Streptomyces sphericalus OPF-9, its composition, fermentation method and use

Abstract

The invention discloses Streptomyces sphericalus OPF-9, a composition thereof, a fermentation method and uses. The Streptomyces sphericalus OPF-9 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO:M 2024905. The aseptic fermentation liquid of the strain is a product obtained by fermenting and culturing Streptomyces sphericalus OPF-9 in a soybean cake powder soluble starch culture liquid. The aseptic fermentation liquid of 10% of the Streptomyces can inhibit a variety of pathogenic fungi and has a wide antibacterial spectrum. Therefore, the aseptic fermentation liquid of Streptomyces sphericalus OPF-9 with a content of 10% is used for the biological control of kiwifruit soft rot. It can also be used for the prevention and control of other plant diseases caused by Botrytis cinerea, Alternaria pedunculata, Alternaria interlaca and Phomopsis spp.

Description

Technical Field

[0001] The invention belongs to the technical field related to biological agriculture, and in particular relates to a new strain and its composition, a fermentation method, and the use thereof in biological control of plant diseases. Background Art

[0002] Kiwifruit soft rot is a disease that seriously affects the kiwifruit industry. It is caused by a variety of microorganisms, among which fungal diseases are the most common. The disease causes kiwifruit to rot, seriously affecting the quality and yield of the fruit, and causing huge economic losses to fruit farmers. At present, the prevention and control of kiwifruit soft rot mainly relies on chemical pesticides, but the long-term and large-scale use of chemical pesticides will not only pollute the environment, but may also cause pathogens to develop drug resistance, reducing the prevention and control effect.

[0003] As people's awareness of food safety and environmental protection increases, biological control, as an environmentally friendly and sustainable control strategy, has gradually gained attention. Biological control uses biological resources, such as microorganisms and plant extracts, to control or prevent pests and diseases, which has the advantages of reducing the use of chemical pesticides and reducing environmental pollution.

[0004] In the field of microbial control, actinomycetes, as a group of microorganisms with a wide range of biological activities, have been proven to have great potential in disease control. Actinomycetes can produce a variety of antibiotics and bioactive substances, which have inhibitory effects on a variety of pathogens.

[0005] Actinomycetes are Gram-positive rods that are non-spore-free, non-motile, non-acid-fast, and branched or club-shaped. They are a type of prokaryotic microorganism with filamentous branching cells and asexual spores. They are named because their colonies grow radially. They are a special group of prokaryotes that mainly reproduce by spores and grow in a mycelial form. They are Gram-positive bacteria with a very high G+C content. So far, almost all the actinomycetes found are Gram-positive bacteria. Actinomycetes are widely distributed in the soil, and the vast majority of actinomycetes are beneficial bacteria. Actinomycetes are a very important type of microbial resource, which is closely related to human life and production, and has made significant contributions to human life, especially in medical treatment.

[0006] The biocontrol mechanisms of actinomycetes mainly include antibiotic action, hyperparasitism, competition, growth promotion and induced disease resistance. In the natural environment, actinomycetes do not exert biocontrol effects in a single form but rather a variety of biocontrol mechanisms in a coordinated manner, such as hyperparasitism and antibiotic action. Some microorganisms will produce metabolites that destroy the cell wall structure of pathogens, thereby facilitating their hyperparasitism.

[0007] Therefore, discovering new strains and developing their uses in plant disease prevention and control, especially kiwifruit soft rot, is of great significance to agricultural production. Summary of the invention

[0008] The purpose of the present invention is to provide Streptomyces sphericalus OPF-9, its composition, fermentation method and use, especially the use of the sterile fermentation liquid of Streptomyces sphericalus OPF-9 in preventing and controlling kiwifruit soft rot.

[0009] According to the first main aspect of the present invention, a strain of globosus is provided, which is classified and named as Streptomyces globosus OPF-9, with a preservation number of CCTCC NO: M 2024905, a preservation date of May 11, 2024, and a preservation unit of China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, zip code: 430072, telephone: 027-68754052). The test result on May 18, 2024 was: survival.

