Actinomyces, compound and preparation method and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a kind of actinomycete, compound and its preparation method and application. The actinomycete is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.20306. The actinomycete can be separated and purified to obtain kailing ketone mycin. The compound has obvious stomach poison activity on greenhouse whitefly adults. When the compound concentration is 100 μg / mL and treated for 72 h, the corrected mortality rate reaches 90.30%, and the compound also has touch kill activity on greenhouse whitefly. When the concentration is 100 μg / mL and treated for 72 h, the corrected mortality rate reaches 82.55%.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a kind of actinomycete, compound and its preparation method and application. BACKGROUND

[0002] The greenhouse whitefly (Trialeurodes vaporariorum Westwood) is a worldwide pest, which seriously damages fruit trees, vegetables and flowers, and can cause direct damage by sucking plant sap and indirect damage by transmitting multiple plant viruses, resulting in reduced yield or even crop failure. Chemical control is the most commonly used method for controlling greenhouse whitefly in the field, such as organophosphorus lipids, pyrethroid, and neonicotinoid insecticides. However, with the extensive use of chemical pesticides, greenhouse whitefly has developed resistance to almost all major conventional insecticides to varying degrees. Therefore, different chemical pesticides have different effects on the same species of greenhouse whitefly cryptic species, and are no longer suitable for the control of greenhouse whitefly. At the same time, different cryptic species show different degrees of resistance to the same chemical pesticide. Therefore, new biological insecticides are needed to delay the development of insecticide resistance and extend the effectiveness of insecticides.

[0003] Natural products have been used as insecticides or as lead compounds for the synthesis of insecticides for a long time, and are widely used as the basis for creating new insecticides due to their wide range of biological activities, structural diversity, and environmental friendliness. Therefore, it is particularly important to develop a natural compound that can effectively control greenhouse whitefly. SUMMARY

[0004] The present application develops a kind of actinomycete and its fermentation preparation compound, which has obvious stomach poison activity and contact toxicity activity on greenhouse whitefly adults. Therefore, the secondary metabolite compound (Kailing ketone mycin) prepared by the actinomycete of the present application can be used for controlling greenhouse whitefly or preparing pesticides for preventing greenhouse whitefly, which can effectively reduce the problem of resistance caused by the use of traditional chemical pesticides in China, and effectively reduce the pollution of ecological environment.

[0005] In one aspect, the present application provides a kind of actinomycete (Streptomyces tianzhuensis), which is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.20306.

[0006] In another aspect, the present application provides a composition comprising one or more combinations of the following substances: (a) the actinomycete of the present application; (b) the lysate of the actinomycete of the present application; (c) the culture of the actinomycete of the present application; (d) the fermentation broth of the actinomycete of the present application.

[0007] In another aspect of the present application, a compound is provided, which has the structure shown in formula I,

[0008]

[0009] In another aspect of the present application, a preparation method of the compound is provided, which comprises: fermenting the actinomycete of the present application.

[0010] In another aspect of the present application, a preparation method of the compound is provided, which comprises: fermenting the actinomycete of the present application.

[0011] In some embodiments, the preparation is a tablet, a pill, a capsule, a powder, a gel, a granule or a liquid.

[0012] In another aspect of the present application, the actinomycete of the present application, the composition of the present application or the compound of the present application is used as or prepared into a pesticide for controlling greenhouse whitefly.

[0013] The actinomycete of the present application is preserved in the following information: Preserving organization: China General Microbiological Culture Collection Center (CGMCC); Preserving address: No. 3, Beichen West Road, Chaoyang District, Beijing; Preserving date: July 7, 2020; Preserving number: CGMCC No. 20306; Classification and naming: Streptomyces tianzhuensis.

