A soluble solution of roseoviridis streptomyces nkz-259, its preparation method and application

Abstract

The present application relates to the field of bio-agriculture, and particularly relates to a soluble solution of Streptomyces roseofulvus NKZ-259, a preparation method and application thereof. The present application screens different types of chemical surfactants, and finally screens two safe and compatible synergistic adjuvants, alkyl polyglucoside and / or fatty alcohol polyoxyethylene ether sodium sulfate. In the formula provided by the present application, alkyl polyglucoside and / or fatty alcohol polyoxyethylene ether sodium sulfate are used as interface performance synergistic agents, which can significantly improve the wetting spreading and adhesion capacity of Streptomyces roseofulvus NKZ-259 on crop leaves; alkyl polyglucoside and fatty alcohol polyoxyethylene ether sodium sulfate can realize significant synergistic effect after being combined with Streptomyces roseofulvus NKZ-259. Moreover, the soluble solution provided by the present application has good compatibility and good storage stability, and can significantly improve the disease resistance of Streptomyces roseofulvus NKZ-259.

Description

Technical Field

[0001] This invention relates to the field of bio-agricultural technology, and in particular to a soluble form of Streptomyces roseum NKZ-259, its preparation method, and its application. Background Technology

[0002] Streptomyces roseoflavus NKZ-259 is a biocontrol fungus isolated from soil in the Qilian Mountains region of Qinghai Province by the Agricultural Antibiotics Group of the National Key Laboratory of Pest and Disease Control, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 2, 2016, with accession number CGMCC NO.13416. Streptomyces roseoflavus NKZ-259 colonies are pale yellow to pink, with well-developed aerial hyphae, and produce chain-like spores. It is Gram-positive and aerobic. It is highly adaptable, able to colonize in various soil environments, and form a symbiotic relationship with plants. Studies have found that this strain has a significant promoting effect on plant growth. During fermentation, it produces various secondary metabolites and secretes plant hormones (such as IAA) or siderophores, promoting root development and nutrient absorption, playing an important role in promoting plant growth. Currently, the application of this strain still relies on fermenting the strain, filtering the fermentation liquid, concentrating it, and then spraying it onto crops.

[0003] Currently, *Streptomyces roseum* NKZ-259 can be formulated into both soluble and wettable powder products. Since this strain is specifically used in the preparation of biopesticides, the active ingredients of these biopesticides mainly consist of live bacteria and their metabolites. To protect its biological activity, there are no reported methods for adding chemical surfactants to the formulation to achieve synergistic effects, and there is also no research on the strain's resistance to plant viruses. Summary of the Invention

[0004] The purpose of this invention is to provide a soluble form of Streptomyces roseum NKZ-259, its preparation method, and its application, thereby addressing the problems existing in the prior art. This invention, through screening a large number of different types of chemical surfactants, ultimately selected two safe and compatible synergistic adjuvants. When combined with Streptomyces roseum NKZ-259 in the formulation, these adjuvants exhibit a significant synergistic effect and provide a new research direction for the application of other types of biological pesticides, which is of great significance.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a soluble concentrate of Streptomyces roseoflavus NKZ-259, comprising the following components in weight percentage:

[0007] The formula consists of 0-80% Streptomyces roseum NKZ-259 mother liquor, 5%-15% synergistic surfactant, 5%-20% cosolvent, 0.1-0.5% defoamer, 3-5% antifreeze, and the balance being water.

[0008] The mass percentage of the mother liquor of Streptomyces roseum NKZ-259 is not 0.

[0009] Preferably, the Streptomyces roseum NKZ-259 soluble concentrate comprises the following components in weight percentage:

[0010] The formula consists of 50% Streptomyces roseum NKZ-259 mother liquor, 10% synergistic surfactant, 10% cosolvent, 0.2% defoamer, 5% antifreeze, and the balance being water.

