Use of streptomyces sdu1201 and / or its secondary metabolites for controlling plant pests

Abstract

The application belongs to the technical field of biological control of plant diseases and insect pests, and relates to application of streptomyces sdu1201 and / or secondary metabolites thereof in control of plant pests. The application finds that the streptomyces sdu1201 and the secondary metabolites thereof have the effects of controlling the two-spotted spider mite and the root-knot nematode.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological control of plant pests, and relates to application of Streptomyces sdu1201 and / or secondary metabolites thereof in control of plant pests. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general context of the present application and does not necessarily constitute an acknowledgement or a suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

[0003] Plant pest control is an integral part of agricultural production, and is an important link for ensuring the yield and quality of agricultural products, and the agricultural ecological environment. At present, chemical control is the main measure for controlling plant pests in agricultural production. Chemical control can rapidly achieve control effect, but in the process of controlling pests, it also brings problems of pest resistance, re-rampage and pesticide residues. Biological control is widely promoted due to its safety to humans and animals, non-pollution to the environment, and non-resistance to drugs, and is an important way to solve the problems of agricultural product safety and residues. Therefore, the use of microorganisms and their secondary metabolites to control plant diseases has broad application prospects. SUMMARY

[0004] Streptomyces not only can colonize various plant roots, stems, leaves and other parts in the form of mycelium to play a role, but also produces metabolites with the function of promoting plant growth. Unique ecological environments (such as oceans, deserts, polar regions, volcanoes, plant endophytes and insect symbiosis) give birth to a variety of microbial populations, and their metabolic ways have specificity, can produce natural active substances with unique structure and function, and provide rich resources for the discovery of various active lead compounds, and have important development value. In the previous research of the present application, a strain of Streptomyces sdu1201 was obtained, which was preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 16, 2024, and the address is No. 3, Beichen West Road, Haidian District, Beijing, China, and the address is Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No. 32223. And on November 4, 2022, it was disclosed in Biocontrol of strawberry gray mold caused by Botrytis cinerea with the termite associated Streptomyces sp. sdu1201 and actinomycin D. Front Microbiol. 2022;13:1051730. Through further research, the present application found that the Streptomyces sdu1201 and its secondary metabolites have the effects of preventing and treating two-spotted spider mites and root-knot nematodes. Based on the above research results, the present application provides a Streptomyces sdu1201 and / or its secondary metabolites for preventing and treating plant pests.

[0005] Specifically, the present application provides the following technical solutions:

[0006] In a first aspect, the application provides a Streptomyces sdu1201 and / or its secondary metabolites for preventing and treating plant pests, the preservation number of the Streptomyces sdu1201 is CGMCC No. 32223, and the name is Streptomyces sp. sdu1201, and the pests are two-spotted spider mites and / or root-knot nematodes.

[0007] In some embodiments, the fermentation method of the Streptomyces sdu1201 is secondary fermentation. Specifically, a monoclonal strain is inoculated in a seed culture medium for seed culture to obtain a seed liquid, and then the seed liquid is inoculated in a fermentation culture medium for fermentation culture, and then the fermentation culture medium is obtained. The seed culture medium is TSB culture medium; in the seed culture, the temperature is 28-32℃, and the time is 30-40h. The fermentation culture medium is ISP-7 fermentation culture medium; in the fermentation culture, the inoculation amount is 2% (mass fraction), the temperature is 28-32℃, and the time is 7-9 days (d).

[0008] In some implementations, the method for separating the secondary metabolites of Streptomyces sdu1201 is as follows: XAD16 non-ionic macroporous resin is added to the fermentation broth of Streptomyces sdu1201 for adsorption, and then collected by centrifugation.

[0009] In some embodiments, the secondary metabolites of Streptomyces sdu1201 include actinomycin Y6, actinomycin D, and actinomycin X. 0β One or more of actinomycin G6.

[0010] The application of this invention in the prevention and control of plant pests can be either a method for preventing and controlling plant pests or a product for preparing plant pest control products.