[0014] The actinomycete (Streptomyces tianzhuensis) provided by the present application can isolate and purify kailinketonin, which has obvious stomach poison activity on greenhouse whitefly adults, and the corrected mortality rate reaches 90.30% when the compound concentration is 100 μg / mL and the treatment time is 72 h. In addition, kailinketonin also has touch killing activity on greenhouse whitefly, and the corrected mortality rate reaches 82.55% when the concentration is 100 μg / mL and the treatment time is 72 h. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The colony morphology of the actinomycete SA61 on ISP1-ISP7 medium;

[0016] Figure 2 High-resolution mass spectrum of the secondary metabolite kailinketonin of the actinomycete;

[0017] Figure 3 Nuclear magnetic resonance hydrogen spectrum of the secondary metabolite kailinketonin of the actinomycete;

[0018] Figure 4The HSQC spectrum of the secondary metabolite of Streptomyces, kaillingkoniomycin;

[0019] Figure 5 The HSQC spectrum of the secondary metabolite of Streptomyces, kaillingkoniomycin;

[0020] Figure 6 The 1H-1HCOSY spectrum of the secondary metabolite of Streptomyces, kaillingkoniomycin;

[0021] Figure 7 The HMBC spectrum of the secondary metabolite of Streptomyces, kaillingkoniomycin;

[0022] Figure 8 The single crystal diffraction result of the secondary metabolite of Streptomyces, kaillingkoniomycin;

[0023] Figure 9 The schematic diagram of the glass tube double-pass method;

[0024] Figure 10 The stomach toxicity activity effect of the secondary metabolite of Streptomyces, kaillingkoniomycin, on greenhouse whitefly;

[0025] Figure 11 The contact toxicity activity effect of the secondary metabolite of Streptomyces, kaillingkoniomycin, on greenhouse whitefly. DETAILED DESCRIPTION

[0026] The embodiments are provided for better illustrating the present application, but are not intended to limit the present application to the embodiments only. Therefore, the skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above description, which still belong to the protection scope of the present application.

[0027] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless there is a clear different meaning in the context, the singular form includes the plural form. As used herein, it should be understood that terms such as "include", "have", "contain", etc. are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present application are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations can exist or can be added. As used herein, " / " can be interpreted as "and" or "or" depending on the circumstances.

[0028] The present application provides a Streptomyces tianzhuensis (hereinafter also referred to as Streptomyces SA61), which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 20306.

[0029] It should be noted that the actinomycete preservation information in the present application is as follows: preservation agency: China General Microbiological Culture Collection Center (CGMCC); preservation address: No. 1, Beichen West Road, Hua-yuan District, Beijing; preservation date: July 7, 2020; preservation number: CGMCC No. 20306; classification and naming: Streptomyces tianzhuensis.

[0030] In some embodiments, the 16S rDNA sequence comprises the sequence shown as SEQ ID NO. 1.

[0031] Another embodiment of the present application provides a composition comprising one or more of the following in combination: (a) the actinomycete of the present application; (b) the lysate of the actinomycete of the present application; (c) the culture of the actinomycete of the present application; (d) the fermentation broth of the actinomycete of the present application.

[0032] It should be noted that in the above composition, as described above, when the actinomycete is prepared into a composition, the bacteria can be directly introduced into the composition in the form of live bacteria to play a role, or the bacteria can be inactivated by existing technical means and introduced into the composition in the form of inactivated bacteria to play a role; the lysate of the bacteria can also be introduced into the composition to play a role; the protein, peptide, secretion or metabolite and other products obtained by culturing the bacteria can also be introduced into the composition to play a role; and the fermentation broth after fermentation of the bacteria can also be introduced into the composition to play a role. In the specific use process, different forms of the bacteria can be selected according to the specific needs to prepare the composition to play a role.

[0033] Still another embodiment of the present application provides a compound, the structure of which is shown in the following formula I,

[0034]

[0035] Still another embodiment of the present application provides a preparation method of the above-mentioned compound, characterized in that it comprises: fermentation by the actinomycete of the present application.

[0036] In some embodiments, the above preparation method comprises: (1) activating the actinomycete and then culturing it at a constant temperature; mixing the culture with the culture medium, sterilizing, and continuing to culture to obtain a primary seed liquid; (2) mixing the primary seed liquid with the sterilized culture medium and culturing to obtain a secondary seed liquid; (3) mixing the secondary seed liquid with sterilized Amberlite XAD-16 resin and culture medium, removing the bacteria after culturing, collecting the resin, and drying the resin; (4) using methanol to elute the resin, collecting the eluate, and distilling under reduced pressure to obtain a methanol extract; (5) dissolving the methanol extract in a methanol aqueous solution, using dichloromethane for extraction, concentrating and collecting the dichloromethane fraction to obtain a dichloromethane extract of the actinomycete.