[0011] Preferably, the synergistic surfactant comprises alkyl polyglucosides and / or sodium fatty alcohol polyoxyethylene ether sulfate;

[0012] And / or, the co-solvent includes acetophenone and / or N,N-dimethylacetamide;

[0013] And / or, the defoamer includes trisiloxane defoamers;

[0014] And / or, the antifreeze includes 1,2-propanediol or glycerol.

[0015] The present invention provides a method for preparing the above-mentioned Streptomyces roseum NKZ-259 soluble agent, comprising the step of mixing the mixture of the synergistic surfactant, the cosolvent, the defoamer, the antifreeze and the water with the Streptomyces roseum NKZ-259 mother liquor.

[0016] As an additional option, the present invention also provides an interface synergistic method for Streptomyces roseum NKZ-259, comprising the step of mixing a mixture of the synergistic surfactant, the cosolvent, the defoamer, the antifreeze and the water with the Streptomyces roseum NKZ-259 mother liquor.

[0017] This invention provides the application of the above-mentioned Streptomyces roseum NKZ-259 soluble concentrate in the control of plant pathogens.

[0018] Preferably, the plant pathogens include Botrytis cinerea and / or tobacco mosaic virus.

[0019] This invention provides the application of the above-mentioned Streptomyces roseum NKZ-259 soluble concentrate in the preparation of biological agents for the control of plant pathogens.

[0020] This invention provides a biological agent for controlling plant pathogens, including the above-mentioned Streptomyces roseum NKZ-259 soluble agent.

[0021] More preferably, the plant pathogens include gray mold and / or tobacco mosaic virus.

[0022] This invention provides a method for controlling plant pathogens, comprising the steps of treating the plants to be treated with the above-mentioned Streptomyces roseum NKZ-259 soluble agent or a biological agent prepared using the above-mentioned Streptomyces roseum NKZ-259 soluble agent.

[0023] More preferably, the plant pathogens include gray mold and / or tobacco mosaic virus.

[0024] The present invention discloses the following technical effects:

[0025] This invention, through screening different types of chemical surfactants, ultimately identified two safe and compatible synergistic adjuvants—alkyl polyglucoside and sodium fatty alcohol polyoxyethylene ether sulfate (SOE). In the formulation provided by this invention, using alkyl polyglucoside and SOE as interfacial performance synergists significantly improves the wetting, spreading, and adhesion ability of *Streptomyces roseum* NKZ-259 on crop leaves. The combination of alkyl polyglucoside and SOE with *Streptomyces roseum* NKZ-259 achieves a significant synergistic effect. Furthermore, the soluble concentrate provided by this invention exhibits good compatibility among its components, good storage stability, and significantly enhances the disease resistance of *Streptomyces roseum* NKZ-259. This invention's method of using chemical surfactants in biopesticide formulations to achieve synergistic effects provides a valuable reference for the research and application of other types of biopesticides, offering a new direction for their development and application, and is of great significance.

[0026] This invention discloses for the first time a novel application of this soluble agent in the control of plant pathogens. Experiments have demonstrated that the formulation provided by this invention can better exert its effect in controlling plant pathogens. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The image shows the sample for Comparative Example 2; where A is the top view and B is the front view.

[0029] Figure 2 The image shows sample 3 for comparison; where A is the top view and B is the front view.

[0030] Figure 3 The image shows the dynamic surface tension curves of four experimental samples related to the 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate in Example 4. Specifically, 4#375× represents a 375-fold dilution of sample #4, 4#750× represents a 750-fold dilution of sample #4, 1#375× represents a 375-fold dilution of sample #1, 1#750× represents a 750-fold dilution of sample #1, 2#375× represents a 375-fold dilution of sample #2, 2#750× represents a 750-fold dilution of sample #2, 3#375× represents a 375-fold dilution of sample #3, and 3#750× represents a 750-fold dilution of sample #3.

