doi: 10.15389/agrobiology.2019.3.494eng

UDC: 632:632.937.15:579.64

Supported financially by the project of applied research and experimental development (PNIER) 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 RFMEFI60717X0178


POLYFUNCTIONAL PROPERTIES OF THE Bacillus thuringiensis var. thuringiensis INDUSTRIAL STRAIN 800/15

S.D. Grishechkina1, V.P. Ermolova1, T.K. Kovalenko2,
K.S. Antonets1, M.Е. Belousova1, V.V. Yaкhno1, A.A. Nizhnikov1

1All-Russian Research Institute for Agricultural Microbiology, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail (✉ corresponding author),,,,,;
2Far-Eastern Research Institute of Plant Protection, 42-a, ul. Mira, s. Kamen-Rybolov, Primorskii Krai, 692682 Russia, e-mail

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

Received September 23, 2018


Crop losses caused by pests can reach 40-50 % and even more. Application of biological methods for regulation of the harmful species is promising and providing ecological safety. Biological preparations based on the living cultures of microorganisms and their metabolites meet these requirements. Currently, the crystal-forming bacterium Bacillus thuringiensis is considered to be the most important species for production of biological insecticides, since this bacterium exhibits high specificity in relation to the target pathogens, safety for humans and the environment. At ARRIAM, the biological preparation based on the Bacillus thuringiensis var. thuringiensis (BtH1) 800/15 strain was developed. The strain was isolated from larvae of the Colorado potato beetle (Leptinotarsa deсemlineаta Say.) in the Leningrad region, studied for culture-morphological, biochemical and serological properties and identified according to the classification of De Barjac and Bonnefoi. Sequencing of the genes encoding 16S RNA and B-subunit of the DNA-gyrase (GyrB) confirmed that the isolated strain 800/15 belongs to Bacillus thuringiensis var. thuringiensis. The BtH1 800/15 strain was deposited in the Russian Collection of Agricultural Microorganisms (RCAM) under the registration number 611 (Patent of the Russian Federation RU 2514211 C1 of 27.04.2014). This paper is the first to report that the BtH1 800/15-based biologicals increases the germination of seeds, the height of seedlings and the root length of various crops, and also revealed the inhibitory activity against phytopathogenic fungi. The goal of this study was to investigate whether the biological preparation based on the BtH1 800/15 strain has multifunctional properties including entomocidal activity against mass insect pests of crops, growth-stimulating effect on economically significant plant species and antifungal activity against phytopathogenic fungi. The preparation based on the BtH1 800/15 strain is a liquid that is easily diluted with water to the required concentration and contains the components of the cultural medium, spores and entomocidal exo- and endotoxins. The initial values of the biological activity of the preparation were as follows: titer was 3.5×109 CFU/ml, exotoxin content for the Musca domestica Linn. larvae in LC50 was 3.1 μl/g of feed, entomocidal activity for the larvae of the Colorado beetle Leptinotarsa decemlineata Say. in LC50 was 0,28 %. The paper presents the data of field trials of the effectiveness of the preparation carried out on different agricultural crops in the period of 2014-2017 in the Leningrad Region, Krasnodar and Primorsky Krai against phytophagous insects, the Colorado beetle (L. decemlineata Say.), the 28-spotted potato ladybird (Henosepilachna vigintioctomaculata Motsch.), the diamondback moth (Plutella xylostella L.), the cabbage white and the small white (Pieris brassica L., P. rapae L.), cabbage moth (Barathra brassicae L.), gooseberry sawfly (Pteronidea ribesii Scop.), red spider mite (Tetranichus urtica Koch.) and whitefly (Trialeurodes vaporariorum West.). Field tests demonstrated the effectiveness of this biological preparation against harmful phytophagous insects (66.7-100 %). The laboratory tests revealed that the preparation did not exhibit phytotoxicity, moreover, it showed a growth-stimulating effect on the seed germination (up to 32 %), as well as the height of seedlings and root length (up to 52 %). The efficacy of the preparation against phytopathogenous fungi did not exceed 54 % and was inferior to the preparation based on the BtH10 strain 56. The combined use of the biological preparation based on the BtH1 800/15 strain with the chemical insecticide Decis Extra, CE (emulsion concentrate) on potato against H. vigintioctomaculata Motsch. was very efficient (100 %) even if the application rates were reduced 2 and 3 times, respectively. This combination of biological and chemical insecticides is economically valuable and can be successfully used in potato fields when they are pest-infected, with the predominance of larvae of older ages and imago, which allows a significant reduction of the pesticide load. Overall, data obtained show that the biological preparation based on the Bacillus thuringiensis var. thuringiensis strain 800/15 has multifunctional properties, including entomocidal, antifungal and growth-stimulating activities, and is also promising for joint use with chemical insecticides.

