A preparation and method for improving the control effect of entomopathogenic nematodes against thrips

Abstract

The present invention discloses a preparation and method for improving the control effect of entomopathogenic nematodes on thrips. The preparation includes entomopathogenic nematodes and carvacrol. The combined use of entomopathogenic nematodes and carvacrol for controlling thrips, under the condition of releasing the same number of Steinernema carpocapsae All (abbreviated as SC) entomopathogenic nematodes, the lethality rate of the treatment with low-concentration carvacrol added to the pupal stage thrips is significantly higher than that of the treatment using only entomopathogenic nematodes, the control effect on thrips is significantly improved, and it can quickly achieve the control effect on thrips. Compared with the single use of entomopathogenic nematodes, it overcomes the limitations of slow effect and low virulence on thrips. At the same time, carvacrol has no problems such as environmental pollution and drug resistance compared with chemical agent control, can greatly reduce the dosage of chemical agents, and is more green and safe. Therefore, the preparation and method of the present invention have broad application prospects in agricultural pest control.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological control, and particularly relates to a preparation and method for improving the control effect of entomopathogenic nematodes on thrips. Background Art

[0002] With the growth of the global population, food security is of utmost importance. Thrips are important agricultural pests belonging to the order Thysanoptera. Due to their tiny size, they are easy to hide, have strong reproductive ability, short growth cycles, and severe generation overlapping, making them difficult to control. They have gradually become one of the most important groups of agricultural pests in the world. Thrips feed on the sap of plants with rasping-sucking mouthparts, causing plant wilting, shriveled grains, and seriously affecting the yield and quality of crops. For their control, chemical control is currently mainly used. However, the long-term and excessive use of chemical pesticides not only easily causes excessive pesticide residues but also causes serious environmental pollution, kills natural enemies, and makes thrips develop drug resistance and a series of other problems. Therefore, today when it is extremely urgent to seek environmentally friendly control strategies, biological control has broad application prospects in the control of thrips pests. As a natural plant-derived pesticide, thymol is difficult for pests to develop resistance to. In addition, plant-derived pesticides are less likely to cause phytotoxicity to receptor plants compared to chemical pesticides and are also easily coordinated with other organisms in the environment. Currently, the biological control of thrips mainly includes pathogenic microorganisms, entomopathogenic nematodes, predatory mites, Orius spp., and plant-derived insecticides, etc., but the effect of using them alone is very limited. Therefore, the biological control measure of combining natural enemies with plant-derived agents has become an important measure today.

[0003] Entomopathogenic nematodes (EPNs) are important biological natural enemies, with advantages such as actively searching for hosts, highly efficiently killing hosts, and having a wide host range. They are safe for non-target organisms and the environment, are easy to mass culture, convenient to use, and can be recycled in the environment. They have been widely used in the biological control of underground pests. However, their application effect in thrips control still needs to be further studied. Previous studies have found that entomopathogenic nematodes have problems such as low lethality and slow onset against Megalurothrips usitatus. In recent years, the mixed use of entomopathogenic nematodes and chemical pesticides has achieved good control effects in the management of various pests, such as Bradysia odoriphaga, Holotrichia diomphalia, and Agrotis ypsilon, etc. At the same time, there is currently no control and evaluation in China on the combined use of entomopathogenic nematodes and thymol for thrips control. In order to obtain better control effects and meet the requirements of green prevention and control, the combined use of entomopathogenic nematodes and thymol for thrips control is an essential control measure. Summary of the Invention

[0004] The object of the present invention is to provide a preparation and method for improving the control effect of entomopathogenic nematodes on thrips. There is no report on the screening and application of entomopathogenic nematode synergists for controlling thrips. Through indoor experiments, it is found that the control effect of using an aqueous solution of entomopathogenic nematodes to control thrips is low and the action is slow. The method of the present invention significantly improves the control effect on thrips, can quickly achieve the control effect on thrips, and when the same number of Steinernema carpocapsae All (abbreviated as SC) entomopathogenic nematodes are released, the addition of a low-concentration carvacrol treatment has a significantly higher lethality rate on pupal thrips than the treatment using nematodes alone. Compared with the single use of entomopathogenic nematodes, the combined use of carvacrol can overcome its limitation of poor control effect on thrips. At the same time, compared with chemical agents, carvacrol belongs to natural products and has the advantages of being environmentally friendly, not easily generating drug resistance, and being safe for crops, greatly reducing the dosage of chemical agents and being more green and safe.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] First, provide a preparation for improving the control effect of entomopathogenic nematodes on thrips, which preparation comprises entomopathogenic nematodes and carvacrol.

