doi: 10.15389/agrobiology.2019.6.1267eng

UDC: 579.64:632.937.15

The work was carried out on the equipment of the ARRIAM Center for Genomic Technologies, Proteomics and Cell Biology.
Supported financially by the project of applied research and experimental development (PNER) batch 2017-14-579-0030 on the topic “Creation of microbiological preparations for expanding the adaptive capacity of agricultural crops for nutrition, resistance to stress and pathogens” (code of the application 2017-14-579-0030-013), Agreement No. 14.607.21.0178, a unique identifier (project) RFMEFI60717X0178


INSECTICIDAL PROPERTIES of Bacillus thuringiensis var. israelensis.

V.P. Ermolova1, S.D. Grishechkina1, A.M. Rakhman2, K.S. Antonets1,
M.E. Belousova1, V.V. Yakhno1, A.A. Nizhnikov1

1All-Russian Research Institute for Agricultural Microbiology, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail,,,,, (✉, corresponding author);
2AD Station of preventive disinfection, 47 B, 4-N, ul. Chernyakhovskogo, St. Petersburg, 191119 Russia, e-mail

Ermolova V.P.
Belousova M.V.
Grishechkina S.D.
Yaкhno V.V.
Rahman A.M.
Nizhnikov A.A.
Antonets K.S.

Received August 2, 2019


Blood-sucking mosquitoes and blackflies belonging to the order Diptera cause sanitary-epidemiological and veterinary-epizootological damages in humans and animals and serve as the carriers of various dangerous transmissible diseases. The productivity and milk yield of livestock as well as egg production in poultry birds are significantly affected by harmful Diptera species. The problem of bioprotection from harmful insects is extremely relevant in crop production, including the cultivation of seeded fodder crops. Currently, the spore-forming bacterium Bacillus thuringiensis Berliner (Bt) represents the most common agent for biological control of the number of insect species and is considered as the basis for the production of insecticides. Biological preparations are of particular importance due to their significant advantages over chemical pesticides and are considered in modern agricultural systems as environmentally and socially beneficial alternatives to agrochemicals. A liquid form of larvicidal preparation against blood-sucking and herbivorous mosquitoes based on the original strain Bacillus thuringiensis var. israelensis 7-1/23А was developed at the ARRIAM. Under registration number RCAM 00626 (RF patent 2539732 dated 12/09/2014), the strain was deposited in the ARRIAM Collection of beneficial agricultural microorganisms (RCAM). The composition of the liquid form of the biopreparation includes a spore-crystalline complex, 5 % sodium chloride, 2 % coniferous extract (as a preservative and perfume, respectively), and the remains of a nutrient medium. The titer was 4.25×109 CFU/ml. Larvicidal activity expressed in LC50 for Aedes aegypti IV instar larvae was 0.11×10-3 % (biotest proposed by the World Health Organization). The strain was previously isolated from an anophelogenic reservoir in the Leningrad region, studied for physiological and cultural features, and characterized according to Н. De Barjac’s and А. Bonnefoi’s classification. In this work, for the first time, a comprehensive analysis of a larvicidal preparation based on BtH14 7-1/23A was performed, including the molecular characterization of the strain, determination of its larvicidal activity against a number of harmful dipterans, and an assessment of its effects on the growth and development of non-target objects (oyster mushroom mycelium and champignon). Sequencing of the gene encoding B subunit of the DNA gyrase (GyrB) confirmed that the isolated strain belongs to B. thuringiensis var. israelensis. The presence of the cry4 and cry11 genes encoding protein insecticidal toxins was detected by PCR analysis. We also studied the spectrum of action of the larvicidal biopreparation against the blood-sucking mosquitoes of the genera Aedes, Anopheles, Culex, and harmful herbivorous dipterans: rice (Сricotopus sylvestris Fabr.) and mushroom (Lycoriella fucorum Frey) flies. The analysis was carried out in laboratory and field conditions (2014-2016, Leningrad and Moscow regions, Krasnodar Territory, aedogenic, anofelogenic ponds, raw basement rooms, rice checks) and also included an assessment of the effect of the preparation on the growth of mycelium of oyster mushrooms (Pleurotus ostreatus) and champignon (Agaricus campestris) as bio-eco indicators. LC50 values in laboratory tests were 0.11×10-3 and 0.12×10-3 % for Culex and Аеdes, respectively, and 0.29×10-3 % for Anopheles maculipennis Meigen. Field tests in water reservoirs against malarial (Anopheles maculipennis) and non-malarial mosquitoes of the genus Aedes communis, dorsalis, punctor, caspius, and flavescens; in moist basement rooms (Culex pipiens Linnaeus f. molestus Forskål), rice fields (Сricotopus sylvestris Fabr.) showed 90.2-100 % mortality of larvae. The effect of the biological preparation on the growth of mycelium of oyster mushrooms and champignon was studied in three concentrations (5, 10, and 20 %). The most efficient was 5 % concentration which stimulated the growth of fungal mycelium by 28.2-32.5 %. The analysis of the separate and combined applications of the 7-1/23А-based biopreparation and the chemical insecticide, chitin synthesis inhibitor Dimilin («Arysta LifeScience S.A.S.», France) against the larvae of the mushroom fly of the family Lycoriidae (L. fucorum Frey) demonstrated that combined use of these insecticides in 4-8 times reduced dozes caused death of 97.2 % larvae and led to an increase in the yield by 38.6 %. Thus, a preparation based on the BtH14 7-1/23A is promising for sanitary ecology and veterinary. In addition, it is advisable to study the possibilities of its use against insect pests of seeded forage crops.

