doi: 10.15389/agrobiology.2023.3.416eng

UDC: 632.7:579.64

Supported by the Ministry of Science and Higher Education of the Russian Federation (agreement № 075-15-2021-1055 dated September 28, 2021 on providing a grant in the form of subsidies from the Federal budget of the Russian Federation). The grant was provided for the implementation of the project: “Mobilization of the genetic resources of microorganisms on the basis of the Russian Collection of Agricultural Microorganisms (RCAM) at the All-Russia Research Institute for Agricultural Microbiology (ARRIAM) according to the network principle of organization”.
The authors declare no conflict of interests



S.D. Grishechkina1 , T.K. Kovalenko2, T.V. Kirpicheva3,
K.S. Antonets1, 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 — Branch of the Chaika Federal Research Center of Agricultural Biotechnology of the Far East, 42-a, ul. Mira, s. Kamen’-Rybolov, Primorsky Krai, 692682 Russia, e-mail;
3Ekaterininskaya Experimental Station — Branch of the Federal Research Center Vavilov All-Russian Institute of Plant Genetic Resources, Ekaterininsky experimental station, s. Ekaterino, Nikiforovsky District, Tambov Province, 393023 Russia, e-mail

Grishechkina S.D.
Antonets K.S.
Kovalenko T.K.
Nizhnikov A.A.
Kirpicheva T.V.
Tikhomirova N.Yu.

Final revision received October 16, 2022
Accepted February 28, 2023

One of the trends in the biological control of pests is the use of bacteria belonging to the genus Bacillus and, first of all, entomopathogenic strains of Bacillus thuringiensis. Of great interest to industrial biotechnology are studies related to the search for optimal cultivation conditions that can improve the manufacturability of the production of microbiological preparations and their effectiveness. Previously, the nutrient media for the production of microbiological preparations based on B. thuringiensis which include natural organic components have been developed. Nevertheless, during the production of biopreparations based on this bacterium, the foaming of the culture frequently occurs and expensive filters of bioreactors have to be replaced. Also, during the treatment of plants, working solutions containing organic components of the liquid medium can clog the nozzles. This effect complicates the treatment process. In addition, organic cultural media components are not standard and depend on the quality and source origin. In this regard, it is important to carry out the screening of optimal synthetic media that could eliminate these shortcomings. Our study was aimed at selecting the optimal synthetic media and evaluating the effectiveness of the obtained preparation samples in laboratory and field conditions. The objects of study were the cultures of B. thuringiensis var. thuringiensis 800/15 (BtH1 800/15) and B. thuringiensis var. darmstadiensis 25 (BtH10 25). The composition of the culture media was as follows: CCY medium — 0.5 mM MgCl2·6H2O, 0.01 mM MnCl2·4H2O, 0.05mM FeCl3·6H2O, 0.05 mM ZnCl2, 0.2 mM CaCl2·6H2O, 13 mM KH2PO4, 26 mM K2HPO4, 20 mg /l glutamine, 1 g/l casein hydrolysate, 0.4 g/l yeast extract, 0.6 g/l glycerol; MBt medium: 7 g/l casein hydrolyzate, 6.8 g/l KH2PO4, 0.12 g/l MgSO4·7H2O, 0.0022 g/l MnSO4·4H2O, 0.014 g/l ZnSO4·7H2O, 0.02 g/l Fe2(SO4)3, 0.18 g/l CaCl2·4H2O; LB medium: 10 g/l trypton, 5 g/l yeast extract, 10 g/l NaCl; modified semi-synthetic medium MMBt (modified MBt): 7 g/l casein hydrolyzate, 6.8 g/l KH2PO4, 0.12 g/l MgSO4·7H2O, 0.0022 g/l MnSO4·4H2O, 0.014 g/l ZnSO4·7H2O, 0.02 g/l Fe2(SO4)3, 0.18 g/l CaCl2·4H2O (25), glucose (1.0 %), Na citrate (2 g/l). Yeast polysaccharide media (YPM) for BtH1 and BtH10 served as a reference. Bt strains were cultivated in 750 ml Erlenmeyer flasks filled with 40-50 ml of medium on a shaker at 220 rpm and 29 °C for 48-72 h until the maturation of culture, accompanied by the formation of