A Streptomyces noursei S83 nanoemulsion, its preparation method and application

Abstract

The present invention relates to the field of biological control, and particularly to a nanoemulsion of Streptomyces noelsii S83, a preparation method thereof, and an application thereof. Through strain screening, a biocontrol bacterium for controlling target spot disease, Streptomyces noelsii S83, was obtained. The preservation number of this strain is CGMCC No. 31252. The present invention uses a polysaccharide material as a carrier to screen and prepare a nano-pesticide of Streptomyces noelsii S83. While improving the control effect of Streptomyces noelsii S83 on tobacco target spot disease, this nanoemulsion significantly promotes the healthy growth of tobacco.

Description

Technical Field

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

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

[0003] Over-reliance on chemical pesticides to control tobacco diseases has led to problems such as a decline in tobacco leaf quality, resistance, and 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, which is a non-toxic, residue-free, and environmentally friendly method, meeting the green development needs of the tobacco industry. The metabolites of Streptomyces have functions such as promoting growth and disease resistance, and have important resource exploration and application value for the control of tobacco diseases.

[0004] With the coordinated development of nanotechnology and interdisciplinary disciplines, nano-pesticides 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 nano-pesticides. Chitosan and its derivatives can not only be used as carriers, but also show induction resistance and promotion effects on plant growth. At present, there are few reports on the preparation of nano-biological pesticides based on Streptomyces using chitosan-based nanomaterials to control 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 described in the present invention can effectively control tobacco target spot disease.

[0006] To achieve the above purpose, 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 includes the following steps:

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

[0009] The Streptomyces noursei S83 - quaternary ammonium chitosan 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 - quaternary ammonium chitosan solution to the Streptomyces noursei S83 - carboxymethyl chitosan solution is 1:1.

[0011] Preferably, the concentration of quaternary ammonium chitosan in the Streptomyces noursei S83 - quaternary ammonium chitosan 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 present invention provides a method for preparing a Streptomyces noursei S83 nanoemulsion. The fermentation broth of Streptomyces noursei S83 is mixed with quaternary ammonium chitosan and carboxymethyl chitosan respectively to obtain a Streptomyces noursei S83 - quaternary ammonium chitosan solution and a Streptomyces noursei S83 - carboxymethyl chitosan solution; the preservation number of 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 - quaternary ammonium chitosan solution to the Streptomyces noursei S83 - carboxymethyl chitosan solution is 1:1.

[0016] Preferably, the concentration of quaternary ammonium chitosan in the Streptomyces noursei S83 - quaternary ammonium chitosan 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 noelsii S83 in the fermentation broth is 2.8×10 4 CFU / mL.

[0018] The present invention provides the application of the above-mentioned Streptomyces noelsii S83 nanoemulsion in preventing and controlling tobacco target spot disease.

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

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

[0021] Through strain screening, the present invention has obtained a biocontrol bacterium for preventing and controlling target spot disease, Streptomyces noelsii S83, and used a polysaccharide material as a carrier to screen the formulation to prepare a nano-pesticide. While improving the control effect of Streptomyces noelsii S83 on tobacco target spot disease, it significantly promotes the healthy growth of tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is the morphological characteristic diagram of Streptomyces 83; among them, A is the morphological characteristic diagram of Streptomyces 83 in the early growth stage; B is the morphological characteristic diagram of Streptomyces 83 in the late growth stage; C is the hyphal morphological diagram of Streptomyces 83; D is the spore morphological diagram of Streptomyces 83;

[0024] Figure 2 It is the whole genome map of Streptomyces noelsii S83;

[0025] Figure 3 It is the Tyndall effect refraction result diagram of Streptomyces noelsii S83 nanoemulsion;

[0026] Figure 4 It is the transmission electron microscope diagram of Streptomyces noelsii S83 nanoemulsion; among them, A is the transmission electron microscope diagram of the nanoemulsion without adding the fermentation broth of Streptomyces noelsii S83, and B is the transmission electron microscope diagram of Streptomyces noelsii S83 nanoemulsion;

[0027] Figure 5 It is the contact angle change diagram of Streptomyces noelsii S83 nanoemulsion after cold and heat storage; among them, H5C5, H5C2 and H2C2 are the nanoemulsions H prepared with the fermentation broth of Streptomyces noelsii S83 as the solvent in sequence 5 C5 , H 5 C 2 and H 2 C 2 ;

