Streptomyces roseoflavus NKZ-259 soluble concentrate as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological agriculture, in particular to a streptomyces roseoflavus NKZ-259 soluble concentrate as well as a preparation method and application thereof. According to the invention, by screening different types of chemical surfactants, two safe and compatible synergistic aids, namely alkyl polyglucoside and / or fatty alcohol-polyoxyethylene ether sodium sulfate, are finally screened out. In the formula provided by the invention, alkyl polyglucoside and / or fatty alcohol-polyoxyethylene ether sodium sulfate are / is used as an interface performance synergist, so that the wetting, spreading and adhesion capabilities of the streptomyces roseoflavus NKZ-259 on crop leaves can be remarkably improved; after the alkyl polyglucoside and the fatty alcohol-polyoxyethylene ether sodium sulfate are combined with the streptomyces roseoflavus NKZ-259, a remarkable synergistic effect can be realized. Moreover, the soluble concentrate provided by the invention is good in compatibility of all components and good in storage stability, and the disease-resistant effect of the streptomyces roseoflavus NKZ-259 can be remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bio-agriculture, in particular to a soluble solution of Streptomyces roseoflavus NKZ-259, a preparation method and application thereof. BACKGROUND

[0002] Streptomyces roseoflavus NKZ-259 is a biocontrol strain isolated from the soil of Qilian Mountain in Qinghai Province by the Agricultural Antibiotics Group of the State Key Laboratory of Plant Protection of the Chinese Academy of Agricultural Sciences, and has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 2, 2016, with the preservation number of CGMCC NO. 13416. The colony of Streptomyces roseoflavus NKZ-259 is light yellow to pink, with developed aerial hyphae and chain spores. It is gram-positive and grows aerobically. It has strong adaptability and can colonize in various soil environments and form a symbiotic relationship with plants. Studies have found that the strain has a significant promoting effect on plant growth. It can produce various secondary metabolites during fermentation, and also secrete plant hormones (such as IAA) or siderophores to promote root development and nutrient absorption, and plays an important role in promoting plant growth. At present, the application of the strain still relies on fermentation of the strain, filtration of the fermentation broth, and concentration treatment for spraying on crops.

[0003] At present, Streptomyces roseoflavus NKZ-259 can be prepared into two dosage forms of soluble solution or wettable powder. Since the strain is applied to prepare biopesticides, the active ingredients of the biopesticides mainly include live bacteria and metabolites, and there is no report on adding chemical surfactants to achieve synergistic effect in the formula to protect the biological activity, and there is also no research on the strain against plant viruses. SUMMARY

[0004] The purpose of the present application is to provide a soluble solution of Streptomyces roseoflavus NKZ-259, a preparation method and application thereof, to solve the problems existing in the prior art. Through screening a large number of different types of chemical surfactants, two safe and compatible synergistic adjuvants are finally selected, which have a significant synergistic effect after being combined with Streptomyces roseoflavus NKZ-259 in the formula, and provide a new research direction for the application of other types of biopesticides, which has important significance.

[0005] To achieve the above purpose, the present application provides the following scheme:

[0006] The present application provides a soluble solution of Streptomyces roseoflavus NKZ-259, which comprises the following components in mass percentage:

[0007] Streptomyces roseus NKZ-259 mother liquor 0-80%, synergistic surfactant 5%-15%, cosolvent 5%-20%, antifoaming agent 0.1-0.5%, antifreezing agent 3-5%, and the rest water;

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

[0009] Preferably, the Streptomyces roseus NKZ-259 soluble liquid includes the following components with the mass percentage content:

[0010] Streptomyces roseus NKZ-259 mother liquor 50%, synergistic surfactant 10%, cosolvent 10%, antifoaming agent 0.2%, antifreezing agent 5%, and the rest water.

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

[0012] Preferably, the cosolvent includes phenylacetone and / or N, N-dimethylacetamide;

[0013] Preferably, the antifoaming agent includes a polysiloxane antifoaming agent;

[0014] Preferably, the antifreezing agent includes 1,2-propanediol or glycerol.

[0015] The application provides a preparation method of the above-mentioned Streptomyces roseus NKZ-259 soluble liquid, which includes the step of mixing the mixture of the synergistic surfactant, the cosolvent, the antifoaming agent, the antifreezing agent, and the water with the Streptomyces roseus NKZ-259 mother liquor.

[0016] As an additional option, the application also provides an interface synergistic method of Streptomyces roseus NKZ-259, which includes the step of mixing the mixture of the synergistic surfactant, the cosolvent, the antifoaming agent, the antifreezing agent, and the water with the Streptomyces roseus NKZ-259 mother liquor.

