PL236518B1 - Protein insecticide composition - Google Patents

Description

Description of the invention

The subject of the invention is an insecticide preparation containing the insecticidal proteins Cry2_B5 and Vip3Aa59 Bacillus thuringiensis and its use in the control of pests of the order Lepidoptera, in particular Spodoptera exigua.

Insects of the species S. exigua are economically important pests of crops, feeding, among others, on many types of vegetables and cereals. They are also proven model organisms used in bioinsecticide activity tests.

The insecticidal activity of the Vip3Aa59 protein on the larvae of the earthworm (Spodoptera exigua, Hubner) is known and it was described in the scientific publication by Baranek et al. (2017, BioControl 62: 649-658). The determined LC50 value against S. exigua after 10 days of exposure to Vip3Aa59 was 17 ng / cm ² . The Cry2_B5 protein is the subject of patent application P.428223.

The aim of the invention was to develop an insecticidal mixture with high insecticidal activity, effective at low concentrations, in a short time and causing high insect mortality.

In the course of our own research, it was found that the composition of the new Cry2_B5 protein (disclosed in patent application P.428223) and the Vip3Aa59 protein is characterized by high activity against Spodoptera exigua larvae.

The subject of the invention is mixtures of Cry2_B5 and Vip3Aa59 proteins, containing from one to three parts of Cry2_B5 and from one to three parts of Vip3Aa59, which due to the synergy effect are characterized by much higher insecticidal activity than its individual components administered to insects separately and a simple sum of the activities of both its components. In particular, the mixtures of Cry2_B5 and Vip3Aa59 in a ratio of 1: 1 to 2: 1 are characterized by the highest activity.

Obtaining the Vip3Aa59 and Cry2_B5 proteins

The Vip3Aa59 protein was obtained according to the procedure described by Baranek et al. (2017, BioControl 62: 649-658). In order to confirm the obtaining of the Vip3Aa59 toxin, the electrophoretic separation of the proteins of the solution containing the expected toxin was performed in parallel with the control sample and the molecular weight marker (ThermoFisherScientific, catalog number 26614). SDS-PAGE electrophoretic separation was performed according to the procedure "SDS-Polyaclylamide gel electrophoresis of proteins" described in Appendix 8: Commonly Used Techniques in Molecular Cloning (Sambrook J, Russell D (2001) Molecular Cloning: A Laboratory Manual, 3rd edition Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.). Upon analysis of the electropherogram shown in Figure 1, it was found that the test sample contained an additional protein compared to the control, which protein has a molecular weight of approximately 85 kDa, which is consistent with the theoretical molecular weight of the Vip3Aa59 protein. Figure 1 shows the electropherogram of the obtained protein solution (right lane), K is the control and M is the molecular weight marker (ThermoFisherScientific, cat no. 26614). The numbers on the left represent the molecular weight of the marker proteins in kilodaltons (kDa). The arrow indicates the electropherogram band corresponding to the Vip3Aa59 protein, with a mass of approximately 85 kDa.

The Cry2_B5 protein was obtained identically as described in the patent application No. P.428223. In order to confirm the obtaining of the Cry2_B5 protein, the electrophoretic separation of the proteins of the solution containing the expected toxin was performed in parallel with the control sample and the molecular weight marker (ThermoFisherScientific, catalog no. 26614). SDS-PAGE electrophoretic separation was performed according to the procedure "SDS-Polyaclylamide gel electrophoresis of proteins" described in Appendix 8: Commonly Used Techniques in Molecular Cloning (Sambrook J, Russell D (2001) Molecular Cloning: A Laboratory Manual, 3rd edition Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.). After analysis of the electropherogram shown in Figure 2, it was found that the test sample contained additional protein compared to the control, this protein having a molecular weight of about 65 kDa, which is in agreement with the theoretical molecular weight of the Cry2_B5 protein. Figure 2 shows the electropherogram of the obtained protein solution (left lane), K is the control, M is the molecular weight marker (ThermoFisherScientific, cat no. 26614). The numbers on the left indicate the molecular weight of the marker proteins in kilodaltons (kDa). The arrow indicates the electropherogram band corresponding to the Cry2_B5 protein, with a mass of approximately 65 kDa.

