RU2559548C2 - Strain of bacteria bacillus thuringiensis to control colorado potato beetle - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of Bacillus thuringiensis 16T100/18 (Russian National Research Institute of Plant Protection) has high insecticidal activity against the larvae and imago of Colorado potato beetle. Culture fluid of the strain is active against larvae of 1-2 age and young imago. The strain is deposited in the National Collection of Microorganisms pathogenic to plants and plant pests (State Research Institute of Russian National Research Institute of Plant Protection) under registration number B 94-1 RNRIPP.

EFFECT: invention enables to reduce the number of imago of Colorado potato beetle.

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Description

The invention relates to the microbiological industry, can be used in the manufacture of preparations for controlling pests of crops, in particular the Colorado potato beetle, and is a new strain of crystal-forming bacteria.

Ensuring food security is largely associated with the protection of crops from harmful arthropods, which destroy up to 30% of the crop. The widespread use of chemical insecticides is harmful to the environment and leads to the formation of resistant pests. Biological preparations based on the bacteria Bacillus thuringiensis are safer.

A known strain of B. thuringiensis var. thuringiensis - producer of an industrial drug - bitoxibacillin (Kandybin, 1972). The lack of strain B. thuringiensis var. thuringiensis - low pathogenicity for the Colorado potato beetle.

A known strain of bacteria Bacillus thuringiensis spp. thuringiensis 16-T50 for the production of exotoxin-containing bioinsecticides, selected by multi-stage selection based on the signs of exotoxin formation, thermal stability of spores and phage resistance (RF patent No. 2061376, 1996). Bacteria B. thuringiensis subspecies thuringiensis I serotype, in addition to endotoxin, produce a thermostable exotoxin (synonyms beta-exotoxin, fly factor), which is an atypical nucleotide that contains residues of adenine, ribose, glucose, alloslyzum and phosphoric acid. Endotoxin Bac. thuringiensis subsp. thuringiensis is toxic to representatives of the order Lepidoptera. Exotoxin has a wide spectrum of action for insects, since it can be used as intestinal and contact poison, however, according to modern concepts, it is unsafe for warm-blooded people.

The closest technical solution (prototype) to the present invention is a strain of bacteria Bacillus thuringiensis used to control beetle insects (RF patent No. 2204598, 2003). The active principle of insecticidal activity in this strain is protein crystalline endotoxin, which has a high specificity of action against insects. The disadvantage of the prototype is the insufficiently high strain activity against older larvae and adults of the Colorado potato beetle. The survival of the larvae 10 days after treatment with the liquid formulation was 50.0%. In relation to the imago of the Colorado potato beetle, the strain was not active.

The objective of the invention is to obtain a new strain with high entomocidal activity against Colorado potato beetle. The strain was obtained in 2012 by selection of B. thuringiensis 16T100 strain.

The problem is solved by obtaining a new strain of B. thuringiensis 16T100 / 18 by selection of the known strain of B. thuringiensis 16T100 to increase insecticidal activity against older larvae and adults of the Colorado potato beetle. The resulting strain is registered in the collection of microorganisms VIZR under the number 16T100 / 18.

Nomenclature data: Genus: Bacillus, Cohn, year of registration - 1872.

Species: B. thuringiensis.

Strain B. thuringiensis 16T100 / 18 is characterized by the following cultural-morphological and physiological-biochemical characteristics.

Cultural and morphological features

Vegetative cells are gram-positive motile, single or in chains spore-forming rods of size (2.6-5.0) × (0.7-1.0) microns, peritrichi. The spores are oval, in size (1.0-1.6) × (0.6-1.1), sporangia do not expand. Disputes are centrally located. With sporulation, parasporal inclusions of various shapes are formed - oval, round, cubic, irregular tetrahedrons. Oval-round crystals predominate.

It grows well on meat-peptone agar (MPA), fish agar (RA) and other nutrient media with the addition of peptone and yeast or corn extract.

On RA forms a brown pigment. The colonies are flat with uneven lobed edges of a pasty consistency. The pigment does not form pigment on the vitelline, sliced potatoes and the medium with the addition of BVK. The optimum growth temperature is 28-30 ° C.