[0037] In some embodiments, the above preparation method further comprises: (6) gradient elution is performed on the dichloromethane-methanol dichloromethane extract of actinomycete with dichloromethane-methanol as the eluent, and the elution conditions are as follows: V (二氯甲烷) :V (甲醇) = 100:0, 100:1, 100:2, 100:4, 100:8, 100:16 and 1:1, respectively; after primary silica gel column chromatography, similar components are combined according to the thin layer chromatography results, and four components A-D are obtained; (7) column chromatography is performed on the A component with 200-300 mesh silica gel, and isocratic gradient elution is performed with petroleum ether-ethyl acetate = 100:1 as the eluent, and after the A3 component is combined with the B component, the next step of separation and purification is performed; (8) isocratic gradient elution is performed on the B component with petroleum ether: ether = 100:1 as the eluent, and the fractions obtained by elution are detected by thin layer chromatography, and after similar components are combined, seven components B1-B7 are obtained; the B3 component is further purified by Sephadex LH-20 gel column with a system of petroleum ether:dichloromethane:methanol = 2:1:1 to remove impurities, and the B3A component is obtained; (9) the B3A component is separated and purified by using semi-preparative high performance liquid chromatography, the peaks in the liquid chromatogram that may be target compounds are collected, and the target compounds are determined by comparing the thin layer chromatography detection results, the preparation conditions are adjusted according to the thin layer chromatography detection results, and then the liquid phase preparation is performed; the target peaks in the liquid chromatogram are collected, concentrated and dried to obtain the compound shown in formula I.

[0038] In some embodiments, the above preparation method comprises:

[0039] (1) the actinomycete SA61 stored in a-80℃ refrigerator is activated on a Gao's first plate, and is incubated at 28℃ for 7d; 5mL of ISP2 culture solution is added to a 25mL test tube, and is sterilized at 121℃ for 30min; in a sterile operation table, a well-grown colony is picked into 5mL of ISP2 culture solution, and is cultured at 28℃ and 180r / min for 2d, which is a primary seed solution;

[0040] (2) 50mL of ISP2 culture solution is added to a 250mL flask, and is sterilized at 121℃ for 30min; in a sterile operation table, the primary seed solution is transferred into the flask, and is cultured at 28℃ and 180r / min for 2d, which is a secondary seed solution;

[0041] (3) In a 2L flask, 400ml F medium and 16g Amberlite XAD-16 resin were added, sterilized at 121℃ for 30min; in a sterile operation table, the secondary seed liquid was transferred into the 2L flask, cultured at 28℃, 180r / min for 7d, and the total fermentation was 48L; after the fermentation was completed, water was added to the flask, repeatedly washed to remove the bacteria, and the resin was collected; the resin was dried in an oven at 30℃ to remove water;

[0042] (4) The resin was eluted with methanol for 4 times. The eluate was collected and distilled under reduced pressure to obtain a methanol extract;

[0043] (5) The methanol extract was redissolved in 50% methanol aqueous solution (0.6L), and extracted with the same volume of dichloromethane for 4 times. The collected dichloromethane fraction was concentrated to obtain a dichloromethane extract of actinomycete SA61.

[0044] (6) The dichloromethane crude extract (dichloromethane extract of actinomycete SA61) was separated by silica gel column chromatography in a dry column loading and dry sample loading mode. The dichloromethane-methanol system was used to gradient elute the dichloromethane extract of actinomycete SA61, and the elution conditions were as follows: V (二氯甲烷) :V (甲醇) =100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 1:1, eluted for 2.5L respectively; after the primary silica gel column chromatography, similar components were combined according to the thin layer chromatography results, and 4 components A-D were obtained;

[0045] (7) The 200-300 mesh silica gel was selected for column chromatography of component A, and the petroleum ether-ethyl acetate=100:1 system was used as the eluent for isocratic elution. After the components with similar polarity were combined, 5 components A1-A5 were obtained. Component A3 was combined with component B for further separation and purification;