[0017] The application provides the application of the above-mentioned Streptomyces roseus NKZ-259 soluble liquid in preventing and controlling plant pathogenic fungi.

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

[0019] The application provides the application of the above-mentioned Streptomyces roseus NKZ-259 soluble liquid in preparing a biological preparation for preventing and controlling plant pathogenic fungi.

[0020] The application provides a biological preparation for preventing and controlling plant pathogenic bacteria, and the biological preparation comprises the soluble solution of rosea streptomyces NKZ-259.

[0021] Further preferably, the plant pathogenic bacteria comprise botrytis and / or tobacco mosaic virus.

[0022] The application provides a method for preventing and controlling plant pathogenic bacteria, and the method comprises the steps of treating the plant to be treated by using the soluble solution of rosea streptomyces NKZ-259 or the biological preparation prepared by using the soluble solution of rosea streptomyces NKZ-259.

[0023] Further preferably, the plant pathogenic bacteria comprise botrytis and / or tobacco mosaic virus.

[0024] The application discloses the following technical effects:

[0025] The application screens different types of chemical surfactants, and finally screens two safe and compatible synergistic adjuvants, i.e., alkyl polyglucoside and sodium fatty alcohol polyoxyethylene ether sulfate. In the formula provided by the application, alkyl polyglucoside and sodium fatty alcohol polyoxyethylene ether sulfate are used as interface performance synergistic agents, so that the wetting, spreading and adhesion capacity of rosea streptomyces NKZ-259 on crop leaves can be significantly improved. After alkyl polyglucoside and sodium fatty alcohol polyoxyethylene ether sulfate are combined with rosea streptomyces NKZ-259, a significant synergistic effect can be achieved. Moreover, the soluble solution provided by the application has good compatibility and good storage stability, and can significantly improve the disease resistance of rosea streptomyces NKZ-259. The method for achieving synergistic effect by applying chemical surfactants in biological pesticide preparation can provide reference and reference for the research and application of other types of biological pesticides, provide a new direction for the research and application of other types of biological pesticides, and has important significance.

[0026] The application first discloses the new application of the soluble solution in preventing and controlling plant pathogenic bacteria. Experiments prove that the soluble solution can better play a role in preventing and controlling plant pathogenic bacteria under the formula provided by the application. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0028] Figure 1 It is a top view and B is a front view;

[0029] Figure 2 For the sample figure of Comparative Example 3; wherein A is a top view and B is a front view;

[0030] Figure 3 For the dynamic surface tension curve figure of 4 experimental samples related to 0.5wt% Streptomyces roseus NKZ-259 soluble solution in Example 4; wherein 4#375x is 375 times dilution of 4# sample to be tested, 4#750x is 750 times dilution of 4# sample to be tested, 1#375x is 375 times dilution of 1# sample to be tested, 1#750x is 750 times dilution of 1# sample to be tested, 2#375x is 375 times dilution of 2# sample to be tested, 2#750x is 750 times dilution of 2# sample to be tested, 3#375x is 375 times dilution of 3# sample to be tested, 3#750x is 750 times dilution of 3# sample to be tested;

[0031] Figure 4 For the inhibition effect figure of 4 experimental samples related to 0.5wt% Streptomyces roseus NKZ-259 soluble solution in Example 5 on Botrytis cinerea; wherein A is 1# inhibition effect figure, the left side is CK and the right side is 0.5wt% Streptomyces roseus NKZ-259 soluble solution stock solution prepared in Example 1; B is 2# inhibition effect figure, the left side is CK and the right side is 0.5wt% Streptomyces roseus NKZ-259 soluble solution stock solution prepared in Example 2; C is 3# inhibition effect figure, the left side is CK and the right side is 0.5wt% Streptomyces roseus NKZ-259 soluble solution stock solution prepared in Example 3; D is 4# inhibition effect figure, the left side is CK and the right side is 0.5wt% Streptomyces roseus NKZ-259 soluble solution stock solution prepared in Example 4;

[0032] Figure 5The inhibition effect diagram of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution (1#) prepared in Example 1 on Botrytis cinerea; from left to right, each hole is CK, 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 1, 10-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 1, 20-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 1, 50-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 1, 100-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 1, 200-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 1, and 500-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 1;

[0033] Figure 6 The inhibition effect diagram of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution (2#) prepared in Example 2 on Botrytis cinerea; from left to right, each hole is CK, 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 2, 10-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 2, 20-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 2, 50-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 2, 100-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 2, 200-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 2, and 500-fold dilution of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution stock solution prepared in Example 2;

[0034] Figure 7 The antiviral effect diagram of 5 experimental groups related to 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution on TMV; wherein, A is a staining diagram; B is a statistical diagram; * is significantly different from CK, and n.s. is not significantly different from CK. DETAILED DESCRIPTION

[0035] The following detailed description of various exemplary embodiments of the application will not be considered limiting of the application, but rather a description of certain aspects, features, and embodiments of the application.

