RU2033721C1 - Strain of bacterium bacillus thuringiensis var kurstaki for bioinsecticide preparing - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: strain of bacterium Bacillus thuringiensis var. kurstaki 7A is obtained by chemical mutagenesis using ethylmethanesulfonate and deposited in All-Russian Collection of Industrial Microorganisms at number B-5155. For bioinsecticide preparing strain is grown on the medium containing food yeast, corn flour and tap water. Activity of cultural fluid with respect to gypsy moth larvae is 0.044%. EFFECT: preparing of bacterial strain indicated above. 3 tbl

Description

 The invention relates to the microbiological industry and is a new, non-spore forming strain of Bacillus thuringiensis (BT) III serotype (NS) var. kurstaki 7A (VKPM-5155) to produce an environmentally friendly, live-spore-free entomocidal biological product for controlling lepidopteran insect pests in agriculture.

 Currently available entomopathogenic preparations based on Bac. thuringiensis contain a mixture of dry spores and δ-endotoxin crystals. Although it is known that the main active principle is the protein insecticidal δ-endotoxin and the presence of spores is optional. The titer of viable spores in the preparations reaches 60-100 billion / g. With the use of such drugs, protein crystals decompose and disappear after some time, while spores, due to their stability, continue to exist for a long time and can grow and multiply under favorable conditions.

 In this regard, it is promising to create indisputable bioinsecticides even more environmentally friendly than existing plant protection products (plant protection products).

 Known strain VT-H3 Z-52 (base analog) producer of δ-endotoxin (ed. St. USSR N 769787, 1984).

 However, the disadvantage of pcs. Z-52 is the formation in addition to δ-endotoxin of a significant number of spores that can germinate, multiply and pollute the environment.

 Known strain VT-H4 var. dendrolimus IPM-2a asphorogenic producer of δ-endotoxin (ed. St. USSR N 1367484, 1985).

 However, the disadvantage of this strain is lower entomocidal activity and lower yield of δ-endotoxin than that of the proposed strain.

 Known strain BT var / kurstaki IPM-13A asporogenous mutant.

 However, the disadvantage of this strain is also lower insecticidal activity.

 To achieve the goal, an aporogenic strain of Bacillus thuringiensis var.kurstaki 7A is proposed. The strain is stored in the culture collection of the All-Russian Research Institute of Applied Microbiology and deposited in the All-Union Collection of Industrial Microorganisms (collection number VKPM N V-5155).

 Strain 7A was obtained from strain Z-52 by chemical mutagenesis using ethyl methane sulfonate and subsequent selection based on the absence of spore formation.

 Strain 7A is characterized by the following features.

 Morphological and cultural characteristics.

Aerobe. Gram-positive. Optimum growth of 29-32 ^o C. Vegetative cells large mobile sticks with rounded ends, arranged singly, in pairs or chains. Cell size (1.5-1.7) (3.6-6.2) microns. In the stationary phase of development, rhomboid-shaped protein crystalline inclusions of size (0.6-0.8) ˙ (1.3-2.0) microns are produced, spores are not formed, since sporulation is blocked at stage IV.

 The nature of growth in various nutrient media.

 On meat-peptone agar, after 1 day of incubation, roundish grayish-white colonies with a diameter of 3-5 mm with a slightly wavy edge, a dull fine-grained surface, and a pasty consistency are formed. After 3-5 days, the colonies become gray, smooth, a transparent border appears along the edge.

 On dry nutrient agar, growth is plentiful, colonies are rounded, creamy white, dull, fine-grained with a slightly rugged edge, pasty consistency.

 On milk agar, growth is plentiful, colonies are white, smooth, matte with an enlightenment zone of up to 18-20 mm.

 On potato agar, growth is plentiful, the colonies are round, white, smooth with a slightly rugged edge.

 Growth is plentiful on slices of potato, the raid is fine-grained, opaque, the pigment is not formed.

 On the meat-peptone broth, a uniform turbidity is formed.

 Yolk medium after 1 day. incubations form rounded serotova-white colonies with a diameter of 2-6 mm with a slightly wavy edge, matte, with a whitening zone of the medium around the colonies up to 5 mm.

 Physiological and biochemical characteristics.

 Relation to carbon sources. Uses glucose, maltose, starch, cellobiose, salicin, esculin. It does not utilize sucrose mannose, mannitol, lactose.