[0046] (8) The petroleum ether: ether=100:1 system was used as the eluent for isocratic elution of component B. The fractions eluted were detected by thin layer chromatography, and the components with similar detection results were combined to obtain 7 components B1-B7. Component B3 was further purified by Sephadex LH-20 gel column with the system of petroleum ether:dichloromethane:methanol=2:1:1 to obtain component B3A;

[0047] (9) The semi-preparative high performance liquid chromatography was used to separate and purify component B3A. The peaks in the liquid chromatogram that might be target compounds were collected, and the target compounds were determined by comparing the thin layer chromatography detection results. The preparation conditions were adjusted according to the thin layer chromatography detection results, and then the liquid phase preparation was carried out;

[0048] Liquid phase preparation condition: chromatographic column: C18 reversed-phase chromatographic column, 250mm x 9.4mm i.d., 5μm;

[0049] Flow phase: 0-35min, 84% CH3OH; detection wavelength: 210nm; sample concentration: 10mg / mL;

[0050] Flow rate: 3.0mL / min; retention time: 30.8min;

[0051] Collect the target peak in the liquid chromatogram, concentrate and dry to obtain the polyketide compound kaillingkoni.

[0052] Another embodiment of the present application provides a preparation comprising the actinomycete of the present application or the composition of the present application or the compound of the present application; and a carrier.

[0053] In some specific embodiments, the preparation described above is a tablet, a pill, a capsule, a powder, a gel, a granule or a liquid.

[0054] It should be noted that the carrier can be known in the art and can need to be selected according to the dosage form, such as diluents (e.g. starch, dextrin, sucrose or glucose, etc.), absorbents (calcium sulfate, calcium hydrogen phosphate or light magnesium oxide, etc.), binders (povidone, syrup or hydroxypropyl methyl cellulose, etc.), wetting agents (water, etc.) or disintegrants (dry starch, sodium hydroxymethyl starch or cross-linked povidone, etc.) for preparing tablets, and such as extenders, suspending agents, emulsifiers or colorants for preparing liquid preparations.

[0055] Another embodiment of the present application provides the use of the actinomycete of the present application or the composition of the present application or the compound of the present application as or in the preparation of a pesticide for controlling greenhouse whitefly.

[0056] It should be noted that the actinomycete (Streptomyces tianzhuensis) provided by the present application can be separated and purified to obtain kaillingkoni, which has obvious stomach poison activity on greenhouse whitefly adults, and the corrected mortality rate reaches 90.30% when the compound concentration is 100μg / mL and the treatment time is 72h, and it also has contact toxicity activity on greenhouse whitefly, and the corrected mortality rate reaches 82.55% when the concentration is 100μg / mL and the treatment time is 72h.

[0057] In order to better understand the present application, the content of the present application will be further illustrated below in combination with specific examples, but the content of the present application is not limited to the following examples.

[0058] In the following examples, the medium formula is shown in Table 1.

[0059] Table 1 Medium and formula

[0060]

[0061]

[0062] Note: In Table 1, the solvent is water.

[0063] Example 1

[0064] In a clean bench, the earthworms were surface-disinfected with 75% alcohol, and then the earthworm surface was washed several times with sterile water; the treated earthworms were placed in a sterile culture dish, and the intestinal contents were taken out with a sterile scalpel and placed in a mortar, 1 mL of sterile water was added, and it was ground thoroughly; by gradient dilution method, the grinding liquid was diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 times, and 100 μL of the grinding liquid was evenly coated on Gao's No. 1 medium, and incubated at 28°C; different morphological single colonies were picked up with an inoculation needle, and repeatedly purified to obtain actinomycete SA61; the medium was prepared according to Table 1. Strain SA61 was inoculated into strains ISP1-ISP17 medium (see Table 1 below), and incubated at 28°C in the dark for two weeks, and then the colony characteristics, colony growth, etc. were observed (as shown in Table 1 below); it was preliminarily determined as Streptomyces mycelium characteristics according to morphological characteristics. Figure 1

[0065] Then, 16S rDNA method was used to extract DNA from the bacterial suspension of the target strain, and the extracted DNA sequence was amplified by PCR instrument, and the length of the target strain sequence obtained was 1433 bp, which was uploaded to NCBI, and the Genbank accession number was OR126165.

