Streptomyces noursei S83 nanoemulsion as well as preparation method and application thereof

Abstract

The invention relates to the field of biological prevention and treatment, in particular to a streptomyces noursei S83 nanoemulsion as well as a preparation method and application thereof. According to the invention, a biocontrol bacterium streptomyces noursei S83 for preventing and treating target leaf spot is obtained through strain excavation and screening, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.31252. According to the invention, a polysaccharide material is adopted as a carrier to carry out formula screening to prepare the streptomyces noursei S83 nano-pesticide, and the nano-emulsion obviously promotes the healthy growth of tobacco while improving the prevention effect of the streptomyces noursei S83 on the tobacco target leaf spot.

Description

Technical Field

[0001] The invention relates to the field of biological control, and in particular to a Streptomyces noursei S83 nanoemulsion and a preparation method and application thereof. Background Art

[0002] The annual yield loss caused by tobacco diseases accounts for about 8% of the total tobacco production, resulting in huge economic losses. Tobacco target spot is a new tobacco disease reported in my country in recent years. The disease is caused by Rhizoctonia solani, whose hyphae can survive in the soil for many years and produce sclerotia, making common prevention and control measures have little effect on the disease. Therefore, the disease has caused huge losses to both tobacco leaf quality and yield.

[0003] Excessive reliance on chemical pesticides to control tobacco diseases has led to problems such as reduced tobacco leaf quality and resistance, as well as environmental pollution. Therefore, it is urgent to develop safe and effective green control measures. As an important measure for the green development of agriculture, biological control uses biocontrol bacteria to control tobacco diseases. It is a non-toxic, residue-free and environmentally friendly method that meets the green development needs of the tobacco industry. Streptomyces metabolites have the functions of promoting growth and resisting diseases, and have important resource mining and application value for the prevention and control of tobacco diseases.

[0004] With the coordinated development of nanotechnology and interdisciplinary disciplines, nanopesticides have emerged in the field of pesticide formulations with their unique advantages. Among them, polysaccharide materials, especially chitosan-based materials, play a very important role in the preparation of nanopesticides. Chitosan and its derivatives can not only be used as carriers, but also have an inducing and promoting effect on plant growth. At present, there are few reports on the use of chitosan-based nanomaterials to prepare Streptomyces-based nanobiopesticides for the prevention and control of tobacco target spot disease. Summary of the invention

[0005] The purpose of the present invention is to provide a Streptomyces noursei S83 nanoemulsion and its preparation method and application to solve the problems existing in the above-mentioned prior art. The Streptomyces noursei S83 nanoemulsion of the present invention can effectively prevent and treat tobacco target spot disease.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a Streptomyces noursei S83 nanoemulsion, and the preparation method of the Streptomyces noursei S83 nanoemulsion comprises the following steps:

[0008] The fermentation liquid of Streptomyces noursei S83 is mixed with chitosan quaternary ammonium salt and carboxymethyl chitosan respectively to obtain a Streptomyces noursei S83-chitosan quaternary ammonium salt solution and a Streptomyces noursei S83-carboxymethyl chitosan solution; the deposit number of the Streptomyces noursei S83 is CGMCC No.31252;

[0009] The Streptomyces noursei S83-chitosan quaternary ammonium salt solution and the Streptomyces noursei S83-carboxymethyl chitosan solution are mixed to obtain the Streptomyces noursei S83 nanoemulsion.

[0010] Preferably, the volume ratio of the Streptomyces noursei S83-chitosan quaternary ammonium salt solution to the Streptomyces noursei S83-carboxymethyl chitosan solution is 1:1.

[0011] Preferably, the concentration of chitosan quaternary ammonium salt in the Streptomyces noursei S83-chitosan quaternary ammonium salt solution is 2-5 mg / mL; the concentration of carboxymethyl chitosan in the Streptomyces noursei S83-carboxymethyl chitosan solution is 1-2 mg / mL.

[0012] Preferably, the effective viable count of Streptomyces noursei S83 in the fermentation broth is 2.8×10 4 CFU / mL.

[0013] The invention provides a method for preparing a nanoemulsion of Streptomyces noursei S83. The method comprises mixing fermentation liquid of the Streptomyces noursei S83 with chitosan quaternary ammonium salt and carboxymethyl chitosan respectively to obtain a Streptomyces noursei S83-chitosan quaternary ammonium salt solution and a Streptomyces noursei S83-carboxymethyl chitosan solution. The deposit number of the Streptomyces noursei S83 is CGMCC No.31252.

[0014] The Streptomyces noursei S83-carboxymethyl chitosan solution and the Streptomyces noursei S83-carboxymethyl chitosan solution are mixed to obtain the Streptomyces noursei S83 nanoemulsion.

[0015] Preferably, the volume ratio of the Streptomyces noursei S83-chitosan quaternary ammonium salt solution to the Streptomyces noursei S83-carboxymethyl chitosan solution is 1:1.