[0028] Figure 6 Figure showing the morphological effect of Streptomyces nogalater S83 nanoemulsion on the hyphae of tobacco target spot pathogen; among them, A is the water control; B - D are the nanoemulsions of blank materials using water as the solvent, H 5 C 5 and H 5 C 2 and H 2 C 2 ; E is the fermentation broth of Streptomyces nogalater S83; F - H are the nanoemulsions prepared using the fermentation broth of Streptomyces nogalater S83 as the solvent, H 5 C 5 and H 5 C 2 and H 2 C 2 ; The scale bar is 100 μm;

[0029] Figure 7 Figure for evaluating the nano - induced resistance performance of Streptomyces nogalater S83; among them, A is the statistical chart of POD activity detection; B is the statistical chart of SOD activity detection; C is the detection result of CAT activity; D is the detection result of PPO activity. Detailed implementation manners

[0030] Now, various exemplary implementation manners of the present invention will 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, characteristics, and implementation schemes of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0032] Unless otherwise noted, 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 invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0033] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0034] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] 1. Screening of biocontrol Streptomyces

[0037] 1.1 Screening of fermentation broth against tobacco target spot pathogen

[0038] Seventeen biocontrol Streptomyces strains (see Table 1 for strain names) with relatively good antagonistic effects preliminarily screened according to the Rhodotorula experiment were inoculated into the fermentation medium (20 g of soybean powder, 30 g of corn flour, 20 g of glucose, 4 g of sodium chloride, 3 g of calcium carbonate, 1 L of water) at an inoculation amount of 1%, and cultured in a shaker at 220 rpm and 28 °C for 72 h. The fermentation broth of the seventeen biocontrol Streptomyces strains was filtered to obtain the fermentation filtrates of the seventeen biocontrol Streptomyces strains. The effective viable count of the fermentation filtrates was 2.8×10 4 CFU / mL. A tobacco target spot pathogen cake with a diameter of 0.6 cm was punched out using a punch and placed in the center of a PDA medium plate. An Oxford cup was placed 2 cm to the left and right of the tobacco target spot pathogen cake, and 200 μL of the fermentation broth of different biocontrol Streptomyces strains was added. There were a total of 17 biocontrol Streptomyces treatments, that is, each fermentation broth of biocontrol Streptomyces was treated once, and each treatment was repeated 3 times. At the same time, a water treatment was used as the control group; when the colonies in 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 1 The 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 taxonomic name was Streptomyces noursei. It was deposited in the China General Microbiological Culture Collection Center (CGMCC) on July 10, 2024. The deposit address was the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number was CGMCC No. 31252.

[0049] The analysis results of the antiSMASH database showed that there were 36 secondary metabolite gene clusters in total. The types of gene clusters included: nucleosides, polyketides, non-ribosomal peptides, butyrolactones, lantibiotics, etc. The base positions and gene cluster types of specific secondary metabolite gene clusters are shown in Tables 2 and 3.

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

[0051]

[0052] Table 3 Prediction of secondary metabolite biosynthetic 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 the physiological and biochemical characteristics of Streptomyces noursei S83

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

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

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

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

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

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

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

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

[0064] Siderophore-producing medium: 20 g of agar powder, 100 mL of 10× buffer (sterilized), 0.06 g of chrome azurol S (CAS); 0.0027 g of ferrous chloride hexahydrate, 0.073 g of cetyltrimethylammonium bromide (MDTMA), 1 L of water;

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

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

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

[0068] Gelatin liquefaction test: Streptomyces nogalater S83 strain was stab inoculated into a test tube containing gelatin liquefaction medium. After culturing at 28 °C for 10 d, the test tube was placed in a 4 °C refrigerator for 0.5 h, and the degree of gelatin liquefaction was observed. If the medium was partially or completely liquefied, it was a positive reaction; otherwise, it was a negative reaction.

[0069] Milk coagulation and peptization: Streptomyces nogalater S83 strain was stab inoculated into a test tube containing milk coagulation and peptization medium. After culturing at 28 °C for 7 d, if milk clots appeared, it was a coagulation phenomenon. If it became liquid again after continuous culturing, it was a peptization phenomenon, that is, a positive reaction; otherwise, it was a negative reaction.

[0070] Hydrogen sulfide production: Streptomyces nogalater S83 strain was inoculated on hydrogen sulfide medium. After culturing at 28 °C for 5 d, the color change around the bacterial mass was observed. If the area around the bacterial mass turned black, it indicated the ability to produce hydrogen sulfide, which was a positive reaction; otherwise, it was a negative reaction.