[0066] Further, the Neighbor-Join method was used to construct a phylogenetic tree, and it was found that the sequence homology of the strain was the highest (98.3%) with Streptomyces longispororuber, and was 97.63% with Streptomyces ambofaciens in the same branch; according to the phylogenetic analysis and physiological and biochemical analysis, the target strain SA61 was determined as a potential new strain, named Streptomyces sp. SA61, and the gene sequencing result was as follows (SEQ ID NO. 1):

[0067] ​

[0068] Example 2

[0069] The actinomycete SA61 obtained in Example 1 was subjected to 48L fermentation treatment, and its fermentation crude extract was obtained by macroporous resin adsorption, methanol immersion and dichloromethane extraction; then the actinomycete SA61 fermentation crude extract was separated and purified to obtain the polyketide compound kailongmycin; the specific process is as follows:

[0070] The actinomycete SA61 stored in a refrigerator at -80°C was activated on a Gao's first flat plate and incubated at 28°C for 7 days; 5mL of ISP2 culture solution (see Table 1) was added to a 25mL test tube, and sterilized at 121°C for 30min; in a sterile operation table, the well-grown colonies were picked into 5mL of ISP2 culture solution, and shaken at 180rpm for 2 days, at which time it was a primary seed solution;

[0071] 50mL of ISP2 culture solution was added to a 250mL flask, and sterilized at 121°C for 30min; in a sterile operation table, the primary seed solution was transferred into the flask, and shaken at 180r / min at 28°C for 2 days, to obtain a secondary seed solution;

[0072] 400mL of F culture solution (see Table 1) and 16g of Amberlite XAD-16 resin were added to a 2L flask, and sterilized at 121°C for 30min; in a sterile operation table, the secondary seed solution was transferred into the 2L flask, and shaken at 180r / min at 28°C for 7 days, for a total of 48L fermentation; after the fermentation was completed, water was added to the flask, and the bacteria were repeatedly washed and discarded, and the resin was collected; the resin was dried in a 30°C oven to remove water; the resin was eluted with methanol 4 times; the eluate was collected and distilled under reduced pressure to obtain a methanol extract; the methanol extract was redissolved in 50% methanol aqueous solution (0.6L), and extracted with the same volume of dichloromethane four times; the collected dichloromethane fraction was concentrated to obtain 20g of dichloromethane extract of actinomycete SA61;

[0073] Silica gel column chromatography was used to separate the dichloromethane crude extract, and dry column packing and dry sample loading were selected. The crude extract was gradient eluted with a dichloromethane-methanol system, and the elution conditions were as follows: V (二氯甲烷) :V (甲醇) =100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 1:1, respectively, eluted for 2.5L; after the first silica gel column chromatography, similar components were combined according to the thin layer chromatography results, and four components A (1.2012g), B (1.0211g), C (4.1774g), and D (4.7280g) were obtained;

[0074] The A group was separated by column chromatography using 200-300 mesh silica gel, and eluted by isocratic elution with petroleum ether-ethyl acetate = 100:1 as eluent. The A1 (486 mg), A2 (22.5 mg) and A3 (48.4 mg) components were obtained by combining the components with similar polarity. The A3 component was combined with the B component for the next separation and purification.

[0075] The B component was eluted by isocratic elution with petroleum ether: diethyl ether = 100:1 as eluent. The B1-B77 components were obtained by combining the components with similar detection results by thin layer chromatography. The B3 (412.2 mg) component was further purified by Sephadex LH-20 gel column with a system of petroleum ether: dichloromethane: methanol = 2:1:1 to remove impurities, and the B3A (359.7 mg) component was obtained.

[0076] The B3A component was separated and purified by semi-preparative high performance liquid chromatography. The peaks possibly being target compounds in the liquid chromatogram were collected, and the target compounds were determined by comparing the thin layer chromatography detection results. The preparation conditions were adjusted according to the thin layer chromatography detection results, and then the liquid phase preparation was performed to obtain the polyketide compound kaillingkoni.

[0077] The semi-preparative high performance liquid chromatography conditions were as follows: a C18 reversed-phase chromatographic column with a size of 250 mm x 9.4 mm i.d. and a particle size of 5 μm; a mobile phase of 84% CH3OH for 0-35 min; a detection wavelength of 210 nm; a sample concentration of 10 mg / mL; a flow rate of 3.0 mL / min; and a retention time of 30.8 min.