[0016] Preferably, the concentration of chitosan quaternary ammonium salt in the Streptomyces noursei S83-chitosan quaternary ammonium salt solution is 2-5 mg / mL; the concentration of carboxymethyl chitosan in the Streptomyces noursei S83-carboxymethyl chitosan solution is 1-2 mg / mL.

[0017] Preferably, the effective viable count of Streptomyces noursei S83 in the fermentation broth is 2.8×10 4 CFU / mL.

[0018] The present invention provides application of the Streptomyces noursei S83 nanoemulsion in preventing and treating tobacco target spot disease.

[0019] The present invention provides a preparation for preventing and treating tobacco target spot disease, wherein the preparation comprises the above-mentioned Streptomyces noursei S83 nanoemulsion.

[0020] The present invention discloses the following technical effects:

[0021] The present invention screened out a biocontrol bacterium for preventing and controlling target spot disease, namely, Streptomyces noursei S83, through strain mining, and prepared a nanopesticide by using a polysaccharide material as a carrier for formula screening, which not only improved the prevention effect of Streptomyces noursei S83 on tobacco target spot disease, but also significantly promoted the healthy growth of tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 83; A is a morphological characteristic diagram of Streptomyces 83 in the early growth stage; B is a morphological characteristic diagram of Streptomyces 83 in the late growth stage; C is a mycelium morphology diagram of Streptomyces 83; D is a spore morphology diagram of Streptomyces 83;

[0024] Figure 2 This is the complete genome map of Streptomyces noursei S83;

[0025] Figure 3 This is the refraction result of Tyndall effect of Streptomyces noursei S83 nanoemulsion;

[0026] Figure 4 : are transmission electron micrographs of nanoemulsions of Streptomyces noursei S83; wherein A is a transmission electron micrograph of a nanoemulsion without the addition of fermentation broth of Streptomyces noursei S83, and B is a transmission electron micrograph of a nanoemulsion of Streptomyces noursei S83;

[0027] Figure 5 The contact angle change diagram of Streptomyces noursei S83 nanoemulsion after cold and hot storage; wherein, H5C5, H5C2 and H2C2 are nanoemulsions H5C5, H5C2 and H2C2 prepared using the fermentation liquid of Streptomyces noursei S83 as solvent respectively;

[0028] Figure 6 The figure shows the effect of nanoemulsion of Streptomyces noursei S83 on the morphology of tobacco target spot pathogen hyphae; A is a water control; BD are blank material nanoemulsions H5C5, H5C2 and H2C2 prepared with water as solvent; E is the fermentation broth of Streptomyces noursei S83; FH are nanoemulsions H5C5, H5C2 and H2C2 prepared with the fermentation broth of Streptomyces noursei S83 as solvent; the scale bar is 100 μm;

[0029] Figure 7 It is a graph for evaluating the nano-induced resistance performance of Streptomyces noursei S83; wherein, A is a statistical graph for POD activity detection; B is a statistical graph for SOD activity detection; C is a result of CAT activity detection; and D is a result of PPO activity detection. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] 1. Screening of biocontrol Streptomyces

[0037] 1.1. Screening of tobacco spot pathogens using fermentation broth

[0038] According to the red yeast experiment, 17 biocontrol Streptomyces with good antagonistic effects (the strain names are shown in Table 1) were inoculated with 1% inoculation into fermentation medium (20 g soybean powder, 30 g corn powder, 20 g glucose, 4 g sodium chloride, 3 g calcium carbonate, 1 L water), and cultured in a shaker at 220 rpm and 28 °C for 72 h. The fermentation broth of the 17 biocontrol Streptomyces was filtered to obtain the fermentation filtrates of the 17 biocontrol Streptomyces. The effective viable bacterial counts of the fermentation filtrates were all 2.8 × 10 4 CFU / mL. Use a hole puncher to punch a tobacco target spot pathogen cake with a diameter of 0.6 cm and place it in the center of the PDA medium plate. Place an Oxford cup 2 cm to the left and right of the tobacco target spot pathogen cake and add 200 μL of different biocontrol Streptomyces fermentation liquid. A total of 17 biocontrol Streptomyces treatments were performed, that is, each biocontrol Streptomyces fermentation liquid was treated once, and each treatment was repeated 3 times. At the same time, the water treatment was used as the control group. When the colony of the control group grew to 2 / 3 of the entire plate, the inhibition zone was observed and measured, and the inhibition rate was calculated. The results are shown in Table 1.

[0039] Table 1 Inhibition zone diameter and inhibition rate of Streptomyces antagonistic to tobacco target spot

[0040]

[0041] The antibacterial activity of fermented broths of 17 Streptomyces species preserved in the laboratory against tobacco target spot pathogen was determined using the Oxford cup method. The results showed that the fermented broths of 14 Streptomyces species had inhibitory effects on tobacco target spot disease, among which Streptomyces 83 had the highest inhibition rate against tobacco target spot disease, reaching 64.6% (Table 1).

