RU2061376C1 - Strain of bacterium bacillus thuringiensis spp thuringiensis for production of exotoxin-containing bioinsecticides - Google Patents

Abstract

FIELD: microbiological industry, bacterial insecticides. SUBSTANCE: strain of bacterium Bacillus thuringiensis 16-150 is isolated from the strain 98 by multistep selection for exotoxin formation features, spore thermostability and phase-resistance. Strain is deposited in ВКПМ at number B-6404. At strain cultivation on the yeast-polysaccharide medium the obtained cultural fluid shows cell titer 3.2 billion cells/ml, thermostable spore content is 0.5%, thermolabile spore content is 95%, exotoxin level is 1.8 mg/ml. EFFECT: increased exotoxin yield, strain indicated above. 3 tbl

Description

 The invention relates to the microbiological industry and biotechnology, in particular to the production of bacterial insecticides for controlling plant pests: aphids, spider mites, cotton scoops, cabbage fly and larvae of Colorado flour, as well as against larvae of bestial and synanthropic flies.

 Food supply is directly dependent on the preservation of crops from pests that destroy up to 30% of the crop. The use of chemical insecticides in view of their danger to the environment is impractical. Organic insecticides based on the bacteria Vaci11us thuringiensis are environmentally friendly. The active principle of insecticidal activity is protein crystalline endotoxin, which has a high specificity of action. The bacteria is you. in addition to endotoxin, thermostable exotoxin (synonyms beta-exotoxin, flu factor), which is an atypical nucleotide, which contains the remains of adenine, ribose, glucose, alloslyzum and orthophosphoric acids, is produced by thuringiensis subspecies of the thuringiensis I serotype. Exotoxin is relatively safe for warm-blooded and human, and its spectrum of action for insects is almost unlimited, it can be used both intestinal and contact poison. Endotoxin bacteria you. thuringiensis subsp.thuringiensis toxic to representatives of the order Lepidoptera.

 Currently based on the bacteria Vasillus thuringiensis subsp. thuringiensis is produced by two domestic insecticidal preparations: Bitoxibacillin containing exo- and endotoxins and Thuringin based on beta-exotoxin.

 As the closest analogue, the producer of Bitoxibacillin - Vacillus thuringiensis subsr. thuringiensis 98 [1] secreting exotoxin in a relatively low concentration of 0.6 to 0.8 mg / ml. Another significant drawback of this strain is sensitivity to phages [2] The high spore-forming ability of the known strain with prolonged use of a bacterial protective agent can lead to the accumulation of spores in the soil and cause a shift in the ecological balance of soil microorganisms.

 The technical result of the claimed invention consists in obtaining a bacterial strain of Bac. Thuringiensis subsp.thuringiensis, which produces higher concentration of exotoxin and endotoxin with activity not lower than the strain described in the closest analogue in the culture fluid. While maintaining the level of spore formation, it is proposed to reduce the thermal stability of spores.

 This goal is achieved by using as a producer of strain Bac. thuriпgiensis NI Scientific Research Institute of Genetics 16-T5O (VKPM B-6404), which has higher technological characteristics: increased concentration of exotoxin, comparative thermolability of spores, active protein endotoxin and phage resistance.

 The inventive strain was isolated by multistage selection from strain 98 on the grounds of exotoxin formation, spore heat lability and phage resistance, has the following characteristics.

 1. Cultural and morphological characters. Gram-positive moving sticks, 1.2-1.6 x 2.5-3.0 microns in size.

On Hottinger agar at 28 ^° C, after 24 hours of growth, it forms round colonies with uneven edges and a fine-grained surface with a diameter of 4-5 mm. When growing in Hottinger broth at 28 ^° C after 18-24 hours, a moderately turbid suspension of cells forms, when standing precipitates, single spores form after 72 hours.

In the yeast-polysaccharide medium (DPS) of the following composition, g / l: fodder yeast -30, corn flour -15, tap water up to 1 l, pH 7.0 7.2, after 48 h of incubation at 30 ^o C the titer of viable cells is 3.2 billion / ml, thermostable spores of 0.05 form crystalline rhomboid inclusions of 2 x 0.6 microns and thermostable exotoxin.

 2. Biochemical signs.

Strain 16-T50 does not ferment mannose, salicin, esculin, does not form lecithinase, hydrolyzes starch, ferments sucrose. The strain assimilates ammonium ions and glutamic acid. The cultivation mode is 28-34 ^o C, the optimum growth is 30 ^o C. The pH of the nutrient medium for optimal cultivation is 6.8-7.0.

 3. Antigenic properties.

 Vegetative cells of the strain NIIgenetika 16-T50 give a positive agglutination reaction with serum H1.

 4. Antagonistic properties.

 Strain 16-T50 inhibits the growth of a number of bacterial species: Sarcina f1ava, Bac. Subti1is and Bac. Thuringiensis of other subspecies.

 5. Genetic characteristics.

 The strain is resistant to penicillin (in μg / ml) -100, polymyxin -100, tetracycline 25. The crystals of the strain consist of a protein with a molecular weight of -130 kDa.

 6. Insecticidal properties.

 The strain is highly active against dipterans (Musca domestic, Aedos aegirtu), coleopteran insects (Colorado potato beetle), aphids, cabbage flies due to exotoxin; The protein crystal of the strain is active against unpaired silkworm.

 7. Phagosensitivity.

 Strain 16-T5O is resistant to the phages Tg4, Tg13, Tg81, K, M, E-5, I-77,2,3, 4,5 and limits the development of phages of the BTB group by 8 orders of magnitude.

 8. Storage.

 The strain is recommended to be stored in lyophilized form in sealed glass ampoules or as a spore preparation in a liquid or agarized DPS medium.