 Attitude to nitrogen sources. Absorbs ammonium forms of nitrogen. Restores nitrates. Milk pentonizes. It dilutes gelatin. Urea decomposes.

 Forms acetylmethylcarbinol and lecithinase.

 Antigenic properties. Belongs to the III serotype of Bac.thuringiensis var.kurstaki (НЗа3в).

 Pathogenic properties. Does not produce thermostable exotoxin. Pathogenic for many species of Lepidoptera insects.

 Not toxic to warm-blooded animals.

 Relation to phages. Resistant to phages I-77. 11, 25/16. Sensitive to phages s / n, 17, 22. Does not isolate its own virulent phages.

 The essence of the invention is as follows.

Strain 7A is grown for 12-16 hours on beveled MPA. The resulting culture inoculate a fermentation medium of the following composition, wt. feed yeast (BVK) 3.0, corn flour 1.5, tap water the rest. pH after sterilization of 6.8-7.2. Cultivation was carried out in 750 ml flasks with 30 ml culture medium on a rotary shaker at a speed of rotation of 260 rev / min at 29-13 ^{° C} for 48 h. The resulting QOL heated at 70 ^{° C} for 20 min and plated on 0.1 ml per MPa. Visulano no growth of the colonies of the strain is observed, indicating the absence of viable spores in the culture fluid.

PRI me R 1. 5 ml of a suspension of spores (10 spores / ml) of strain Z-52 in phosphate buffer (0.1 M, pH 7.0) is heated in a water bath at 70 ^about C for 20 minutes After cooling to room temperature the suspension was added 0.1 ml of ethyl methanesulfonate and incubated at ²⁸ ° C on a rotary shaker for 15-18 hours. The mixture was then centrifuged at 4000 rev / min for 20 min at 5 ° ^C. The precipitate was washed twice with saline and transferred to 2.5 ml of phosphate buffer. 100 μl of the resulting suspension are plated on Petri dishes with MPA and incubated at 30 ^about C for 2-3 days. The grown colonies are examined using phase control microscopy and clones are selected without sporulation.

PRI me R 2. The culture of strain 7A is grown on beveled MPA at 30 ^about C for 12-18 hours and used for inoculation of 300 ml of fermentation medium in a flask of 2 l. Seed culture was grown in a flask shaker at ³⁰ ° C for 7 hours, checking the absence of the foreign microflora and the phage and used to inoculate the fermenter.

 The fermentation medium has the following composition, wt. feed yeast (BVK) 3, corn flour 1.5, tap water the rest.

Fermentation is carried out until complete cell lysis and the presence of 50-70% free crystals in the medium, which is controlled by phase contrast microscopy. Get a culture fluid containing crystals of δ-endotoxin, a small admixture of non-lysed vegetative cells and the remains of a nutrient medium. The insecticidal activity of the culture fluid in relation to unpaired silkworm larvae (based on LC ₅₀ ) is 0.022-0.066%
The tables below show the properties of the proposed strain and preparations based on it in comparison with prototypes.

 In the table. 1 shows comparative data on the insecticidal activity of the culture fluid and the productivity of the proposed pcs. 7A, prototypes (pcs. IPM-13a, pcs. IPM-2a) and analogue (pcs. Z-52).

 As follows from the data given in table. 1 PC. 7A superior to pcs. IPM-13a, pcs. IPM-2a and pcs. Z-52 for insecticidal activity, respectively, at 1.88; 27.0 and 1.57 times.

 In the table. 2 presents comparative data on sensitivity to phage strains.

 The results presented in table. 2, show that by phage resistance pcs. 7A, pcs. IPM-13a and pcs. IPM-2a are on the same level: all strains are sensitive to three of the six phages isolated in the production of plant protection products.

 In the table. 3 presents comparative data on insecticidal activity and the content of δ-endotoxin in samples of preparations based on the proposed strain and prototype strains obtained according to the regulations for the production of concentrated lepidocide.

 From the data presented in table. 3, it follows that the insecticidal activity of a sample of the drug based on the proposed pcs. 7A, obtained by traditional technology, surpasses that of drugs based on prototype strains.

Claims (1)

 The bacterial strain Bacillus thuringiensis var. Kurstaki VKPM B-5155 for the production of bioinsecticide.

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