[0010] According to the second main aspect of the present invention, a composition is provided, which comprises the aforementioned Streptomyces sphericalus OPF-9 or its fermentation product, and may also include necessary auxiliary agents or carriers. These auxiliary agents or carriers include but are not limited to the following one or a combination of two or more:

[0011] Surfactant: Increase the adhesion of fermentation liquid on the plant surface and improve its bioavailability.

[0012] Stabilizer: Prevents the active ingredients in the fermentation broth from decomposing or becoming inactive, thus prolonging their effective time.

[0013] Dispersant: Ensure that the fermentation broth is evenly dispersed before final use to avoid local concentrations that are too high or too low.

[0014] Sustained-release agent: controls the release rate of active ingredients in the fermentation broth so that it continues to work over a period of time.

[0015] Solvent or diluent: Adjust the concentration of the fermentation broth to make it suitable for field application.

[0016] Preservatives: protect the composition from microbial contamination during storage.

[0017] pH adjuster: adjusts the pH of the fermentation broth to optimize its biological activity.

[0018] Nutritional additives: provide nutrients required for microbial growth and enhance their biological activity.

[0019] Carrier material: such as starch, clay or other porous materials, used to adsorb and carry the fermentation liquid.

[0020] Emulsifiers: If the fermentation broth needs to be made into an emulsion, emulsifiers can help form a stable emulsion.

[0021] Thickener: Increases the viscosity of the fermentation liquid, making it easier to form a protective film on the surface of plants.

[0022] Antioxidant: Prevents the active ingredients in the fermentation broth from becoming ineffective due to oxidation.

[0023] Penetrants: Help the fermented liquid to better penetrate into plant tissues, improving its therapeutic effects.

[0024] Adsorbent: such as activated carbon or other porous materials, used to adsorb and enrich active ingredients in the fermentation broth.

[0025] Protective colloid: forms a protective layer to reduce the impact of environmental factors on the active ingredients in the fermentation broth.

[0026] As a preferred embodiment of the present invention, the metabolites of Streptomyces sphericalensis OPF-9 include sterile fermentation broth.

[0027] Furthermore, the sterile fermentation liquid is a product obtained by fermenting the spherical Streptomyces OPF-9 in a soybean cake powder soluble starch culture liquid.

[0028] In the above scheme, the aforementioned soybean cake powder soluble starch culture solution is composed of the following components in parts by weight: 20 parts of glucose, 15 parts of soybean cake powder, 10 parts of soluble starch, 4 parts of sodium chloride, 2.5 parts of ammonium sulfate, 1 part of calcium carbonate, and 0.2 parts of potassium dihydrogen phosphate.

[0029] Among them, glucose, as a carbon source, provides energy and carbon skeleton required for microbial growth. Soybean cake powder provides nitrogen source and growth factors to promote the growth and metabolism of microorganisms. Soluble starch is used as another carbon source for Streptomyces spheronata OPF-9, because Streptomyces spheronata OPF-9 is a strain that decomposes starch. Sodium chloride is used to maintain the osmotic pressure balance of the culture medium. Ammonium sulfate, as an inorganic nitrogen source, provides nitrogen required for microbial growth. Calcium carbonate is used to adjust the pH value of the culture medium or as a buffer for microbial growth. Potassium dihydrogen phosphate provides phosphorus and potassium, which are the main nutrients necessary for microbial growth.

[0030] The soybean cake powder soluble starch culture medium contains a variety of nutrients that can meet the metabolic needs of different microorganisms. By adjusting the ratio of each component, the culture medium can be optimized to adapt to the growth of specific microorganisms. Calcium carbonate and potassium dihydrogen phosphate help maintain the chemical stability of the culture medium. In the soybean cake powder soluble starch culture medium, a specific combination of nitrogen and carbon sources can promote the growth and metabolic activity of the new strain.

[0031] According to the third main aspect of the present invention, there is provided a method for preparing the aforementioned sterile fermentation broth by using Streptomyces sphericalus OPF-9, comprising at least the following steps: inoculating OPF-9 at an amount of 1% into a soybean cake powder soluble starch culture broth, placing it in a shaking incubator at 28°C and 200rpm / min for 4 days to obtain the fermentation broth, centrifuging the fermentation broth in a centrifuge at 24°C and 4000rpm / min for 30 minutes, filtering the supernatant three times with a 0.22μm microporous filter membrane to obtain a sterile fermentation broth free of bacteria.

