Combined application of nuclear polyhedrosis virus and bacillus thuringiensis in preparation of preparation for preventing and treating agrotis ypsilon larvae

Abstract

The invention discloses combined application of nuclear polyhedrosis virus and bacillus thuringiensis in preparation of a preparation for preventing and treating black cutworm larvae, and relates to the technical field of black cutworm larva prevention and treatment. The matching experiment of different types and different concentrations of nuclear polyhedrosis viruses and bacillus thuringiensis shows that compared with single use of nuclear polyhedrosis viruses or single use of bacillus thuringiensis, the mixed combination of the nuclear polyhedrosis viruses and the bacillus thuringiensis has an obvious synergistic effect in prevention and treatment of young cutworms; the death rate of indoor and field black cutworm larvae can be remarkably increased, the prevention and treatment effect is improved, and a theoretical basis is provided for biological prevention and treatment of black cutworms. Meanwhile, the nuclear polyhedrosis virus and the bacillus thuringiensis are combined for use, so that the problem of < 3 > R caused by chemical pesticide prevention and control can be avoided from the source, and the method is environment-friendly and beneficial to long-term development.

Description

Technical Field

[0001] The invention relates to the technical field of preventing and controlling small cutworm larvae, and more specifically to application of nuclear polyhedrosis virus and bacillus thuringiensis in preparing a preparation for preventing and controlling small cutworm larvae. Background Art

[0002] Cutworms belong to the family Noctuidae of the order Lepidoptera. They are also known as earthworms, earthworms, black earthworms, black earthworms, ground shears, and root cutters. They have a varied diet and a wide range of hosts. Their larvae harm a variety of vegetables, flowers, field crops, tobacco, fruit trees, forest seedlings, and weeds. Young larvae harm the heart leaves or tender leaves of crops, causing holes or notches. After the third instar, they cut off the tender stems of crop seedlings near the ground, causing missing seedlings and broken ridges. In severe cases, they destroy the seeds and re-sow them. They are the most serious underground pests. There are 170 species of cutworms recorded in agricultural areas in my country, and there are about 10 species of cutworms that commonly harm crops. Among them, small cutworms and yellow cutworms are the most widely distributed and the most harmful. White-edged cutworms, large cutworms, warning-marked cutworms, and eight-character cutworms often cause harm in local areas.

[0003] At present, my country mainly adopts chemical control to control cutworms. In view of the fact that the long-term large-scale use of chemical pesticides will bring about "3R" and other problems, it is urgent to establish a green control technology system for cutworms with biological control as the main method. Indoor bioassay is an important means to screen and clarify the effects of different biological control agents.

[0004] Therefore, how to provide a method for effectively preventing and controlling cutworm larvae is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0005] In view of this, the present invention provides the use of nuclear polyhedrosis virus and Bacillus thuringiensis in the preparation of a preparation for preventing and controlling black cutworm larvae.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In one aspect, an embodiment of the present invention provides the use of nuclear polyhedrosis virus and Bacillus thuringiensis in combination in the preparation of a preparation for controlling cutworm larvae.

[0008] Preferably, the nuclear polyhedrosis virus includes: cabbage armyworm nuclear polyhedrosis virus MbMNPV, Spodoptera litura nuclear polyhedrosis virus SpltMNPV and beet armyworm nuclear polyhedrosis virus SeMNPV. Preferably, the virus is SpltMNPV.

[0009] Preferably, the volume ratio of the nuclear polyhedrosis virus and the liquid of Bacillus thuringiensis is 1:1; wherein the concentration of Bacillus thuringiensis is 1×10 2~1×10 6 IU / mL; the concentration of nuclear polyhedrosis virus is 1×10 4 ~1×10 8 OBs / mL.

[0010] A second aspect of an embodiment of the present invention provides a preparation for controlling black cutworm larvae, the preparation comprising a nuclear polyhedrosis virus liquid and a Bacillus thuringiensis suspension, and the volume ratio of the two is 1:1.