[0031] Figure 4 The images show the antibacterial effects of four experimental samples related to the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate against the pathogen *Botrytis cinerea* in Example 5. A represents the antibacterial effect of sample #1, with the left side representing the control (CK) and the right side representing the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate stock solution prepared in Example 1; B represents the antibacterial effect of sample #2, with the left side representing the control (CK) and the right side representing the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate stock solution prepared in Example 2; C represents the antibacterial effect of sample #3, with the left side representing the control (CK) and the right side representing the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate stock solution prepared in Example 3; and D represents the antibacterial effect of sample #4, with the left side representing the control (CK) and the right side representing the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate stock solution prepared in Example 4.

[0032] Figure 5This is a graph showing the antibacterial effect of 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate (1#) prepared in Example 1 of Example 5 against *Botrytis cinerea* pathogen. From left to right, each well represents: CK, the stock solution of 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate prepared in Example 1, a 10-fold dilution of the stock solution of 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate prepared in Example 1, and the stock solution of 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate prepared in Example 1. The following solutions were prepared: a 20-fold dilution of the liquid, a 50-fold dilution of the 0.5 wt% Streptomyces roseum NKZ-259 soluble stock solution prepared in Example 1, a 100-fold dilution of the 0.5 wt% Streptomyces roseum NKZ-259 soluble stock solution prepared in Example 1, a 200-fold dilution of the 0.5 wt% Streptomyces roseum NKZ-259 soluble stock solution prepared in Example 1, and a 500-fold dilution of the 0.5 wt% Streptomyces roseum NKZ-259 soluble stock solution prepared in Example 1.

[0033] Figure 6 This is a graph showing the antibacterial effect of the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate (2#) prepared in Example 2 of Example 5 against *Botrytis cinerea* pathogen. From left to right, each well represents: CK, the stock solution of the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate prepared in Example 2, a 10-fold dilution of the stock solution of the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate prepared in Example 2, and the stock solution of the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate prepared in Example 2. The following solutions were prepared: a 20-fold dilution of the liquid, a 50-fold dilution of the 0.5 wt% Streptomyces roseum NKZ-259 soluble stock solution prepared in Example 2, a 100-fold dilution of the 0.5 wt% Streptomyces roseum NKZ-259 soluble stock solution prepared in Example 2, a 200-fold dilution of the 0.5 wt% Streptomyces roseum NKZ-259 soluble stock solution prepared in Example 2, and a 500-fold dilution of the 0.5 wt% Streptomyces roseum NKZ-259 soluble stock solution prepared in Example 2.

[0034] Figure 7 The antiviral effects of five experimental groups on TMV associated with 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate are shown in the figure. A is a staining graph; B is a statistical graph; * indicates a significant difference compared with CK, and ns indicates no significant difference compared with CK. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] In preparing a soluble concentrate containing 0.5 wt% *Streptomyces roseum* NKZ-259, the selection of various raw material components is crucial to the successful preparation of a stable and synergistically effective soluble concentrate. Therefore, the inventors screened and compared multiple raw materials, with specific experiments as follows:

[0044] Example 1: Preparation of a 0.5 wt% soluble concentrate of Streptomyces roseoflavus NKZ-259

[0045] Preparation of 1% Streptomyces roseum NKZ-259 stock solution: Streptomyces roseum NKZ-259 was inoculated onto MS medium and cultured at 28°C in the dark for 7 days. A 1cm sample was scraped off using a sterile toothpick. 2 Microbial blocks of varying sizes were inoculated into fermentation medium and cultured at 28℃ and 220 r / min on a shaker for 96 h. After filtration through filter paper, a 1% mother liquor of *Streptomyces roseoflavus* NKZ-259 was obtained. The accession number for *Streptomyces roseoflavus* NKZ-259 is CGMCC NO.13416. This strain, classified as *Streptomyces roseoflavus*, was deposited on December 2, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. This strain is described in authorized patent ZL201710044242.4, entitled "A *Streptomyces roseoflavus* strain and its application."