Physiological and biochemical characteristics

The strain produces catalase, hydrolyzes starch, casein, and dilutes gelatin. Forms acetylmethylcarbinol, lecithinase, urease. Does not decompose esculin and salicin. Does not form acids and gases from maltose, lactose and sucrose. Absorbs glucose, mannose to form acid without gas. Hydrogen sulfide and indole does not form.

Attitude to nitrogen sources

Uses ammonium and nitrate forms of nitrogen. Nitrate restores. Absorbs nitrogen compounds that are part of the corn extract, meat water and peptone.

Storage conditions - in the form of freeze-dried culture in sealed ampoules.

Physiologically different from B. thuringiensis var. thuringiensis (H-I) because it does not absorb esculin does not form acid from sucrose. The pigment forms. According to insecticidal activity, it differs in that the strain acts on older larvae and adults of the Colorado potato beetle.

The invention is illustrated by the following specific examples of the use of strain B. thuringiensis 16T100 / 18.

Example 1

The strain B. thuringiensis 16T100 / 18 was stored on beveled meat and peptone agar (MPA) (Zhukova et al., 1978) at room temperature with periodic reseeding on fresh medium. Before use, a 3-fold passage of culture was performed on MPA containing 1.0% glucose; the grown culture was checked for purity by plating on MPA medium. The inoculum suspension was prepared from a 5-day culture based on the inoculum dose of 10 ⁶ spores / ml sterile culture medium according to the turbidity standard.

The morphology of cells, spores and crystals was studied using optical optical microscopy according to the Smirnoff method [Smirnoff, 1962].

The cultivation of BT strain in order to obtain culture fluid and preparation of the preparative forms was carried out in a sterile liquid nutrient medium of the following composition (g / l): sprat pancreatic hydrolyzate - 10.05, sodium chloride - 4.95, water up to 1.0 l, pH = 7.2 ± 0.2, in Erlenmeyer flasks with a capacity of 750 cm ³ containing 50 and 100 ml of medium, at 28-30 ° C with constant stirring on a rocking chair (n = 250 rpm). The cultivation duration was 72-96 hours. For inoculation used a 1-day culture of the studied strain. The sterility of the process was monitored by light microscopy and by direct plating on agar medium in Petri dishes. The titer of viable cells was determined for 1-4 days by plating 6-8 dilution of the culture fluid on the surface of the MPA. In the fermentation process, crystal formation was monitored using light-optical methods and sterility by light microscopy and by direct plating on agarized nutrient medium in Petri dishes. The process of crystal formation ended by 72 hours of strain growth. The resulting culture fluid was lyophilized (FreeZone dryer -105 ° C 4.5L, bench-top, to -105 ° C, Labconco), (Diaem Catalog 2012).

Example 2

The insecticidal activity of the strain was evaluated in laboratory conditions using larvae of the Colorado potato beetle Leptinotarsa decemlineata (Colorado potato beetle) of the 1st-2nd generation of beetle eggs originating from eggs laid by young natural beetles of the 1st summer generation (Steps and et al., 1992).