spores and crystalline endotoxin. On the basis of BtH1 800/15 and BtH10 25 strains, batches of liquid preparations were obtained, the effectiveness of which was evaluated in 2020 and 2021 on potatoes (Solanum tuberosum L.) of the Yantar variety in the Far East (Ussuri district of Primorsky Krai) against Henoseplachna vigintioctomaculata Motsch and on potatoes of the Emelya variety in the Tambov region against Leptinotarsa decemleniata Say. In the experiments, liquid preparations obtained on YPM and MMBt were used, which were used at consumption rates of 15 and 20 l/ha. The biological effectiveness of the preparations was calculated according to the formula W.S. Abbot. The antifungal activity of the preparation BtH10 25, obtained on MMBt and YPM, was determined by the method of agar blocks in vitro in Petri dishes. The control medium was used without the addition of drugs. Fungi Botrytis cinerea Pers (strain C-5) and Bipolaris sorokiniana (Sacc.) Shoemaker (strain C-20) served as test cultures. The inhibitory activity was calculated according to the W.S. Abbot. Cultivation of BtH1 800/15 and BtH10 25 strains on different nutrient media showed that on semi-synthetic media MBt and LB CFU titers were 2 times lower than on YPM, while on CCY medium they were 10 times lower. Their activity, determined by the content of exotoxin, was also lower, but on the MBt medium it was slightly inferior to YPM for BtH1 800/15. Therefore, MBt medium was chosen for further studies, and the composition of this medium was modified by adding glucose (1.0 %) and Na citrate (2 g/l). The resulting MMBt medium made it possible to achieve a significant increase in titers, activity, and the rate of culture development compared to the initial MBt. In 2020 in the Tambov region, the effectiveness of the preparation based on BtH1 800/15 obtained on DPS was high against the Colorado potato beetle and on the 5th day was 95.3 %, slightly inferior to the chemical standard. In the case of preparation obtained on MMBt, it was slightly lower (83.3 %), but the protective effect lasted longer, and on day 15 the efficiency was 73.7 %. In 2021, the efficacy of BtH1 800/15 was lower than in 2020. In the preparation obtained on MMBt, it was slightly inferior to the effectiveness of the preparation obtained on YPM, amounting to 75.3 and 67.7 %, respectively, on the 5th day after treatment. The effect of the BtH10 25 preparation obtained on MMBt was weaker than in the variant with BtH1 800/15 (47.7 % on day 5). In Primorsky Krai, the high efficacy of liquid preparations against H. vigintioctomaculata was also noted. In 2020, at a rate of application of the BtH1 800/15 preparation of 15 l/ha, the effectiveness in the YPM and MMBt variants was 60.5 and 63.9 %, respectively, on day 5. Similar data was obtained in 2021. The inhibitory activity of the BtH10 25 preparation obtained on MMBt was 12 % higher on day 5 than that of the preparation obtained on YPM, and was 72.3 and 60.8 % for B. sorokiniana and 78.9 and 67.4% for B. cinerea. On day 10, this trend persisted, but for the preparation produced on YPM, a decrease in the inhibition of the growth of B. sorokiniana and B. cinerea colonies, respectively, to 57.3 and 44.3% was noted. Thus, preparations based on Bacillus thuringiensis obtained on the MMBt medium were only slightly inferior in terms of effectiveness against pests to preparations obtained on YPM, while their effectiveness against phytopathogens was higher than that of preparations with YPM. The MMBtmedium is promising for agricultural biotechnology, since its use reduces the time required for the formation of spores and crystalline protein endotoxin by increasing the growth rate of the B.thuringiensis culture. Thus, on the MMBt medium, this process ends after 48 h, and on the YPM medium, after 72 h, which makes it possible to reduce energy consumption.