[0007] Preferably, the preparation is an aqueous solution, wherein the concentration of carvacrol is 0.0104 - 0.0208 g / L (1 / 10RC), and the concentration of entomopathogenic nematodes is 12000 - 24000 IJs / mL.

[0008] Preferably, the species of the entomopathogenic nematodes is Steinernema carpocapsae All.

[0009] Secondly, provide a method for improving the control effect of entomopathogenic nematodes on thrips, comprising the following steps:

[0010] 1) Prepare a mixed solution containing entomopathogenic nematodes and carvacrol;

[0011] 2) Spray, immerse or soak the thrips with the said mixed solution.

[0012] Preferably, in step 1), the concentration of carvacrol in the mixed solution is 0.0104 - 0.0208 g / L (1 / 10RC), and the concentration of entomopathogenic nematodes is 12000 - 24000 IJs / mL.

[0013] Preferably, in step 2), the dosage of the mixed solution used for spraying, immersing or soaking is calculated according to the quantity ratio of entomopathogenic nematodes to thrips being 600:1 to 1200:1.

[0014] Preferably, the entomopathogenic nematode species is Steinernema carpocapsae All, and the thrips is in the pupal stage.

[0015] Preferably, step 2) includes the following steps: In a six-well plate, the diameter of each well is about 35 mm, and the overall plate size is about 85 mm × 125 mm × 15 mm; three layers of filter paper with a diameter of 35 mm are laid in each well; about 5-mm-long cowpea segments are placed in each well, and then 10 thrips in the pupal stage are placed; subsequently, 0.5 mL of the mixed solution in step 1) is added to each well of the plate in sequence.

[0016] Preferably, the dosage of the 0.5 mL of the mixed solution in step 1) is such that the ratio of the number of infective entomopathogenic nematodes to thrips in the pupal stage is 1200:1, 1000:1, 800:1, or 600:1.

[0017] Finally, provide the application of the above preparation in controlling thrips.

[0018] Beneficial effects: Compared with chemical control, the combination of entomopathogenic nematodes and carvacrol in the present invention is more environmentally and crop-friendly, and has the advantages of protecting natural enemy insects, reducing drug resistance, and reducing pesticide residues; compared with physical control methods for Megalurothrips usitatus, the combination of entomopathogenic nematodes and carvacrol has the advantages of low input, environmental friendliness, and quick and convenient operation; compared with the sole use of entomopathogenic nematodes, the combination of entomopathogenic nematodes and carvacrol can enhance the pathogenicity of entomopathogenic nematodes, increase the mortality of Megalurothrips usitatus, and has obvious and rapid control effects. Therefore, the preparation and method of the present invention have broad application prospects in the control of agricultural pests and diseases. Description of the Drawings

[0019] Figure 1 Effect of different insecticides on nematode survival under RC and 1 / 10RC conditions, nematode mortality rate (Mean ± SE) %;

[0020] Figure 2 Effect of different insecticides on nematode infection when mixed with nematodes under RC and 1 / 10RC conditions, corrected mortality rate of Tenebrio molitor (Mean ± SE) %;

[0021] Figure 3 Ratio of nematode infection in dissected dead Tenebrio molitor, Tenebrio molitor infection rate (Mean ± SE) %. Detailed Embodiments