[0042] 2. Identification and characterization of Streptomyces 83

[0043] 2.1 Observation of mycelial morphology

[0044] Gao's No. 1 culture medium (brand name: Aoboxing, item number: S1090198) was heated to melt and then cooled inverted. A sterilized cover glass was inserted into the culture medium at an angle of about 40-45 degrees. Streptomyces 83 was picked up with an inoculation loop and a line was drawn at the junction of the culture medium and the cover glass. The culture was inverted. The hyphae and spore morphology were observed under a microscope. The results were as follows. Figure 1The results showed that the surface of the colony of Streptomyces 83 strain was dry, opaque, and tightly velvety, and it was tightly bound to Gao's medium No. 1; the colony was white at the beginning, and gray spores began to be produced on the 4th to 5th day of culture at 28℃, and the colony gradually turned gray. Under the microscope, it can be observed that the hyphae are multi-branched and unbroken. From the 4th day of culture, the hyphae partially differentiated into compact spore threads.

[0045] 2.2. Whole genome sequencing of Streptomyces 83

[0046] The whole genome of Streptomyces 83 was sequenced and the sequencing results were analyzed. The metabolites of 83 were analyzed using the antiSMASH database. The whole genome map of the strain is shown in Figure 2 The analysis results of the antiSMASH database are shown in Table 2.

[0047] The sequencing and assembly results showed that the total length of the whole genome was 8212422bp and the GC content was 72.54%. At the same time, the whole genome was annotated with gene functions in combination with NR, COG, KEGG and GO databases. A total of 8998 open reading frames were predicted in the whole genome of the strain, accounting for 86.9% of the total genome length. The coding genes, repetitive sequences, non-coding RNAs, etc. were predicted to obtain the composition of the Streptomyces 83 genome. The genome encodes 71 tRNAs (total length 5385bp), 7 5S rRNAs (total length 812bp), 7 16S rRNAs (total length 10619bp), 7 23S rRNAs (total length 21836bp) and 16 sRNAs (total length 2197bp).

[0048] The blast results showed that Streptomyces 83 was Streptomyces noursei S83, and its classification name was Streptomyces noursei. It was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection on July 10, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31252.

[0049] The analysis results of the antiSMASH database showed that there were 36 secondary metabolic gene clusters, including nucleosides, polyketides, non-ribosomal peptides, butyrolactones, lactic acid bacteria peptides, etc. The base positions and gene cluster types of specific secondary metabolic gene clusters were (Tables 2 and 3).

[0050] Table 2 Prediction of secondary metabolism biosynthesis gene clusters in the whole genome of Streptomyces noursei S83

[0051]

[0052] Table 3 Prediction of secondary metabolism biosynthesis gene clusters in the whole genome of Streptomyces noursei S83

[0053]

[0054] Note: The numbers are connected to the above table.

[0055] 2.3. Determination of physiological and biochemical characteristics of Streptomyces noursei S83

[0056] The culture medium formula used in the experiment is:

[0057] Gelatin liquefaction medium: 20 g glucose, 5 g peptone, 120 g gelatin, 1 L water, sterilized at 115 °C for 15 min;

[0058] Milk culture coagulation and peptone medium: 200g skim milk powder, 0.2g calcium carbonate, 1L water, sterilized at 115℃ for 15min;

[0059] Hydrogen sulfide medium: peptone 10g, ferric citrate 0.5g, agar powder 17g, water 1L;

[0060] Nitrate reduction medium: 0.5 g magnesium sulfate, 1 g potassium nitrate, 0.5 g potassium dihydrogen phosphate, 0.5 g sodium chloride, 20 g sucrose, 1 L water;

[0061] Gao's medium No. 1 containing starch: 0.5 g sodium chloride, 20 g soluble starch, 1 g potassium nitrate, 0.5 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 18 g agar, 1 L water;

[0062] Cellulose hydrolysis liquid medium: sodium chloride 0.5g, soluble starch 20g, potassium nitrate 1g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.5g, water 1L;

[0063] Phosphate-dissolving medium: glucose 10.0 g, ammonium sulfate 0.5 g, potassium chloride 0.3 g, sodium chloride 0.3 g, manganese sulfate 0.03 g, ferrous sulfate heptahydrate 0.03 g, magnesium sulfate 0.3 g, calcium phosphate 5.0 g, agar 20.0 g, water 1 L, pH 7.0-7.5;

[0064] Iron carrier production medium: agar powder 20g, 10× buffer (sterilized) 100mL, chrome azurol S (CAS) 0.06g; ferrous chloride hexahydrate 0.0027g, hexadecyltrimethylammonium bromide (MDTMA) 0.073g, water 1L;

[0065] Dextran agar medium: 4.0g Poria powder, 17.9g disodium hydrogen phosphate, 6.8g potassium dihydrogen phosphate, 6.7g yeast powder, 0.06g aniline blue, 15g agar powder, 1L water;

[0066] IAA production liquid medium: glucose 20.0 g / L, peptone 6.0 g / L, yeast extract 6.0 g / L, NaCl 10.0 g / L;

[0067] Carbon source utilization basic culture medium: 1 g diammonium hydrogen phosphate, 1 g sodium chloride, 0.2 g magnesium sulfate heptahydrate, 0.5 g potassium dihydrogen phosphate, 20 g agar, and 1 L distilled water.