Keywords: biopreparation, Bacillus thuringiensis var. thuringiensis, Bacillus thuringiensis var. darmstadiensis, Bipolaris sorokiniana, Botrytis cinerea, Colorado potato beetle, potato ladybug, inhibitory activity, cultural medium.



  1. Navon A. Bacillus thuringiensis insecticides in crop protection — reality and prospects. Crop Protection, 2000, 19(8-10): 669-670 CrossRef
  2. Kalmykova G.V., Gorobey I.M., Osipova G.M. Biotekhnologiya,2016, 4: 12-19 (in Russ.).
  3. De Maagd R.A., Bravo A., Crickmore N. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends in Genetics, 2001, 17(4): 193-199 CrossRef
  4. Marroquin L.D., Elyassnia D., Griffitts J.S., Feitelson J.S., Aroian R.V. Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans. Genetics, 2000, 155(4): 1693-1699 CrossRef
  5. Grishechkina S.D. Mechanism and activity spectrum of microbiological preparation batsikol with phytoprotective action. Sel'skokhozyaistvennaya biologiya [Agricultural Biology], 2015, 50(5): 685-693 CrossRef
  6. Schnepf E., Crickmore N., Van Rie J., Lereclus D., Baum J., Feitelson J., Zeigler D.R., Dean D.H. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews,1998, 62(3): 775-806 CrossRef
  7. Siegel J.P. The mammalian safety of Bacillus thuringiensis —based insecticides. Journal of Invertebrate Pathology,2001, 77(1): 13-21 CrossRef
  8. Raymond B., Federici B.A. In defence of Bacillus thuringiensis, the safest and most successful microbial insecticide available to humanity — a response to EFSA. FEMS Microbiology Ecology, 2017, 93(7):fix084 CrossRef
  9. Belousova M.E., Malovichko Yu.V., Shikov A.E., Nizhnikov A.A., Antonets K.S. Dissecting the environmental consequences of Bacillus thuringiensis application for natural ecosystems. Toxins, 2021, 13(5): e355 CrossRef
  10. Grishechkina S.D., Smirnov O.V., Kandybin N.V. Mikologiya i fitopatologiya, 2002, 36(1): 58-62 (in Russ.).
  11. Smirnov O.V., Grishechkina S.D. Polyfunctional activity of Bacillus thuringiensis Berliner. Sel'skokhozyaistvennaya biologiya, 2011, 3: 123-126.
  12. Grishechkina S.D., Ermolova V.P., Kovalenko T.K., Antonets K.S., Belousova M.E., Yakhno V.V., Nizhnikov A.A. Polyfunctional properties of theBacillus thuringiensis var. thuringiensis industrial strain 800/15. Sel'skokhozyaistvennaya biologiya [Agricultural Biology],2019, 54(3): 494-504 CrossRef
  13. 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): 347 CrossRef
  14. Mordkovichskiy K.Z. Zashchita i karantin rasteniy, 2016, 3: 36-38 (in Russ.).
  15. Kovalenko T.K., Matsishina N.V. Chteniya pamyati A.I. Kurentsova [Readings in memory of A.I. Kurentsov]. Vladivostok, 2015, vyp. KhKhVI: 128-136 (in Russ.).
  16. Volkov O.G., Smirnov Yu.V., Kovalenko T.K. Karantin rasteniy. Nauka i praktika, 2012, 1(1): 41-44 (in Russ.).
  17. Grishechkina S.D., Ermolova V.P. Efficiency of batsikol based on a new strain Bacillus thuringiensis var. darmstadiensis № 25 for biocontrol of phytophagous pests and phytopathogens. Sel'skokhozyaistvennaya biologiya [Agricultural Biology], 2015, 50(3): 361-368 CrossRef
  18. Kamoun F., Zouari F.N., Saagdaoui I., Jaoua S. Improvement of Bacillus thuringiensis bacteriocin production through culture conditions optimization. Preparative Biochemistry & Biotechnology, 2009, 39(4): 400-412 CrossRef
  19. Martínez-Cardeñas J.A., de la Fuente-Salcido N.M., Salcedo-Hernández R., Bideshi D.K., Barboza-Corona J.E. Effects of physical culture parameters on bacteriocin production by Mexican strains of Bacillus thuringiensis after cellular induction. Journal of Industrial Microbiology and Biotechnology, 2012, 39(1): 183-189 CrossRef
  20. Prabakaran G., Balaraman K., Hoti S.L., Manonmani A.M. A cost — effective medium for the large-scale production of. B. sphaericus H5a5b (VCRC) for mosquito control. Biological Control,2007, 41(3): 379-383 CrossRef
  21. Pearson D., Ward O.P. Effect of culture conditions on growth and sporulation of Bacillus thuringiensis subsp. israelensis and development of media for production of the protein crystal endotoxin. Biotechnol. Lett., 1988, 10: 451-456 CrossRef
  22. Kalmykova G.V., Cheshkova A.F., Akulova N.I. Sibirskiy vestnik sel’skokhozyaystvennoy nauki,2020, 50(2): 44-51 CrossRef (in Russ.).
  23. Smith R.A. Effect of strain and medium variation on mosquito toxin production by Bacillus thuringiensis var. israelensis. Canadian Journal of Microbiology,1982, 28(9): 1089 CrossRef
  24. Gladstone G.P., Fildes P.A. A simple culture medium for general use without meat extract or peptone. British Journal of Experimental Pathology, 1940, 21(4): 161-173.