[0011] It can be known from the above technical scheme that compared with the prior art, the present invention conducts a ratio experiment by setting different types and concentrations of nuclear polyhedrosis virus and Bacillus thuringiensis, and finds that compared with the single use of nuclear polyhedrosis virus or the single use of Bacillus thuringiensis, the mixed use of the two has an obvious synergistic effect in the control of small cutworm larvae, which can significantly increase the mortality of indoor and field small cutworm larvae, improve the control effect, and provide a theoretical basis for the biological control of small cutworms. At the same time, the combination of nuclear polyhedrosis virus and Bacillus thuringiensis can avoid the "3R" problem caused by chemical pesticide control from the root, which is environmentally friendly and conducive to long-term development. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0013] Figure 1 The corrected mortality rates of different nuclear polyhedrosis viruses and different concentrations against cutworms are shown in the figure.

[0014] Figure 2 These are the survival curves of black cutworms under different nuclear polyhedrosis viruses and different concentrations; A: MbMNPV, B: SpltMNPV, C: SeMNPV.

[0015] Figure 3 The survival rates of the second and third instar larvae of cutworms treated with different Bacillus thuringiensis; A, B and C are the survival rates of the second instar larvae treated with Bt LJT, Bt WD and Bt ZB, respectively; D, E and F are the survival rates of the third instar larvae treated with Bt LJT, BtWD and Bt ZB, respectively.

[0016] Figure 4The survival curves of the third-instar larvae of black cutworms after the combination of NPVs and Bt ZB; A, B and C are the third combination of BtZB with MbMNPV, SpltMNPV and SeMNPV, respectively. Survival curves A2 to A6 (MbMNPV+1×10 2 ~1×10 6 IU / mL Bt ZB), B2~B6 (Spl tMNPV+1×10 2 ~1×10 6 IU / mL Bt ZB), C2~C6 (SeMNPV+1×10 2 ~1×10 6 IU / mL Bt ZB).

[0017] Figure 5 The survival rate curve of the third instar larvae of black cutworm when NPV and Bt LJT were used in combination D2-D6 (MbMNPV+1×10 2 ~1×10 6 IU / mL Bt LJT), E2~E6 (SpltMNPV+1×10 2 ~1×10 6 IU / mL Bt LJT), F2~F6(SeMNPV+1×10 2 ~1×10 6 IU / mL Bt LJT).

[0018] Figure 6 The synergistic effect of NPV combined with Bt ZB on third-instar larvae; A: MbMNPV; B: SpltMNPV; C: SeMNPV.

[0019] Figure 7 The synergistic effect of the combination of NPV and Bt LJT on third-instar larvae; D: MbMNPV; E: SpltMNPV; F: SeMNPV.

[0020] Figure 8 This is the control effect of SpltMNPV (500 million PIB / g) and LJT strain (16000 IU / mg) in combination in the field against cutworms. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1 Biological Activity of Nuclear Polyhedrosis Virus against Cutworm Larvae

[0023] The 3rd instar larvae of the black cutworm that had been reared for more than 5 generations were randomly selected and placed in a 12-well cell culture plate, with one larva in each well, and starved for 12 h. The stock solutions of the cabbage armyworm nuclear polyhedrosis virus MbMNPV, the Spodoptera litura nuclear polyhedrosis virus SpltMNPV, and the beet armyworm nuclear polyhedrosis virus SeMNPV were diluted to 1×10 4 OBs / mL, 1×10 5 OBs / mL, 1×10 6 OBs / mL, 1×10 7 OBs / mL and 1×10 8 OBs / mL, 10μL of virus suspension was respectively added to fresh artificial feed (0.2cm×0.2cm×0.2cm), and the feed was placed in a 12-well plate after natural air drying, and the third-instar larvae were infected by feeding poisoning. Six replicates were set for each treatment, with 12 larvae in each replicate, and the feed with distilled water added was used as a control. The number of deaths of larvae in each treatment was observed and recorded every day, and the 7-day corrected mortality rate was calculated to draw a survival curve. The toxicity equation was calculated using the Probit regression model.