[0022] The present invention will be described in detail below in conjunction with embodiments. These embodiments are for illustrative purposes only and are not limited to the application scope of the present invention. The present invention is not limited to the following embodiments or implementation manners. Any modifications and deformations made without departing from the spirit of the present invention shall be included within the scope of the present invention; unless otherwise specified, the experimental materials or reagents used in the following embodiments are all commercially available. The entomopathogenic nematode species used in the following embodiments is Steinernema carpocapsae All (abbreviated as SC), which was purchased from Weifang Hongrun Agricultural Technology Co., Ltd.; the Megalurothrips usitatus used was collected from the fields in Huaxi District, Guiyang City, Guizhou Province and brought back to the laboratory and placed in an incubator for subsequent experiments. The following embodiments are included:

[0023] 1. Treatment of entomopathogenic nematode suspension

[0024] The sponge block containing the entomopathogenic nematodes purchased was placed on a sieve and soaked with pure water for 1 h using the soaking method. Then, the nematodes were collected and precipitated in a conical flask, and then repeatedly washed 3 times with pure water. After removing the supernatant, the nematode mother liquor was obtained and stored at 4 °C. Before use, 0.5 ml of the nematode mother liquor was counted for concentration under a stereomicroscope. After calculating the concentration of the entomopathogenic nematode mother liquor, the nematode mother liquor was diluted with pure water by an appropriate multiple as needed to prepare a nematode suspension with a corresponding concentration.

[0025] 2. Compatibility determination of entomopathogenic nematodes and insecticides

[0026] 2.1 Effect of insecticides on the survival of EPNs

[0027] The tested insecticide products are shown in Table 1, a total of 6 kinds. The insecticidal active ingredients in the products are pyrethrins, matrine, chamaejasmine, carvacrol, veratramine, and imidacloprid respectively, and the mass percentage contents of the active ingredients are 0.5%, 0.5%, 1.6%, 5%, 0.1%, and 70% respectively. The recommended application rates of the products are 240 - 480 g per mu, 45 - 60 mL per mu, 50 - 100 mL per mu, 120 - 180 mL per mu, 120 - 140 g per mu, and 2 - 4 g per mu respectively. The usually recommended water consumption in the field is 45 - 60 kg per mu, that is, the product is added to 45 - 60 kg of water according to the recommended application rate per mu, and the application concentration of the insecticidal active ingredient obtained thereby is the field recommended application concentration (RC). In subsequent experiments, 1 / 10RC represents 1 / 10 times the field recommended application concentration. Taking carvacrol as an example: The concentration of carvacrol obtained by adding 120 - 180 mL of the 5% carvacrol product to 45 - 60 kg of water is the field recommended application concentration (RC) of carvacrol, which is 0.104 - 0.208 g / L. The specific calculation method is as follows: First, accurately measure a certain volume of the 5% carvacrol product and accurately weigh the mass with an analytical balance, then calculate the concentration of the active ingredient carvacrol in the product according to the mass percentage, which is 0.052 g / mL. Finally, calculate the field recommended application concentration of carvacrol according to the field recommended application rate (120 - 180 mL) of the product and the field water consumption (45 - 60 kg), which is 0.104 - 0.208 g / L. 1 / 10RC of carvacrol is 0.0104 - 0.0208 g / L.

[0028] In all the examples in this embodiment part, the RC of each insecticide active ingredient prepared and obtained is: Take the middle value of the product recommended application rate (for example, for the active ingredient carvacrol, take the middle value of the product application rate, that is, 150 mL / mu), and the water consumption per mu is 50 kg, that is, the product and the water consumption are prepared according to this ratio to obtain RC, and diluting RC by 10 times can obtain 1 / 10RC.

