argentina de Microbiología. 2017. Vol. 49, no. 3. P. 264–272. DOI: 10.1016/j.ram.2016.12.010.Perez M.P., Sauka D.H., Onco M.I., Berretta M.F., Benintende G.B. Selection of Bacillus thuringiensis strains toxic to cotton boll weevil (Anthonomus grandis, Coleoptera: Curculionidae) larvae. Revista Argentina de Microbiología. 2017;49(3):264-272. DOI: 10.1016/j.ram.2016.12.010.Soleymani S., Sarrafzadeh M.-H., Mostoufi N. Modeling of fermentation process of Bacillus thuringiensis as a sporulating bacterium // Chemical Product and Process Modeling. 2019. Vol. 14, no. 2. P. 2018000. DOI: 10.1515/cppm-2018-0007.Soleymani S., Sarrafzadeh M.-H., Mostoufi N. Modeling of fermentation process of Bacillus thuringiensis as a sporulating bacterium. Chemical Product and Process Modeling. 2019;14(2):2018000. DOI: 10.1515/cppm-2018-0007.Guttmann D.M., Ellar D.J. Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors // FEMS Microbiology Letters. 2000. Vol. 188, no. 1. P. 7–13. DOI: 10.1111/j.1574-6968.2000.tb09160.x.Guttmann D.M., Ellar D.J. Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors. FEMS Microbiology Letters. 2000;188(1):7-13. DOI: 10.1111/j.1574-6968.2000.tb09160.x.Hansen B.M., Hendriksen N.B. Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis // Applied and Environmental Microbiology. 2001. Vol. 67, no. 1. P.185–189. DOI: 10.1128/AEM.67.1.185-189.2001.Hansen B.M., Hendriksen N.B. Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis. Applied and Environmental Microbiology. 2001. Vol. 67, no. 1. P.185–189. DOI: 10.1128/AEM.67.1.185-189.2001.Palma L., Muñoz D., Berry C., Murillo J., Caballero P. Bacillus thuringiensis toxins: an overview of their biocidal activity // Toxins (Basel). 2014. Vol. 6, no. 12. P. 3296–3325. DOI: 10.3390/toxins6123296.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. DOI: 10.3390/toxins6123296.Fang Y., Li Z., Liu J., Shu C., Wang X., Zhang X., et al. A pangenomic study of Bacillus thuringiensis // Journal of Genetics and Genomics. 2011. Vol. 38, no. 12. P. 567–576. DOI: 10.1016/j.jgg.2011.11.001.Fang Y., Li Z., Liu J., Shu C., Wang X., Zhang X., et al. A pangenomic study of Bacillus thuringiensis. Journal of Genetics and Genomics. 2011;38(12):567- 576. DOI: 10.1016/j.jgg.2011.11.001.Lee M.K., Walters F.S., Hart H., Palekar N., Chen J.-S. The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin // Applied and Environmental Microbiology. 2003. Vol. 69, no. 8. P. 4648–4657. DOI: 10.1128/AEM.69.8.4648-4657.2003.Lee M.K., Walters F.S., Hart H., Palekar N., Chen J.-S. The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin. Applied and Environmental Microbiology. 2003;69(8):4648-4657. DOI: 10.1128/AEM.69.8.4648-4657.2003.Sena J.A., Hernández-Rodríguez C.S., Ferré J. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites // Applied and Environmental Microbiology. 2009. Vol. 75, no. 7. P. 2236–2237. DOI: 10.1128/AEM.02342-08.Sena J.A., Hernández-Rodríguez C.S., Ferré J. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites. Applied and Environmental Microbiology. 2009;75(7):2236-2237. DOI: 10.1128/AEM.02342-08.Bravo A., Likitvivatanavong S., Gill S.S., Soberón M. Bacillus thuringiensis: a story of a successful bioinsecticide // Insect Biochemistry and Molecular Biology. 2011. Vol. 41, no. 7. P. 423–431. DOI: 10.1016/j.ibmb.2011.02.006.Bravo A., Likitvivatanavong S., Gill S.S., Soberón M. Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochemistry and Molecular Biology. 2011;41(7):423- 431. DOI: 10.1016/j.ibmb.2011.02.006.Crickmore N., Zeigler D.R., Schnepf E., Van Rie J., Lereclus D., Baum J., et al. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins // Microbiology and Molecular Biology Reviews. 1998. Vol. 62, no. 3. P. 807–813. DOI: 10.1128/MMBR.62.3.807-813.1998.Crickmore N., Zeigler D.R., Schnepf E., Van Rie J., Lereclus D., Baum J., et al. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiology and Molecular Biology Reviews. 1998;62(3):807- 813. DOI: 10.1128/MMBR.62.3.807-813.1998.Van Frankenhuyzen K. Cross-order and cross-phylum activity of Bacillus thuringiensis pesticidal proteins // Journal of Invertebrate Pathology. 2013. Vol. 114, no. 1. P. 76–85. DOI: 10.1016/j.jip.2013.05.010.Van Frankenhuyzen K. Cross-order and crossphylum activity of Bacillus thuringiensis pesticidal proteins. Journal of Invertebrate Pathology. 2013;114(1):76-85. DOI: 10.1016/j.jip.2013.05.010.Ben-Dov E. Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins // Toxins (Basel). 2014. Vol. 6, no. 4. P. 1222–1243. DOI: 10.3390/toxins6041222.Ben-Dov E. Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins. Toxins (Basel). 2014;6(4):1222-1243. DOI: 10.3390/toxins6041222.De Maagd R.A., Bravo A., Berry C., Crickmore N., Schnepf H.E. Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria // Annual Review of Genetics. 2003. Vol. 37. P. 409–433. DOI: 10.1146/annurev.genet.37.110801.143042.De Maagd R.A., Bravo A., Berry C., Crickmore N., Schnepf H.E. Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annual Review of Genetics. 2003;37:409-433. DOI: 10.1146/annurev.genet.37.110801.143042.Chougule N.P., Bonning B.C. Toxins for transgenic resistance to hemipteran pests // Toxins (Basel). 2012. Vol. 4, no. 6. P. 405–429. DOI: 10.3390/toxins4060405.Chougule N.P., Bonning B.C. Toxins for transgenic resistance to hemipteran pests. Toxins (Basel). 2012;4(6):405-429. DOI: 10.3390/toxins4060405.Ohba M., Mizuki E., Uemori A. Parasporin, a new anticancer protein group from Bacillus thuringiensis // Anticancer Research. 2009. Vol. 29, no. 1. P. 427– 433.Ohba M., Mizuki E., Uemori A. Parasporin, a new anticancer protein group from Bacillus thuringiensis. Anticancer Research. 2009;29(1):427- 433.Zheng J., Gao Q., Liu L, Liu H., Wang Y., Peng D., et al. Comparative genomics of Bacillus thuringiensis reveals a path to specialized exploitation of multiple invertebrate hosts // mBio. 2017. Vol. 8, no. 4. P. e00822-17. DOI: 10.1128/mBio.00822-17.Zheng J., Gao Q., Liu L, Liu H., Wang Y., Peng D., et al. Comparative genomics of Bacillus thuringiensis reveals a path to specialized exploitation of multiple invertebrate hosts. mBio. 2017;8(4):e00822-17. DOI: 10.1128/mBio.00822-17.Ye J., Coulouris G., Zaretskaya I., Cutcutache I., Rozen S., Madden T.L. Primer–BLAST: a tool to design target-specific primers for polymerase chain reaction // BMC Bioinfomatics. 2012. Vol. 13. P. 134. DOI: 10.1186/1471-2105-13-134.Ye J., Coulouris G., Zaretskaya I., Cutcutache I., Rozen S., Madden T.L. Primer–BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinfomatics. 2012;13:134. DOI: 10.1186/1471-2105-13-134.Franco-Rivera A., Benintende G., Cozzi J., Baizabal-Aguirre V.M., Valdez-Alarcón J.J., López-Meza J.E. Molecular characterization of Bacillus thuringiensis strains from Argentina // Antonie Van Leeuwenhoek. 2004. Vol. 86, no. 1. P. 87–92. DOI: 10.1023/B:ANTO.0000024913.94410.05.Franco-Rivera A., Benintende G., Cozzi J., BaizabalAguirre V.M., Valdez-Alarcón J.J., López-Meza J.E. Molecular characterization of Bacillus thuringiensis strains from Argentina. Antonie Van Leeuwenhoek. 2004;86(1):87-92. DOI: 10.1023/B:ANTO.0000024913.94410.05.Willumsen P.A., Johansen J.E., Karlson U. Isolation and taxonomic affiliation of N-heterocyclicaromatic hydrocarbon-transforming bacteria // Applied Microbiology and Biotechnology. 2005. Vol. 67, no. 3. P. 420–428. DOI: 10.1007/s00253-004-1799-8.Willumsen P.A., Johansen J.E., Karlson U. Isolation and taxonomic affiliation of N-heterocyclicaromatic hydrocarbon-transforming bacteria. Applied Microbiology and Biotechnology. 2005;67(3):420-428. DOI: 10.1007/s00253-004-1799-8.Bravo A., Sarabia S., Lopez L., Ontiveros H., Abarca C., Ortiz A., et al. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection // Applied and Environmental Microbiology. 1998. Vol. 64, no. 12. P. 4965–4972. DOI: 10.1128/AEM.64.12.4965-4972.1998.Bravo A., Sarabia S., Lopez L., Ontiveros H., Abarca C., Ortiz A., et al. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Applied and Environmental Microbiology. 1998;64(12):4965-4972. DOI: 10.1128/AEM.64.12.4965-4972.1998.Jain D., Sunda S.D., Sanadhya S., Nath D.J., Khandelwal S.K. Molecular characterization and PCRbased screening of cry genes from Bacillus thuringiensis strains // 3 Biotech. 2017. Vol. 7, no. 1. P. 4. DOI: 10.1007/s13205-016-0583-7.Jain D., Sunda S.D., Sanadhya S., Nath D.J., Khandelwal S.K. Molecular characterization and PCR-based screening of cry genes from Bacillus thuringiensis strains. 3 Biotech. 2017;7(1):4. DOI: 10.1007/s13205-016-0583-7.PCR primer design / ed. A. Yuryev. Totowa: Humana Press, 2007. 431 p.Yuryev A. PCR primer design. Totowa: Humana Press; 2007, 431 p.Хайлафян А.А. Современные статистические методы медицинских исследований: монография. М.: Ленанд, 2014. 320 с.Khalafyan A.A. Modern statistical methods of medical research. Moscow: Lenand; 2014, 320 p. (In Russian).Natingga D. Data science algorithms in a week. Birmingham – Mumbai: Packt Publishing, 2018. 214 p.Natingga D. Data science algorithms in a week. Birmingham – Mumbai: Packt Publishing; 2018, 214 p.Galea A. Applied data science with Python and Jupyter. Birmingham – Mumbai: Packt Publishing, 2018. 174 p.Galea A. Applied data science with Python and Jupyter. Birmingham – Mumbai: Packt Publishing; 2018, 174 p.

The authors declare that there are no conflicts of interest present.