[0046] Weigh out the following ingredients by mass percentage: 50% of 1% Streptomyces roseum NKZ-259 stock solution, 10% of the synergistic surfactant Agnique PG 8107 (alkyl polyglucoside, BASF), 10% of the solubilizer acetophenone (analytical grade, Shanghai Maclean Biotechnology Co., Ltd.), 0.2% of the defoamer SAG 1522 (trisiloxane, Momentive Silicon Materials (Shanghai) Co., Ltd.), and 5% of the antifreeze 1,2-propanediol (analytical grade, Shanghai Maclean Biotechnology Co., Ltd.). Make up the total volume to 100% with deionized water (tertiary water, laboratory-made).

[0047] The preparation method of soluble solvent is as follows:

[0048] (1) Add deionized water, synergistic surfactant Agnique PG 8107, cosolvent acetophenone, antifreeze 1,2-propanediol and defoamer SAG 1522 to the reaction vessel in proportion and stir until uniform;

[0049] (2) Add 1% Streptomyces roseum NKZ-259 mother liquor to the above mixed solution in proportion and stir evenly to obtain 0.5wt% Streptomyces roseum NKZ-259 soluble agent.

[0050] All preparation processes were carried out at room temperature.

[0051] Example 2: Preparation of 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate

[0052] The preparation of the 1% Streptomyces roseum NKZ-259 mother liquor was the same as in Example 1.

[0053] Weigh out the following components by mass percentage: 50% of 1% Streptomyces roseum NKZ-259 mother liquor, 10% of synergistic surfactant W1635 (sodium fatty alcohol polyoxyethylene ether sulfate, Shanghai Lanheng Chemical Technology Co., Ltd.), 10% of cosolvent N,N-dimethylacetamide (analytical grade, Shanghai Maclean Biotechnology Co., Ltd.), 0.2% of defoamer SAG 1522 (trisiloxane, Momentive Silicon Materials (Shanghai) Co., Ltd.), and 5% of antifreeze glycerol (analytical grade, Shanghai Maclean Biotechnology Co., Ltd.), and bring the total volume to 100% with deionized water.

[0054] The preparation method of soluble solvent is as follows:

[0055] (1) Add deionized water, synergistic surfactant W1635, cosolvent N,N-dimethylacetamide, antifreeze glycerol and defoamer SAG 1522 to the reaction vessel in proportion and stir until uniform;

[0056] (2) Add 1% Streptomyces roseum NKZ-259 mother liquor to the above mixed solution in proportion and stir evenly to obtain 0.5wt% Streptomyces roseum NKZ-259 soluble agent.

[0057] All preparation processes were carried out at room temperature.

[0058] Preparation of 10.5 wt% soluble concentrate of Streptomyces roseum NKZ-259 (Comparative Example)

[0059] The synergistic surfactant Agnique PG 8107 in Example 1 was replaced with Tween 80 (sorbitan monooleate polyoxyethylene ether, Xingtai Yancheng Chemical Additives Co., Ltd.), while other components and preparation methods remained unchanged. As a result, the sample became viscous and had no inhibitory effect on gray mold pathogens and tobacco virus TMV. For details on the inhibitory effects on pathogens and viruses, please refer to the efficacy results of Comparative Example 1 (3#) in Examples 5 and 6.

[0060] Preparation of 20.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (Comparative Example)

[0061] In Example 2, the synergistic surfactant Agnique PG 8107 was replaced with a surfactant, while other components and preparation methods remained unchanged. After sample preparation, white, blocky flocculent matter appeared, such as... Figure 1 As shown.

[0062] Comparative Example 3

[0063] The synergistic surfactant Agnique PG 8107 in Example 1 was replaced with T-70F (sodium dioctyl sulfonate, Xingtai Yancheng Chemical Additives Co., Ltd.), while other components and preparation methods remained unchanged. The sample, after preparation, separated into layers, indicating incompatibility with this system. Figure 2 As shown.

[0064] Example 3: Stability verification experiment of 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate with different adjuvants mixed with different formulations of insecticides and fungicides in tanks.

[0065] Different formulations of fungicides and insecticides, including microemulsions, water-in-oil emulsions, suspensions, emulsifiable concentrates, wettable powders, soluble concentrates, dispersible oil suspensions, and water-dispersible granules, were selected and mixed in tanks with 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate at various dilution ratios. The stability of the tank mixture was observed (30 min). The 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate was diluted 375 times.