[0011] In some embodiments, the product is a pesticide composition. Specifically, the pesticide composition includes pesticide adjuvants. These adjuvants include dispersants, emulsifiers, solvents, carriers, fillers, stabilizers, controlled-release adjuvants, synergists, wetting agents, penetrants, binders, etc.

[0012] In some implementations, the plant is an agricultural crop. Specifically, the agricultural crop includes vegetables, peanuts, corn, sorghum, apples, pears, peaches, apricots, plums, cherries, grapes, cotton, beans, etc. More specifically, the plant is a loofah.

[0013] Secondly, the application of a compound in the preparation of pesticides for controlling plant pests, said compound being actinomycin Y6, actinomycin D, or actinomycin X. 0β One or more of actinomycin G6, wherein the pest is two-spotted spider mite and / or root-knot nematode.

[0014] In some implementations, when the pest is the two-spotted spider mite, the compound is actinomycin D or actinomycin Y6. Studies have shown that actinomycin D or actinomycin Y6 (especially actinomycin D) is more effective against the two-spotted spider mite.

[0015] In some implementation schemes, when the pest is a root-knot nematode, the compound is actinomycin Y6 or actinomycin X. 0β And / or actinomycin G6. Studies have shown that the compounds are actinomycin Y6 and actinomycin X. 0β And / or actinomycin G6 (especially actinomycin Y6 or actinomycin X) 0β It is more effective against root-knot nematodes.

[0016] In some embodiments, the pesticide includes an active ingredient and pesticide adjuvants, wherein the active ingredient is the compound. The pesticide adjuvants include dispersants, emulsifiers, solvents, carriers, fillers, stabilizers, controlled-release adjuvants, synergists, wetting agents, penetrants, binders, etc.

[0017] In some embodiments, the compound is prepared by adding XAD16 non-ionic macroporous resin to the fermentation broth of Streptomyces sdu1201 for adsorption, collecting by centrifugation, and then obtaining the compound by methanol extraction, forward silica gel column separation and liquid phase separation.

[0018] The beneficial effects of this invention are as follows:

[0019] The Streptomyces sdu1201 and / or its secondary metabolites provided by this invention have a broad spectrum of action and can effectively control the two-spotted spider mite and root-knot nematode of loofah. They can be used as biocontrol agents to replace chemical pesticides, thereby improving the safety of agricultural products and alleviating the development of resistance. At the same time, Streptomyces sdu1201 has the characteristics of rapid growth, large sporulation, and large amount of secondary metabolites, thus it has good prospects for industrial application. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 This is an LC-HRMS data graph of compound 1 prepared in Example 1 of the present invention.

[0022] Figure 2 This is an LC-HRMS data graph of compound 2 prepared in Example 1 of the present invention.

[0023] Figure 3 This is an LC-HRMS data graph of compound 3 prepared in Example 1 of the present invention.

[0024] Figure 4 This is an LC-HRMS data graph of compound 4 prepared in Example 1 of the present invention.

[0025] Figure 5 Compound 1 prepared in Example 1 of the present invention in CD3OD 1 H NMR spectrum.

[0026] Figure 6 Compound 1 prepared in Example 1 of the present invention in CD3OD 13 C NMR spectrum.

[0027] Figure 7 Compound 2 prepared in Example 1 of this invention in CD3OD 1 H NMR spectrum.

[0028] Figure 8 Compound 2 prepared in Example 1 of this invention in CD3OD 13 C NMR spectrum.

[0029] Figure 9 H NMR spectrum of compound 3 prepared for Example 1 of the present application in CDCl3. 1 H NMR spectrum.

[0030] Figure 10 H NMR spectrum of compound 3 prepared for Example 1 of the present application in CDCl3. 13 C NMR spectrum.

[0031] Figure 11 H NMR spectrum of compound 4 prepared for Example 1 of the present application in CD3OD. 1 H NMR spectrum.

