Application of the combination of nuclear polyhedrosis virus and Bacillus thuringiensis in the preparation of a preparation for controlling Agrotis ypsilon larvae

Abstract

The present invention discloses the application of the combination of nuclear polyhedrosis virus and Bacillus thuringiensis in the preparation of a preparation for controlling Agrotis ypsilon larvae, and relates to the technical field of controlling Agrotis ypsilon larvae. Through the ratio experiments of nuclear polyhedrosis virus and Bacillus thuringiensis with different types and different concentrations, it is found that, compared with the single use of nuclear polyhedrosis virus or the single use of Bacillus thuringiensis, the combined use of the two has an obvious synergistic effect in the control of Agrotis ypsilon larvae, can significantly increase the mortality rate of Agrotis ypsilon larvae indoors and in the field, improve the control effect, and provides a theoretical basis for the biological control of Agrotis ypsilon. At the same time, the combined use of nuclear polyhedrosis virus and Bacillus thuringiensis can avoid the "3R" problems brought by chemical pesticide control from the root, is environmentally friendly, and is conducive to long-term development.

Description

Technical Field

[0001] The present invention relates to the technical field of controlling Agrotis ypsilon larvae, and more specifically to the application of the combination of nucleopolyhedrovirus and Bacillus thuringiensis in the preparation of a preparation for controlling Agrotis ypsilon larvae. Background Art

[0002] Agrotis belongs to the family Noctuidae of the order Lepidoptera, and has aliases such as cutworm, earthworm, black cutworm, black earthworm, earth clipper, root cutter, etc. It has a miscellaneous diet and a very wide host range. The larvae damage a variety of vegetables, flowers, field crops, tobacco, fruit trees, forest seedlings and weeds. The young larvae damage the heart leaves or young leaves of crops, causing holes or notches. After the 3rd instar, they cut off the tender stems of crop seedlings near the ground, causing seedlings to be missing and ridges to be broken. In severe cases, the seeds need to be replanted, and it is the most serious underground pest. There are 170 species of Agrotis recorded in the agricultural areas of our country, and there are about more than 10 common Agrotis species that damage crops. Among them, Agrotis ypsilon and Agrotis segetum have the widest distribution and the most serious damage. Agrotis albovenosa, Agrotis tokionis, Agrotis exclamationis and Agrotis c-nigrum often occur and cause damage in local areas.

[0003] At present, chemical control methods are mainly adopted in our country to control Agrotis. In view of the problems such as "3R" brought about by the long-term large-scale use of chemical pesticides, it is urgent to establish a green prevention and control technology system for Agrotis mainly based on biological control. Indoor bioassay is an important means to screen and clarify the action effects of different biocontrol preparations.

[0004] Therefore, how to provide an effective method for controlling Agrotis larvae is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides the application of the combination of nucleopolyhedrovirus and Bacillus thuringiensis in the preparation of a preparation for controlling Agrotis ypsilon larvae.

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

[0007] One aspect of the embodiments of the present invention provides the application of the combination of nucleopolyhedrovirus and Bacillus thuringiensis in the preparation of a preparation for controlling Agrotis ypsilon larvae.

[0008] Preferably, the nucleopolyhedrovirus includes: Mamestra brassicae nucleopolyhedrovirus MbMNPV, Spodoptera litura nucleopolyhedrovirus SpltMNPV and Spodoptera exigua nucleopolyhedrovirus SeMNPV. Preferably, the virus is SpltMNPV.

[0009] Preferably, the volume ratio of the liquid of the nucleopolyhedrovirus to 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] In the second aspect of the embodiments of the present invention, a preparation for controlling Agrotis ypsilon larvae is provided. The preparation includes a nuclear polyhedrosis virus solution and a Bacillus thuringiensis suspending agent, and the volume ratio of the two is 1:1.

[0011] It can be seen from the above technical solutions that, compared with the prior art, through the ratio experiments of nuclear polyhedrosis viruses of different types and different concentrations and Bacillus thuringiensis in the present invention, it is found that, compared with the single use of nuclear polyhedrosis virus or the single use of Bacillus thuringiensis, the combined use of the two has an obvious synergistic effect in the control of Agrotis ypsilon larvae, can significantly increase the mortality rate of Agrotis ypsilon larvae indoors and in the field, improve the control effect, and provide a theoretical basis for the biological control of Agrotis ypsilon. At the same time, the combined use of nuclear polyhedrosis virus and Bacillus thuringiensis can avoid the "3R" problems brought by chemical pesticide control from the root, is environmentally friendly, and is conducive to long-term development. Description of the Drawings

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0013] Figure 1 It is the corrected mortality rate of Agrotis ypsilon under different nuclear polyhedrosis viruses and different concentrations of the present invention.