[0068] Gelatin liquefaction test: Inoculate the S83 strain of Streptomyces noursei into a test tube containing gelatin liquefaction medium, culture at 28℃ for 10 days, then place the test tube in a 4℃ refrigerator for 0.5 hours to observe the degree of gelatin liquefaction. If the medium is partially or completely liquefied, it is a positive reaction; otherwise, it is a negative reaction.

[0069] Milk coagulation and peptonization: Inoculate the Streptomyces noursei S83 strain into a test tube containing milk culture medium by puncture. After culturing at 28°C for 7 days, if clots appear in the milk, it is coagulation. If it becomes liquid with continued culturing, it is peptonization, which is a positive reaction. Otherwise, it is a negative reaction.

[0070] Production of hydrogen sulfide: Inoculate the S83 strain of Streptomyces noursei on hydrogen sulfide culture medium, culture at 28℃ for 5 days, and observe the color change around the bacterial block. If the area around the bacterial block turns black, it means that hydrogen sulfide can be produced, which is a positive reaction; otherwise, it is a negative reaction.

[0071] Nitrate reduction test: Inoculate the Streptomyces noursei S83 strain into nitrate reduction medium, incubate at 28°C for 7-14 days, and then perform the test according to the operating procedures of the nitrate reduction kit.

[0072] Starch hydrolysis test: Inoculate the S83 strain of Streptomyces noursei into Gao's medium No. 1 containing starch, and culture it at 28℃ for 7 days. After the S83 strain of Streptomyces noursei grows well, drip iodine solution around the colony. If amylase is produced, the starch will be converted into dextrin or absorbed, and it will not turn blue when it encounters iodine solution, forming a transparent circle; otherwise, the colony will turn blue when it encounters iodine solution.

[0073] Cellulose hydrolysis test: Inoculate the S83 strain of Streptomyces noursei in a cellulose hydrolysis liquid medium with a filter paper strip, culture at 220 rpm at 28°C for 3 days and observe. If the filter paper breaks or becomes thinner, it means that the S83 strain of Streptomyces noursei can produce cellulase that can hydrolyze the filter paper, otherwise it can't.

[0074] Phosphate solubilization ability test: Inoculate the S83 strain of Streptomyces noursei on the phosphate solubilization medium and culture it at 28℃ for 5-7 days. Observe whether there is an obvious transparent circle around the colony, which means that the S83 strain of Streptomyces noursei has the ability to solubilize phosphate, otherwise it does not.

[0075] Siderophore production ability test: The Streptomyces noursei S83 strain was inoculated on the siderophore production medium and cultured at 28°C for 5-7 days. The formation of an orange halo around the colony was observed, indicating that the Streptomyces noursei S83 had the ability to synthesize siderophores, otherwise it did not.

[0076] Glucanase capacity test: Inoculate the Streptomyces noursei S83 strain on the glucan agar medium and culture it at 28°C for 5 days. If the transparent circle continues to increase with the growth of the Streptomyces noursei S83 colony, it means that glucanase is produced, otherwise it is not.

[0077] IAA production ability test: The Streptomyces noursei S83 strain was inoculated into the IAA-producing liquid culture medium, and cultured at 28°C, 220 rpm in a shaking incubator for 3 days. Then, an equal volume of Salkowski colorimetric solution (50 mL 35% HCl + 1 mL 0.5 mol / LFeCl3) was added, the mixture was allowed to stand in the dark for 3 minutes, and a color reaction was performed. If the fermentation broth of the Streptomyces noursei S83 strain turned pink, it proved that the secondary metabolism of the strain could produce IAA, otherwise it could not produce IAA.

[0078] Carbon source utilization test: 1% of galactose, maltose, sucrose, xylose, mannitol, glucose, lactose, rhamnose and fructose were added to the carbon source utilization basal culture medium respectively, and the Streptomyces noursei S83 strain was inoculated into the culture medium containing only one carbon source and cultured at 28°C. After 7 days, whether the Streptomyces noursei S83 strain can grow normally was observed. If it grows normally, the Streptomyces noursei S83 strain can utilize the carbon source, otherwise it cannot.

[0079] The survey results are shown in Table 4.

[0080] Table 4 Physiological and biochemical characteristics of Streptomyces noursei S83

[0081]

[0082] Note: + in the table indicates a positive reaction.