[0036] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values of the parameter are combined with a stated value of the parameter in range form. These are only exemplary of the various preferred embodiments and are not intended to be limiting of the application. Other embodiments will occur to those skilled in the art upon consideration of this disclosure. Additionally, other combinations, permutations, applications and modifications of the application will occur to those skilled in the art upon consideration of this disclosure. The application is not to be limited, therefore, by the illustrative embodiments and logical modifications thereof.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not construed as an admission that it is prior art with respect to the present application.

[0038] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only and are not intended to be limiting.

[0039] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0040] The application will be further described in the following detailed description of specific embodiments with reference to the attached drawings, of which:

[0041] The experimental methods in the following examples are routine methods unless otherwise specified.

[0042] The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0043] In the preparation of the soluble liquid formulation containing 0.5 wt% of Streptomyces roseus NKZ-259, the selection of the types of various raw material components is crucial to the successful preparation of the soluble liquid formulation with stable properties and synergistic effects. Therefore, the inventors compared and screened a plurality of raw materials, and the specific experiments are as follows:

[0044] Example 10. Preparation of 5 wt% Streptomyces roseoflavus NKZ-259 soluble concentrate

[0045] 1% Streptomyces roseoflavus NKZ-259 mother liquor preparation: Streptomyces roseoflavus NKZ-259 was inoculated on MS medium and cultured at 28°C in the dark for 7 days, and 1 cm 2 sized mycelium block was inoculated into fermentation medium and cultured at 28°C with 220 r / min shaking for 96 h. After filtration with filter paper, Streptomyces roseoflavus NKZ-259 mother liquor was obtained, and 1% Streptomyces roseoflavus NKZ-259 mother liquor was obtained. The preservation number of Streptomyces roseoflavus NKZ-259 is CGMCC NO. 13416, and the classification and naming of the strain is Streptomyces roseoflavus. The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 2, 2016, and the address is No. 1, Beichen West Road, Chaoyang District, Beijing, China. The strain is recorded in the authorized patent ZL201710044242.4, and the invention name is: a strain of Streptomyces roseoflavus and its application.

[0046] 1% Streptomyces roseoflavus NKZ-259 mother liquor: 50%, synergistic surfactant Agnique PG 8107: 10% (alkyl polyglucoside, BASF), cosolvent phenacetin: 10% (analytical pure, Shanghai Maikelin Biochemical Technology Co., Ltd.), defoamer SAG 1522: 0.2% (silicone, Momentive Organics Materials (Shanghai) Co., Ltd.), and antifreeze 1,2-propanediol: 5% (analytical pure, Shanghai Maikelin Biochemical Technology Co., Ltd.) were weighed by mass percentage, and deionized water (third grade water, laboratory self-made) was added to 100%.

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

[0048] (1) Deionized water, synergistic surfactant Agnique PG 8107, cosolvent phenacetin, antifreeze 1,2-propanediol and defoamer SAG 1522 were added to the reaction kettle in proportion, and stirred until uniform;

[0049] (2) 1% Streptomyces roseoflavus NKZ-259 mother liquor was added to the above mixed solution in proportion, and stirred uniformly to obtain 0.5 wt% Streptomyces roseoflavus NKZ-259 soluble concentrate.

[0050] The preparation process was carried out at room temperature.

[0051] Example 20. Preparation of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution

[0052] 1% Streptomyces roseosporus NKZ-259 stock solution was prepared according to Example 1.

[0053] 1% Streptomyces roseosporus NKZ-259 stock solution was prepared according to Example 1.

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

[0055] (1) Deionized water, synergistic surfactant W1635, cosolvent N,N-dimethylacetamide, antifreeze glycerol and defoamer SAG 1522 were added to the reaction kettle in proportion, and stirred until uniform;

[0056] (2) 1% Streptomyces roseosporus NKZ-259 stock solution was added to the above mixed solution in proportion, and stirred uniformly to obtain 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution.

[0057] The preparation process was carried out at room temperature.

[0058] Example 20. Preparation of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution

[0059] The synergistic surfactant Agnique PG 8107 in Example 1 was replaced with Tween 80 (sorbitan monooleate polyoxyethylene ether, Xingtai Yancheng Chemical Auxiliary Co., Ltd.), and other components and preparation methods remained unchanged. The result sample became viscous, and had no inhibitory effect on Botrytis cinerea and tobacco virus TMV. The pathogen and virus inhibition effect is shown in Example 5 and Example 6, and the sample 3# in Comparative Example 1.