[0071] Nitrate reduction test: Streptomyces nogalater S83 strain was inoculated into nitrate reduction medium and cultured statically at 28 °C for 7 - 14 d. Then, it was detected according to the operation steps of the nitrate reduction kit.

[0072] Starch hydrolysis test: The Streptomyces nogalater S83 strain was inoculated into Gause's No. 1 medium containing starch and cultured at 28 °C for 7 d. After the Streptomyces nogalater S83 strain grew well, iodine solution was dropped around the colony. If amylase was produced, the starch would be turned into dextrin or utilized and absorbed, and it would not turn blue when encountering iodine solution, forming a transparent circle; otherwise, the colony would turn blue when encountering iodine solution.

[0073] Cellulose hydrolysis test: The Streptomyces nogalater S83 strain was inoculated into a cellulose hydrolysis liquid medium with filter paper strips and observed after culturing at 28 °C and 220 rpm for 3 d. If the filter paper broke or became thinner, it indicated that the Streptomyces nogalater S83 strain could produce cellulase to hydrolyze the filter paper; otherwise, it could not.

[0074] Phosphate-solubilizing ability test: The Streptomyces nogalater S83 strain was inoculated on a phosphate-solubilizing medium and cultured at 28 °C for 5 - 7 d. Whether an obvious transparent circle appeared around the colony was observed. If so, it indicated that the Streptomyces nogalater S83 had phosphate-solubilizing ability; otherwise, it did not.

[0075] Siderophore-producing ability test: The Streptomyces nogalater S83 strain was inoculated on a siderophore-producing medium and cultured at 28 °C for 5 - 7 d. If an orange-yellow halo formed around the colony, it indicated that the Streptomyces nogalater S83 had the ability to synthesize siderophores; otherwise, it did not.

[0076] Glucanase ability test: The Streptomyces nogalater S83 strain was inoculated on a dextran agar medium and cultured at 28 °C for 5 d. If the transparent circle continuously enlarged as the Streptomyces nogalater S83 colony grew, it indicated that glucanase was produced; otherwise, it was not.

[0077] IAA-producing ability test: The Streptomyces nogalater S83 strain was inoculated into an IAA-producing liquid medium and cultured on a shaker at 28 °C and 220 rpm for 3 d. Then, an equal volume of Salkowski colorimetric solution (50 mL of 35% HCl + 1 mL of 0.5 mol / L FeCl 3 ) was added and the mixture was allowed to stand for 3 min under dark conditions for color development reaction. If the fermentation broth of the Streptomyces nogalater S83 strain turned pink, it proved that the strain could produce IAA through secondary metabolism; otherwise, it could not produce IAA.

[0078] Carbon source utilization test: Add 1% of nine carbon sources, namely galactose, maltose, sucrose, xylose, mannitol, glucose, lactose, rhamnose, and fructose, into the basal medium for carbon source utilization respectively. Inoculate Streptomyces noursei S83 strain into the medium containing only one carbon source and culture it at 28 °C. After 7 days, observe whether Streptomyces noursei S83 strain can grow normally. If it can grow normally, it means that Streptomyces noursei S83 strain can utilize this carbon source; otherwise, it cannot utilize it.

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

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

[0081]

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

[0083] The results show that: After Streptomyces noursei S83 strain was inoculated into the detection plate or test tube and cultured for 5 days, a yellow halo formed by iron ions appeared on the detection plate; and it had the ability of nitrate reduction and starch hydrolysis, indicating that Streptomyces noursei S83 can secrete amylase and nitrate reductase; it did not have the ability to secrete dextranase, the ability to dissolve organic phosphorus, the ability of milk coagulation and peptone formation, and the ability of cellulose hydrolysis; the result of gelatin liquefaction reaction was negative; the result of hydrogen sulfide reaction was negative. Streptomyces noursei S83 strain had a relatively 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. Formulation optimization of Streptomyces noursei S83 nanoemulsion

[0086] Using sterile water as the solvent, prepare chitosan quaternary ammonium salt (HTCC) solution and carboxymethyl chitosan (CMCS) solution with concentrations of 1 - 5 mg / mL respectively. Taking the chitosan quaternary ammonium salt solution as the main body, slowly add the carboxymethyl chitosan solution drop by drop while shaking, and stop adding when it turns milky white (the volume ratio of chitosan quaternary ammonium salt solution to carboxymethyl chitosan solution is 1:1). After standing for 24 hours, observe the stability of the emulsion. Use the mycelial growth rate method to detect the antibacterial activity of different concentration combinations with tobacco target spot pathogen as the target.