[0083] The results showed that after the S. noursei S83 strain was inoculated into the test plate or test tube and cultured for 5 days, a yellow halo formed by iron ions appeared on the test plate; it had the ability to reduce nitrate and hydrolyze starch, indicating that the S. noursei S83 strain could secrete amylase and nitrate reductase; it did not have the ability to secrete glucanase, dissolve organic phosphorus, coagulate and peptone milk, or hydrolyze cellulose; the gelatin liquefaction reaction result was negative; the hydrogen sulfide reaction result was negative. The S. noursei S83 strain had a high utilization rate of lactose, rhamnose, glucose, and fructose (Table 4).

[0084] Example 2 Preparation and formulation optimization of Streptomyces noursei S83 nanoemulsion

[0085] 1. Optimization of the formula of Streptomyces noursei S83 nanoemulsion

[0086] Using sterile water as solvent, chitosan quaternary ammonium salt (HTCC) and carboxymethyl chitosan (CMCS) were prepared into 1-5 mg / mL chitosan quaternary ammonium salt solution and carboxymethyl chitosan solution, respectively. Chitosan quaternary ammonium salt solution was used as the main component, and carboxymethyl chitosan solution was added dropwise while shaking. When milky white appeared, the addition was stopped (the volume ratio of chitosan quaternary ammonium salt solution to carboxymethyl chitosan solution was 1:1). After standing for 24 hours, the stability of the emulsion was observed. The mycelium growth rate method was used to detect the antibacterial activity of different concentration combinations with tobacco target spot pathogen as the target.

[0087] 2. Preparation and characterization of Streptomyces noursei S83 nanoemulsion

[0088] 2.1. Preparation of Streptomyces noursei S83 nanoemulsion

[0089] After determining the optimal combination based on the above two methods, the fermentation filtrate of Streptomyces noursei S83 (with an effective viable count of 2.8×10 4 CFU / mL) was used as a solvent to form a nanoemulsion of Streptomyces noursei S83, and the result was evaluated. The preparation method of the nanoemulsion of Streptomyces noursei S83 was as follows: using the fermentation broth of Streptomyces noursei S83 as a solvent, HTCC and CMCS were prepared into 2 mg / mL or 5 mg / mL of Streptomyces noursei S83-chitosan quaternary ammonium salt solution and Streptomyces noursei S83-carboxymethyl chitosan solution respectively; using the quaternary ammonium salt solution of Streptomyces noursei S83 with a concentration of 2 mg / mL or 5 mg / mL as the main body, adding 2 mg / mL of quaternary ammonium salt solution into the solution dropwise while shaking. or 5 mg / mL of Streptomyces noursei S83-carboxymethyl chitosan solution, and the addition was stopped when a milky white color appeared (the volume ratio of Streptomyces noursei S83-chitosan quaternary ammonium salt solution to Streptomyces noursei S83-carboxymethyl chitosan solution was 1:1), and the obtained Streptomyces noursei S83 nanoemulsions were recorded as H2C2, H5C2 and H5C5, respectively; using the fermentation broth of Streptomyces noursei S83 as the solvent, HTCC was prepared into a Streptomyces noursei S83-chitosan quaternary ammonium salt solution with a concentration of 5 mg / mL, and the obtained Streptomyces noursei S83 nanoemulsion was recorded as H5; wherein, H a C b Represents the Streptomyces noursei S83 nanoemulsion composed of amg / mL HTCC and bmg / mL CMCS in a 1:1 volume ratio.

[0090] 2.2 Characterization and evaluation of Streptomyces noursei S83 nanoemulsion

[0091] 2.2.1 Tyndall effect

[0092] An infrared laser pen is used to utilize the light scattering phenomenon to observe the "pathway" of a beam of light passing through the colloid to determine the formation of nanoparticles in the nanoemulsion and its Tyndall phenomenon.

[0093] 2.2.2 Zeta potential and particle size analysis of Streptomyces noursei S83 nanoemulsion

[0094] The hydrated particle size and potential of the Streptomyces noursei S83 nanoemulsion prepared under different conditions were measured using a nanoparticle size analyzer; the particle size of the nanoemulsion was analyzed using a transmission electron microscope.

[0095] 2.2.3 Determination of the wetting properties of Streptomyces noursei S83 nanoemulsion on tobacco leaf surfaces

[0096] The dynamic contact angle of the nanoemulsion on the tobacco leaf surface was measured by a contact angle meter to characterize the wetting and spreading performance. The surface tension of the Streptomyces noursei S83 nanoemulsion was measured by a surface tension meter using a platinum plate method.

[0097] 2.2.4 Effect of Streptomyces noursei S83 nanoemulsion on hyphae morphology of tobacco target spot disease

[0098] The Streptomyces noursei S83 nanoemulsion was added to PDA, and a piece of cellophane paper was spread on the culture medium. A 5 mm diameter block of tobacco target spot pathogen was placed in the center of the PDA culture medium and cultured in a constant temperature incubator at 28°C for 3 days. At the same time, clean water was used as the clean water control group, and the mycelial growth of the control group and the treatment group was observed under an optical microscope.

