doi: 10.15389/agrobiology.2020.1.87eng

UDC: 635.21:632.7:632.937.15

Whole genome sequencing was financially supported by the Ministry of Science and Higher Education of the Russian Federation. Annotation of toxins and virulence factors was financially supported by the Russian Science Foundation, grant No. 18-76-00028.



M.E. Belousova1, S.D. Grishechkina1, V.P. Ermolova1, K.S. Antonets1,
A.V. Mardanov2, A.L. Rakitin2, A.V. Beletsky2, N.V. Ravin2,
A.A. Nizhnikov1

1All-Russian Research Institute for Agricultural Microbiology, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail (✉ corresponding author),,,,;
2Institute of Bioengineering, Research Center of Biotechnology RAS, 33/2, Leninskii prosp., Moscow, 119071 Russia, e-mail,,,

Belousova M.V.
Rakitin A.L.
Grishechkina S.D.
Beletsky A.V.
Ermolova V.P.
Ravin N.V.
Antonets K.S.
Nizhnikov A.A.
Mardanov A.V.

Received September 9, 2019


Multifunctional microbiological preparations are promising for use in plant protection due to their diverse effects including growth-promoting effect and complex antifungal and insecticidal activity. One of the key microorganisms used as the basis of biological preparations production is the gram-positive spore-forming bacterium Bacillus thuringiensis (Bt). The high specificity of the action and the environmental safety of Bt-based preparations contribute to maintain biocenosis balance and to reduce the number of treatments as well as to obtain environmentally friendly products. Previously, Bacillus thuringiensis var. darmstadiensis 56 (BtH10 56) strain was isolated and selected at the All-Russian Research Institute of Agricultural Microbiology. It possesses insecticidal effect for the larval stages of leaf-eating insect pests, growth-promoting activity for potatoes and antifungal effect against various phytopathogenic fungi. This paper presents the first data on sequencing and annotation of the whole genome of the BtH10 56 industrial strain; the factors responsible for the insecticidal and antifungal activity of this strain are identified, and the high efficiency of the biological preparation based on this strain is demonstrated under the field conditions against the Colorado potato beetle (Leptinotarsa decemlineata Say.). The goal of the work was to identify the molecular determinants of the insecticidal properties of the industrial strain Bacillus thuringiensis var. darmstadiensis 56 as well as to test its activity in the field. Field trials of the effectiveness of the biological preparation based on BtH10 56 against the Colorado potato beetle was carried out on potatoes (Solanum tuberosum L.) of the Vineta and Rocco varieties in 2018 and 2019 (MTS-Agro LLC, Voronezh Province) in the area of 1 ha. To evaluate the entomocidal activity, we used a liquid form of the preparation based on the strain produced by the Ekos branch of ARRIAM (the spore titer was 2.12-2.3×109 CFU/ml) in yeast-polysaccharide medium in a 100 l bioreactor. The application rate of the preparation was 20 l/ha. The potato plantings were treated using an OPG-2000 sprayer (Zarya LLC, Russia). As a chemical standard, the insecticidal preparations Cepellin, EC and Colorado, SC (Agro Expert Group LLC, Russia) at 100 g/l and 0.1 l/ha doses, respectively, were used as the chemical standards. The counts were carried out in 5, 10 and 14 days after treatment. The biological effectiveness of the preparation was determined by analyzing a decrease in the number of pests according to the Abbott formula. According to the test results, the high efficiency of the developed preparation against the Colorado potato beetle was established. This efficiency varied from 83.8 to 87.8 % and did not differ from the chemical standards. Using Illumina and Oxford Nanopore technology, we obtained the whole genome sequence of the ВtH10 56 strain. After assembly and annotation of the genome, a search for toxins was conducted. The CryProcessor and BtToxin_scanner programs were used to search and classify genes encoding the Bt insecticidal toxins. As a result, a gene belonging to the cry1E group, cry1Ea7, was found. The toxins belonging to this group are characterized by activity against various Lepidoptera pests. It was found that the genome of the strain does not contain genes encoding Vip, Sip and Cyt. toxins, however, it harbors several genes encoding synthetases of non-ribosomally synthesized peptides (nrp) that may explain its multifunctional properties. Thus, considering the data obtained the liquid form of the biological preparation based on BtH10 56, can be recommended for use in the industry and organic farming.

Keywords: Bacillus thuringiensis var. darmstadiensis, insecticidal activity, exotoxin, endotoxin, Oxford Nanopore, Illumina, Cry toxin, Bt, Colorado potato beetle, Leptinotarsa decemlineata.



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