[0014] Figure 2 It is the survival curve of Agrotis ypsilon under different nuclear polyhedrosis viruses and different concentrations; A: MbMNPV, B: SpltMNPV, C: SeMNPV.

[0015] Figure 3 It is the survival rate of the second-instar and third-instar larvae of Agrotis ypsilon under different treatments of Bacillus thuringiensis; A, B, and C are the survival rates of the second-instar larvae after treatment with Bt LJT, Bt WD, and Bt ZB respectively; D, E, and F are the survival rates of the third-instar larvae after treatment with Bt LJT, Bt WD, and Bt ZB respectively.

[0016] Figure 4Survival rate curves of the 3rd instar larvae of Agrotis ypsilon after the combination of NPVs and Bt ZB; A, B, and C are the third combinations of BtZB with MbMNPV, SpltMNPV, and SeMNPV, respectively. Survival rate curves A2 - 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 Survival rate curves of the 3rd instar larvae of Agrotis ypsilon when NPV is combined with Bt LJT; 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 Synergistic effect of the combination of NPV and Bt ZB on the 3rd instar larvae; A: MbMNPV; B: SpltMNPV; C: SeMNPV.

[0019] Figure 7 Synergistic effect of the combination of NPV and Bt LJT on the 3rd instar larvae; D: MbMNPV; E: SpltMNPV; F: SeMNPV.

[0020] Figure 8 Control effect of the field combined use of SpltMNPV (500 million PIB / g) and strain LJT (16000 IU / mg) on Agrotis ypsilon. Detailed implementation mode

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] Biological Activity of Nucleopolyhedrovirus against Agrotis ypsilon Larvae in Example 1

[0023] Randomly select the early 3rd instar larvae of Agrotis ypsilon that have been continuously reared for more than 5 generations and place one larva in each well of a 12-well cell culture plate, and subject them to starvation treatment for 12 h. Dilute the stock solutions of Mamestra brassicae nucleopolyhedrovirus (MbMNPV), Spodoptera litura nucleopolyhedrovirus (SpltMNPV), and Spodoptera exigua nucleopolyhedrovirus (SeMNPV) with distilled water 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. Respectively, pipette 10 μL of the virus suspension and add it to fresh artificial feed (0.2 cm × 0.2 cm × 0.2 cm). After natural air drying, put the feed into the 12-well plate, and infect the 3rd instar larvae by the method of feeding the virus. Set 6 replicates for each treatment, with 12 larvae in each replicate, and use the feed with distilled water added as the control. Observe and record the number of dead larvae in each treatment every day, calculate the corrected mortality rate at 7 d, and draw the survival curve. Use the Probit regression model to calculate the virulence equation.

[0024] Table 1 Virulence of Different Nucleopolyhedroviruses against Agrotis ypsilon

[0025]

[0026] The results are shown in Figure 1 and Figure 2 , and the three nucleopolyhedroviruses at different concentrations can all cause different degrees of death of Agrotis ypsilon larvae, and there are significant differences among different concentrations and different viruses. Among them, MbMNPV has the highest biological activity against Agrotis ypsilon. The corrected mortality rates of Agrotis ypsilon at 7 d with 1×10 5 OBs / mL, 1×10 6 OBs / mL, 1×10 7 OBs / mL, and 1×10 8 OBs / mL treatments are 84.62%, 91.07%, 100%, and 98.21% respectively, and its survival curve also has differences from the treatment with 1×10 4 OBs / mL. The biological activity of SpltMNPV against Agrotis ypsilon ranks second. The corrected mortality rates of Agrotis ypsilon at 7 d with 1×10 6 OBs / mL, 1×10 7 OBs / mL, and 1×10 8 OBs / mL treatments are 42.86%, 92.86%, and 94.64% respectively, and its survival curve also has differences from the treatment with 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, with the 7-day mortality rate not exceeding 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 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 third-instar larvae of the black cutworms that had been reared for more than five generations were randomly selected and placed in a 12-well cell culture plate, with one larva per well, and starved for 12 hours. 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 black cutworm was determined by feeding method. 10 μL of Bt suspension was respectively aspirated and added dropwise to fresh artificial diet (0.2 cm × 0.2 cm × 0.2 cm). After natural air drying, the diet was placed in a 12-well plate. Each treatment was repeated 6 times, with 12 larvae in each repeat. The control was fed with diet dripped with distilled water. Based on the results of Bt toxicity test 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 on the larvae of the black cutworm, its bioactivity test 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 toxicity equation was calculated using the Probit regression model.