[0099] 3. Results

[0100] 3.1. Optimization of the formula of Streptomyces noursei S83 nanoemulsion

[0101] Nanoemulsion preparation and formula screening were carried out based on the two indicators of antibacterial rate and observation of whether nanoemulsion was produced. The results showed that nanoemulsion could be produced within the range of 2-5 mg / mL HTCC and 1-2 mg / mL CMCS, and no precipitation was produced after static observation, indicating that it had a certain antibacterial effect.

[0102] 3.2 Tyndall Effect

[0103] The results of the Tyndall effect test are as follows Figure 3 As shown, the results showed that: through observation, it was found that the three combinations of H5C5, H5C2 and H2C2 had obvious and clear non-dispersed pathways.

[0104] 3.3 Zeta potential and particle size analysis of Streptomyces noursei S83 nanoemulsion

[0105] The stability of the nanoemulsion was determined based on the changes in particle size and zeta potential. As shown in Table 5, the hydrated particle size distribution of H5C2 and H2C2 was uniform, and the absolute value of Zeta potential was high, indicating that the system was relatively stable. Figure 4 It can be seen that the nanoemulsion particle size is relatively small, less than 500nm.

[0106] Table 5 Particle size of nanoemulsions of Streptomyces noursei S83 with different emulsifying material ratios

[0107]

[0108] 3.4 Determination of the wetting properties of Streptomyces noursei S83 nanoemulsion on tobacco leaf surfaces

[0109] pass Figure 5 It can be seen that the contact angle of the tobacco leaves treated with the nanoemulsion of Streptomyces noursei S83 was significantly reduced, and through the measurement of surface tension, the surface tension value of the nanoemulsion treatment was much lower than that of water, reaching 40mN / m, indicating that the nanoemulsion of Streptomyces noursei S83 can better wet and adhere to the surface of tobacco leaves (Table 6).

[0110] Table 6 Surface tension measurements of Streptomyces noursei S83 nanoemulsion

[0111]

[0112] Note: CK is the clean water control; CK+ is the fermentation broth of Streptomyces noursei S83 alone.

[0113] 3.5 Effect of Streptomyces noursei S83 nanoemulsion on the hyphae morphology of tobacco target spot disease

[0114] The hyphae morphology was observed under a microscope. The hyphae of the water control group were thick and strong, with longer internodes. The angle between the newly grown hyphae and the parent hyphae was about 30°-45°, and they grew regularly. The remaining treatment groups showed many branches, deformities, short and thick branches, etc. ( Figure 6 ).

[0115] Example 3 Effect of Streptomyces noursei S83 Nanoemulsion on Tobacco Growth

[0116] According to the criteria for the safety evaluation of pesticides on crops (NY / T1965.1-2010), the potted method was used to spray tobacco when it grew to 3-4 leaves. The sprayed preparation was a nanoemulsion of Streptomyces noursei S83 (H a C b, the preparation method of the nanoemulsion of Streptomyces noursii S83 is the same as that of Example 2), the dosage is 5 mL / plant, 1 clear water control and 1 fermentation liquid control of Streptomyces noursii S83 are set, 10 plants are treated each time, and 3 repetitions are performed for each treatment. When the tobacco leaves are 3-4, 5 tobacco plants with relatively consistent growth are selected in each repetition, and the height from the soil surface to the tobacco growth point is measured as the plant height, and the plant height growth rate and plant height growth inhibition rate are calculated. The calculation formula of the plant height growth rate is plant height growth rate (mm / d) = (plant height after application - plant height before application) / plant height before application × interval time before and after application; the calculation formula of the plant height growth inhibition rate is plant height growth inhibition rate = (blank control plant height growth rate - agent treatment plant height growth rate) / blank control plant height growth rate × 100%. 7, 14, and 21 days after spraying, tobacco plants were investigated for symptoms of phytotoxicity such as discoloration, necrosis, wilting, deformity, growth stagnation, and the number of affected plants. The results are shown in Table 7.

[0117] Table 7 Effects of Streptomyces noursei S83 nanoemulsion on tobacco plant growth

[0118]

[0119] Note: CK: clean water control; CK+: fermentation broth of Streptomyces noursei S83 control; the data in the table are mean ± standard error.

[0120] Its safety was evaluated according to the criteria for pesticide safety assessment on crops NY / T1965.1-2010, and it was found that the nanoemulsion of Streptomyces noursei S83 had no inhibitory effect on tobacco growth, no phytotoxicity occurred, and had a promoting effect on plant growth (Table 7).

[0121] Example 4 Indoor control effect of Streptomyces noursei S83 nanoemulsion on tobacco target spot

[0122] When the tobacco seedlings transplanted in the greenhouse grew to the 6-leaf stage, a protective test was conducted. First, the pesticide was sprayed on the leaves of the tobacco plants, and 6 treatments were set up, with 6 pots of tobacco in each treatment.