[0024] Table 1 Virulence of different nuclear polyhedrosis viruses to cutworms

[0025]

[0026] Results Figure 1 and Figure 2 Different concentrations of the three nuclear polyhedrosis viruses can cause different degrees of death of small cutworm larvae, and there are significant differences between different concentrations and different viruses. Among them, the cabbage armyworm nuclear polyhedrosis virus MbMNPV has the highest biological activity against small cutworms, 1×10 5 OBs / mL, 1×10 6 OBs / mL, 1×10 7 OBs / mL and 1×10 8 The corrected mortality rates of cutworms at 7 days were 84.62%, 91.07%, 100% and 98.21% when treated with OBs / mL, respectively. The survival curves were also similar to those of 1×10 4 There were differences in OBs / mL treatment. SpltMNPV had the second highest biological activity against cutworms, 1×10 6 OBs / mL, 1×10 7 OBs / mL and 1×10 8 The corrected mortality rates of cutworms at 7 days were 42.86%, 92.86% and 94.64% when treated with OBs / mL, and their survival curves were also similar to those of 1×10 4OBs / mL and 1×10 5 There were differences in OBs / mL treatment. The beet armyworm nuclear polyhedrosis virus SeMNPV had the lowest biological activity against small cutworms, and its 7-day mortality rate did not exceed 30%. The toxicity test also confirmed the biological activity of different viruses against small cutworms. The LC of MbMNPV, SpltMNPV and SeMNPV viruses against small cutworms was 25 They are 6.01×10 3 , 3.69×10 5 and 4.49×10 7 OBs / mL (Table 1).

[0027] Example 2 Biological Activity of Bacillus thuringiensis against Cutworm Larvae

[0028] The 3rd instar larvae of the black cutworms that were reared for more than 5 generations were randomly selected and placed in a 12-well cell culture plate, with one larva in each well, and starved for 12 h. The Bt suspensions LJT (the original strain was from Shandong Lukang Biological Pesticide Co., Ltd.), ZB (the original strain was from Hanwei Biotechnology Co., Ltd.), and WD (the original strain was from Wuhan Kono Biotechnology Co., Ltd.) were diluted with distilled water to 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 and 1×10 2 IU / mL. The pathogenicity to the third-instar larvae of the small cutworm was determined by feeding method. 10 μL of Bt suspension was respectively aspirated and added to fresh artificial feed (0.2 cm×0.2 cm×0.2 cm). After natural air drying, the feed was placed in a 12-well plate. Six replicates were set for each treatment, with 12 worms in each replicate. The feed with distilled water added was used as a control. According to the results of Bt toxicity determination on the third-instar larvae (the mortality rate of the third-instar larvae did not exceed 50%), in order to further determine the toxicity of Bt to the larvae of the small cutworm, its biological activity detection on the second-instar larvae was supplemented, and the implementation process was the same as that of the third-instar larvae. The number of deaths of larvae in each treatment was observed and recorded every day, and the 7-day corrected mortality rate was calculated to draw a survival curve. The Probit regression model was used to calculate its toxicity equation.

[0029] Results Figure 3 As shown in Table 2, the three Bt strains at different concentrations can cause the death of the 2nd and 3rd instar larvae of the small cutworm, but there are certain differences in their mortality rates. As the age of the small cutworm larvae increases or the Bt concentration decreases, the mortality rate caused by the Bt strains decreases significantly. At the same time, the mortality rate of small cutworms in each treatment group gradually increases over time, reflecting the characteristics of biological control. The LC of WD treatment on the 3rd instar larvae 25 3.02×10 5IU / mL, LC treated with LJT 25 4.20×10 5 IU / mL, ZB-treated LC 25 1.51×10 7 IU / mL. LC of 2nd instar larvae treated with WD 25 6.38×10 5 IU / mL, LC treated with LJT 25 8.10×10 4 IU / mL, ZB-treated LC 25 5.64×10 6 IU / mL. The mortality of 2nd and 3rd instar larvae did not exceed 40% at all concentrations. It can be concluded that Bt alone is not very effective against cutworms.

[0030] Table 2 Pathogenicity of three Bt strains to cutworm larvae

[0031]

[0032] Example 3 Synergistic Effects of Nuclear Polyhedrosis Virus and Bacillus thuringiensis on Cutworm Larvae

[0033] The three virus suspensions (MbMNPV, SpltMNPV and SeMNPV) were diluted to 1×10 5 OBs / mL, and the two Bt suspensions (LJT and ZB) were diluted with distilled water to 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 and 1×10 2 IU / mL, the virus suspension and different concentrations of Bt suspension were mixed in a 1:1 ratio to form 6 series of gradient mixtures. The 3rd instar larvae of the small cutworm that were subcultured for more than 5 generations were randomly selected and placed in a 12-well cell culture plate, 1 head per well, and starved for 12 hours. The pathogenicity of the NPV and Bt mixture to the 3rd instar larvae of the small cutworm was determined by feeding. 10 μL of the mixture was respectively drawn and added to fresh artificial feed (0.2 cm × 0.2 cm × 0.2 cm), and the feed was placed in a 12-well plate after it was naturally air-dried. Six replicates were set for each treatment, with 12 larvae in each replicate, and the feed with distilled water added was used as a control. The number of deaths of larvae in each treatment was observed and recorded every day, and the 7-day corrected mortality rate was calculated to draw a survival curve.