Keywords: Bacillus thuringiensis var. israelensis (BtH14), titer, larvicidal activity, growth-promoting activity, biologiсal preparation.



  1. Akbaev M.Sh., Vodyanov A.N., Kosminkov N.E., Yatusevich A.I., Pashkin P.I., Vasilevich F.I. Parazitologiya i invazionnye bolezni zhivotnykh [Parasitology and invasive animal diseases]. Moscow, 2000 (in Russ.).
  2. Kandybin N.V., Patyka T.I, Ermolova V.P., Patyka I.F. Mikrobiokontrol' chislennosti nasekomykh i ego dominanta Bacillus thuringiensis [Insect microbiocontrol and its dominant Bacillus thuringiensis]. St. Petersburg—Pushkin, 2009 (in Russ.).
  3. Hemingway J., Hawkes N.J., McCarroll L., Ranson H. The molecular basis of insecticide resistance in mosquitoes. Insect Biochemistry and Molecular Biology, 2004, 34(7): 653-665 CrossRef
  4. Lacey L.A. Bacillus thuringiensis serovariety israelensis and Bacillus sphaericus for mosquito control. Journal of the American Mosquito Control Association, 2007, 23(sp2): 133-163 CrossRef
  5. Boisvert M., Boisvert J. Effects of Bacillus thuringiensis var. israelensis on target and nontarget organisms: a review of laboratory and field experiments. Biocontrol Science and Technology, 2000, 10(5): 517-561 CrossRef
  6. Bravo A., Gill S.S., Soberón M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 2007, 49(4), 423-435 CrossRef
  7. Choi Y.S., Cho E.S., Je Y.H., Roh J.Y., Chang J.H., Li M.S., Seo S.J., Sohn H.D., Jin B.R. Isolation and characterization of a strain of Bacillus thuringiensis subsp. morrisoni PG-14 encoding delta-endotoxin Cry1Ac. Current Microbiology, 2004, 48(1): 47-50 CrossRef
  8. Kim Y.T., Huang H.T. The β-exotoxins of Bacillus thuringiensis. I. Isolation and characterization. Journal of Invertebrate Pathology, 1970, 15(1): 100-108 CrossRef
  9. Raddadi N., Cherif A., Ouzari H., Marzorati M., Brusetti L., Boudabous, A., Daffonchio D. Bacillus thuringiensis beyond insect biocontrol: plant growth promotion and biosafety of polyvalent strains. Annals of Microbiology, 2007, 57(4): 481-494 CrossRef
  10. Donmez V.F., Uysal B.D., Erkol E.F., Sezai C.R. Biological control of root rot disease caused by Rhizoctonia solani Kuhn on potato and bean using antagonist bacteria. Acta scientiarum Polonorum. Hortorum cultus = Ogrodnictwo, 2015, 14(5): 29-40.
  11. Malovichko Y.V., Nizhnikov A.A., Antonets K.S. Repertoire of the Bacillus thuringiensis virulence factors unrelated to major classes of protein toxins and its role in specificity of host-pathogen interactions. Toxins, 2019, 11(6): e11060347 CrossRef
  12. Choudhary D.K., Johri B.N. Interactions of Bacillus spp. and plants — with special reference to induced systemic resistance. Microbiol. Res., 2009, 164(5): 493-513 CrossRef
  13. Kumar P., Dubey R.C., Mahshwari D.K. Bacillus strain isolated from rhizosphere showed plant growth promoting and antagonistic activity against phythopathogens. Microbiol. Res., 2012, 167(8): 493-499 CrossRef