[0123] Among them, the agent used in treatment 1 is Streptomyces noursei S83 nanoemulsion H5C5; the agent used in treatment 2 is Streptomyces noursei S83 nanoemulsion H5C2; the agent used in treatment 3 is Streptomyces noursei S83 nanoemulsion H2C2; treatment 4 is a positive control, and the agent used is Streptomyces noursei S83 fermentation broth (CK+); treatment 5 is a negative control blank material, that is, the agent used is a nanoemulsion (CK-) prepared from chitosan quaternary ammonium salt (HTCC) with a concentration of 5 mg / mL and carboxymethyl chitosan with a concentration of 2 mg / mL; treatment 6 is a clear water control (CK); the preparation method of Streptomyces noursei S83 nanoemulsion is the same as that in Example 2.

[0124] 24 hours after spraying, the leaves were scraped and inoculated with tobacco boot spot pathogenic bacteria cakes with a diameter of 6 mm; the third and fourth true leaves of each plant were inoculated, and each leaf was inoculated with 6 bacterial cakes, which were moisturized with sterilized cotton. 48 hours after inoculation, the bacterial cakes and cotton were removed, and the pesticide was sprayed again. After 7 days, the incidence rate and disease index were investigated, and the control effect was calculated. The results are shown in Table 8.

[0125] Table 8 Control effect of Streptomyces noursei S83 nanoemulsion on tobacco target spot disease in potted plants

[0126]

[0127] Note: CK: clean water control; CK-: blank material; CK+: fermentation broth of Streptomyces noursei S83 control.

[0128] The plate assay determined that the nanoemulsion of Streptomyces noursii S83 had a good antibacterial effect on tobacco target spot. The results of the indoor pot test showed that the control effect of the combination of Streptomyces noursii S83 nanoemulsion was higher than that of the single treatment of Streptomyces noursii S83 fermentation liquid, the combination of H5C5 had a higher control effect, and the color of the tobacco leaves was greener after the combination of Streptomyces noursii S83 fermentation liquid and Streptomyces noursii S83 nanoemulsion was applied. At the same time, the chlorophyll and nitrogen content of tobacco leaves were measured using the chlorophyll meter of Zhejiang Top Yunnong Technology Co., Ltd., and the results are shown in Table 9.

[0129] Table 9 Effect of application of Streptomyces noursei S83 nanoemulsion on chlorophyll and nitrogen content in tobacco

[0130]

[0131] Note: CK: clean water control; CK-: blank material; H a C b : (i.e., nanoemulsion prepared from chitosan quaternary ammonium salt (HTCC) with a concentration of a and carboxymethyl chitosan with a concentration of b); CK+: fermentation broth of Streptomyces noursei S83 control.

[0132] The chlorophyll and nitrogen were measured, and it was found that the chlorophyll and nitrogen contents were significantly increased after using the fermentation broth of Streptomyces noursei S83 and the nanoemulsion of Streptomyces noursei S83, which were higher than those of the clear water control and the blank material, indicating that spraying the fermentation broth of Streptomyces noursei S83 and the nanoemulsion of Streptomyces noursei S83 can promote plant growth and photosynthesis (Table 9).

[0133] Afterwards, the enzyme activity kit was used to detect the activity of disease-resistant enzymes (CAT, POD, SOD, and PPO of tobacco) in the nanoemulsion of Streptomyces noursei S83. The results were as follows Figure 7As shown in the figure, the activities of CAT, POD, SOD, and PPO in the plants treated with the three combinations of Streptomyces noursei S83 nanoemulsions were all higher than those in the other treatments, and the enzyme activity was significantly increased, so that the tobacco plants showed stronger defense ability when infected by target spot pathogens.

[0134] Example 5 Field efficacy of Streptomyces noursei S83 nanoemulsion

[0135] The tobacco variety used for the test was Yunyan 87. The test site was set up in Fuquan City, Qiannan Prefecture, Guizhou Province.

[0136] The experiment was set up with 6 treatment groups, with the spraying of sterile water and Jinggangmycin as the control treatment, treatment 1 as blank material (same as Example 4); treatment 2 as Noursella S83 nanoemulsion H5C5, the preparation method of Noursella S83 nanoemulsion was the same as Example 2; each treatment was repeated 3 times, with a total of 15 plots. Each plot had 15 tobacco strains, arranged in random blocks, and each plot was about 30m 2 Use a backpack sprayer to spray the front and back sides of the leaves evenly so that the leaves are covered with fog spots. Each spraying should be carried out in sunny weather.

[0137] Conduct two surveys and make statistical records based on the lesion grading standards. Observe and record the disease situation 7 days before the first application and 7 days after the last application, and calculate the disease index and control effect based on the statistical disease level. In the fixed-plant survey, select 5 plants in each plot, use the percentage of the lesion area on the leaves to the total leaf area as the grading standard, and record the total number of leaves and the number of diseased leaves at each level.