PL 236 518 B1

BIOLOGICAL TESTS

Biological tests were performed to determine the insecticidal activity of Cry2_B5 and Vip3Aa59 proteins and their mixtures against insect larvae. Furthermore, the type of interaction (antagonism / addition / synergy) that occurs between the Cry2_B5 and Vip3Aa59 proteins in the mixtures was determined.

The insecticidal tests were carried out on larvae of Spodoptera exigua insects in the L1 larval stage. The caterpillars of S. exigua came from a standardized laboratory breeding of an insect at the Department of Microbiology at the Faculty of Biology of the University of Adam Mickiewicz in Poznań. Both the cultivation of the insects and the tests of the biological activity of the insecticidal preparations were carried out on a culture medium with the composition according to McGuire et al. (1997, in: Lacey, LA (Ed.), Manual of Techniques in Insect Pathology. Academic Press, San Diego, p. 91 -99).

Insect culture medium according to McGuire was formed into disks 10 mm in diameter and 3 mm high and placed individually in closed polystyrene wells with free access to air. The preparations containing the proteins Cry2_B5, Vip3Aa59 or their mixtures according to the following variants were applied to the medium discs:

Variant 1 • 1: 1 mixture of Cry2_B5 and Vip3Aa59 proteins at the concentration of 100 ng / cm ² medium • Cry2_B5 protein at a concentration of 50 ng / cm ² medium • Vip3Aa59 protein at the concentration of 50 ng / cm ² medium

Variant 2 • mixture of Cry2_B5 and Vip3Aa59 proteins in the proportion of 2: 1 at the concentration of 100 ng / cm ^{2 of the} medium • Cry2_B5 protein at the concentration of 66.67 ng / cm ^{2 of the} medium • protein Vip3Aa59 at the concentration of 33.33 ng / cm ^{2 of the} medium

Variant 3 • a mixture of Cry2_B5 and Vip3Aa59 proteins in the ratio of 1: 2 at the concentration of 100 ng / cm ^{2 of the} medium • Cry2_B5 protein at the concentration of 33.33 ng / cm ^{2 of the} medium • protein Vip3Aa59 at the concentration of 66.67 ng / cm ^{2 of the} medium

Variant 4 • mixture of Cry2_B5 and Vip3Aa59 proteins in the proportion of 10: 1 at the concentration of 100 ng / cm ^{2 of the} medium • Cry2_B5 protein at the concentration of 90.9 ng / cm ^{2 of the} medium • protein Vip3Aa59 at the concentration of 9.1 ng / cm ^{2 of the} medium

Variant 5 • a mixture of Cry2_B5 and Vip3Aa59 proteins in the proportion 1:10 at the concentration of 100 ng / cm ^{2 of the} medium • Cry2_B5 protein at the concentration of 9.1 ng / cm ^{2 of the} medium • Vip3Aa59 protein at the concentration of 90.9 ng / cm ^{2 of the} medium

Variant 6 • a mixture of Cry2_B5 and Vip3Aa59 proteins in the proportion of 4: 1 at the concentration of 100 ng / cm ^{2 of the} medium • Cry2_B5 protein at the concentration of 80 ng / cm ^{2 of the} medium • Vip3Aa59 protein at the concentration of 20 ng / cm ^{2 of the} medium

Variant 7 • a mixture of Cry2_B5 and Vip3Aa59 proteins in the proportion of 1: 4 at the concentration of 100 ng / cm ^{2 of the} medium • Cry2_B5 protein at the concentration of 20 ng / cm ^{2 of the} medium • Vip3Aa59 protein at the concentration of 80 ng / cm ^{2 of the} medium

Additionally, for comparative purposes, the insecticidal activity of the preparation containing the Cry2_B5 protein at a concentration of 350 ng / cm ^{2 was} determined (Variant 8). As a negative control, the insecticidal activity of the preparation containing native proteins isolated from E. coli BL21 (DE3) cells at a concentration of 100 ng / cm ^{2 was} determined, whereby the proteins were derived from non-toxin producing cells Cry2_B5_B5 and Vip3Aa59 (Variant 9).

Each of the preparations mentioned in the individual variants was applied to the medium in a volume of 30 g per one disc, and after the preparations were dry, S. exigua larvae were placed on the medium on each disc. The test was performed in duplicate, so there were 48 larvae in total for each preparation (Table 1).