[0032] Figure 12 H NMR spectrum of compound 4 prepared for Example 1 of the present application in CD3OD. 13 C NMR spectrum. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0034] The Streptomyces sp. sdu1201 used in the following examples was deposited with the China General Microbiological Culture Collection Center on October 16, 2024, and the deposit number is CGMCC No. 32223, and was disclosed in Biocontrol of strawberry gray mold caused by Botrytis cinerea with the termite associated Streptomyces sp. sdu1201 and actinomycin D. Front Microbiol. 2022; 13: 1051730.

[0035] Example 1 Isolation and purification of four secondary metabolites in Streptomyces sp. sdu1201

[0036] (1) Enrichment of crude extract

[0037] Streptomyces sp. sdu1201 was fermented using ISP-7 medium to obtain 40 L of fermentation broth, which was used to extract, isolate and purify active secondary metabolites. After extraction and rotary evaporation under reduced pressure, 36 g of crude extract was obtained.

[0038] (2) Normal phase silica gel column separation

[0039] The crude extract was separated by normal-phase silica gel column chromatography. The eluent ratio and eluent volume are shown in Table 1.

[0040] (3) HPLC-MS detection

[0041] The range of the target compound was analyzed by HPLC-MS, with one sample selected at five flow-through intervals for HPLC-MS analysis. The results showed that the target compound was found in the elution gradients of CH2Cl2-MeOH with eluents of 50:1, 30:1 and 10:1, and was designated as Frs1 to Frs3.

[0042] Table 1. Elution Buffer Ratio and Elution Volume

[0043]

[0044] (4) HPLC separation

[0045] Frs1–3 were further separated and purified using semi-preparative high-performance liquid chromatography (ODS, Bruker ZOR-BAX SB-C18, 5 μm, 250 × 10 mm). Frs1 was isocratically eluted using ultrapure water as mobile phase A and chromatographic methanol as mobile phase B (MeOH-H2O, 92:8, 2 mL / min). -1 Compound 1 (61.9 mg, t) was obtained. R =10.5min) and compound 2 (93.8mg,t) R =11.2 min). Frs2 was further separated and purified under the above conditions to obtain compound 3 (49.1 mg, t). R =9.7 min). Frs3 was isocratically eluted using ultrapure water as mobile phase A and chromatographic methanol as mobile phase B (MeOH-H2O, 90:10, 2 mL / min). -1 Compound 4 (53.1 mg, t) was obtained. R =10.1 min). HPLC-MS results of compounds 1–4 are shown in [Figure number missing]. Figures 1-4 .

[0046] (5) NMR testing

[0047] Take appropriate amounts of compounds 1, 2, 3 and 4 for nuclear magnetic resonance (NMR) testing. 1 H and 13 C) Testing. The deuterated solvent used for compounds 1, 2, and 4 was CD3OD, and the deuterated solvent used for compound 3 was CDCl3. The NMR results for compounds 1-4 are shown below. Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11and Figure 12 .

[0048] The four compounds were identified as actinomycin Y6, actinomycin D, actinomycin X and actinomycin G6 respectively by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy data analysis. 0β

[0049] Example 2 Toxicity determination of four secondary metabolites on Tetranychus viridiflavus

[0050] 1 Test method

[0051] The test adopted the leaf mite slide immersion method of agricultural industry standard NY / T 1154.12-2008 and was improved. The female adult mites on the silk gourd leaves were picked up with a brush pen and then the active and uniform female adult mites were selected and their backs were stuck to the adhesive tape with a brush pen, 20 per slide. The mite-sticking slides were placed in a porcelain dish and cultured at 25℃. The dead and inactive individuals were removed before treatment and marked. The slides with mites were immersed in prepared drug solution (2mg / mL, 1mg / mL, 0.5mg / mL, 0.25mg / mL, 0.125mg / mL, 0.0625mg / mL) for 8-10s, then the mite bodies and the excess drug solution around them were quickly absorbed with filter paper, placed in a porcelain dish, covered with plastic wrap to maintain humidity, and cultured at 25℃ under light cycle L:D=(16:8) conditions. The mite death was checked after 24h and 48h. The mite body was gently touched with a brush pen and the mites without movement were considered dead. The water treatment was used as a control. Three parallel tests were set for each concentration and the test was repeated three times.