[0029] Table 1 Tested insecticide products

[0030]

[0031]

[0032] Six kinds of insecticides (Table 1) and entomopathogenic nematodes were separately formulated into mixed solutions with pure water as the liquid medium. The final concentrations of the active ingredients of the insecticides were RC and 1 / 10RC, and the concentration of entomopathogenic nematodes was 1000 IJs per 10 mL of the mixed solution. The prepared mixed solution was added to petri dishes (d = 9 cm), 10 mL was added to each dish, and 10 mL of pure water containing 1000 IJs but no insecticide was used as a control. Each treatment had 4 replicates. After sealing the petri dishes with parafilm, they were placed in a dark environment with a relative humidity of 50% - 70% and a temperature of 25 ± 1 °C for 24 h. Then, 0.1 mL of the suspension was randomly taken, and the survival number of nematodes was observed and counted under a stereomicroscope. Referring to the response criteria of entomopathogenic nematodes to pesticides by Wei Hongyi et al. (Table 2), no response after probing with the tip of a dissecting needle was considered death, and the mortality rate of EPNs was calculated. If the mortality rate of the pesticide against nematodes was less than 10%, the effect of the pesticide on the infection rate of nematodes could be further determined.

[0033] Table 2 Response criteria of entomopathogenic nematodes to pesticides

[0034]

[0035] The results are as Figure 1 shown. In the nematode survival experiment, the highest mortality rate of the 6 common insecticides against nematodes was that of chamaejasmine, followed by pyrethrum extract. The mortality rates of the other insecticides against nematodes were all less than 10%. Therefore, the other 4 insecticides were selected to continue the entomopathogenic nematode infection experiment.

[0036] 2.2 Entomopathogenic nematode infection experiment

[0037] 1.5 mL of a nematode suspension containing 750 IJs was added to a petri dish (d = 9 cm) lined with 2 layers of filter paper, and then 10 9 - 11 instar larvae of Tenebrio molitor (body length 2 - 3 cm) were placed in each petri dish. The treatments included separate EPNs, EPNs - insecticide mixtures, and separate insecticide dilutions (the final concentrations of the active ingredients of the insecticides were RC and 1 / 10RC), with clean water as the control treatment. Each treatment had 4 replicates (dishes). The petri dishes were sealed with parafilm and placed at 25 ± 1 °C and 65% RH for 72 h, and observed every 24 h to check and record the number of dead Tenebrio molitor. The dead larvae were placed in a clean petri dish for cultivation, and after 3 d, they were dissected to evaluate the infection rate of EPNs.

[0038] The results are as Figure 2 shown. In the Tenebrio molitor infection experiment, at the RC and 1 / 10RC concentrations, the corrected mortality rates of the mixed treatments of matrine, imidacloprid, carvacrol, veratridine, and SC nematodes against Tenebrio molitor were not significantly different from those of the SC nematodes alone or slightly higher than those of the SC nematodes alone. But as Figure 3As shown in the figure, through the dissection of Tenebrio molitor, it was found that at the RC concentration, the infection rate of Matrine, Imidacloprid and SC mixed treatment on Tenebrio molitor had no significant difference from that of the single SC nematode treatment, while Carvacrol and Veratridine only had no significant difference from the single SC nematode treatment at the 1 / 10RC concentration. Therefore, in order to reduce costs and minimize the impact on the environment, a 1 / 10RC concentration insecticide and SC nematode mixed treatment was selected for the toxicity determination experiment of Megalurothrips usitatus.

[0039] 2.3 Toxicity determination of insecticide-EPNs mixture against Megalurothrips usitatus

[0040] 2.3.1 The ratio of infective-stage entomopathogenic nematodes to Megalurothrips usitatus is 100:1

[0041] Experimental method: Line the inside of a plastic seasoning cup (d = 4 cm, h = 2.5 cm) with three layers of filter paper, and place 1 cm long cowpea segments into the plastic seasoning cup. Gently place 10 late second-instar red-brown Megalurothrips usitatus larvae on the filter paper with a small brush. Add 0.5 mL of insecticide (final concentration of active ingredient: 1 / 10RC), EPNs-insecticide (final concentration: 1000 IJs, 1 / 10RC) mixture, single EPNs suspension (final concentration: 1000 IJs), and pure water to each dish. Pure water is used as the control treatment, and each treatment has 4 replicates (dishes). After the operation, seal the culture dishes with sealing film and place them at 25 ± 1 °C and 65% RH for 144 hours. Check and record the number of dead Megalurothrips usitatus larvae every 24 hours. Place the dead larvae in a clean six-well plate and place the six-well plate in an incubator at 25 ± 1 °C and 65% RH. Observe under a microscope in a timely manner whether there is nematode infection in the Megalurothrips usitatus larvae.