Keywords: Bacillus thuringiensis, phytophagous insects, phytopathogens, polyfunctional properties, entomocidal, antifungal and growth-stimulating activity.



  1. Olson S. An analysis of the biopesticide market now and where it is going. Outlooks on Pest Management, 2015, 26(5): 203-206 CrossRef
  2. Siegel J.P. The Mammalian Safety of Bacillus thuringiensis- Based Insecticides. Journal of Invertebrate Pathology, 2001, 77(1): 13-21 CrossRef
  3. Raymond B., Federici B.A. In deference of Bacillus thuringiensis, the safest and most success the microbial insecticide available to humanity — a response to EFSA. FEMS Microbiology Ecology, 2017, 93(7): fix084 CrossRef
  4. Kandybin N.V., Patyka T.I, Ermolova V.P., Patyka I.F. Mikrobiokontrol' chislennosti nasekomykh i ego dominanta Bacillus thuringiensis. St. Petersburg—Pushkin, 2009 (in Russ.).   
  5. Polanczyk R.A., Pires da Silva R.F., Fiuza L.M. Effectiveness of Bacillus thuringiensis against Spodoptera frugipera (Lepidoptera: Noctuidae). Brazilian Journal of Microbiology, 2000, 31(3): 165-167 CrossRef
  6. Eswarapriya B., Gopalsamy B., Kameswari B., Meera R., Devi P. Insecticidal activity of Bacillus thuringiensis IBt-15 strain against Plutella xylostella. International Journal of PharmTech Research, 2010, 2(3): 2048-2053.
  7. Seo D.J. Nguyen D.M.S., Song Y.S., Jung W.J. Induction of defense response against Rhizoctonia solani in cucumber plants by endophytic bacterium Bacillus thuringiensis GS1. J. Microbiol. Biotechnol., 2012, 22(3): 407-415 CrossRef
  8. Martinez-Absalón S., Rojas-Solís D., Hernández-León R., Prieto-Barajas C., Orozco- Mosqueda M., Peña-Cabriales J.J., Sakuda S., Valencia-Cantero E., Santoyo G. Potential use and mode of action of the new strain Bacillus thuringiensis UM96 for the biological control of the grey mould phytopathogen Botrytis cinerea. Biocontrol Science and Technology, 2014, 24(12): 1349-1362 CrossRef
  9. Heydari A., Pessaraki M. A review on biological control of fungal plant pathogens using microbial antagonists. Journal of Biological Sciences, 2010, 1(4): 273-290 CrossRef
  10. Mohammed S.H., El Saedy M.A., Enan M.R., Nasser E.I., Ghareeb A., Salah A.M. Biocontrol efficiency of Bacillus thuringiensis toxins against root-knot nematode, Meloidogyne incognita. J. Cell Mol. Biol., 2008, 7(1): 57-66.
  11. 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. Biocontrol Science and Technology, 2012, 22(12): 1373-1388 CrossRef
  12. Akram W., Mahboob A., Jave A.A. Bacillus thuringiensis strain 199 can induce systemic resistance in tomato against Fusarium wilt. European Journal of Microbiology and Immunology, 2013, 3(4): 275-280 CrossRef
  13. 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 flag smut. Biocontrol Science and Technology, 2014, 24(8): 901-924 CrossRef
  14. Smirnov O.V. Patotipy Bacillus thuringiensis i ekologicheskie osnovy ikh ispol'zovaniya v zashchite rastenii. Avtoreferat doktorskoi dissertatsii [Pathotypes of Bacillus thuringiensis and the ecological basis of their use in plant protection. DSc Thesis]. Petersburg—Pushkin, 2000 (in Russ.).   
  15. Shrestha A., Sultana R., Chae J.-C., Kim K., Lee K.-J. Bacillus thuringiensis C25 which is rich in cell wall degrading enzymes efficiently controls lettuce drop caused by Sclerotinia minor. Eur. J. Plant Pathol., 2015, 142(3): 577-589 CrossRef
  16. Loseva O., Ibrahim M., Candas M., Koller C.N., Bauer L.S., Bulla Jr L.A. Changes in protease activity and Cry3Aa toxin binding in the Solorado potato beetle: implications for insect resistance to Bacillus thuringiensis toxins. Insect Biochemistry and Molecular Biology, 2002, 32(5): 567-577 CrossRef