During the bioassay, the insects were incubated at 26 ° C, 40-60% relative air humidity, and a 16: 8 photoperiod (light: dark). The number of dead insect larvae was determined on day 7 post

PL 236 518 Β1 administration of preparations (7 DAT; day aftertreatmenf). On the basis of the number of dead insects after exposure to the preparations at various concentrations, the mortality and the proportion of larvae surviving were calculated.

Table 1 shows the results obtained. The abbreviations in the table description mean:

• Concentration - amount of proteins per cm ^{2 of} medium;

• OBS - empirically obtained observed values;

• P OBS - the observed proportion of insects that survived the exposure to the individually administered Cry2_B5 protein or Vip3Aa59 protein;

• Mortality - percentage of dead insects;

• Control - native proteins isolated from E. coli BL21 (DE3) cells not containing the Cry2_B5 and Vip3Aa59 proteins.

Table 1

Variant Preparation Concentration (ng / cm ² ) The larvae together Number of dead OBS larvae Number of live OBS larvae Mortality OBS P. OBS 1 Cry2B5 + Vip3Aa59 (1: 1) 100 48 31 17 65% Cry2B5 50 48 5 43 10% 0.90 Vip3Aa59 50 48 17 31 35% 0.65 2 Cry2 B5 + Vip3Aa59 (2: 1) 100 48 40 8 83% Cry2_B5 66.67 48 4 44 8% 0.92 Vip3Aa59 33.33 48 16 32 33% 0.67 3 Cry2_B5 + Vip3Aa59 (1: 2) 100 48 25 23 52% Cry2 B5 33.33 48 3 45 6% 0.94 Vip3Aa59 66.67 48 28 twenty 58% 0.42 4 Cry2 B5 + Vip3Aa59 (10: 1) 100 48 12 36 25% Cry2_B5 90.9 48 9 39 19% 0.81 Vip3Aa59 9.1 48 8 40 17% 0.83 5 (Ty2 B5 + Vip3Aa59 (1:10) 100 48 26 22 54% Cry2_B5 9.1 48 0 48 0% 1.00 Vip3Aa59 90.9 48 33 15 69% 0.31 6 Cry2 B5 + Vip3Aa59 (4: 1) 100 48 25 23 52% Cry2 B5 80 48 7 41 15% 0.85 Vip3Aa59 twenty 48 16 32 33% 0.67 7 Cry2 B5 + Vip3Aa59 (1: 4) 100 48 34 14 71% Cry2_B5 twenty 48 1 47 2% 0.98 Vip3Aa59 80 48 32 16 67% 0.33 8 Games2 B5 350 48 22 26 46% 9 Control 100 48 0 48 0% 1.00

PL 236 518 B1

The nature of the interaction (addition / synergism / antagonism) between the insecticidal proteins Cry2_B5 and Vip3Aa59 was determined by the method of Fernandez-Luna et al. [Journal of Invertebrate Pathology 2010, 104, 231-233]. It consists in calculating the expected (theoretical) insect mortality that is expected after exposure to a given mixture of toxins. The expected mortality of insects after exposure to the mixture is determined on the basis of the observed (empirical) survival of insects after exposure to individual components of the mixture at a concentration identical to that present in the mixture, according to the formula:

SATURDAY = [1- (PaOBS x PbOBS)] x 100%

SWABOCZ - expected percentage of insect mortality after exposure to the a + b mixture PaOBS - observed proportion of surviving insects a PbOBS - observed proportion of surviving insects b

Next, it is determined whether the observed mortality differs statistically significantly from the theoretical mortality using the Fisher's Exact Test using the Statistica program (StatSoft). The observed number of dead and alive insects and the expected number of dead and alive insects for each mixture are entered into the calculator. The Fisher's Test determines the probability ("p" factor) of the absence of significant differences between the observed and theoretical mortality. The "p" coefficient of 0.05 or less means that with 95% similarity we reject the hypothesis that there are no statistical differences between the two results.

If the difference between the observed and expected mortality is not statistically significant (p> 0.05), it is considered that the two components of the mixture act by addition.