[0034] Results Figure 4 and Figure 5, different nuclear polyhedrosis viruses and different concentrations of Bt can all cause the death of small cutworms to a certain extent. Among them, SpltMNPV can play a synergistic role when mixed with different concentrations of LJT and ZB strains. The survival curve of small cutworms when the two are combined is significantly different from that of SpltMNPV or Bt alone, showing a significant synergistic effect. When MbMNPV is mixed with high concentrations of Bt strains, the survival curve of small cutworms when the two are combined is significantly different from that of SpltMNPV or Bt alone, and the death rate of small cutworms is faster; when it is mixed with low concentrations of Bt strains, the survival curve of small cutworms when the two are combined is not significantly different from that of SpltMNPV or Bt alone. SeMNPV can play a synergistic role to a certain extent when mixed with different concentrations of Bt strains, but the mortality rate of small cutworms is low. The results show that the combination of two NPVs (MbMNPV and SpltMNPV) and Bt biological pesticides can effectively control small cutworm larvae.

[0035] Example 4 Synergistic coefficient of NPV combined with Bt ZB against 3rd instar larvae of cutworm

[0036] The MbMNPV, SpltMNPV and SeMNPV virus suspensions were diluted with distilled water to 1 × 10 5 OBs / mL, dilute the ZB Bt suspension with distilled water to 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 and 1×10 2 IU / mL, the virus suspension and different concentrations of Bt suspension were mixed in a 1:1 ratio to form a gradient mixture. The 3rd instar larvae of small cutworms that were subcultured for more than 5 generations were randomly selected and placed in a 12-well cell culture plate, 1 head per well, and starved for 12 hours. The pathogenicity of the NPV and Bt mixture to the 3rd instar larvae of small cutworms was determined by feeding. 10 μL of the mixture was respectively drawn and added to fresh artificial feed, and the feed was placed in a 12-well plate after it was naturally air-dried. Six replicates were set for each treatment, with 12 worms in each replicate, and feed dripped with distilled water was used as a control. The mortality of larvae in each treatment was observed and recorded every day. The synergistic coefficient of the mixed agent treatment was calculated based on this. The formula is as follows:

[0037] Theoretical mortality rate of the mixed group = 1-(1-NPV treatment mortality rate) × (1-Bt treatment mortality rate).

[0038] Synergistic coefficient = (actual mortality rate of the mixed group - theoretical mortality rate of the mixed group) / theoretical mortality rate of the mixed group × 100%.

[0039] The synergistic coefficients of mixing different virus suspensions with ZB Bt suspensions are as follows Figure 6 . A synergistic coefficient value greater than 20 indicates that there is a synergistic effect between the two mixtures. A synergistic coefficient value <-20 indicates that there is an antagonistic relationship between the two mixtures, and -20≤synergistic coefficient≤20 indicates that there is an additive effect between the two mixtures. SpltMN PV can play a combined synergistic role when mixed with different concentrations of ZB strains. When the two are used together to treat small cutworms, their synergistic coefficients are much greater than 20, showing a significant synergistic effect. When MbMNPV is mixed with different concentrations of Bt strains, its synergistic coefficient shows a significant additive effect. SeMNPV and 1×10 6 , 1×10 5 and 1×10 2 When ZB Bt strains with a concentration of 1×10 IU / mL were mixed, the synergistic coefficient was greater than 20, showing a synergistic effect; 4 and 1×10 3 When ZB Bt strains with a concentration of 100 IU / mL were mixed, an additive effect was observed. The results showed that SpltMNPV virus had the most significant synergistic effect on ZB Bt strains.