[0029] See the results Figure 3 As shown in Table 2, the three Bt strains at different concentrations can all cause the death of the second and third instar larvae of the cutworm, but there are certain differences in their mortality rates. As the age of the 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 the cutworms in each treatment group gradually increases over time, reflecting the characteristics of biological control. 25 3.02×10 5IU / mL, LC treated with LJT 25 was 4.20×10 5 IU / mL, LC treated with ZB 25 was 1.51×10 7 IU / mL. The LC 25 of WD treatment on 2nd instar larvae 5 was 6.38×10 25 IU / mL, and the LC 4 of LJT treatment was 8.10×10 25 IU / mL, and the LC 6 of ZB treatment was 5.64×10

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

[0031]

[0032] Example 3 Synergistic effect of nucleopolyhedrovirus and Bacillus thuringiensis on Agrotis ipsilon larvae

[0033] Dilute 3 virus suspensions (MbMNPV, SpltMNPV and SeMNPV) with distilled water to 1×10 5 OBs / mL, and dilute two Bt suspensions (LJT, ZB) with distilled water to 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 and 1×10 2 IU / mL. Mix the virus suspension and Bt suspensions at different concentrations at a ratio of 1:1 to form 6 series of gradient mixtures. Randomly select newly hatched 3rd instar larvae of Agrotis ipsilon that have been reared for more than 5 generations and place one larva in each well of a 12-well cell culture plate. Starve the larvae for 12 h. Use the feeding method to determine the pathogenicity of the NPV and Bt mixture to 3rd instar larvae of Agrotis ipsilon. Pipette 10 μL of the mixture and add it to fresh artificial feed (0.2 cm×0.2 cm×0.2 cm). After natural drying, place the feed in the 12-well plate. Set 6 replicates for each treatment, with 12 larvae in each replicate. Use the feed added with distilled water as the control. Observe and record the number of dead larvae in each treatment every day, calculate the corrected mortality rate at 7 d, and draw the survival curve.

[0034] The results are shown in Figure 4 and Figure 5, Mixing different nucleopolyhedroviruses with different concentrations of Bt can, to a certain extent, cause the death of Agrotis ypsilon. Among them, mixing SpltMNPV with different concentrations of LJT and ZB strains can play a synergistic effect. When the two are jointly treated, the survival curve of Agrotis ypsilon is significantly different from that of using SpltMNPV or Bt alone, showing a significant synergistic effect. When MbMNPV is mixed with a high concentration of Bt strain, the survival curve of Agrotis ypsilon is significantly different from that of using SpltMNPV or Bt alone when the two are jointly treated, and the death rate of Agrotis ypsilon is faster; while when it is mixed with a low concentration of Bt strain, the survival curve of Agrotis ypsilon is not significantly different from that of using SpltMNPV or Bt alone when the two are jointly treated. Mixing SeMNPV with different concentrations of Bt strains can play a synergistic role to a certain extent, but the mortality rate of Agrotis ypsilon is relatively low. The results show that mixing two NPVs (MbMNPV and SpltMNPV) with Bt biopesticide can effectively control the larvae of Agrotis ypsilon.

[0035] Example 4 Synergistic coefficient of the combination of NPV and Bt ZB against the 3rd instar larvae of Agrotis ypsilon

[0036] Dilute the virus suspensions of MbMNPV, SpltMNPV and SeMNPV 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, mix the virus suspension and different concentrations of Bt suspension in a 1:1 ratio to form gradient mixtures. Randomly select the newly hatched 3rd instar larvae of Agrotis ypsilon that have been reared for more than 5 generations and place them in a 12-well cell culture plate, 1 larva per well, and starve them for 12 h. Use the feeding method to determine the pathogenicity of the NPV and Bt mixture against the 3rd instar larvae of Agrotis ypsilon. Pipette 10 μL of the mixture and add it to the fresh artificial diet respectively. After natural drying, put the diet into the 12-well plate. Set 6 replicates for each treatment, 12 larvae per replicate, and use the diet added with distilled water as the control. Observe and record the death of larvae in each treatment every day. Calculate the synergistic coefficient of the chemical mixture treatment according to the following formula:

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

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

[0039] The synergistic coefficient of the mixture of different virus suspensions and ZB Bt suspension is as follows Figure 6 . A synergistic coefficient value greater than 20 indicates a synergistic effect between the two mixtures. A synergistic coefficient value < -20 indicates an antagonistic relationship between the two mixtures, and -20 ≤ synergistic coefficient ≤ 20 indicates an additive effect between the two mixtures. Mixing SpltMN PV with ZB strains at different concentrations can all play a combined synergistic role. When the two are jointly treated against Agrotis ypsilon, their synergistic coefficients are all much greater than 20, showing a significant synergistic effect. When MbMNPV is mixed with Bt strains at different concentrations, its synergistic coefficient shows a significant additive effect. When SeMNPV is mixed with ZB Bt strains at concentrations of 1×10 6 , 1×10 5 and 1×10 2 IU / mL, its synergistic coefficients are all greater than 20, showing a synergistic effect; while when mixed with ZB Bt strains at concentrations of 1×10 4 and 1×10 3 IU / mL, it shows an additive effect. The results show that the SpltMNPV virus has the most significant synergistic effect on the ZB Bt strain

[0040] Synergistic coefficient of the combination of NPV and Bt LJT against the 3rd instar larvae of Agrotis ypsilon

[0041] Dilute 3 virus suspensions 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, and mix the virus and Bt suspensions at different concentrations in a 1:1 ratio to form gradient mixtures. Use the feeding method to determine the pathogenicity of the NPV and Bt mixture against the 3rd instar larvae of Agrotis ypsilon. Respectively suck 10 μL of the mixture and drop it onto the fresh artificial feed. After natural air drying, put the feed into a 12-well plate. Set 6 replicates for each treatment, with 12 insects in each replicate, and use the feed dropped with distilled water as the control. Observe and record the death situation of the larvae in each treatment every day. Calculate the synergistic coefficient of the medicament mixture treatment accordingly

[0042] The synergistic coefficient of the mixture of NPVs and LJT Bt suspension is as follows Figure 7。When SpltMNPV is mixed and used with different concentrations of LJT strains, it can also play a synergistic effect. When the two are jointly used to treat Agrotis ypsilon, the synergistic coefficient is far greater than 20, showing an obvious synergistic effect. When MbMNPV is mixed and used with different concentrations of LJT strains, it shows a significant additive effect. When SeMNPV is mixed and used with a low concentration of LJT Bt strain, the synergistic coefficient is greater than 20, showing a synergistic effect; while when it is mixed and used with a high concentration of LJ T Bt strain, it shows an additive effect. The results show that the SpltMNPV virus has the most significant synergistic effect on the LJ T Bt strain.

[0043] Example 6 Field Control Effect of Nucleopolyhedrovirus and Bacillus thuringiensis on Agrotis ypsilon

[0044] Select tobacco fields where Agrotis ypsilon occurred severely in previous years for field plot trials. During the tobacco transplanting period, corresponding doses of NPV virus and Bt were mixed into the soil at the root base. SpltMNPV nucleopolyhedrovirus and Bacillus thuringiensis LJT strain, which have good application potential and significant synergistic effects, were used as control agents for a preliminary exploration of field effects. The NPV virus and Bt were evenly mixed with the soil and applied by mixing with the soil at the roots. The application rate of SpltM NPV (500 million PIB / g) was 1000 g / mu, and the application rate of LJT strain (16000 IU / mg) was 2500 g / mu. Bt and the virus were used alone and conventional control were used as controls. About 100 tobacco plants were planted in each plot, and 3 replicates were carried out. At 7 days and 14 days after final thinning, 5-point sampling in a Z-shape was carried out in each plot to investigate the damage of 50 tobacco plants in the plot, and the percentage of damaged plants was counted. On this basis, the relative control effect was calculated by comparing with the control area.

[0045] Control effect (%) = (Percentage of damaged plants in the blank control area - Percentage of damaged plants in the medicament treatment area) / Percentage of damaged plants in the blank control area × 100%.

[0046] The results are shown in Figure 8 , as can be seen from Figure 8 , there are significant differences in the control effects of different medicaments on Agrotis ypsilon. The control effects of Agrotis ypsilon in each treatment group were significantly improved over time, which reflects the characteristics of the action of biological agents. Among them, the highest control effect of Agrotis ypsilon in the treatment group using SpltMNPV and LJT strain jointly at 14 days was 73.57%, which was significantly higher than the control effects of using the medicament alone and at 7 days. Field trials show that the combined use of SpltMNPV virus and LJT strain has a good control effect on Agrotis ypsilon, and the two have a synergistic effect.

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

[0048] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein can 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 is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a nuclear polyhedrosis virus and Bacillus thuringiensis in combination in the preparation of a preparation for controlling Agrotis ypsilon larvae, characterized in that, The nucleopolyhedrovirus is Spodoptera litura nucleopolyhedrovirus SpltMNPV.

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

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