  14. Chatterjee S.N., Bhattacharya T., Dangar T.K., Chandra G. Ecology and diversity of Bacillus thuringiensis in soil environment. African Journal of Biotechnology, 2007, 6(13): 1587-1591.
  15. Das J., Dangar T.K. Diversity of Bacillus thuringiensis in the rice field soils of different ecologies in India. Indian J. Microbiol., 2007, 47(4): 364-368 CrossRef
  16. Ramalakshmi A., Udayasuriyan V. Diversity of Bacillus thuringiensis isolated from Western Ghats of Tamil Nadu state, India. Current Microbiology, 2010, 61(1): 13-18 CrossRef
  17. Pane C., Villecco D., Campanile F., Zaccardelli M. Novel strains of Bacillus isolated from compost and compost-amended soils as biological control agents against soil-borne phytopathogenic fungi. Biol. Sci. Technol., 2012, 22(12): 1373-1388 CrossRef
  18. Akram W., Mahboob A., Javed A.A. Bacillus thuringiensis strain 199 can induce systemic resistance in tomato against Fusarium wilt. Eur. J. Microbiol. Immunol., 2013, 3(4): 275-280 CrossRef
  19. Tao A., Pang F., Huang S., Yu G., Li B., Wang T. Characterization of endophytic Bacillus thuringiensis strains isolated from wheat plants as biocontrol agents against wheat flagsmut. Biocontrol. Sci. Technol., 2014, 24(8): 901-924 CrossRef
  20. Lacey L.A., Grywaczet D., Shapiro-Ilan D., Frutos R., Brownbridge M., Goettel M.S. Insect pathogens as biological control agents: back to the future. Journal of Invertebrate Pathology, 2015, 132: 1-41 CrossRef
  21. Eswarapriya B., Gopalsamy B., Kameswari B., Meera R., Devi P. Insecticidal activity of Bacillus thuringiensis IBt-15 strain against Plutella xylostella. Int. J. Pharm. Tech. Res., 2010, 2(3): 2048-2053.
  22. Patel K.D., Bhanshali F.C., Chaudhary A.V., Ingle S.S. A new enrichment method for isolation of Bacillus thuringiensis from diverse sample types. Appl. Biochem. Biotechnol., 2013, 170: 58-66 CrossRef
  23. Rakhmanin Yu.A., Novikov S.M., Rumyantsev G.I., Ivanov S.I. Gigiena i sanitariya, 2006, 5: 10-13 (in Russ.).
  24. Raddadi N., Cherif A., Ouzari H., Marzorati M., Brusetti L., Boudabous A., Daffonchio D. Bacillus thuringiensis beyond insect biocontrol: plant growth promotion and biosafety of polyvalent strains. Annals of Microbiology, 2007, 57(4): 481-494 CrossRef
  25. Narayanasamy P. Biological management of diseases of crops. Progress in biological control (Book 15). Springer, Dordrecht, 2013: 295-429 CrossRef
  26. Patel K.D., Bhanshali F.C., Ingle S.S. Diversity and characterization of Bacillus thuringiensis isolates from alluvial soils of Mahi river basin, India. J. Adv. Dev. Res., 2011, 2(1): 14-20.
  27. Ermolova V.P. Bacillus thuringiensis strains from natural sources in the Leningrad region: isolation and identification. Agricultural Biology [Sel’skokhozyaistvennaya Biologiya], 2016, 51(1): 128-131 CrossRef
  28. 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 CrossRef