[0066] Samples to be tested: 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (1#) prepared in Example 1, 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (2#) prepared in Example 2, 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (3#) prepared in Comparative Example 1, and 0.5 wt% Streptomyces roseum NKZ-259 mother liquor (made from 1% Streptomyces roseum NKZ-259 mother liquor and deionized water, 4#).

[0067] Tank-mixed pesticides: 10% pyraclostrobin microemulsion, 20% pyraclostrobin·fluopyram water emulsion, 30% difenoconazole suspension, 40% isoprothiolane emulsifiable concentrate, 60% carbendazim·thiram wettable powder, 75% azoxystrobin·tebuconazole water dispersible granules, 30% difenoconazole·acetamiprid suspension, 20% dinotefuran·spirotetramat suspension, 5.6% cymoxanil soluble concentrate, 1% emamectin benzoate microemulsion, and 30% tetrazolium·fluopyram suspension.

[0068] The results of the tank mixing experiment are shown in Table 1. The results show that the 0.5wt% Streptomyces roseum NKZ-259 soluble concentrate exhibited good compatibility with various formulations of conventional chemical pesticides in tank mixing, with no flocculation or precipitation.

[0069] Table 1. Stability of four experimental samples related to Streptomyces roseum NKZ-259 in tank mixing with various formulations of conventional chemical pesticides.

[0070] Serial Number Names and formulations of bactericides Dilution factor Test sample 1 10% pyraclostrobin microemulsion 230 All of #1-#4 are in good condition. 2 20% Pyrimethanil·Fluorazole Emulsion 2000 All of #1-#4 are in good condition. 3 30% difenoconazole suspension 4000 All of #1-#4 are in good condition. 4 40% Isoprothiolane EC 267 All of #1-#4 are in good condition. 5 60% Carbendazim·Thiram Wettable Powder 300 All of #1-#4 are in good condition. 6 75% azoxystrobin·tebuconazole water dispersible granules 3500 All of #1-#4 are in good condition. 7 30% Butyrazosulfuron-methyl·Acetamide Suspension Concentrate 2000 All of #1-#4 are in good condition. 8 20% fipronil·spirotetramat suspension 2000 All of #1-#4 are in good condition. 9 5.6% Cymoxanil soluble solution 1500 All of #1-#4 are in good condition. 10 1% abamectin microemulsion 1500 All of #1-#4 are in good condition. 11 30% Tetraazolidinyl·Fluoropyram Suspension Concentrate 1500 All of #1-#4 are in good condition.

[0071] Example 4: pH and interfacial performance evaluation of 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate with different additives.

[0072] Experimental objective: To evaluate the physicochemical properties and interfacial performance of each experimental sample by measuring its pH, dynamic surface tension, static surface tension, contact angle, wetting and penetration, drug retention, spreading properties, adhesion tension, and adhesion work.

[0073] Samples to be tested: 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (1#) prepared in Example 1, 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (2#) prepared in Example 2, 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (3#) prepared in Comparative Example 1, and 0.5 wt% Streptomyces roseum NKZ-259 mother liquor (made from 1% Streptomyces roseum NKZ-259 mother liquor and deionized water, 4#).

[0074] Sample preparation: Dilute the sample to be tested with water 100 times and measure the pH. Dilute it 375 times and 750 times and measure other interfacial performance indicators.

[0075] Experimental methods:

[0076] (1) pH index: The four test samples were diluted 100 times with freshly boiled and cooled to room temperature grade III water, shaken evenly, and then measured with a pH meter (FE-28, Mettler Toledo).

[0077] (2) Dynamic surface tension: The dynamic surface tension data of the test samples at specific dilution ratios were measured from 10ms to 5000ms using a Kruss dynamic surface tension meter (BP-100, CRUSS).

[0078] (3) Static surface tension: The static surface tension data of the sample under test at a specific dilution factor were determined using the platinum plate method and a static surface tension meter (JK-99CM, Beijing Zhongyi Kexin Technology Co., Ltd.).