[0032] According to a fourth main aspect of the present invention, there is provided a use of a composition containing Streptomyces sphericalus OPF-9 or a fermentation product thereof in controlling plant diseases, wherein the composition acts as an antibacterial active substance.

[0033] Among them, the aforementioned plant diseases include kiwifruit soft rot.

[0034] Furthermore, the composition has an antagonistic effect on Botrytis cinerea, Alternaria pedunculata, Alternaria alternata, and Phomopsis spp.

[0035] In particular, the OPF-9 sterile fermentation broth had the best antibacterial effect on Botrytis cinerea, with an inhibition rate of 95.9%, followed by long-stalked chain spores, cross-linked chain spores, and phomopsis, with inhibition rates of 78.64%, 77.36%, and 70.70%, respectively.

[0036] The 10% aseptic fermentation liquid of Streptomyces can inhibit a variety of pathogenic fungi and has a broad antibacterial spectrum. Therefore, the aseptic fermentation liquid of Streptomyces sphericalus OPF-9 with a content of 10% is used for biological control of kiwi fruit soft rot. It can also be used for the prevention and control of other plant diseases caused by Botrytis cinerea, Alternaria pedunculata, Alternaria alternata, and Phomopsis spp.

[0037] Advantages and beneficial effects of the present invention:

[0038] The sterile fermentation liquid obtained by fermenting the strain OPF-9 under optimal conditions has a good inhibitory effect on a variety of pathogenic fungi. The use of the sterile fermentation liquid with a content of 10% OPF-9 can not only effectively prevent and control diseases, but also avoid environmental pollution and residue problems that may be caused by chemical pesticides. Compared with chemical pesticides, the use of OPF-9 sterile fermentation liquid as a biological control agent has less impact on the environment, which meets the current demand for green agriculture and sustainable development. The present invention not only provides an efficient, broad-spectrum, and environmentally friendly biological control method, but also provides a new solution for the prevention and control of plant diseases, and has important practical value and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0040] Figure 1 The morphological characteristics of colonies, hyphae, spore hyphae and spores of the antagonistic bacteria OPF-9 in one embodiment of the present invention are shown;

[0041] Figure 2 A maximum likelihood phylogenetic tree constructed based on 16S rDNA in one embodiment of the present invention is shown;

[0042] Figure 3 The effect of different culture media on the antibacterial activity of the aseptic fermentation liquid of strain OPF-9 in one embodiment of the present invention is shown;

[0043] Figure 4 The effect of different temperatures on the antibacterial activity of the fermentation broth of strain OPF-9 in one embodiment of the present invention is shown;

[0044] Figure 5 The effect of different rotation speeds on the antibacterial activity of the fermentation liquid of strain OPF-9 in one embodiment of the present invention is shown;

[0045] Figure 6 The effect of different inoculation amounts on the antibacterial activity of the fermentation broth of strain OPF-9 in one embodiment of the present invention is shown;

[0046] Figure 7 The effect of different fermentation days on the antibacterial activity of the fermentation liquid of strain OPF-9 in one embodiment of the present invention is shown;

[0047] Figure 8 The relative protective effects of the fermentation broth of strain OPF-9 under different temperature treatments in one embodiment of the present invention are shown;

[0048] Fig. 9 The relative protective effect of the fermentation liquid of strain OPF-9 under different ultraviolet irradiation time treatment in one embodiment of the present invention is shown;

[0049] Fig.10 The relative protective effects of the fermentation broth of strain OPF-9 under different pH treatments in one embodiment of the present invention are shown;

[0050] Fig.11 The relative protective effects of the fermentation broth of strain OPF-9 treated with different proteases in one embodiment of the present invention are shown;

[0051] Fig.12The antifungal spectrum of strain OPF-9 in one embodiment of the present invention is shown. DETAILED DESCRIPTION

[0052] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings so that the purpose, features and advantages of the present invention can be more clearly understood. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

[0053] In the following description, certain specific details are set forth for the purpose of illustrating the various disclosed embodiments to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with the present application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0054] References throughout the specification to "one embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0055] The antagonistic bacteria mentioned below are the generic names of strain OPF-9 before the name is determined.