  29. Jouzani G.S., Valijanian E., Sharafi R. Bacillus thuringiensis: a successful insecticide with new environmental features and tidings. Appl. Microbiol. Biotechnol., 2017, 101(7): 2691-2711 CrossRef
  30. Al-KHamada A.D. Vydelenie entomopatogenov Vacillus thuringiensis (Bt) iz regiona Deir Ezzor Sirii i ikh biotestirovanie. Vestnik zashchity rastenii, 2009, 4: 54-62.
  31. Grishechkina S.D., Ermolova V.P., Romanova T.A., Nizhnikov A.A. Search for natural isolates of Bacillus thuringiensis for development of ecologically friendly biologicals. Agricultural Biology [Sel’skokhozyaistvennaya Biologiya], 2018, 53(5): 1062-1069 CrossRef
  32. de Barjac H., Bonnefoi A. A classification of strains of Bacillus thuringiensis Berliner with a key to their differentiation. Journal of Invertebrate Pathology, 1968, 11: 335-347.
  33. Hansen B.M., Hendriksen N.B. Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis. Applied and Environmental Microbiology, 2001, 67(1): 185-189 CrossRef
  34. Sanger F., Nicklen S., Coulson A.R. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA. 1977, 74(12): 5463-5467 CrossRef
  35. Smirnoff U.A. The formation of crystals in Bacillus thuringiensis var thuringiensis Berliner before sporulation of temperature inculcation. J. Insect Pathol., 1965, 2: 242-250.
  36. Kollektsiya shtammov bakterii-simbiontov vrednykh nasekomykh i gryzunov, prigodnykh dlya biokontrolya chislennosti vreditelei sel'skokhozyaistvennykh rastenii [Collection of bacterial symbionts of harmful insects and rodents suitable for biocontrol of the number of pests of agricultural plants].St. Petersburg, 2014 (in Russ.).
  37. Metodicheskie ukazaniya po primeneniyu i metodam kontrolya kachestva insektitsidnogo mikrobiologicheskogo sredstva «Baktitsid» [Guidelines for the use and quality control methods of the insecticidal microbiological agent «Bacticid»]. Moscow, 2001 (in Russ.).
  38. Mokrousova E.P., Glazunova I.N., Kandybin N.V., Ermolova V.P. V sbornike: Problemy entomologii v Rossii [In: Entomological problems in Russia]. St. Petersburg, 1998, tom 2: 40-41 (in Russ.).
  39. Dospekhov B.A. Metodika polevogo opyta [Methods of field trials]. Moscow, 1973 (in Russ.).
  40. Punina N.V., Zotov V.S., Parkhomenko A.L. Genetic diversity of Bacillus thuringiensis from different geo-ecological regions of Ukraine by analyzing the 16S rRNA and gyrB genes and by AP-PCR and saAFLP. Acta Naturae, 2013, 5(1): 90-100.
  41. Bravo A., Sarabia S., Lopez L., Ontiveros H., Abarca C., Ortiz A., Quintero R. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Applied and Environmental Microbiology, 1998. 64(12): 4965-4972.
  42. Ben-Dov E., Zaritsky A., Dahan E., Barak Z., Sinai R., Manasherob R., Margalith Y. Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis. Applied and Environmental Microbiology, 1997, 63(12): 4883-4890.