[0078] The secondary metabolite of the actinomycete was structurally identified According to Figure 2 the high resolution mass spectrum shown in FIG. 1, the molecular weight of the compound was determined to be 334 (m / z 357.2041 [M+Na] + ), the molecular formula was C 20 H 30 O4, and it contained 6 degrees of unsaturation; according to Figures 3-7 the data shown in FIG. 1 and Table 1, 1 H NMR, 13 C NMR, HSQC, 1 H- 1 H COSY and HMBC, and comparison with the literature, the chemical structure of the compound was deduced; and finally, the absolute configuration was determined by X-ray single crystal diffraction results Figure 8 .

[0079] Table 2 Different characterization data of the compound

[0080]

[0081] Based on the comprehensive data, the structure of the secondary metabolite of the actinomycete is identified as:

[0082] Example 3

[0083] The sterile 30% sucrose water containing 0.5% acetone and 0.5% Tween-80 is used as the diluent; the compound kailinketone is diluted with the solvent to obtain the test agents with the concentration gradient of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.25 μg / mL and 6.125 μg / mL; the diluent is used as the blank control; the standard agent is the acetamiprid solution with the concentration of 2.5 μg / mL diluted with the diluent as the feeding liquid; the main body of the test device is a glass double-tube (d=3 cm, h=6 cm), one end of the double-tube is sealed with the fully stretched sealing film, 200 μL of the corresponding agent is placed on the surface of the fully stretched sealing film, and another fully stretched sealing film is sealed on the agent to avoid the overflow or evaporation of the agent (see Figure 9 ); 30 greenhouse whitefly adults (emerged for 2-3 days) are inoculated in the glass double-tube for each test repetition, in order to avoid the escape of the test insects and maintain the air permeability of the device, the other end of the glass double-tube is fully sealed with the sterile cotton; 3 groups of controls are prepared for each concentration, the stomach toxicity test is repeated for 3 times, and the observation is performed once every 24 h for 3 times; the results are shown in Table 2 and Figure 10 .

[0084] Table 2 Stomach toxicity activity test with acetone, Tween-8 and sucrose water as the diluent

[0085]

[0086] Note: the data in the table is the average corrected mortality rate ± standard error (P>0.05); PC (standard agent); CK (blank control);

[0087] The above results show that kailinketone has obvious toxic effect on the greenhouse whitefly adults, and has obvious concentration effect. At the concentration of 6.125 μg / mL, the corrected mortality rate of the greenhouse whitefly treated for 72 h is 33.37%, and at the concentration of 100 μg / mL, the corrected mortality rate treated for 72 h reaches 90.30%, the LC 50 is 18.10 μg / mL, kailinketone has strong insecticidal activity on the greenhouse whitefly adults, and the corrected mortality rate of the greenhouse whitefly is obviously increased with the increase of the concentration and the extension of the treatment time.

[0088] Example 4

[0089] Glass tube medicine film method was used. Kailinketongmycin was dissolved in acetone to prepare 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.25 μg / mL and 6.125 μg / mL liquid medicine. The standard medicine was acetamiprid solution diluted with diluent to a concentration of 2.5 μg / mL as feeding liquid. 300 μL was taken and added into a glass tube (d = 3 cm, h = 6 cm). The glass tube was rotated at a uniform speed to make the acetone completely evaporate, and kailinketongmycin was uniformly attached to the inner wall of the glass tube. After standing for 3 h, activity determination was performed. CK was 300 μL of acetone. One end of the glass tube was sealed with parafilm sealing film. 180 μL of nutrient solution containing 30% sucrose was added on the film, and three layers of parafilm sealing film were overlaid. The greenhouse whitefly adults (2-3 d after emergence) were transferred to the glass tube after being frozen at 4°C. There were 30 in each tube, and each treatment was repeated 3 times. The other end of the glass tube was sealed with gauze, and the outer wall was wrapped with aluminum foil paper. The end with the nutrient solution was placed upward in a constant temperature and humidity light incubator with 27±1°C, RH (60±5)%, and light cycle L / / D = 14h / / 10h. Observation was performed once every 24 h, a total of 3 times, and the results are shown in Table 3 and Figure 11

[0090] Table 3 Contact activity test of acetone as a solvent

[0091]

[0092] Note: The data in the table is the average corrected mortality rate ± standard error (P>0.05); PC (standard medicine); CK (blank control).