[0060] Example 20. Preparation of 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution

[0061] The synergistic surfactant Agnique PG 8107 in Example 2 was replaced with surfactin, and other components and preparation methods remained unchanged. After the sample was prepared, white block flocculation appeared, as shown in Figure 1

[0062] Comparative Example 3​

[0063] Example 1, other components and preparation method remain unchanged, after sample preparation, stratification, incompatible with the system, such as Figure 2

[0064] Example 3: 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution with different adjuvants added and different formulations of insecticides and fungicides were used for verification of tank mixing stability experiment

[0065] Select microemulsion, emulsion, suspension, emulsion, wettable powder, soluble solution, dispersible oil suspension and water dispersible granules and other different formulations of fungicides and insecticides were tank mixed with 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution at the use dilution, and the tank mixing stability was observed (30min), wherein the 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution was diluted 375 times.

[0066] Test samples: 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution prepared in Example 1 (1#), 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution prepared in Example 2 (2#), 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution prepared in Comparative Example 1 (3#) and 0.5wt% Streptomyces roseosporus NKZ-259 stock solution (made from 1% Streptomyces roseosporus NKZ-259 stock solution and deionized water, 4#).

[0067] Tank mixing agents: 10% pyraclostrobin microemulsion, 20% penthiopyrad·fluquinconazole emulsion, 30% difenoconazole suspension, 40% isoprothiolane emulsion, 60% carbendazim·thiram wettable powder, 75% trifloxystrobin·tebuconazole water dispersible granules, 30% diafenthiuron·chlorantraniliprole suspension, 20% dinotefuran·spirodiclofen suspension, 5.6% xycimbam soluble solution, 1% emamectin benzoate microemulsion and 30% tetraconazole·fluazinam suspension.

[0068] The results of the tank mixing experiment are shown in Table 1. The results show that 0.5wt% Streptomyces roseosporus NKZ-259 soluble solution has good compatibility performance in tank mixing with each formulation of conventional chemical pesticides, and there is no flocculation and precipitation phenomenon.

[0069] Table 1: Tank mixing stability performance of 4 experimental samples related to Streptomyces roseosporus NKZ-259 and each formulation of conventional chemical pesticides

[0070] Serial number Fungicide name and dosage form Dilution ratio Sample to be tested 1 10% pyraclostrobin microemulsion 230 1#-4# are all good 2 20% pyithiadin · flutriafol emulsion in water 2000 1#-4# are all good 3 30% benzoxaole suspension concentrate 4000 1#-4# are all good 4 40% isoprothiolane emulsion 267 1#-4# are all good 5 60% carbendazim · thiram wettable powder 300 1#-4# are all good 6 75% trifloxystrobin · tebuconazole water dispersible granules 3500 1#-4# are all good 7 30% diafenthiuron · chlorfenapyr suspension concentrate 2000 1#-4# are all good 8 20% dinotefuran · spirotetramat suspension concentrate 2000 1#-4# are all good 9 5.6% xycimbamine soluble concentrate 1500 1#-4# are all good 10 1% emamectin benzoate microemulsion 1500 1#-4# are all good 11 30% tetraconazole · fluopicolide suspension concentrate 1500 1#-4# are all good

[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. The agent was sprayed again 1-2 days after the 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 components include the following components by mass percentage: 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. The mass percentage of the mother liquor of Streptomyces roseum NKZ-259 is not 0.

2. The soluble form of Streptomyces roseum NKZ-259 according to claim 1, characterized in that, The Streptomyces roseum NKZ-259 soluble concentrate comprises 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 balance being water.

3. The soluble form of Streptomyces roseum NKZ-259 according to claim 1, characterized in that, 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.

4. The method for preparing the soluble form of Streptomyces roseum NKZ-259 according to claims 1-3, 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.

5. The application of the soluble Streptomyces roseum NKZ-259 according to any one of claims 1-3 in the control of plant pathogens.

6. The application according to claim 5, characterized in that, The plant pathogens include gray mold and / or tobacco mosaic virus.

7. The use of the soluble Streptomyces roseum NKZ-259 according to any one of claims 1-3 in the preparation of biological agents for the control of plant pathogens.

8. The application according to claim 7, characterized in that, The plant pathogens include gray mold and / or tobacco mosaic virus.

9. A biological agent for controlling plant pathogens, characterized in that, The soluble form of Streptomyces roseum NKZ-259 as described in any one of claims 1-3.

10. A method for controlling plant pathogens, characterized in that, The procedure includes treating the plants to be treated with a soluble agent of Streptomyces roseum NKZ-259 as described in any one of claims 1-3 or a biological agent prepared using a soluble agent of Streptomyces roseum NKZ-259 as described in claim 9.

Images