Application of streptomyces sdu1201 and / or secondary metabolite thereof in prevention and control of plant pests

Abstract

The invention belongs to the technical field of biological prevention and control of plant pests, and relates to application of streptomyces sdu1201 and / or secondary metabolites in prevention and control of plant pests. Research finds that the streptomyces sp. Sdu1201 and the secondary metabolite thereof have the effect of preventing and treating tetranychus urticae and root-knot nematode.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control of plant diseases and insect pests, and relates to application of Streptomyces sdu1201 and / or its secondary metabolites in controlling plant pests. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Plant pest control is an integral part of agricultural production and an important link in ensuring the output and quality of agricultural products, as well as the agricultural ecological environment. At present, chemical control is the main measure to control plant pests and diseases in agricultural production. Chemical control can quickly achieve control effects, but while controlling pests and diseases, it also brings about problems such as pest resistance, resurgence and pesticide residues. Biological control is widely promoted because of its advantages such as safety for humans and animals, no pollution to the environment, and low resistance to pesticides. It is also 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 of the invention

[0004] Streptomyces can not only colonize in the roots, stems, leaves and other parts of various plants in the form of mycelium to play a role, but also the metabolites produced have functions such as promoting plant growth. Unique ecological environments (such as oceans, deserts, polar regions, volcanoes, plant endophytes and insect symbiosis) breed a variety of microbial populations, which have specific metabolic modes and can produce natural active substances with unique structures and functions, providing rich resources for the discovery of various active lead compounds, and have important development value. In the previous research of the present invention, a strain of Streptomyces sdu1201 was obtained, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (referred to as CGMCC, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on October 16, 2024, with a deposit number of CGMCC No. 32223. It was disclosed in Biocontrol of strawberry gray mold caused by Botrytis cinerea with thetermite associated Streptomyces sp.sdu1201 and actinomycin D.Front Microbiol.2022;13:1051730. on November 4, 2022. After further research, the present invention found that Streptomyces sdu1201 and its secondary metabolites have the effect of preventing and controlling two-spotted spider mites and root-knot nematodes. Based on the above research results, the present invention provides an application of Streptomyces sdu1201 and / or its secondary metabolites in preventing and controlling plant pests.

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

[0006] In a first aspect, a use of Streptomyces sdu1201 and / or its secondary metabolites in controlling plant pests, wherein the Streptomyces sdu1201 has a preservation number of CGMCC No. 32223 and is named Streptomyces (Streptomyces cess p.) sdu1201, and the pests are two-spotted spider mites and / or root-knot nematodes.

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

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

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

[0010] The application of the present invention in preventing and controlling plant pests can be a method for preventing and controlling plant pests, or can be used to prepare a product for preventing and controlling plant pests.

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

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

[0013] In the second aspect, a compound is used in the preparation of a pesticide for controlling plant pests, wherein the compound is actinomycin Y6, actinomycin D, actinomycin X 0β , actinomycin G6, and the pests are two-spotted spider mites and / or root-knot nematodes.

[0014] In some embodiments, when the pest is two-spotted spider mite, the compound is actinomycin D or actinomycin Y6. Studies have shown that actinomycin D or actinomycin Y6 (especially actinomycin D) has a better control effect on two-spotted spider mite.

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

[0016] In some embodiments, the pesticide includes an active ingredient and a pesticide adjuvant, wherein the active ingredient is the compound. The pesticide adjuvant includes a dispersant, an emulsifier, a solvent, a carrier, a filler, a stabilizer, a controlled release adjuvant, a synergist, a wetting agent, a penetrant, an adhesive, and the like.

[0017] In some embodiments, the preparation method of the compound is: adding XAD16 non-ionic macroporous resin to the fermentation broth of Streptomyces sdu1201 for adsorption, collecting by centrifugation, and then extracting with methanol, separating by forward silica gel column and liquid phase separation to obtain the compound.