  17. Zhong C.H., Ellar D.J., Bishop A., Johnson C., Lin S.S., Hart E.R. Characterization of Bacillus thuringiensis δ-endotoxin which is toxic to insects in three orders. Journal of Invertebrate Pathology, 2000, 76(2): 131-139 CrossRef
  18. Aronson A.I., Shai Y. Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action. FEMS Microbiology Letters, 2001, 195(1): 1-8 CrossRef
  19. Knaak N., Rohr A., Fiuza L. In vitro effect of Bacillus thuringiensis strains and Cry proteins in phytopathogenic fungi of paddy rice-field. Brazil. J. Microbiol., 2007, 38(3): 526-530 CrossRef
  20. 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.).   
  21. Wagner W., Möhrlen F., Schnetter W. Characterization of the proteolytic enzymes in the midgut of the European Cockchafer, Melolontha melolontha (Coleoptera: Scarabaidae). Insect Biochemistry and Molecular Biology, 2002, 32(7): 803-814 CrossRef
  22. Smirnov O.V., Grishechkina S.D. Polyfunctional activity of Bacillus thuringiensis Berliner. Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2011, 3: 123-126 (in Russ.).
  23. Reyes‐Ramírez A., Escudero-Abarca B.I., Aguilar-Uscanga G., Hayward-Jones P.M., Barboza-Corona J.E. Antifungal activity of Bacillus thuringiensis chitinase and its potential for the biocontrol of phytopathogenic fungi in soybean seeds. Journal of Food Science, 2004, 69(5): M131-M134 CrossRef
  24. Xiao L., Xie C.-C., Cai J., Lin Z.-J., Chen Y.-H. Identification and characterization of chitinase producing Bacillus slowing significant antifungal activity. Curr. Microbiol., 2009, 58(5): 528-533 CrossRef
  25. Kim P.I., Bai H., Bai D., Chae H., Chung S., Kim Y., Park R., Chi Y.-T. Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26. Journal of Applied Microbiology, 2004, 97(5): 942-949 CrossRef
  26. Yánez-Mendizábal V., Zeriouh H., Viñas I., Torres R., Usall J., de Vicente A., Pérez-García A., Teixidó N. Biological control of peach brown rot (Monilinia spp.) by Bacillus subtilis CPA-8 is based on production of fengycin-like lipopeptides. Eur. J. Plant Pathol., 2011, 132(4): 609-619 CrossRef
  27. Elkahoui S., Djébalin N., Karkouch I., Hadj Ibrahim A., Kalai L., Bachkouel S., Tabbene O., Limam F. Mass spectrometry identification of antifungal lipopeptides from Bacillus sp. BCLRB2 against Rhizoctonia solani and Sclerotinia sclerotiorum. Appl. Biochem. Microbiol., 2014, 50(2): 161-165 CrossRef
  28. Choudhary D.K., Johri B.N. Interactions of Bacillus spp. and plants — With special reference to induced systemic resistance (ISP). Microbiological Research, 2009, 164(5): 493-513 CrossRef
  29. Kumar P., Dubey R.C., Maheshwari D.K. Bacillus strain isolated from rhizosphere showed plant growth promoting and antagonistic activity against phytopathogens. Microbiological Research, 2012, 167(8): 493-499 CrossRef
  30. Chebotar' V.K., Naumkina T.S., Borisov A.Yu. Kompleksnoe mikrobnoe udobrenie «Bisolbimiks» /Pod redaktsiei A.Yu. Borisova [Complex microbial fertilizer Bisolbimix. A.Yu. Borisov (ed.)]. St. Petersburg, 2015 (in Russ.).   
  31. Partida-Martínez L.P., Heil M. The microbe-free plant root: fact or artifact? Frontiers in Plant Science, 2011, 2(100): 1-16 CrossRef
  32. Grishechkina S.D., Ermolova V.P., Minina G.N., Safronova V.I., Bologova E.V. Metodika. Kollektsiya shtammov bakterii-simbiontov vrednykh nasekomykh i gryzunov, prigodnykh dlya biokontrolya chislennosti vreditelei sel'skokhozyaistvennykh rastenii [Collection of bacterial symbionts in harmful insects and rodents, suitable for biocontrol of agricultural plant pests]. St. Petersburg, 2014 (in Russ.).   