[0042] The results are shown in Table 3. In the mixed treatment of SC nematodes and insecticides, as time increased, the mortality rate of Megalurothrips usitatus gradually increased, but did not reach the expected death effect. Among them, at 144 h, in the mixed treatment of entomopathogenic nematodes and insecticides, the highest mortality rate of Megalurothrips usitatus was for Imidacloprid and Carvacrol. After the mixed treatment, their mortality rates were higher than those of the CK and the two insecticides treated alone. Therefore, increasing the concentration of entomopathogenic nematodes and mixing them with 1 / 10RC Imidacloprid and 1 / 10RC Carvacrol were selected to explore whether the mortality rate of Megalurothrips usitatus could be increased.

[0043] Table 3 Mortality rate of Megalurothrips usitatus larvae (Mean ± SE) %

[0044]

[0045] 2.3.2 The ratio of infective-stage entomopathogenic nematodes to Megalurothrips usitatus is 1200:1

[0046] The experimental method was the same as that in 2.3.1, and the late second-instar red-brown Megalurothrips usitatus larvae were replaced with pupal-stage Megalurothrips usitatus.

[0047] The experimental results are shown in Table 4. At 24 h, the mortality rate of Megalurothrips usitatus treated with carvacrol and SC nematodes reached 80%, which was significantly higher than that of other treatments. As time gradually increased, the mortality rate also gradually increased. At 48 h and 72 h, the mortality rates of Megalurothrips usitatus were 90% and 97% respectively, which were higher than those of the mixture of imidacloprid and SC nematodes. Therefore, the concentration of entomopathogenic nematodes was reduced, and the combination of carvacrol and entomopathogenic nematodes was continued to determine the virulence against Megalurothrips usitatus.

[0048] Table 4 Mortality rate of pupal-stage Megalurothrips usitatus (Mean±SE) %

[0049]

[0050]

[0051] 2.3.3 The ratio of entomopathogenic nematodes to thrips at the infective stage was 1200:1, 1000:1, 800:1, 600:1

[0052] Line the inside of a six-well plate (d = 3.5 cm) with three layers of filter paper, and place 0.5-cm-long cowpea segments into the six-well plate. Prepare a mixed solution with the selected insecticide and entomopathogenic nematodes, and adjust the final concentration of the insecticidal active ingredient in the mixed solution to 1 / 10 RC. Each 0.5 mL of the mixed solution contains 12000 IJs, 10000 IJs, 8000 IJs, and 6000 IJs respectively. Gently place 10 pupal-stage Megalurothrips usitatus on the filter paper with a small brush. Add 0.5 mL of 1 / 10 RC carvacrol, EPNs-1 / 10 RC carvacrol mixed solution, single EPNs suspension, and pure water to each dish. Pure water is used as the control treatment, and each treatment has 4 replicates (dishes). After the operation, seal the culture dish with a sealing film, place it at 25±1 °C and 65% RH, check and record the number of dead pupal-stage Megalurothrips usitatus every 24 h. Place the dead larvae in a clean six-well plate, and place the six-well plate in an incubator at 25±1 °C and 65% RH. Observe under a microscope in a timely manner whether there is nematode infection in the larvae of Megalurothrips usitatus.

[0053] From χ 2 The independence test was used to judge the type of combined action between entomopathogenic nematodes and carvacrol, and the mortality rate of the tested Megalurothrips usitatus in each treatment was converted into the corrected mortality rate.

[0054] M = [M N + M I (1 - M N )]; M E = M × N; χ 2 = (M NI-M E ) 2 / M E

[0055] Among them, M and M E respectively represent the expected mortality rate and the number of dead individuals of the tested Megalurothrips usitatus when entomopathogenic nematodes are mixed with carvacrol; M N and M I are the corrected mortality rates of the tested Megalurothrips usitatus when treated with entomopathogenic nematodes and carvacrol alone, respectively; M NI is the actual corrected number of dead individuals of the tested Megalurothrips usitatus when entomopathogenic nematodes are mixed with carvacrol; N is the total number of the tested Megalurothrips usitatus.

