Application of vanillylamine in preparation of preparation for preventing and treating rice stem borers

Abstract

The invention discloses application of vanillylamine in preparation of a preparation for preventing and treating rice stem borers, and belongs to the technical field of microbial pesticides. The invention finds that the enterococcus metabolite vanillylamine, namely 4-(aminomethyl)-2-methoxyphenol, has insecticidal activity on rice stem borers, and particularly has excellent insecticidal activity on chilo suppressalis, so that a new biocontrol resource is provided for prevention and treatment of the rice pest chilo suppressalis.

Description

Technical Field

[0001] The invention relates to the technical field of microbial pesticides, and in particular to application of vanillylamine in the preparation of a preparation for preventing and controlling rice stem borers. Background Art

[0002] Microbial pesticides mainly include living microbial pesticides (such as fungi, bacteria, viruses, nematodes, etc.) and microbial metabolite pesticides. Among them, microbial metabolite pesticides (MMPs) use metabolites produced by microbial fermentation as active ingredients. In the past two decades, bacterial metabolites have been used to control pests such as diseases, insects and weeds or to regulate plant growth and development. However, the application of new active natural products from bacteria needs to be further explored and developed. In recent years, the application of new interdisciplinary methods and technologies (such as genomics, metabolomics, etc.) has significantly improved our ability to discover and apply new bacterial natural products, and has also rekindled the interest of scientific researchers in the research and development and application of bacterial natural products.

[0003] Rice is an important food crop, and rice stem borers are important pests that affect rice yield and quality. Although traditional chemical control methods are effective, they have problems such as environmental pollution and increased pest resistance. Therefore, biological control technology, especially the use of microbial metabolites to control rice stem borers, has gradually become the main technology and research hotspot for pest control. However, there is no report in the prior art on the use of vanillin to control rice stem borers. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an application of vanillylamine in the preparation of a preparation for controlling rice stem borers.

[0005] The first object of the present invention is to provide an application of vanillylamine in the preparation of a preparation for controlling rice stem borers.

[0006] The beneficial effects of the present invention are as follows: Vanillylamine is a common name for 4-(aminomethyl)-2-methoxyphenol (Vanillylamine, VA), which can be obtained by direct purchase, chemical synthesis or biosynthesis. In biosynthesis, it can be obtained by microbial fermentation, and can be obtained from enterococci such as Enterococcus fusogenus ( Enterococcus casseliflavus ), Enterococcus faecalis ( Enterococcus faecalis ) and Enterococcus monteri ( Enterococcus mundtii ) is obtained from the metabolites produced by fermentation. The present invention uses vanillin to control rice borers, and has excellent control effect.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the rice stem borer is Chilo suppressalis or Tryporyza incertulas.

[0008] The second object of the present invention is to provide a preparation for controlling rice stem borers, which preparation comprises vanillamine.

[0009] Furthermore, the above-mentioned preparation further comprises agriculturally acceptable excipients.

[0010] Furthermore, the preparation is an aqueous solution, powder, granule, wettable powder, soluble powder, emulsifiable concentrate or microemulsion.

[0011] The present invention has the following beneficial effects: The present invention discovers that a metabolite of Enterococcus, vanillamine, i.e., 4-(aminomethyl)-2-methoxyphenol, has insecticidal activity against rice stem borers, especially excellent insecticidal activity against Chilo suppressalis, which provides a new biological control resource for the control of the rice pest Chilo suppressalis. Description of the Drawings

[0012] Figure 1 For injection E. casseliflavus 、 E.faecalis and E.mundtii Up-regulated metabolite Venn diagram.

[0013] Figure 2 It is the result of the effect of feeding vanillamine on the body weight of newly hatched larvae of Chilo suppressalis.

[0014] Figure 3 It is the result of vanillamine injection-induced apoptosis of hemolymph cells of Chilo suppressalis. Detailed Embodiments

[0015] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0016] Example 1: Injection of Enterococcus and Analysis of Hemolymph Metabolome The process of injection of Enterococcus and analysis of hemolymph metabolome is as follows: (1) Feed newly hatched larvae of Chilo suppressalis with artificial feed containing antibiotics until the larvae reach the early 4th instar. Disinfect the body surface with 75% alcohol for 3 min and perform cryoanesthesia for standby; Among them, the antibiotic stock solution is prepared by the following method: Weigh 0.1 g each of penicillin, streptomycin, rifampicin, chlortetracycline and gentamicin, add sterile double-distilled water to a total volume of 10 mL, and prepare an antibiotic stock solution of 10 mg / mL; The artificial feed for Chilo suppressalis larvae is prepared according to the formula and method in the invention patent CN200910080336.2.

[0017] (2) Take an appropriate amount of Chilo suppressalis larval artificial diet in a dry and clean beaker, then melt the diet using a microwave oven, and dispense it into a 24-well plate before the diet solidifies. After condensation, it is ready for use.

[0018] (3) Transfer the overnight cultured Enterococcus (Enterococcus casseliflavus ( Enterococcus casseliflavus ), Enterococcus faecalis ( Enterococcus faecalis ), and Enterococcus mundtii ( Enterococcus mundtii )) bacterial liquid into 50 mL of LB liquid medium. When the Enterococcus grows to the logarithmic growth phase, centrifuge the bacterial liquid at high speed (8000 rpm, 4 °C, 30 min), then discard the supernatant, resuspend and dilute the precipitate with sterile PBS solution, and measure OD 600 . Dilute the bacterial liquid concentration to 1×10 4 CFU / μL through the standard curve; Among them, the LB liquid medium includes: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, and 1000 mL of distilled water.