[0138] Grading method: Grade 0: The whole plant (leaves) is disease-free; Grade 1: The area of ​​lesions accounts for less than 1% of the leaf area; Grade 3: The area of ​​lesions accounts for 2%-10% of the leaf area; Grade 5: The area of ​​lesions accounts for 11%-20% of the leaf area; Grade 7: The area of ​​lesions accounts for 21%-50% of the leaf area; Grade 9: The area of ​​lesions accounts for more than 51% of the leaf area.

[0139] The calculation formula of disease index is: disease index = ∑ (number of diseased leaves at each level × relative level value) / total number of leaves investigated × highest disease level × 100;

[0140] The calculation formula for the prevention effect is: Prevention effect = [1-(disease index of the control area before application × disease index of the treatment area after application) / disease index of the control area after application × disease index of the treatment area before application] × 100%.

[0141] The survey results are shown in Table 10.

[0142] Table 10 Field efficacy of Streptomyces S83 nanoemulsion against tobacco target spot

[0143]

[0144] After the indoor prevention test, it was found that the concentration combination of Streptomyces noursii S83 nanoemulsion H5C5 had the best control effect, so this combination was used for field tests, and 8% jinggangmycin, which is commonly used for the prevention and control of tobacco target spot pathogens, was selected as the control agent. The results are shown in Table 10. The prevention effect of Streptomyces noursii S83 nanoemulsion H5C5 on tobacco target spot pathogens reached 72.97%, which was significantly different from the fermentation liquid and blank material treatment groups; the prevention effect of jinggangmycin was 57.52%, which was not significantly different from the prevention effect of Streptomyces noursii S83 nanoemulsion H5C5.

[0145] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Streptomyces noursei S83 nanoemulsion, characterized in that: The preparation method of the Streptomyces noursei S83 nanoemulsion comprises the following steps: The Streptomyces noursei ( Streptomyces noursei ) The fermentation broth of S83 is mixed with chitosan quaternary ammonium salt and carboxymethyl chitosan respectively to obtain Streptomyces noursei S83-chitosan quaternary ammonium salt solution and Streptomyces noursei S83-carboxymethyl chitosan solution; the deposit number of the Streptomyces noursei S83 is CGMCC No.31252; The Streptomyces noursei S83-chitosan quaternary ammonium salt solution and the Streptomyces noursei S83-carboxymethyl chitosan solution are mixed to obtain the Streptomyces noursei S83 nanoemulsion.

2. The Streptomyces noursei S83 nanoemulsion according to claim 1, characterized in that The volume ratio of the Streptomyces noursei S83-chitosan quaternary ammonium salt solution to the Streptomyces noursei S83-carboxymethyl chitosan solution is 1:

1.

3. The Streptomyces noursei S83 nanoemulsion according to claim 1, characterized in that: The concentration of chitosan quaternary ammonium salt in the Streptomyces noursei S83-chitosan quaternary ammonium salt solution is 2-5 mg / mL; the concentration of carboxymethyl chitosan in the Streptomyces noursei S83-carboxymethyl chitosan solution is 1-2 mg / mL.

4. The Streptomyces noursei S83 nanoemulsion according to claim 1, characterized in that: The effective viable count of Streptomyces noursei S83 in the fermentation broth was 2.8×10 4 CFU / mL.

5. A method for preparing a nanoemulsion of Streptomyces noursei S83, characterized in that: The fermentation liquid of the Streptomyces noursei S83 is mixed with chitosan quaternary ammonium salt and carboxymethyl chitosan respectively to obtain a Streptomyces noursei S83-chitosan quaternary ammonium salt solution and a Streptomyces noursei S83-carboxymethyl chitosan solution; the deposit number of the Streptomyces noursei S83 is CGMCC No.31252; The Streptomyces noursei S83-chitosan quaternary ammonium salt solution and the Streptomyces noursei S83-carboxymethyl chitosan solution are mixed to obtain the Streptomyces noursei S83 nanoemulsion.

6. The preparation method according to claim 5, characterized in that: The volume ratio of the Streptomyces noursei S83-chitosan quaternary ammonium salt solution to the Streptomyces noursei S83-carboxymethyl chitosan solution is 1:

1.

7. The preparation method according to claim 5, characterized in that: The concentration of chitosan quaternary ammonium salt in the Streptomyces noursei S83-chitosan quaternary ammonium salt solution is 2-5 mg / mL; the concentration of carboxymethyl chitosan in the Streptomyces noursei S83-carboxymethyl chitosan solution is 1-2 mg / mL.

8. The preparation method according to claim 5, characterized in that: The effective viable count of Streptomyces noursei S83 in the fermentation broth was 2.8×10 4 CFU / mL.

9. Use of the Streptomyces noursei S83 nanoemulsion according to any one of claims 1 to 4 in preventing and treating tobacco target spot disease.

10. A preparation for preventing and treating tobacco target spot disease, characterized in that: The preparation comprises the Streptomyces noursei S83 nanoemulsion according to any one of claims 1 to 4.

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