[0040] Example 5 Synergistic coefficient of NPV combined with Bt LJT against 3rd instar larvae of black cutworm

[0041] The three virus suspensions were diluted with distilled water to 1 × 10 5 OBs / mL, dilute the LJT Bt suspension to 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 and 1×10 2 IU / mL, the virus and different concentrations of Bt suspension were mixed in a 1:1 ratio to form a gradient mixture. The pathogenicity of the NPV and Bt mixture to the third-instar larvae of the small cutworm was determined by feeding method. 10 μL of the mixture was respectively aspirated and added to fresh artificial feed, and the feed was placed in a 12-well plate after natural air drying. Six replicates were set for each treatment, with 12 worms in each replicate, and the feed with distilled water added was used as the control. The mortality of larvae in each treatment was observed and recorded every day. The synergistic coefficient of the mixed agent treatment was calculated based on this.

[0042] The synergistic coefficient of mixing NPVs and LJT Bt suspension is as follows Figure 7. SpltMNPV can also play a combined synergistic role when mixed with different concentrations of LJT strains. When the two are used together to treat small cutworms, the synergistic coefficient is much greater than 20, showing an obvious synergistic effect. When MbMNPV is mixed with different concentrations of LJT strains, it shows a significant additive effect. When SeMNPV is mixed with low concentrations of LJT Bt strains, its synergistic coefficient is greater than 20, showing a synergistic effect; when mixed with high concentrations of LJ T Bt strains, it shows an additive effect. The results show that the SpltMNPV virus has the most significant synergistic effect on LJ T Bt strains.

[0043] Example 6 Field control effect of nuclear polyhedrosis virus and Bacillus thuringiensis on cutworm

[0044] Tobacco fields where cutworms were seriously infested in previous years were selected for field plot experiments. During the tobacco transplanting period, the corresponding doses of NPV virus and Bacillus thuringiensis LJT strains were mixed into the soil at the root base. SpltM NPV nuclear polyhedrosis virus and Bacillus thuringiensis LJT strains, which have good application potential and significant synergistic effects, were used as control agents for preliminary field effects. NPV virus and Bt were mixed evenly with the soil and applied to the roots. The application rate of SpltM NPV (500 million PIB / g) was 1000g / mu, and the application rate of LJT strain (16000 IU / mg) was 2500g / mu. Bt and virus were applied alone and conventional control was used as controls. About 100 tobacco plants were planted in each plot, and 3 replicates were performed. Samples were taken at 5 points in a Z shape in each plot 7d and 14d after seedlings were set, and the damage of 50 tobacco plants in the plot was investigated respectively. The rate of damaged plants was calculated, and on this basis, the relative control effect was calculated compared with the control area.

[0045] Control effect (%) = (the rate of damaged plants in the blank control area - the rate of damaged plants in the drug-treated area) / the rate of damaged plants in the blank control area × 100%.

[0046] Results Figure 8 ,Depend on Figure 8 It can be seen that there are significant differences in the control effects of different agents on small cutworms. The control effect of small cutworms in each treatment group increased significantly over time, which reflects the characteristics of the action of biological agents. Among them, the control effect of small cutworms in the treatment group with the combined use of SpltMNPV and LJT strains was the highest at 73.57% for 14 days, which was significantly higher than the control effect of the treatment with the agent alone and 7 days. Field trials have shown that the combination of SpltMNPV virus and LJT strain has a good control effect on small cutworms, and the two have a synergistic effect.

[0047] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0048] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of nuclear polyhedrosis virus and Bacillus thuringiensis in the preparation of preparations for controlling black cutworm larvae.

2. The use according to claim 1, characterized in that: The nuclear polyhedrosis virus includes: cabbage armyworm nuclear polyhedrosis virus MbMNPV, Spodoptera litura nuclear polyhedrosis virus SpltMNPV and beet armyworm nuclear polyhedrosis virus SeMNPV.

3. The use according to claim 2, characterized in that: The nuclear polyhedrosis virus is SpltMNPV.

4. The use according to claim 1, characterized in that: The volume ratio of the liquid of nuclear polyhedrosis virus and Bacillus thuringiensis is 1:1; wherein the concentration of Bacillus thuringiensis is 1×10 2 ~1×10 6 IU / mL; the concentration of nuclear polyhedrosis virus is 1×10 4 ~1×10 8 OBs / mL.

5. A preparation for controlling cutworm larvae, characterized in that: The preparation comprises nuclear polyhedrosis virus liquid and bacillus thuringiensis suspension, and the volume ratio of the two is 1:1.

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