[0093] The above results show that the mortality rate of greenhouse whitefly increases gradually with time and compound concentration, and even all die, showing a clear dose-effect relationship. At a concentration of 6.125 μg / mL, the corrected mortality rate of greenhouse whitefly after 72 h treatment is 17.28%, and at a concentration of 100 μg / mL, the corrected mortality rate after 72 h treatment reaches 82.55%. The LC 50 is 27.80 μg / mL. Kailinketongmycin has strong insecticidal activity on greenhouse whitefly adults, and the corrected mortality rate of greenhouse whitefly increases significantly with increasing concentration and prolonged treatment time.

[0094] The results show that kailinketongmycin has strong insecticidal activity on greenhouse whitefly adults, and the corrected mortality rate of greenhouse whitefly increases significantly with increasing concentration and prolonged treatment time. Kailinketongmycin has potential value in developing into a new type of environment-friendly pesticide lead compound, which can be used for preventing and controlling greenhouse whitefly or preparing pesticides for preventing and controlling greenhouse whitefly.

[0095] ​Finally, it is to be explained that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. A compound, characterized in that, Its structure is shown in Equation I below.

2. The method for preparing the compound according to claim 1, characterized in that, include: It was prepared by fermentation of the actinomycete (Streptomyces tianzhuensis), which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 20306.

3. The preparation method according to claim 2, characterized in that, include: (1) After activating the actinomycetes, they were cultured at a constant temperature; the culture was mixed with the culture medium, sterilized, and cultured again to obtain the first-stage seed culture. (2) Mix the primary seed culture with the sterilized culture medium and culture to obtain the secondary seed culture; (3) Mix the secondary seed culture with the sterilized Amberlite XAD-16 resin and culture medium, culture to remove bacteria, collect the resin, and dry the resin; (4) Elute the resin with methanol, collect the eluent, and distill under reduced pressure to obtain methanol extract; (5) Dissolve the methanol extract in an aqueous methanol solution, extract with dichloromethane, concentrate the collected dichloromethane fraction, and obtain dichloromethane extract of actinomycetes.

4. The preparation method according to claim 3, characterized in that, Also includes: (6) The dichloromethane extract of dichloromethane-methanol actinomycetes was subjected to gradient elution under the following conditions: V(dichloromethane):V(methanol) = 100:0, 100:1, 100:2, 100:4, 100:8, 100:16 and 1:1 respectively. After primary silica gel column chromatography, similar components were combined according to the thin-layer chromatography results to obtain a total of 4 components AD. (7) 200-300 mesh silica gel was selected for column chromatography of component A. The petroleum ether-ethyl acetate = 100:1 system was used as the eluent for isogradient elution. After combining the components with similar polarity, components A1-A5 were obtained. A3 was combined into component B for further separation and purification. (8) Using a petroleum ether:diethyl ether = 100:1 system as the eluent, fraction B was eluted with an isogradient. The eluted fractions were then analyzed by thin-layer chromatography. Fractions with similar results were combined to obtain fractions B1-B77. Fraction B3 was eluted using a petroleum ether:diethyl ether = 100:1 system. Impurities were further removed using a Sephadex LH-20 gel column with dichloromethane:methanol = 2:1:1 to obtain component B3A; (9) Component B3A was separated and purified using semi-preparative high performance liquid chromatography. Peaks that may be the target compound in the liquid chromatogram were collected. The target compound was then determined by comparing the results of thin-layer chromatography. The preparation conditions were adjusted according to the results of thin-layer chromatography. Liquid preparation was then carried out. The target peaks in the liquid chromatogram were collected, concentrated and dried to obtain the compound shown in Formula I.

5. A formulation, characterized in that, It includes the compound of claim 1; and a carrier.

6. The formulation according to claim 5, characterized in that, The preparations are tablets, pills, capsules, powders, gels, granules, or liquids.

7. The use of the compound of claim 1 as or in the preparation of a pesticide for controlling greenhouse whiteflies.

Images