[0079] (4) Contact angle: The contact angle data of the test sample on the PARAFILM sealing film at a specific dilution ratio were measured using a Kruss contact angle meter (DSA-100, CRUSS).

[0080] (5) Wetting and Penetration: Wetting and penetration were determined using the disc canvas method. A test solution was prepared with distilled water at a specific dilution ratio, placed in a 500mL beaker, and kept in a constant temperature water bath at 20℃ for 20 minutes. A 35mm diameter stiff canvas was used for the experiment. The canvas was placed flat on the liquid surface, and a stopwatch was started when the canvas touched the liquid surface. The canvas sank after wetting and continued until it touched the bottom of the beaker, recording the required time. The shorter the penetration time, the better the wetting and penetration performance of the sample.

[0081] (6) Retention of pesticide solution: Cut the leaves of the pothos into round leaves with a diameter of 4cm, use tweezers to pick up the leaves and immerse them in the liquid to be tested for 3s, then suspend the leaves at 45° and let them stand still. When no more liquid drops fall, weigh the mass of pesticide solution remaining on the leaves Δm. The larger the value of Δm, the greater the retention of pesticide solution.

[0082] (7) Spreadability: Using a high-transparency acrylic plate, place the graph paper under the plate, glue the edges of the two acrylic plates together, place the graph paper in the middle, and place it flat on a horizontal table. Using a 0.9mm inner diameter syringe, keep it at a height of about 5mm, and slowly drip the test liquid at a specific dilution factor onto the acrylic plate. Read the number of grids wetted by the liquid. The more grids wetted, the better the spreadability of the sample.

[0083] (8) Adhesive tension: The physical meaning of adhesive tension is the driving force for the spread of a liquid on a solid surface. It can be calculated using static surface tension and contact angle. For example, if the contact angle of the liquid to be measured is θ and the static surface tension is T, then the adhesive tension F can be expressed as: F = T × cosθ;

[0084] (9) Adhesion work: The physical meaning of adhesion work is to characterize the strength of the bond between the solid and liquid interfaces. It can also be calculated using static surface tension and contact angle. For example, if the contact angle of the liquid to be tested is θ and the static surface tension is T, then the adhesion work can be expressed as: W=T×(1+cosθ).

[0085] Experimental results:

[0086] (1) The pH values ​​of the four system samples are similar and all show weak acidity, indicating that the addition of the synergistic surfactant did not change the acid-base environment of the original system of Streptomyces roseum NKZ-259, which is conducive to maintaining the chemical stability of the active ingredients in the system.

[0087] (2) All four samples exhibited poor wetting and penetration at two specific dilution ratios; static surface tension, contact angle, adhesion tension, and adhesion work were best for sample #2, followed by sample #3; drug retention was best for sample #1; spreading experiment showed best performance for sample #3; dynamic surface tension was best for sample #2 (Table 2 and...). Figure 3 ).

[0088] Table 2. pH and interfacial properties data of four experimental samples related to Streptomyces roseum NKZ-259.

[0089]

[0090]

[0091] Example 5: Inhibition experiment of 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate with different adjuvants on gray mold pathogen.

[0092] Experimental method: Mycelial growth inhibition method.

[0093] Experimental Principle: The growth of grape gray mold strain B05.10, or simply Botrytis cinerea B05.10 (disclosed in the literature "The Effect and Mechanism of Wuyi Mycin in the Control of Grape Gray Mold, with a 20-year commitment to release it to the public") on the culture medium depends on the extension and branching of hyphal tips. When the test substance is mixed with the culture medium, if it has antibacterial activity, it will directly interfere with the cell wall synthesis, membrane integrity, or energy metabolism of the hyphae, leading to the inhibition of colony expansion. Therefore, the antibacterial effect can be evaluated by observing changes in colony growth.

[0094] 1. Experimental materials and preparation:

[0095] Pathogen: Activated Botrytis cinerea B05.10, cultured on PDA medium at 25°C for 5–7 days until sporulation.