[0056] When χ 2 < 3.84 (1 df and P = 0.05), it indicates that the mixture of the two insecticidal factors shows an additive effect;

[0057] When χ 2 > 3.84 (1 df and P = 0.05) and M NI < M E at this time, it indicates that the mixture of the two insecticidal factors shows an antagonistic effect;

[0058] When χ 2 > 3.84 (1 df and P = 0.05) and M NI > M E at this time, it indicates that the mixture of the two insecticidal factors shows a synergistic effect.

[0059] The experimental results are shown in Table 5. By performing a χ 2 analysis on the corrected mortality rate of Megalurothrips usitatus treated with the mixture of entomopathogenic nematodes and carvacrol, it can be obtained that the mixture of SC nematodes (6000 IJs) and 1 / 10 RC carvacrol shows a significant synergistic effect at 24 h (χ 2 = 5.008). At this time, the corrected mortality rate of Megalurothrips usitatus is about 90%. The mixtures of other concentrations and SC nematodes show an additive effect at different times.

[0060] Table 5 Synergistic effect of the mixture of entomopathogenic nematodes and 1 / 10 RC carvacrol on Megalurothrips usitatus

[0061]

[0062] Note: Car, 1 / 10 times the field-recommended concentration of carvacrol; SC, S. carpocapsae All; 1, 2, 3, 4 = 1200, 1000, 800, 600 IJs / larva. Different lowercase letters represent significant differences in the corrected mortality rate of Megalurothrips usitatus in the same column under different treatments, and different uppercase letters represent significant differences in the corrected mortality rate of Megalurothrips usitatus at different treatment times for the same treatment (P < 0.05).

[0063] The conventional technologies and the solutions not described in detail in the above embodiments are well known in the art, so they will not be elaborated here in detail. The above embodiments and / or experimental examples have described in detail the preferred embodiments of the present invention. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A preparation for improving the control effect of entomopathogenic nematodes against thrips, characterized in that, It includes entomopathogenic nematodes and carvacrol; the preparation is an aqueous solution, the concentration of carvacrol is 0.0104 - 0.0208 g / L, the concentration of entomopathogenic nematodes is 12000 - 24000 IJs / mL, and the species of the entomopathogenic nematodes is Steinernema carpocapsae ( Steinernema carpocapsae All); the thrips is Megalurothrips usitatus.

2. A method for improving the control effect of entomopathogenic nematodes on thrips, characterized in that, comprises the following steps: 1) Prepare a mixture containing entomopathogenic nematodes and carvacrol; 2) Spray, dip or soak thrips with the said mixture; In the mixture in step 1), the concentration of carvacrol is 0.0104 - 0.0208 g / L, and the concentration of entomopathogenic nematodes is 12000 - 24000 IJs / mL; In step 2), the dosage of the mixture used for spraying, dipping or soaking is calculated according to the ratio of the number of entomopathogenic nematodes to thrips being 600:1 to 1200:1; The entomopathogenic nematode species is Steinernema carpocapsae ( Steinernema carpocapsae All), and the thrips is Megalurothrips usitatus.

3. The method according to claim 2, wherein Step 2) comprises the following steps: In a six-well plate, each well has a diameter of 35 mm, and the overall plate size is 85 mm × 125 mm × 15 mm; Lay 3 layers of filter paper with a diameter of 35 mm in each well; Put 5-mm-long cowpea segments and then 10 pupal thrips in each well; Subsequently, add 0.5 mL of the mixture in step 1) to each well of the plate in sequence.

4. The method according to claim 3, wherein The dosage of the 0.5 mL of the mixture in step 1) is such that the ratio of the number of infective entomopathogenic nematodes to pupal thrips is 1200:1, 1000:1, 800:1 or 600:

1.

5. Use of the preparation according to claim 1 in controlling thrips, wherein the thrips are Megalurothrips usitatus.

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