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

[0088] 2.1 Preparation of Streptomyces noursei S83 nanoemulsion

[0089] After determining the optimal combination according to the above two methods, using the fermentation filtrate of Streptomyces noelsii S83 (the effective viable count is 2.8×10 4 CFU / mL) as the solvent to form the nanoemulsion of Streptomyces noelsii S83, and then evaluating it. Among them, the preparation method of the nanoemulsion of Streptomyces noelsii S83 is as follows: using the fermentation broth of Streptomyces noelsii S83 as the solvent, preparing the Streptomyces noelsii S83-chitosan quaternary ammonium salt solution and the Streptomyces noelsii S83-carboxymethyl chitosan solution of 2mg / mL or 5mg / mL respectively with HTCC and CMCS; using the Streptomyces noelsii S83-chitosan quaternary ammonium salt solution with a concentration of 2mg / mL or 5mg / mL as the main body, adding the Streptomyces noelsii S83-carboxymethyl chitosan solution with a concentration of 2mg / mL or 5mg / mL drop by drop while shaking, and stopping dropping when it turns milky white (the volume ratio of the Streptomyces noelsii S83-chitosan quaternary ammonium salt solution to the Streptomyces noelsii S83-carboxymethyl chitosan solution is 1:1). The obtained nanoemulsions of Streptomyces noelsii S83 are respectively denoted as H 2 C 2 、H 5 C 2 and H 5 C 5 ; using the fermentation broth of Streptomyces noelsii S83 as the solvent, preparing the Streptomyces noelsii S83-chitosan quaternary ammonium salt solution with a concentration of 5mg / mL with HTCC, and the obtained nanoemulsion of Streptomyces noelsii S83 is denoted as H 5 ; among them, H a C b represents the nanoemulsion of Streptomyces noelsii S83 composed of amg / mL HTCC and bmg / mL CMCS in a volume ratio of 1:1.

[0090] 2.2. Characterization and evaluation of the nanoemulsion of Streptomyces noelsii S83

[0091] 2.2.1. Tyndall effect

[0092] Using an infrared laser pointer and utilizing the light scattering phenomenon, observing the situation of a beam of light passing through the colloid to form an optical "path", and judging the formation of the nanoparticles of this nanoemulsion and its Tyndall phenomenon.

[0093] 2.2.2. Zeta potential and particle size analysis of the nanoemulsion of Streptomyces noelsii S83

[0094] Using a nanoparticle size analyzer to measure the hydrated particle size and potential of the nanoemulsion of Streptomyces noelsii S83 prepared under different conditions; using a transmission electron microscope to analyze the particle size of the nanoemulsion.

[0095] 2.2.3 Determination of the wetting performance of Streptomyces noelsii S83 nanoemulsion on the surface of tobacco leaves

[0096] Using a contact angle measuring instrument, the dynamic contact angle of the nanoemulsion on the tobacco leaf surface was measured to characterize the wetting and spreading performance. Using a surface tension meter, the surface tension of the Streptomyces noelsii S83 nanoemulsion was measured by the platinum plate method surface tension meter.

[0097] 2.2.4 Effect of Streptomyces noelsii S83 nanoemulsion on the hyphal morphology of tobacco target spot pathogen

[0098] Add the Streptomyces noelsii S83 nanoemulsion to PDA, lay a piece of cellophane on the culture medium, punch a 5-mm-diameter fungal block of tobacco target spot pathogen and place it in the center of the PDA culture medium, and culture it in a constant temperature incubator at 28 °C for 3 days. At the same time, use water as the clear water control group, and observe the hyphal growth of the control group and the treatment group with an optical microscope.

[0099] 3. Results

[0100] 3.1 Optimization of the formula of Streptomyces noelsii S83 nanoemulsion

[0101] The preparation and formula screening of the nanoemulsion were carried out through two indexes: the antibacterial rate and observing whether nanoemulsion was produced. The results showed that nanoemulsion could be produced in the range of 2 - 5 mg / mL HTCC and 1 - 2 mg / mL CMCS, and there would be no precipitation after static observation, and it had a certain antibacterial effect.