[0019] (4) Use a micro syringe to inject 1 μL of the bacterial liquid into the hemocoel of the anesthetized larva through the ventral prolegs. After injection, continue to treat the larva on ice for 10 min and then transfer it to a 24-well plate with Chilo suppressalis larval artificial diet, using the injection of an equal volume of sterile PBS solution as a control.

[0020] (5) Inject 40 larvae per replicate, set 3 replicates. After 12 h of injection, use a sterilized insect pin to pierce the ventral prolegs of the larvae to collect hemolymph into a 1.5 mL centrifuge tube. To prevent hemolymph melanization, add a high concentration of phenylthiourea (its final concentration is 1 mM) to the centrifuge tube in advance, and then place the centrifuge tube collecting hemolymph on ice.

[0021] (6) Centrifuge the collected hemolymph at 400×g, 4 °C for 15 min, collect the supernatant for metabolome sequencing and perform metabolome analysis, and the results are shown in Figure 1 and Table 1.

[0022] Table 1 Metabolites that were significantly increased after injection of three Enterococcus strains ( E. casseliflavus , E.faecalis and E.mundtii )

[0023] As can be seen from Table 1 and Figure 1 , compared with the control group, after injection of E. casseliflavus, E.faecalis and E.mundtii, 27 metabolites were all significantly increased, among which 15 metabolites have CAS IDs, and only 9 of them can be purchased on the market. One of the metabolites with the best effect, namely 4-(aminomethyl)-2-methoxyphenol (vanillylamine), was selected for subsequent experiments in the present invention.

[0024] Example 2: Bioassay experiment of vanillylamine on newly hatched larvae of Chilo suppressalis The specific process of the bioassay experiment of vanillylamine on newly hatched larvae of Chilo suppressalis is as follows: (1) Dissolve vanillylamine in dimethyl sulfoxide to prepare a stock solution of 30 mg / mL for later use.

[0025] (2) After melting the artificial diet for Chilo suppressalis larvae in a microwave oven, weigh 4 g of the melted diet into a 50 mL beaker. When it cools to 50 - 60 °C, add the 30 mg / mL vanillylamine stock solution to the diet so that the final concentration of vanillylamine is 0.47 mg / g. Then quickly stir evenly and place it in a 4 °C refrigerator for cooling and standby.

[0026] (3) The difference in the preparation method of the control diet from step (2) is that the vanillylamine stock solution is replaced with an equal volume of dimethyl sulfoxide solvent, and the rest of the process is the same as step (2).

[0027] (4) After the diet has completely cooled, weigh 1 g of the control diet and the artificial diet containing the metabolite respectively, and place them in a bioassay box. Gently press the diet to make it spread out into a thin cake shape, and inoculate newly hatched larvae of Chilo suppressalis. Set 3 biological replicates, with 30 larvae in each replicate. After 7 days, measure the weight of the larvae of Chilo suppressalis. The results are shown in Figure 2 .

[0028] It can be seen from Figure 2 that compared with the control (CK), after feeding vanillylamine for 7 days, the weight of newly hatched larvae of Chilo suppressalis can be significantly reduced (P < 0.0001).

[0029] Example 3: Injection of vanillylamine induces apoptosis of hemolymph cells in Chilo suppressalis The specific process of injecting vanillylamine to induce apoptosis of hemolymph cells in Chilo suppressalis is as follows: (1) Weigh a certain amount of artificial diet for Chilo suppressalis larvae into a sterile beaker and melt it in a microwave oven. When the diet cools to about 50 °C, add the antibiotic stock solution to the diet so that the final concentration of the antibiotic is 500 μg / mL. Stir evenly and place it in a 4 °C refrigerator for standby.

[0030] (2) Feed newly hatched larvae of Chilo suppressalis with the above-mentioned diet containing antibiotics until the early 4th instar.

[0031] (3) Dissolve vanillylamine in dimethyl sulfoxide to prepare a stock solution of 10 mg / mL for later use.

[0032] (4)Pick the early 4th instar larvae, place them on ice for 15 min for freezing anesthesia, and then inject 1 μL of the vanillamine stock solution into each larva using a microinjection system (Nanoject Ⅲ). Inject 1 μL of dimethyl sulfoxide solution into the control group larvae. After 24 h, collect the hemolymphocytes for apoptosis detection (the detection method is carried out according to the instruction manual of the TransGen Biotech kit FA201), and the results are shown in Figure 3 .

[0033] It can be seen from Figure 3 that compared with the control (CK), injecting vanillamine for 24 h can significantly induce apoptosis of the hemolymphocytes of Chilo suppressalis (P < 0.0001).

[0034] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of vanillin in the preparation of preparations for controlling rice stem borers.

2. The use according to claim 1, characterized in that: The rice stem borer is the Chilo suppressalis or Yellow stem borer.

3. A preparation for controlling rice borers, characterized in that: The formulation includes vanillylamine.

4. The preparation for controlling rice stem borers according to claim 3, characterized in that: Also included are agriculturally acceptable adjuvants.

5. The preparation for controlling rice stem borers according to claim 3 or 4, characterized in that: The preparation is aqueous solution, powder, granule, wettable powder, soluble powder, emulsifiable concentrate or microemulsion.

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