When the difference between the observed mortality and the expected mortality is statistically significant (p <0.05), it is assumed that between the components of the mixture there is:

a) synergism when observed mortality is higher than expected b) antagonism when observed mortality is lower than expected

Based on the data in Table 1, it was established:

• expected mortality values of insects • expected number of dead larvae • expected number of live larvae • p-values obtained from the Fisher test and the type of protein interaction was determined. The obtained values are presented in Table 2.

The expected mortality calculations were made using the formula

SATURDAY = [1- (PaOBS x PbOBS)] x 100%

The values from the "P OBS" column for Cry2_B5 were adopted as the PaOBS parameter, and the values from the "P OBS" column for Vip3Aa59 were adopted as the PbOBS parameter.

The expected number of dead insects was then determined by multiplying the total number of larvae (Table 2, "Larvae total" column) by the expected mortality value for the mixture (Table 2, "OCZ Mortality" column). The results are presented in Table 2 in the column "OCZ dead larvae". The expected number of live insects was also determined by subtracting the expected number of dead larvae (Table 2, "Dead larvae" column) from the total number of larvae (Table 2, "Total larvae" column). The results are presented in Table 2 in the column "Live OCZ larvae".

In order to check whether for different variants of preparations being mixtures of Cry2_B5 and Vip3Aa59 proteins, the observed mortality differed in a statistically significant manner from the expected mortality, the Fisher Exact Test with the Statistica program was used. As input data, the above-mentioned of the program, the observed number of dead and live larvae and the expected number of dead and live larvae after exposure to the mixture of toxins Cry2_B5 and Vip3Aa59 (Table 1, columns: "Dead OBS larvae"; "Live OBS larvae"; Table 2, columns: "Dead OCZ larvae" ; "Live OCZ larvae"). As a result, the "p" values for each mixture were obtained and are presented in Table 2 in the column "Fisher's test (p value)". When the p value exceeded 0.05, it was found that the proteins Cry2_B5 and Vip3Aa59 in a given variant act additively. If the p value was less than or equal to 0.05 and the observed mortality (Table 2; "OBS Mortality" column) was higher than the expected mortality (Table 2; "OCZ Mortality" column), the synergistic effect of both proteins in a given mixture was considered. Antagonism was not observed in any variant. The results are listed in Table 2 under the column "Type of interaction".

PL236 518 B1

And one for:

Option 1

Expected mortality SABOCZ = [1- (PaOBS x PbOBS)] x 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1 - (Pcry2_B5OBS X Pvip3Aa59OBS)] X 100% of (Cry2_B5 + Vip3Aa59), OCZ = [1- (0) 9 X 0.65)] X 100% (Cry2_B5 + Vip3Aa59) OCZ = 42%

Number of dead larvae expected: 42% x 48 = 20

Expected number of live larvae: 48 - 20 = 28

Fisher's test

Dead Alive Watched 31 17 Expected twenty 28

Value determined in Fischer's test: p = 0.04

The type of interaction between the components of the mixture

For the mixture Cry2_B5 + Vip3Aa59 (1: 1), the difference between the observed (65%) and expected (42%) mortality is statistically significant (p <0.05). The observed value is 1.55 times higher, which proves the synergistic effect of the Cry2_B5 and Vip3Aa59 proteins in the proportion 1: 1 against S. exigua.

Option 2

Expected mortality

SWABOCZ = [1- (P _a OBS x PbOBS)] x 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1 - (Pcry2_B5OBS X Pvip3Aa59OBS)] X 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1- (0, 92 X 0.67)] X 100% (Cry2_B5 + Vip3Aa59) OCZ = 39%

Number of dead larvae expected: 39% x 48 = 19

Expected number of live larvae: 48 - 19 = 29

Fisher's test

Alive Dead Watched 40 8 Expected 19 29

The value determined in the Fischer's test: p = 0.00002

The type of interaction between the components of the mixture

For the Cry2_B5 + Vip3Aa59 (2: 1) mixture, the difference between the observed (83%) and expected (39%) mortality is statistically significant (p <0.05). The observed value is 2.13 times higher, which proves the synergistic effect of the Cry2_B5 and Vip3Aa59 proteins in the proportion 2: 1 against S. exigua.