[0018] The beneficial effects of the present invention are:

[0019] The Streptomyces sdu1201 and / or its secondary metabolites provided by the present invention have a wide spectrum of action, can effectively prevent and control the two-spotted spider mite and the loofah root-knot nematode, can be used as a biological control agent to replace chemical pesticides, so as to improve the safety of agricultural products and alleviate the resistance generation; at the same time, the Streptomyces sdu1201 has the characteristics of fast growth rate, large spore production, large amount of secondary metabolites, etc., and therefore has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

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

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

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

[0024] Figure 4 This is the LC-HRMS data chart 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 the present invention in CD3OD 1 H NMR spectrum.

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

[0029] Fig. 9 Compound 3 prepared in Example 1 of the present invention in CDCl3 1 H NMR spectrum.

[0030] Fig.10 Compound 3 prepared in Example 1 of the present invention in CDCl3 13 C NMR spectrum.

[0031] Fig.11 Compound 4 prepared in Example 1 of the present invention in CD3OD 1 H NMR spectrum.

[0032] Fig.12 Compound 4 prepared in Example 1 of the present invention in CD3OD 13 C NMR spectrum. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0034] The Streptomyces sdu1201 used in the following examples was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 16, 2024, with a deposit number of CGMCC No. 32223, and was disclosed on November 4, 2022 in Biocontrol of strawberry gray mold caused by Botrytis cinerea with the termite associated Streptomyces sdu1201 and actinomycin D. Front Microbiol. 2022; 13: 1051730.

[0035] Example 1 Isolation and purification of four secondary metabolites from Streptomyces sdu1201

[0036] (1) Enrichment of crude extract

[0037] Streptomyces p.sdu1201 was fermented using ISP-7 medium to obtain 40L of fermentation broth for extraction, separation and purification of active secondary metabolites. 36g of crude extract was obtained after extraction and vacuum rotary evaporation of 40L of fermentation broth.

[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 elution volume are shown in Table 1.

[0040] (3) HPLC-MS detection

[0041] The target compound was analyzed by HPLC-MS, and a sample was selected every 5 flow-throughs for HPLC-MS analysis. The target compound was found in the elution gradient of 50:1, 30:1 and 10:1 of the eluent CH2Cl2-MeOH, and was recorded as Frs1-3.

[0042] Table 1 Eluent ratio and elution volume

[0043]

[0044] (4) HPLC separation

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

[0046] (5) NMR test

[0047] Take appropriate amount of compounds 1, 2, 3 and 4 for NMR ( 1 H and 13 C) test. 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 of compounds 1-4 are shown in Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11and Fig.12 .

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

[0049] Example 2 Toxicity determination of four secondary metabolites to Tetranychus urticae

[0050] 1 Test method

[0051] The experiment adopted the NY / T 1154.12-2008 spider mite slide dipping method in the agricultural industry standard and improved it. Wrap the tape with the back facing up on one end of the slide, pick the female adult mites on the loofah leaves onto white paper with a brush, then select active and uniform female adult mites, and stick their backs on the tape with a brush, 20 per slide. Put the slide with sticky mites in an enamel tray and keep it moist at 25℃. Remove dead and inactive individuals and mark them before treating with the liquid medicine. Immerse the glass slide with mites in the prepared solution (2mg / mL, 1mg / mL, 0.5mg / mL, 0.25mg / mL, 0.125mg / mL, 0.0625mg / mL) for 8-10s and then take it out. Quickly use filter paper to absorb the excess solution around the mite and put it in an enamel dish, cover it with plastic wrap to maintain humidity, and culture it at 25℃ and a light cycle of L:D=(16:8). Check the death of adult mites after 24h and 48h. Touch the mite body with a hair brush. If the mite does not move, it is dead. Use clean water as the control, set 3 parallels for each concentration, and repeat the test 3 times.