[0102] 3.2 Tyndall effect

[0103] The detection results of the Tyndall effect are as Figure 3 shown. The results showed that through observation, it was found that the three combinations of H 5 C 5 , H 5 C 2 and H 2 C 2 had obvious, clear and non-dispersed light paths.

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

[0105] According to the changes in particle size and zeta potential, the stability of the nanoemulsion was judged. It could be found from Table 5 that the two combinations of H 5 C 2 and H 2 C 2 had uniform hydrated particle size distribution and a relatively high absolute value of Zeta potential, indicating that the system was relatively stable. It can be seen from the transmission electron microscope Figure 4 that the particle size of the nanoemulsion was small, with a particle size below 500 nm.

[0106] Table 5 Particle size of Streptomyces noelensis S83 nanoemulsion with different ratios of milking materials

[0107]

[0108] 3.4 Determination of the wetting performance of Streptomyces noelensis S83 nanoemulsion on the surface of tobacco leaves

[0109] Through Figure 5 It can be seen that the contact angle of the tobacco leaves after being treated with Streptomyces noelensis S83 nanoemulsion decreased significantly. And through the measurement of surface tension, the surface tension value of the nanoemulsion-treated is much lower than that of water, reaching 40 mN / m, indicating that Streptomyces noelensis S83 nanoemulsion can wet and adhere well to the surface of tobacco leaves (Table 6).

[0110] Table 6 Measured values of the surface tension of Streptomyces noelensis S83 nanoemulsion

[0111]

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

[0113] 3.5 Effect of Streptomyces noelensis S83 nanoemulsion on the hyphal morphology of tobacco target spot pathogen

[0114] Using a microscope to observe the hyphal morphology, the hyphae in the clear water control group were thick and the internodes were long. The angle between the newly grown hyphae and the maternal hyphae at the branching point was about between 30° and 45°, showing regular growth; the remaining treatment groups showed phenomena such as more branches, deformities, short and thick ( Figure 6 ).

[0115] Example 3 Effect of Streptomyces noelensis S83 nanoemulsion on the growth of tobacco

[0116] According to the pesticide safety evaluation criteria for crops NY / T1965.1-2010, the potting method was adopted. When the tobacco grew to the 3-4 leaf stage, spraying was carried out, and the preparation for spraying was Streptomyces noelensis S83 nanoemulsion (H a C b, the preparation method of the nanoemulsion of Streptomyces nogalater S83 was the same as that in Example 2), with a dosage of 5 mL / plant. One clear water control and one control of the fermentation broth of Streptomyces nogalater S83 were set up, with 10 plants in each treatment and 3 replicates for each treatment. When the tobacco plants were at the 3-4 leaf stage, 5 plants with relatively consistent growth were selected from each replicate, and the height from the soil surface to the growth point of the tobacco was measured as the plant height. The plant height growth rate and the plant height growth inhibition rate were calculated. The formula for the plant height growth rate (mm / d) = (plant height after application - plant height before application) / plant height before application × time interval between before and after application; the formula for the plant height growth inhibition rate = (plant height growth rate of the blank control - plant height growth rate of the medicament treatment) / plant height growth rate of the blank control × 100%. At 7, 14, and 21 days after spraying, the tobacco plants were investigated for phytotoxicity symptoms such as discoloration, necrosis, wilting, malformation, and growth stagnation, as well as the number of damaged plants. The investigation results are shown in Table 7.

[0117] Table 7 Effects of the nanoemulsion of Streptomyces nogalater S83 on the growth of tobacco plants

[0118]

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

[0120] According to the pesticide safety evaluation criteria for crops NY / T1965.1 - 2010, its safety was evaluated. It was found that the nanoemulsion of Streptomyces nogalater S83 had no inhibitory effect on the growth inhibition of tobacco, no phytotoxicity occurred, and it had a promoting effect on the growth of plants (Table 7).

[0121] Example 4 Indoor control efficacy of the nanoemulsion of Streptomyces nogalater S83 against tobacco target spot disease

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

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

[0124] After spraying for 24 h, the tobacco target spot pathogen cake with a diameter of 6 mm was inoculated on the scratched leaves; The 3rd and 4th true leaves of each plant were inoculated, and 6 pathogen cakes were inoculated on each leaf, and moisturized with sterilized cotton. After inoculation for 48 h, the pathogen cakes and cotton were removed, and the agent treatment was sprayed again. After 7 d, 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 the nanoemulsion of Streptomyces nogalater S83 on tobacco target spot disease in pot experiments

[0126]

[0127] Note: CK: water control; CK-: blank material; CK+: control with the fermentation broth of Streptomyces nogalater S83.