  25. Lecadet M.-M., Dedonder R. Biogenesis of the crystalline inclusion of Bacillus thuringiensis during sporulation. European Journal of Biochemistry, 1971, 23(2): 282-294 CrossRef
  26. Bertani G. Studies on lysogenesis I. The mode of phage liberation by lysogenic Escherichia coli. Journal of Bacteriology, 1951, 62(3): 293-300 CrossRef
  27. Tikhonovich I.A., Ermolova V.P., Grishechkina S.D., Romanova T.A. Shtamm Bacillus thuringiensis var. thuringiensis № 800/15 v kachestve sredstva dlya polucheniya еntomotsidnogo preparata. Patent St. Petersburg, GNU VNII sel’skokhozyaystvennoy mikrobiologii RU 2514211 S1. Zayavl. 10.10.2012. Opubl. 27.04.2014. Byul. № 12 [The strain of Bacillus thuringiensis var. thuringiensis No. 800/15 as a means for obtaining an insecticide preparation. Patent Petersburg, GNU All-Russian Research Institute of Agricultural Microbiology RU 2514211 C1. Appl. 10/10/2012. Publ. 04/27/2014. Bull. № 12] (in Russ.).
  28. Tikhonovich I.A., Ermolova V.P., Grishechkina S.D., Romanova T.A. Shtamm Bacillus thuringiensis var. darmstadiensis № 25 v kachestve sredstva kompleksnogo vozdeystviya na vrednykh zhestkokrylykh nasekomykh i fitopatogennye griby. Patent St. Petersburg, GNU VNII sel’skokhozyaystvennoy mikrobiologii RU 2514211 S 1. Zayavl. 26.12.2012. Opubl. 27.04.2014. Byull. № 12 [The strain of Bacillus thuringiensis var. darmstadiensis No. 25 as a means of complex action on harmful beetles and phytopathogenic fungi. Patent Petersburg, GNU All-Russian Research Institute of Agricultural Microbiology RU 2514211 C 1. Appl. 12/26/2012. Publ. 04/27/2014. Bull. № 12](in Russ.).
  29. Grishechkina S.D., Ermolova V.P., Minina G.N., Safronova V.I., Bologova E.V. Metodika. Kollektsiya shtammov bakteriy-simbiontov vrednykh nasekomykh i gryzunov, prigodnykh dlya biokontrolya chislennosti vrediteley sel’skokhozyaystvennykh rasteniy [Methodology. Collection of strains of bacteria-symbionts of harmful insects and rodents suitable for biocontrol of pests of agricultural plants]. St. Petersburg, 2014 (in Russ.).
  30. Abbott W.S. A method for computing the effectiveness of insecticide. Journal of Economic Entomology, 1925, 18(2): 265-267 CrossRef
  31. Metody еksperimental’noy mikologii /Pod redaktsiey V.I. Bilay [Methods of experimental mycology. V.I. Bilay (ed.)]. Kiev, 1982 (in Russ.).
  32. Dospekhov B.V. Metodika polevogo opyta [Methods of field trials]. Moscow, 1985 (in Russ.).
  33. Nickerson K.W., Bulla Jr. L.A. Physiology of sporeforming bacteria associated with insects: minimal nutritional requirements for growth, sporulation, and parasporal crystal formation of Bacillus thuringiensis. Appl. Microbiol., 1974, 28(1): 124-128 CrossRef
  34. Biryukov V.V. Osnovy promyshlennoy biotekhnologii [Fundamentals of industrial biotechnology]. Moscow, 2004 (in Russ.).
  35. 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
  36. Domínguez-Arrizabalaga M., Villanueva M., Escriche B., Ancín-Azpilicueta C., Caballero P. Insecticidal activity of Bacillus thuringiensis proteins against coleopteran pests. Toxins (Basel), 2020, 12(7): 430 CrossRef
  37. Tsarenko I.Yu., Roy A.A., Kurdish I.K. Microbiol. zhurn., 2011, 73(2): 13-19.
  38. Matsumoto T., Sugiura Y., Kondo A., Fukuda H. Efficient production of protopectinases by Bacillus subtilis using medium based on soybean flour. Biochemical Engineering Journal, 2000, 6(2): 81-86 CrossRef
  39. Devidas P.C., Pandit B.H., Vitthalrao P.S. Evaluation of different culture media for improvement in bioinsecticides production by indigenous Bacillus thuringiensis and their application against larvae of Aedes aegypti Scientif. The ScientificWorld Journal, 2014, 2014: 273030 CrossRef
  40. Anderson R.K.I., Jayaraman K. Influence of carbon and nitrogen sources on the growth and sporulation of Bacillus thuringiensis var galleriae for biopesticide production. Chem. Biochem. Eng. Q., 2003, 17(3): 225-231. 
  41. Sarrafzadeh M.H. Nutritional requirements of Bacillus thuringiensis during different phases of growth, sporulation and germination evaluated by Plackett-Burman method. Iran. J. Chem. Chem. Eng., 2014, 31(4): 131-136 CrossRef
  42. Saberi F., Marzban R., Ardjmand M., Shariati F.P., Tavakoli O. Optimization of culture media to enhance the ability of local Bacillus thuringiensis var. tenebrionis. Journal of the Saudi Society of Agricultural Sciences, 2020, 19(7): 468-475 CrossRef
  43. Pustake S.O., Bhagwat P.K., Dandge P.B. Statistical media optimization for the production of clinical uricase from Bacillus subtilis strain SP6. Heliyon, 2019, 5(5): e01756 CrossRef







Full article PDF (Rus)