[0096] Preparation of mycelial cakes: Use a sterile punch (5mm in diameter) to cut mycelial blocks from the edge of the colony (the mycelial cakes must contain mycelia and spores).

[0097] Samples to be tested: 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (1#) prepared in Example 1, 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (2#) prepared in Example 2, 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (3#) prepared in Comparative Example 1, and 0.5 wt% Streptomyces roseum NKZ-259 mother liquor (made from 1% Streptomyces roseum NKZ-259 mother liquor and deionized water, 4#).

[0098] Culture medium: Mix the sample to be tested with sterilized PDA culture medium cooled to 50°C, and quickly pour it into a petri dish (avoid high temperature from destroying drug activity).

[0099] 2. Experimental steps:

[0100] (1) Mushroom cake inoculation method

[0101] Preparation of drug-containing plates: Different test samples were mixed with PDA and poured into sterile petri dishes (approximately 4 mm thick). After solidification, the sample numbers were marked. Blank control group CK: Pure PDA plates without drugs (negative control), PDA plates containing 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate prepared in Example 1 (1#), 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate prepared in Example 2 (2#), and PDA plates containing 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate prepared in Comparative Example 1 (3#) and 0.5 wt% Streptomyces roseum NKZ-259 stock solution (made from 1 wt% Streptomyces roseum NKZ-259 stock solution and deionized water, 4#) (positive control).

[0102] (2) Inoculation with mycelium cake

[0103] Using sterile forceps, gently place the mycelial cake upside down (mycelial side down) in the center of the agar plate, avoiding pressing too deeply. Avoid edge effects (use only the central area of ​​the agar plate).

[0104] (3) Cultivation and Observation

[0105] Invert the plates and incubate them in a constant temperature incubator (25℃±1℃, humidity ≥85%, dark conditions) for 3–7 days. Observe and record the colony growth and inhibition status of each experimental group visually.

[0106] 3. Analysis of Experimental Results

[0107] Figure 4 Images showing the antibacterial effects of different treatments. Figure 4 It can be seen that the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate (1#) prepared in Example 1 and the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate (2#) prepared in Example 2 both have inhibitory effects on the gray mold pathogen. However, the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate (3#) and the 0.5 wt% *Streptomyces roseum* NKZ-259 stock solution (4#) prepared in Comparative Example 1 have no inhibitory effect on the gray mold pathogen.

[0108] Figure 5 The figures show the antibacterial effects of the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate (1#) stock solution, and its 10x, 20x, 50x, 100x, 200x, and 500x dilutions prepared in Example 1. As can be seen from the figures, only the stock solution of the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate (1#) prepared in Example 1 showed inhibitory effects against *Botrytis cinerea*, while the other dilutions had no inhibitory effect.

[0109] Figure 6The figures show the antibacterial effects of the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate (2#), and its 10x, 20x, 50x, 100x, 200x, and 500x dilutions prepared in Example 2. The figures show that the 0.5 wt% *Streptomyces roseum* NKZ-259 soluble concentrate (2#) and its 10x dilution prepared in Example 2 have inhibitory effects on *Botrytis cinerea*, while the other dilutions have no inhibitory effect on *Botrytis cinerea*.

[0110] Example 6: Experiment on the therapeutic effect of 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate with different adjuvants on tobacco mosaic virus (TMV).

[0111] Experimental objective: To evaluate the therapeutic effect of the 0.5 wt% Streptomyces roseum NKZ-259 soluble reagent prepared in Example 1 on tobacco virus (TMV).

[0112] 1. Experimental Materials

[0113] Plant material: 4-6 week old Nicotiana benthamiana (growing conditions: 25±2℃, 16h light / 8h dark).

[0114] Virus strain: Tobacco mosaic virus (TMV), published in the literature "Isolation of a Crystalline Protein Possessing the Properties of Tobacco-Mosaic Virus", with a commitment to distribute it to the public for 20 years.