  43. Guidi V., Patocchi N., Lüthy P., Tonolla M. Distribution of Bacillus thuringiensis subsp. israelensis in soil of a Swiss Wetland reserve after 22 years of mosquito control. Applied and Environmental Microbiology, 2011, 77(11): 3663-3668 CrossRef
  44. Ben-Dov E. Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins. Toxins, 2014, 6(4): 1222-1243 CrossRef
  45. Rakhman A.I., Ermolova V.P. V knige: Infektsionnye bolezni [In: Infectious diseases]. St. Petersburg, 2015: 203-207 (in Russ.).
  46. Kandybin N.V., Stus' A.A., Kuznetsova L.N. Byulleten' VNIISKHM, 1981, 33: 54-57 (in Russ.).
  47. Kryzhko A.V., Kuznetsova L.N. Vestnik Voronezhskogo gosudarstvennogo universiteta, 2017, 4: 51-53 (in Russ.).
  48. Chebotar' V.K., Shcherbakov A.V., Shcherbakova E.N., Maslennikova S.N., ZaplatkinA.N., Mal'fanova N.V. Biodiversity of endophytic bacteria as a promising biotechnological resource. Agricultural Biology [Sel’skokhozyaistvennaya Biologiya], 2015, 50(5): 648-654 CrossRef
  49. Shternshis M.V., Belyaev A.A., Tsvetkova V.P., Shpatova T.V., Lelyak A.A., Bakhvalov S.A. Biopreparaty na osnove bakterii roda Bacillus dlya upravleniya zdorov'em rastenii [Bacillus-based biological products for plant health management]. Novosibirsk, 2016 (in Russ.).
  50. Smirnov O.V., Grishechkina S.D. Polyfunctional activity of Bacillus thuringiensis Berliner. Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2011, 3: 123-126 (in Russ.).
  51. Salama H.S., Foda M.S., Zaki F.N., Moawad S. Potency of combinations of Bacillus thuringiensis and chemical insecticides on Spodoptera littoralis (Lepidoptera: Noctuidae). JournalofEconomicEntomology, 1984, 77(4): 885-890 CrossRef
  52. Grishechkina S.D., Ermolova V.P., Kovalenko T.K., Antonets K.S., Belousova M.E., Yakhno V.V., Nizhnikov A.A. Polyfunctional properties of the Bacillus thuringiensis var. thuringiensis industrial strain 800/15. Agricultural Biology [Sel’skokhozyaistvennaya Biologiya], 2019, 54(3): 494-504 CrossRef
  53. Zhang L., Qiu S., Guan X. Effect of chemical additives on Bacillus thuringiensis (Bacillales: Bacillaceae) against Plutella xylostella (Lepidoptera: Pyralidae). Journal of Economic Entomology, 2013, 106(3): 1075-1080 CrossRef
  54. Tetreau G., Alessi M., Veyrenc S., Périgon S., David J.-Ph., Reynaud S., Després L. Fate of Bacillus thuringiensis subsp. israelensis in the field: evidence for spore recycling and differential persistence of toxins in leaf litter. Applied and Environmental Microbiology, 2012, 78(23): 8362-8367 CrossRef
  55. Derua Y.A., Kweka E.J., Kisinza W.N. Bacterial larvicides used for malaria vector control in sub-Saharan Africa: review of their effectiveness and operational feasibility. Parasites Vectors, 2019, 12: 426 CrossRef
  56. Lacey L.A. Bacillus thuringiensis serovariety israelensis and Bacillus sphaericus for mosquito control. Journal of the American Mosquito Control Association, 2007, 23(2 Suppl): 133-163 CrossRef






Full article PDF (Rus)

Full article PDF (Eng)