Option 3

Expected mortality

SWABOCZ = [1- (P _a OBS x PbOBS)] x 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1 - (Pcry2_B5OBS X Pvip3Aa59OBS)] X 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1- (0, 94 X 0.42)] X 100%

S (Cry2_B5 + Vip3Aa59) OCZ = 61%

Expected dead larvae: 61% x 48 = 29

Expected number of live larvae: 48 - 29 = 19

PL 236 518 Β1

Fisher's test

Alive Dead Watched 25 23 Expected 29 19

The value determined in the Fischer's test: p = 0.537

The type of interaction between the components of the mixture

For the Cry2_B5 + Vip3Aa59 (1: 2) mixture, the difference between the observed (52%) and expected (61%) mortality is not statistically significant (p> 0.05), which proves the additive effect of the Cry2_B5 and Vip3Aa59 proteins in the mixture in the ratio 1: 2 against S. exigua.

Option 4

Expected mortality SABOCZ = [1- (PaOBS x PbOBS)] x 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1 - (Pcry2_B5OBS X Pvip3Aa59OBS)] X 100% of (Cry2_B5 + Vip3Aa59), OCZ = [1- (0) 81 X 0.83)] X 100% (Cry2_B5 + Vip3Aa59) OCZ = 32%

Number of dead larvae expected: 32% x48 = 16

Expected number of live larvae: 48 - 16 = 32

Fisher's test

Alive Dead Watched 12 36 Expected 16 32

The value determined in the Fischer's test: p = 0.503

The type of interaction between the components of the mixture

For the Cry2_B5 + Vip3Aa59 (10: 1) mixture, the difference between the observed (25%) and expected (32%) mortality is not statistically significant (p> 0.05), which proves the additive effect of the Cry2_B5 and Vip3Aa59 proteins in the mixture in the proportion of 10: 1 against S. exigua.

Option 5

Expected mortality

SWABOCZ = [1- (P _a OBS x PbOBS)] x 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1 - (Pcry2_B5OBS X Pvip3Aa59OBS)] X 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1- (1 X 0.31)] X 100%

S (Cry2_B5 + Vip3Aa59) OCZ = 69%

Number of dead larvae expected: 69% x48 = 33

Expected number of live larvae: 48 - 33 = 15

Fisher's test

Alive Dead Watched 26 22 Expected 33 15

Value determined in Fischer's test: p = 0.208 Type of interaction between the components of the mixture

PL 236 518 Β1

For the mixture Cry2_B5 + Vip3Aa59 (1:10), the difference between the observed (54%) and expected (69%) mortality is not statistically significant (p> 0.05), which proves the additive effect of the Cry2_B5 and Vip3Aa59 proteins in the mixture in the ratio 1: 10 against S. exigua.

Option 6

Expected mortality SABOCZ = [1- (PaOBS x PbOBS)] x 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1- (Pcry2_B5OBS X Pvip3Aa59OBS)] X 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1- (0, 85 X 0.67)] X 100% (Cry2_B5 + Vip3Aa59) OCZ = 43%

Number of dead larvae expected: 43% x 48 = 21

Expected number of live larvae: 48-21 = 27

Fisher's test

Alive Dead Watched 25 23 Expected 21 27

The value determined in the Fischer test: p = 0.22

The type of interaction between the components of the mixture

For the Cry2_B5 + Vip3Aa59 (4: 1) mixture, the difference between the observed (52%) and expected (43%) mortality is not statistically significant (p> 0.05), which proves the additive effect of the Cry2_B5 and Vip3Aa59 proteins in the mixture in the proportion of 4: 1 against S. exigua.

Option 7

Expected mortality

SWABOCZ = [1- (P _a OBS x PbOBS)] x 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1 - (Pcry2_B5OBS X Pvip3Aa59OBS)] X 100% of (Cry2_B5 + Vip3Aa59) OCZ = [1- (0, 98 X 0.33) X 100%

S (Cry2_B5 + Vip3Aa59) OCZ = 67%

Expected dead larvae: 67% x 48 = 32

Expected number of live larvae: 48 - 32 = 16

Fisher's test

Alive Dead Watched 34 14 Expected 32 16

The value determined in the Fischer test: p = 0.826 Type of interaction between the components of the mixture

For the Cry2_B5 + Vip3Aa59 (1: 4) mixture, the difference between the observed (71%) and expected (67%) mortality is not statistically significant (p> 0.05), which proves the additive effect of the Cry2_B5 and Vip3Aa59 proteins in the mixture in the ratio 1: 4 against S. exigua.