For testing, a suspension of lyophilized culture fluid of the strain was taken in concentrations of 1.0; 2.0; and 5.0%. The culture fluid was obtained, as in example 1. To keep the insects used plastic Petri dishes with a diameter of 90 mm, having small ledges on the lower edge of the lid, which provides ventilation of the inner volume of the cup. A paper filter was placed at the bottom of the cup, and a sheet or fragments of potato leaves in one layer covering the bottom were placed on the filter. The food for beetles and larvae was served by leaves cut from bushes of potato cultivars of the Charodey variety, favorable for nutrition and development of the Colorado potato beetle, which were grown on the experimental VIZR field, as well as in the greenhouse, in order to provide experimental biomaterial with food in the autumn. Potato leaves were treated with an aqueous suspension, respectively, at concentrations of 1.0%, 2.0% and 5.0% by dipping the sheet in a vessel with the drug for 2 seconds. In control variants, the leaves were dipped in clean water. Then, 15 larvae in 4 replicates were planted in cups with leaves prepared in this way; in total, 60 larvae in each experimental and control variants. The experimental material was kept at room temperature (average 23.5 ° C) in a place protected from direct sunlight. The first feed addition was carried out 2-3 days after replanting the larvae, achieving almost complete eating of the first leaves treated with the studied drugs. When the feed was first added, leaves were placed in the cups treated with the same preparation, but after dipping, they had remained dry in the refrigerator; on subsequent feed shifts, untreated leaves were laid. The number of surviving larvae in each dish was recorded on the 3rd, 5th, and 10th day after the start of the experiment. In all variants and replications, where live larvae or pre-pupae remained, the experiment was conducted until its complete termination — until the imago was inspired or until the biomaterial completely died at various stages. The repetition of the experiment is 4 times. The data obtained in the work were statistically processed using standard methods [Armor, 1985]. The results of the laboratory experiment are shown in table 1.

Table 1 The effect of lyophilized culture fluid of B. thuringiensis 16T100 / 18 strain on the survival of the Colorado potato beetle in laboratory conditions Suspension concentration Original sample L1, ind. % survivors L2-L3 (5th day) % survivors L4 (10th day) % winged imago 1,0% fifteen 73.3 53.3 33.3 fifteen 86.7 66.7 26.7 fifteen 66.7 0 0 fifteen 66.7 46.7 13.3 Total: 60 73.3 ± 4.1 b 41.7 ± 12.5 b 18.3 ± 6.4 b 2.0% fifteen 0 0 0 fifteen 40,0 26.7 6.7 fifteen 46.7 20,0 6.7 fifteen 26.7 0 0 Total: 60 28.3 ± 8.9 s 11.7 ± 5.9 s 3.3 ± 1.7 s 5.0% fifteen 73.3 66.7 60.0 fifteen 46.7 46.7 46.7 fifteen 0 0 0 fifteen 0 0 0 Total: 60 30.0 ± 15.7 s 28.3 ± 14.6 b 26.7 ± 13.5 b The control fifteen one hundred 93.3 60.0 fifteen one hundred 86.7 66.7 fifteen one hundred one hundred 80.0 fifteen 93.3 86.7 66.7 Total: 60 98.3 ± 1.5 a 91.7 ± 2.8 a 68.3 ± 3.6 a a, b, c - differences between options and with control (a) are significant at P> 0.95 ... 0.99

An analysis of the results showed that under laboratory conditions, the lyophilized culture fluid of B. thuringiensis 16T100 / 18 strain showed high insecticidal activity against developing beetle larvae. When using a 1% concentration, 41.7% survived, and 18.3% of the beetles fledged. With an increase in the suspension concentration to 2%, the larvae survival decreased to 11.7%, and the adult yield reached only 3.3%, while in the control more than 90% of the larvae survived, and the adult yield was 68.3%. Increasing the concentration of the suspension to 5% slightly reduced the processing efficiency. In general, the average survival rates of larvae and adult inspiration in experiments with all three concentrations of the preparation were significantly lower than in the control, especially when using a suspension in an optimal concentration of 2%.

Example 3

In example 2, the methodology for keeping larvae and adult beetles in laboratory experiments is preserved, which corresponds to the traditionally used in studies of abiotic or food adaptations of a pest and described in example 1.

Larvae of younger ages (mainly the second one) were put into the experiment, with the mass appearance of which plants are usually treated with insecticides and biological products in the field, as well as adults (adult beetles). Larvae for use in the experiment were hatched from egg clutches collected in the fields of the North Caucasus region. In variants with adults, bugs of the summer (young) generation were used, which, after their winging, underwent bait feeding.

In experimental variants, before planting larvae or adult beetles in cups, the potato leaves were treated with undiluted and 2-fold diluted culture fluid of the strain. The culture fluid was obtained as in example 1. The leaves of the potato were dipped into a vessel with the culture fluid for 2 seconds. In control variants, the leaves were dipped in clean water. Next, insects were planted in plates with treated leaves: larvae — 10–20 specimens (4 replicates, 55–80 larvae in each experimental or control variant), adult beetles — 8 specimens (4 or 8 replicates, total 32 or 64 beetles in each variant).