[0128] It was determined by plate assay that the nanoemulsion of Streptomyces nogalater S83 had a good antibacterial effect on tobacco target spot disease. The results of indoor pot experiments showed that: the control effect of the combination of the nanoemulsion of Streptomyces nogalater S83 was higher than that of the treatment with the fermentation broth of Streptomyces nogalater S83 alone, and the combined control effect of H 5 C 5 was relatively high, and the color of the tobacco leaves was greener after applying the fermentation broth of Streptomyces nogalater S83 and the combination of the nanoemulsion of Streptomyces nogalater S83. At the same time, the chlorophyll meter of Zhejiang Top Cloud-Agri Technology Co., Ltd. was used to measure the chlorophyll and nitrogen content of tobacco leaves, and the results are shown in Table 9.

[0129] Table 9 Results of chlorophyll and nitrogen content in tobacco after applying the nanoemulsion of Streptomyces nogalater S83

[0130]

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

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

[0133] Afterwards, an enzyme activity kit was used to detect the activities of disease-resistant related enzymes (CAT, POD, SOD, PPO in tobacco) of the nanoemulsion of Streptomyces noelsii S83, and the results are as Figure 7 shown. For the three combinations of the nanoemulsion of Streptomyces noelsii S83 treating the plants, the activities of CAT, POD, SOD, and PPO in one combination were higher than those of other treatments, and the enzyme activities increased significantly, so that the tobacco plants showed stronger defense ability when infected by the target spot pathogen.

[0134] Example 5 Field control effect of the nanoemulsion of Streptomyces noelsii S83

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

[0136] The test was set with 6 treatment groups, spraying sterile water and jinggangmycin as the control treatment, Treatment 1 was the blank material (the same as Example 4); Treatment 2 was the nanoemulsion of Streptomyces noelsii S83 H 5 C 5 , and the preparation method of the nanoemulsion of Streptomyces noelsii S83 was the same as that in Example 2; each treatment had 3 replicates, with a total of 15 plots. There were 15 tobacco plants in each plot, arranged in a randomized block design, and each plot was about 30m 2 . A knapsack sprayer was used to evenly spray both the front and back sides of the leaves so that the leaves were covered with mist droplets, and each application was carried out on a sunny day.

[0137] Two investigations were carried out, and statistical records were made according to the disease lesion grading standard. The disease incidence was observed and recorded 7 days before the first application and 7 days after the last application respectively, and the disease index and control effect were calculated based on the statistically counted disease grades. When conducting fixed-plant investigations, 5 plants were selected from each plot, and the percentage of the disease lesion area on the leaves accounting for the total leaf area was used as the grading standard, and the total number of leaves and the number of diseased leaves at each level were recorded.

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

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

[0140] The calculation formula for control effect is: Control effect = [1 - (disease index before spraying in control area × disease index after spraying in treatment area) / (disease index after spraying in control area × disease index before spraying in treatment area)] × 100%.

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

[0142] Table 10 Field control effect of Streptomyces S83 nanoemulsion on tobacco target spot disease

[0143]

[0144] After the indoor control effect test, it was found that the combination of Streptomyces noursei S83 nanoemulsion H 5 C 5 had the best control effect. Therefore, this combination was used for the field test, and 8% jinggangmycin, which is commonly used to control tobacco target spot disease, was selected as the control agent. The results are shown in Table 10. The control effect of Streptomyces noursei S83 nanoemulsion H 5 C 5 against tobacco target spot disease reached 72.97%, showing significant differences compared with the fermentation broth and blank material treatment groups; the control effect of jinggangmycin was 57.52%, showing no significant difference compared with the control effect of Streptomyces noursei S83 nanoemulsion H 5 C 5 .

[0145] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall 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 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 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; 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 effective viable count of Streptomyces noursei S83 in the fermentation broth was 2.8×10 4 CFU / mL.

4. A method for preparing a Streptomyces noursei S83 nanoemulsion, 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.

5. The preparation method according to claim 4, 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.

6. The preparation method according to claim 4, 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.

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

8. Use of the Streptomyces noursei S83 nanoemulsion according to any one of claims 1 to 3 in preventing and treating tobacco target spot disease.

9. 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 3.

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