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

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

[0054] 2 Test results

[0055] As shown in Table 5, the compound has a better control effect on spider mites as time goes by, and the control effect decreases as the concentration of the compound decreases. Actinomycin D has the best indoor control effect on spider mites. The corrected mortality rate of 2mg / mL for 24h and 48h can reach 96.50% and 100%, and the rapid effect is good. The corrected mortality rate of 1mg / mL for 48h can still reach 82.50%. Actinomycin Y6 has the second best effect on spider mites. The corrected mortality rate of 2mg / mL for 24h and 48h is 83.85%-87.88%. The control effects of other treatments are all less than 45%, and the effect is poor. Actinomycin X 0βThe control effects of actinomycin G6 on two-spotted spider mites were the worst, with the highest concentration of 2 mg / mL treating for 48 hours having a control effect of only 51% and 61.5%.

[0056] Table 2 Toxicity test of four secondary metabolites to Tetranychus urticae

[0057]

[0058]

[0059] Example 3 Toxicity determination of four secondary metabolites to second instar larvae of loofah root-knot nematode

[0060] 1 Test method

[0061] The second-instar larvae of the root-knot nematode of loofah that had been hatched were collected in advance and prepared into a suspension of the second-instar larvae of the root-knot nematode for later use. 200 μL of the second-instar larvae suspension and 200 μL of compounds of different concentrations (2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL) were added to a 24-well plate, whipped to mix, and placed in a 25°C incubator in the dark for incubation. The number of dead second-instar larvae in each treatment was counted at 24 h (rigid nematodes were considered dead), and the corrected mortality rate was calculated.

[0062] 2 Test results

[0063] As can be seen from Table 6, with the decrease of compound concentration, the control effect gradually decreased; after 24h treatment, 1mg / mL actinomycin Y6, actinomycin X 0β The best control effects of actinomycin G6 on the second instar larvae of loofah root-knot nematodes were 85.29%, 84.03% and 74.78% respectively; while the control effect of 1mg / mL actinomycin D on the second instar larvae of loofah root-knot nematodes was poor, only 58.39%, significantly lower than the treatments of other compounds at the same concentration. When treated with a concentration of 0.5mg / mL, the control effects of the four compounds were 60.49%-72.68%.

[0064] Table 3 Toxicity of four secondary metabolites to the second instar larvae of loofah root-knot nematode

[0065]

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A use of Streptomyces sdu1201 and / or its secondary metabolites in controlling plant pests, wherein the Streptomyces sdu1201 has a deposit number of CGMCC No. 32223 and is named Streptomyces (Streptomyces cess p.) sdu1201, and the pests are two-spotted spider mites and / or root-knot nematodes.

2. The use according to claim 1, characterized in that: The Streptomyces sdu1201 has a preservation number of CGMCC No.32223.

3. The use according to claim 1, characterized in that: The fermentation method of Streptomyces sdu1201 is secondary fermentation; Alternatively, 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 the products are collected by centrifugation.

4. The use according to claim 1, characterized in that: The secondary metabolites of Streptomyces sdu1201 include actinomycin Y6, actinomycin D, actinomycin X 0β , or one or more of actinomycin G6.

5. The use according to claim 1, characterized in that: The application is a method for controlling plant pests or a product for controlling plant pests.

6. The use according to claim 5, characterized in that: The product is a pesticide composition; preferably, the pesticide composition comprises a pesticide adjuvant.

7. The use according to claim 1, characterized in that: The plant is a crop; preferably, the plant is loofah.

8. Use of a compound in the preparation of a pesticide for controlling plant pests, characterized in that: The compound is actinomycin Y6, actinomycin D, actinomycin X 0β , actinomycin G6, and the pests are two-spotted spider mites and / or root-knot nematodes.

9. The use according to claim 8, characterized in that: When the pest is two-spotted spider mite, the compound is actinomycin D or actinomycin Y6; Or, when the pest is a root-knot nematode, the compound is actinomycin Y6, actinomycin X 0β and / or actinomycin G6; Or, the pesticide comprises an active ingredient and a pesticide adjuvant, and the active ingredient is the compound.

10. The use according to claim 8, characterized in that: The preparation method of the compound comprises the following steps: adding XAD16 non-ionic macroporous resin to the fermentation liquid of Streptomyces sdu1201 for adsorption, collecting by centrifugation, and then extracting with methanol, separating by a normal silica gel column and separating by liquid phase to obtain the compound.

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