  33. Tikhonovich I.A., Ermolova V.P., Grishechkina S.D., Romanova T.A. Shtamm bakterii Bacillus thuringiensis var. thuringiensis №800/15 v kachestve sredstva dlya polucheniya entomotsidnogo biopreparata. Patent RU 2514211 S1. Zayavl. 10.10.2012. Opubl. 27.04.2014. Byul. № 12 [The strain of Bacillus thuringiensis var. thuringiensis №800/15 for entomocidal biopreparation. Patent RU 2514211 S1. Claims 10.10.2012. Publ. 27.04.2014. Bul. № 12] (in Russ.).   
  34. De Barjac H., Bonnefoi A. Essai de classification biochmique et seroloque de 24 coucher de Bacillus du type B. thuringiensis. Entomophaga, 1962, 7(1): 5-31 CrossRef
  35. De Barjac H., Bonnefoi A. A classification strains of Bacillus thuringiensis Berliner with a key to their differentiation. Journal of Invertebrate Pathology, 1968, 11(3): 335-347 CrossRef
  36. De Barjac H., Bonnefoi A. Misse au point sur la classification des Bacillus thuringiensis. Entomophaga, 1973, 18(1): 5-17 CrossRef
  37. Lane D.J. 16S/23S rRNA sequencing. In: Nucleic acid technics in bacterial systematics. E. Stackebrandt, M. Goodfellow (eds.). John Wiley & Sons, Chichester, NY, 1991: 115-175.
  38. Punina N.V., Zotov V.S., Parkhomenko A.L., Parkhomenko T.U., Topunov A.F. 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.
  39. Sanger F., Nicklen S., Coulson A.R. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, 1977, 74(12): 5463-5467 CrossRef
  40. Kol'chevskii A.G., Rybina L.M., Kolomiets V.Ya. Vydelenie i otbor vysokovirulentnykh kul'tur Bacillus thuringiensis var. galleriae. Metodicheskie rekomendatsii /Pod redaktsiei V.A. Pavlyushina [Isolation and selection of highly virulent cultures of Bacillus thuringiensis var. galleriae. Guidelines. V.A. Pavlyushin (ed.)]. Leningrad, 1987 (in Russ.).    
  41. Metody eksperimental'noi mikologii /Pod redaktsiei V.I. Bilai [Methods of experimental mycology. V.I. Bilai (ed.)]. Kiev, 1982 (in Russ.).   
  42. Abbot W.S. A method for computing the effectiveness of insecticide. Journal of Economic Entomology, 1925, 18(2): 265-267 CrossRef
  43. Benken A.A., Khatskevich L.K., Grishechkina S.D. Uskorennyi metod otsenki bolezneustoichivosti sortoobraztsov yachmenya i paraziticheskikh svoistv vozbuditelya fuzarioznoi kornevoi gnili. Informatsionnyi listok [A rapid assessment of disease resistance in barley varieties and parasitic activity of the Fusarium root rot causative agent]. St. Petersburg, 1983 (in Russ.).   
  44. Dospekhov B.A. Metodika polevogo opyta [Methods of field trials]. Moscow, 1985 (in Russ.).   
  45. Mokrousova E.P., Glazunova I.N., Kandybin N.V., Ermolova V.P. Sovmestnoe primenenie ingibitorov sinteza khitina s mikrobiologicheskimi preparatami protiv vreditelei shampin'onov. Problemy entomologii v Rossii [Combined use of chitin synthesis inhibitors with microbiological preparations against champignon pests. Problems of entomology in Russia. Vol. 2]. St. Petersburg, 1998, tom 2: 40-41 (in Russ.).   
  46. Ivantsova E.A. Optimizatsiya fitosanitarnogo sostoyaniya agrobiotsenozov Nizhnego Povolzh'ya. Avtoreferat doktorskoi dissertatsii [Optimization of the phytosanitary state of agrobiocenoses in the condition Lower Volga region. DSc Thesis].Saratov, 2009 (in Russ.).   
  47. Stus' A.A., Ermolova V.P. Kandybin N.V. Byull. VNIISKHM, 1989, 52: 25-27 (in Russ.). 






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