 The invention is illustrated by the following examples.

 Example 1. The cultivation of strain 16-T5O.

The culture of strain 16-T50 is grown 24 hours at a temperature of 28-30 ^o C in test tubes on the mowed joints of Hottinger, transferred to Hottinger broth (5 ml) and incubated for 4 hours, then 5 ml are introduced into flasks with a capacity of 750 ml with 50 ml of DPS medium and cultivated 48 hours at 30 ^o With a rocking chair. The titer of viable cells and the number of thermostable spores are determined by titration of the culture before and after heating at 70 ^{° C.} for 30 minutes. The ratio of vegetative cells, spores and crystals is controlled under an optical microscope.

 The results are presented in table 1.

 After 48 hours of incubation in DPS medium, strains 98 and 16-T5O form the same number of viable cells 3.2 billion / ml, however, the content of thermostable spores differs by 0.5 and 95%, respectively. Light microscopy reveals the same number of spores and crystals in the preparations of strains 98 and 16-T50. Strain 16-T50 forms mainly heat-labile spores and this differs from the analogue strain.

Example 2. Comparison of the proposed strain and strain analogue to the insecticidal activity of exotoxin
To evaluate thermostable exotoxin, autoclaved (110 ^o C, 0.5 atm, 5-7 min) or warmed (100 ^o C, 30 min) culture samples are centrifuged, the supernatant is tested for activity against housefly stage III larvae and the Colorado potato beetle 1-2 age.

Dilutions of the supernatant are added to the feed (wheat bran, milk and water in a ratio of 3 1 6) 1 ml per 10 g, 25 larvae of housefly are placed and incubated at 25 ^o C. The pupae are counted at 4-6, and the imago at 10 day after departure.

 To evaluate the effect against the Colorado potato beetle, the potato leaves are dipped in dilutions of the supernatant, dried in air and placed in Petri dishes with 10 beetle larvae. Incubated at room temperature, after 3 days, count the dead larvae.

LK50 is determined by the formula: log LK50lg Cmax (Li -0.5) [3]
The results are presented in table 2.

 Example 3. Determination of the quantitative content of exotoxin in the culture fluid of the proposed strain 16-T5O and strain analogue 98.

The concentration of exotoxin is determined in the supernatant of the cultures: spectrophotometrically by precipitation of exotoxin with Ca-Ba salts, after removal of the salts by ion exchange resins at a wavelength of 260 nm [4] by anion exchange chromatography [5]
The results are presented in table.2.

 Example 4. The determination of the activity of protein endotoxin in the proposed strain 16-T50 and strain analogue 98.

The insecticidal activity of endotoxin is determined against 2-year-old silkworm caterpillars, against which exotoxin does not have activity. Dilutions of the culture fluid of 2 ml are added to 9 g of feed of the following composition, g: beans 200, agar-agar 15, sucrose 12, medical beer yeast - 15, folic acid 0.001, ascorbic acid 4, benzoic acid 2, diluted in 7 ml of ethyl alcohol K ₂ NRA 4; ml, water 350, linseed oil 2, formalin 5. Distribute into three Petri dishes, in which 10 caterpillars are placed. Incubated at room temperature, after 6 days, counting dead caterpillars is performed. LK50 is determined by the formula (see above).

The insecticidal activity of strain 16-T50 did not differ from the activity of the analogue strain and was 0.035
Example 5. Comparison of phage resistance of strains 16-T50 and 98.

 The sensitivity of strains to phages is determined by titration of phages by a two-layer method on Hottinger agar. Use phages of the BTB group isolated in the production of Bitoxibacillin, as well as phages specific for the bacteria Bac. Thuringiensis of other subspecies.

 The results are shown in table.3. The strain NIIgenetics 16-T5O is resistant to all studied phages of Vas.thuringiensis, while strain 98 is sensitive to phages isolated during industrial fermentation.

 Thus, the inventive strain has the following differences and advantages compared with the strain of the analogue.

 1. A higher content of exotoxin in the culture fluid, which provides a higher insecticidal activity.

 2. Phage resistance to phages of the BTB group.

 3. Thermolability spores.

 The above characteristics of the claimed strain 16-T5O allow us to recommend it for the production of exotoxin-containing bioinsecticides.

LITERATURE
1. Auth. testimonial. 1074138 (1982) Skvortsova M.I. Shashkina N.I. Murzintseva N.B. Drawn L.N. Pakhtuev A.I. Kandybin N, B. Barbashova N.M. Skvortsov G.E. Chegodaev F.N. Strain of VasiIIus thuringiensis subrs. thuringiensis 98 - producer of bitoxibacillin.

 2. Koroleva Yu. V. Grigoryeva TM Azizbekyan R.R. Isolation and characterization of phage-resistant mutants of Vasi11us thuringiensis var. thuringiensis. Biotechnology, 1992, 2: 3-5.

 3. Ashmarin N.P. Vorobiev A.A. Statistical methods in microbiological research. Medghiz. M. 1962.

 4. Danilova E. B. Barbashova N. M. Spectrophotometric determination of beta-exotoxin in the culture fluid of Vasillus thuringiensis. Tr. All-Russian Research Institute of Microbiology, 1985, vol. 55, p. 115-118.

 5. Bubenshchikova S. N. Kagramanova V.G. Baratova L.A. Danilova E.B. Kruglyak Ye. B. Determination of exotoxin by high performance chromatography. Chemistry of Natural Compounds, 1982, 3, 389-392. TTT1 TTT2

Claims (1)

 The bacterial strain Bacillus thuringiensis spp.thuringiensis VKPM B-6404 for the production of exotoxin-containing bioinsecticides.

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