PL 236 518 Β1

Table 2

Variant Preparation Eye mortality The larvae together Dead larvae of the eye Live larvae of OCZ Fisher's test (p value) Interaction type 1 Cry2_B5TVip3Aa59 (1: 1) 42% 48 twenty 28 0.04 SYNERGISM 2 Cry2_B5 + Vip3Aa59 (2: 1) 39% 48 19 29 0.00002 SYNERGISM 3 Cry2_B5 + Vip3Aa59 (1: 2) 61% 48 29 19 0.537 ADDITION 4 Cry2_B5 + Vip3Aa59 (10: 1) 32% 48 16 32 0.503 ADDITION 5 Cry2_B5 + Vip3Aa59 (1:10) 69% 48 33 15 0.208 ADDITION 6 Cry2_B5 + Vip3Aa59 (4: 1) 43% 48 21 27 0.22 ADDITION 7 Cry2_B5 + Vip3Aa59 (1: 4) 67% 48 32 16 0.826 ADDITION

The analysis of the test results presented in Table 2 shows that the mixture of Cry2_B5 and Vip3Aa59 proteins in all the tested proportions (except 10: 1), where the total concentration of both components is 100 ng / cm ^{2 of the} surface available to the insect, causes the death of 52-83% of the larvae . This is a significant reduction in the number of feeding insects. For comparison, the Cry2_B5 protein administered alone (Table 1, Variant 8) causes death of about 50% only at a concentration of 350 ng / cm ² . In addition, mixtures of Cry2_B5 and Vip3Aa59 proteins in the proportion of 1: 1, and in particular 2: 1, show a much higher insecticidal activity than its individual components administered to insects separately, and a simple sum of the activities of both its components - this is due to the synergy of both components. In the case of the mixture in the proportion of 1: 1, the empirically determined insect mortality was approximately 1.5 times higher than the expected theoretical mortality, and for the mixture in the proportion of 2: 1, the effectiveness of the preparation was more than twice as high as expected.

In a second aspect, the invention relates to an insecticide formulation comprising one to three parts Cry2_B5 and one to three parts Vip3Aa59, preferably comprising one to two parts Cry2_B5 and one part Vip3Aa59, most preferably two parts Cry2_B5 and one part Vip3Aa59.

In a third aspect, the subject of the invention is the use of the mixture according to the invention in the control of insect pests of plants, in particular of the order Lepidoptera, in particular of the species S. exigua, in a total amount of proteins Cry2_B5 and Vip3Aa59 not less than 100 ng / cm ^{2 of the} area of plant organs exposed to feeding pests.

Claims (8)

Patent claims A composition of a Cry2_B5 protein and a Vip3Aa59 protein characterized in that it comprises one to three parts Cry2_B5 and one to three parts Vip3Aa59. 2. A composition according to claim 1 The method of claim 1, comprising one to two Cry2_B5 portions and one Vip3Aa59 portion. 3. A composition according to p. The composition of claim 2, wherein the proteins Cry2_B5 and Vip3Aa59 are present in a 2: 1 ratio. 4. An insecticidal preparation containing a mixture of the Cry2_B5 and Vip3Aa59 proteins, characterized in that it comprises one to three parts Cry2_B5 and one to three parts Vip3Aa59. 5. A formulation according to claim 1 The method of claim 4, comprising one to two Cry2_B5 portions and one Vip3Aa59 portion. PL 236 518 Β1 6. A formulation according to claim 1 The method of claim 5, characterized in that it contains the proteins Cry2_B5 and Vip3Aa59 in a 2: 1 ratio. 7. The use of an insecticidal preparation containing the proteins Cry2_B5 and Vip3Aa59 for controlling plant insect pests, characterized in that the preparation according to claim 4 or 5 or 6 is used in an amount not less than 100 ng / cm ^{2 of the} area of the plant organs exposed to the feeding of pests. 8. The use of an insecticidal formulation according to claim 1 6. A method according to claim 7, characterized in that the formulation is used to control insects of the order Lepidoptera, in particular of the species S. exigua.