The experimental material was kept at room temperature (on average, 22 ... 24 ° C) in a place protected from direct sunlight, and in September-October - in a thermostat at a temperature of 24 ... 25 ° C. The first feed addition was carried out 2-3 days after replanting the larvae, achieving almost complete eating of the first leaves treated with the culture fluid of the strain. Subsequently, when changing or adding food, untreated leaves were placed in the cups.

The number of surviving larvae in each dish was recorded on the 3rd, 5th, 7th, and 10th day after the start of the experiment. In addition, in all cases where live larvae or pre-pupae remained, the experiment was continued until it was completely completed — until the imago was fledged or until the biomaterial completely died at various stages. The data are presented in table 2.

table 2 Survival of larvae and young adults of the North Caucasian population of the Colorado potato beetle (northern ecotype of the pest) when exposed to the culture fluid of the strain Bacillus thuringiensis 16T100 / 18 Experience option The initial number of planted beetle larvae (100%) Larvae survived (% of the initial amount) Fledged young beetles (% of the number of planted larvae) On the 3rd day On the 5th day On the 7th day On the 10th day (indicating the phase of development of the larvae) Grubs Control (no processing) 40 (4 × 10) one hundred one hundred one hundred one hundred
4th age + preculture
35.3 ± 2.7 4th age + preculture 92.4 ± 4.2
2.6 ± 1.9 Culture fluid
(dilution × 2) 68 (4 × 17) 72.6 ± 2.4 52.9 ± 2.2 41.2 ± 5.0 IMAGO Control (no processing) 48 (6 × 8) one hundred one hundred one hundred one hundred Culture fluid (dilution × 2) 64 (8 × 8) 42.2 ± 4.8 39.1 ± 5.9 39.1 ± 9.8 37.5 ± 6.0 4th age + preculture

The data in table 2 indicate that the culture fluid of the strain Bacillus thuringiensis 16T100 / 18 was equally active against larvae of 1-2 age and young adults. The survival of the larvae 10 days after treatment with a twice diluted culture fluid was 35.3%; adults - 37.5%, while in the control more than 90% of insects survived. Thus, the insecticidal activity of the proposed strain against larvae of the Colorado potato beetle is 2 times greater than the prototype. Unlike the prototype, the proposed strain is active against adults of the Colorado potato beetle.

Literature

1. Azizbekyan P.P., Kuznetsova N.I., Minenkova I.B., Smirnova T.A., Shagov E.M., Shamshina T.N. The bacterial strain Bacillus thuringiensis H-8, designed to control beetle insects. RF patent No. 2204598, 2003.

2. Zhukova R.A., Kommunarskaya A.D., Pronina M.I., Tereshin I.M., Zhuravleva N.P., Shabas M.N. Selection methods for producers of antibiotics and enzymes. 1978. L.: “Medicine”. S.135.

3. Kandybin N.V. New entomopathogenic preparation "Bitoxibacillin". Bacterial agents and methods of controlling insects and rodents. Abstract, Doctor. dis. - Leningrad, 1972.

4. Diaem 2012 catalog: equipment, plastic, glass, accessories, reagents, kits, Russian, 1300 pages

5. Kuznetsova N.I .; Queen Yu.V .; Grigoryeva T.M .; Azizbekyan P.P .; Debabov V.G. The bacterial strain Bacillus thuringiensis spp. thuringiensis for the production of exotoxin-containing bioinsecticides. RF patent No. 2061376, 1996.

6. Steps EM, Minenkova IB, Konstantinova G.E. 1992. Biotechnology N1: 75-77.

7. Catalog Diaem 2012: equipment, plastic, glass, accessories, reagents, kits, Russian, 1300 pages

Claims (1)

The bacterial strain Bacillus thuringiensis B 94-1 VIZR to combat the Colorado potato beetle.