[0052] Mortality (%) = number of dead insects (heads) / total number of treated insects (heads) x 100

[0053] Corrected mortality (%) = (treatment mortality - control mortality) / (1 - control mortality) x 100

[0054] 2 Test results

[0055] As can be seen from Table 5, the longer the time, the better the effect of the compounds on Tetranychus viridiflavus. With the decrease of the concentration of the compounds, the effect decreases. Actinomycin D has the best indoor control effect on Tetranychus viridiflavus. The corrected mortality rates of 2mg / mL treatment for 24h and 48h can reach 96.50% and 100% respectively, and the quick-acting property is good. The corrected mortality rate of 1mg / mL treatment for 48h can still reach 82.50%. Actinomycin Y6 has the second best effect on Tetranychus viridiflavus. The corrected mortality rates of 2mg / mL treatment for 24h and 48h are 83.85%-87.88%. The effects of other treatments are all less than 45% and the effect is poor. Actinomycin X 0β ​The actinomycin Y6 and the actinomycin X6 have the worst effect on the T. urticae, and the highest concentration effect is only 51% and 61.5% respectively after 48 hours of treatment.

[0056] Table 2 Toxicity determination of four secondary metabolites on T. urticae

[0057]

[0058]

[0059] Example 3 Toxicity determination of four secondary metabolites on the second instar larvae of M. incognita

[0060] 1 Test method

[0061] The hatched second instar larvae of M. incognita were collected in advance and prepared into a second instar larvae suspension. 200 μL of the second instar larvae suspension and 200 μL of the compound with different concentrations (2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL) were added into a 24-well plate, mixed by beating, and placed in a 25°C incubator in the dark. The number of dead second instar larvae (rigid nematodes were considered dead) was counted at 24 hours, and the corrected mortality rate was calculated.

[0062] 2 Test results

[0063] As can be seen from Table 6, the effect gradually decreases with the decrease of the concentration of the compound; after 24 hours of treatment, the actinomycin Y6, the actinomycin X6 and the actinomycin G6 have the best effect on the second instar larvae of M. incognita, which is 85.29%, 84.03% and 74.78% respectively; while the actinomycin D has a poor effect on the second instar larvae of M. incognita, which is only 58.39%, which is significantly lower than the treatment of other compounds with the same concentration. When the concentration is 0.5 mg / mL, the control effect of the four compounds is 60.49%-72.68%. 0β and actinomycin G6 on the second instar larvae of M. incognita, which is 85.29%, 84.03% and 74.78% respectively; while the actinomycin D has a poor effect on the second instar larvae of M. incognita, which is only 58.39%, which is significantly lower than the treatment of other compounds with the same concentration. When the concentration is 0.5 mg / mL, the control effect of the four compounds is 60.49%-72.68%.

[0064] Table 3 Toxicity determination of four secondary metabolites on the second instar larvae of M. incognita

[0065]

[0066] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of a compound in the preparation of pesticides for controlling plant pests, characterized in that, When the pest is Tetranychus urticae Koch, the compound is actinomycin Y6; Or, when the pest is Meloidogyne incognita, the compound is actinomycin Y6 and / or actinomycin G6.

2. The use according to claim 1, wherein the compound is ###0001### or a pharmaceutically acceptable salt thereof. The pesticide comprises an active ingredient and a pesticide adjuvant, and the active ingredient is the compound.

3. Use according to claim 1 or 2, characterised in that A preparation method of the compound is as follows: XAD16 non-ionic macroporous resin is added to a fermentation liquor of Streptomyces sdu1201 for adsorption, centrifugal collection is performed, and then methanol extraction, normal-phase silica gel column separation and liquid phase separation are performed to obtain the compound; the preservation number of the Streptomyces sdu1201 is CGMCC No.32223.

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