[0115] Experimental reagents: 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (1#) prepared in Example 1, 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (2#) prepared in Example 2, 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (3#) prepared in Comparative Example 1, and 0.5 wt% Streptomyces roseum NKZ-259 mother liquor (made from 1% Streptomyces roseum NKZ-259 mother liquor and deionized water, 4#).

[0116] Reagents and instruments: Virus inoculation buffer (0.01M phosphate buffer, pH 7.0); fluorescence microscope (using fluorescently labeled virus).

[0117] 2. Experimental Procedure

[0118] (1) Dilute each of the 1#-4# samples with water 250 times and prepare the dilution reagent;

[0119] (2) The agent was evenly sprayed on the surface of healthy Nicotiana benthamiana. After 6 hours, the virus was inoculated onto the treated tobacco. Spraying was carried out again 1-2 days after virus infection. Water treatment was used as a control (CK). Five Nicotiana benthamiana seedlings were set up for each agent group to replicate the experiment.

[0120] (3) Observe the virus infection status under UV light 7-9 days after inoculation (TMV infection clones contain GFP tags, which can be displayed as green under UV light after infection).

[0121] 3. Experimental Results

[0122] Experimental results are as follows Figure 7 As shown. The results indicate that after treatment with 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (1#) prepared in Example 1 and 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (2#) prepared in Example 2, the area of ​​green parts on Nicotiana benthamiana seedlings was significantly reduced, indicating that both agents can significantly inhibit virus infection. In particular, the 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (1#) prepared in Example 1 showed the most significant effect, with an inhibition rate of nearly 90% compared to the control. Compared with the control, the area of ​​green parts in plants treated with 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (3#) and 0.5 wt% Streptomyces roseum NKZ-259 stock solution (4#) prepared in Comparative Example 1 did not change significantly, indicating that the 0.5 wt% Streptomyces roseum NKZ-259 soluble concentrate (3#) and 0.5 wt% Streptomyces roseum NKZ-259 stock solution (4#) prepared in Comparative Example 1 could not inhibit viral infection. Figure 7 ).

[0123] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A soluble form of Streptomyces roseoflavus NKZ-259, characterized in that, The Streptomyces roseum NKZ-259 soluble concentrate is composed of the following components in weight percentage: The formula consists of 50% Streptomyces roseum NKZ-259 mother liquor, 10% synergistic surfactant, 10% cosolvent, 0.2% defoamer, 5% antifreeze, and the remainder water. The synergistic surfactants include alkyl polyglucosides and / or sodium fatty alcohol polyoxyethylene ether sulfate; And / or, the co-solvent includes acetophenone and / or N,N-dimethylacetamide; And / or, the defoamer includes trisiloxane defoamers; And / or, the antifreeze includes 1,2-propanediol or glycerol.

2. The method for preparing the soluble form of Streptomyces roseum NKZ-259 according to claim 1, characterized in that, The method includes the step of mixing the synergistic surfactant, the cosolvent, the defoamer, the antifreeze and the water with the Streptomyces roseum NKZ-259 mother liquor.

3. The application of the soluble Streptomyces roseum NKZ-259 solution according to claim 1 in the control of plant pathogens, characterized in that, The plant pathogens are Botrytis cinerea and / or tobacco mosaic virus.

4. The application of the soluble Streptomyces roseum NKZ-259 solution according to claim 1 in the preparation of biological agents for controlling plant pathogens, characterized in that, The plant pathogens are Botrytis cinerea and / or tobacco mosaic virus.

5. A biological agent for controlling plant pathogens, characterized in that, The product includes the soluble form of Streptomyces roseum NKZ-259 as described in claim 1; the plant pathogen is Botrytis cinerea and / or Tobacco Mosaic Virus.

6. A method for controlling plant pathogens, characterized in that, The treatment includes the steps of using the soluble form of Streptomyces roseum NKZ-259 as described in claim 1 or the biological agent as described in claim 5 to treat the plants to be treated; the plant pathogen is Botrytis cinerea and / or tobacco mosaic virus.

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