[0056] 1 Isolation, screening and identification of antagonistic bacteria

[0057] 1.1 Isolation and purification of antagonistic bacteria

[0058] The antagonistic bacteria were isolated by dilution and spreading method. 10 g of soil sample was mixed with 90 mL of sterile water in a sterile conical flask and sealed. The flask was fully shaken at 28 °C and 120 rpm for 30 min. The suspension was diluted to 10% with sterile water in a clean bench. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Then, 200 μL of the suspension was evenly spread on the surface of the LB agar medium, sealed and placed in a 28°C constant temperature incubator for 48 h. Each gradient was repeated 3 times.

[0059] 1.2 Isolation and purification of strains: After colonies grow on the culture medium, pick different single colonies according to the different morphological and color characteristics of the colonies and place them on new LB agar medium. Repeat 2 to 3 times to obtain pure colonies. Select pure colonies and culture them in LB medium at 28°C for 48 hours, mix them with 50% glycerol (v / v) in a 1:1 ratio, and store them in a -80°C ultra-low temperature refrigerator.

[0060] 1.3 Initial and rescreening of antagonistic bacteria

[0061] The antagonistic bacteria were screened and rescreened by the plate confrontation method. A 5 mm cake of Alternaria longipes was placed in the center of the PDA plate, and the strain was inoculated in four mutually perpendicular directions 3 cm away from the center. The plate without antagonistic bacteria and only inoculated with Alternaria longipes was used as a control.

[0062] After culturing at 25°C for 5 days, the colony diameter of the pathogen was measured by the cross method, and the inhibition rate was calculated as shown in formula (2-1) to evaluate the antibacterial effect of the isolated strains. The strains with antibacterial effect were selected to repeat the above experiment for rescreening. Each strain was repeated 3 times. The strain with the best antagonistic effect, OPF-9, was selected for subsequent experiments.

[0063]

[0064] 1.4 Identification of antagonistic bacteria

[0065] According to the guidelines developed by the International Streptomyces Project, the microscopic (conventional microscopic observation and scanning electron microscopic observation) and macroscopic morphology (color of aerial hyphae and intrabasal hyphae, soluble pigments, etc.) of the isolate OPF-9 were identified. At the same time, the physiological and biochemical characteristics of the isolate OPF-9 were determined with reference to the "Manual of Streptomyces Identification" (edited by the Actinomycetes Classification Group of the Institute of Microbiology, Chinese Academy of Sciences, 1975) and "Rapid Identification and Systematic Classification of Actinomycetes" (Ruan Jisheng et al., 2011).

[0066] 16S rDNA was selected for molecular identification, and the bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit. The universal primer sequences were: 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-TACGACTTAACCCCAATCGC-3' (Loganathan et al., 2021). The reaction system (25 μL) was as follows: 1 μL DNA template, 1 μL of each primer, 12.5 μL of 2× Taq PCR StarMix (containing loading dye, Sangon, Inc, Shanghai, China), and 9.5 μL of ddH2O.

[0067] The reaction process was as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 52°C annealing for 30 s, 72°C extension for 60 s, a total of 35 cycles, and finally 72°C extension for 10 min, and storage at 4°C. The amplified product was entrusted to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The obtained sequences were compared using the BLAST tool in the NCBI database, and the sequences with the highest homology were downloaded. The phylogenetic tree was constructed using MEGA 11 with the maximum likelihood method.

[0068] 2 Results and analysis

[0069] 2.1 Colony and bacterial morphology

[0070] Its culture and physiological characteristics were determined according to the guidelines developed by the International Streptomyces Project. After 7 days of cultivation on Gao's medium No. 1 plates, strain OPF-9 colonies were round and produced abundant aerial hyphae and basal hyphae. Aerial hyphae were off-white and gray-pink, and basal hyphae were light green-gray to gray ( Figure 1 Part A in the figure). The hyphae are curved and branched, without diaphragms and breaks ( Figure 1 Part B in the figure). Spore filaments are spiral ( Figure 1 The spores are short rod-shaped or cylindrical ( Figure 1 in Section D).

[0071] 2.2 Culture characteristics

[0072] Based on the ISCC-NBS centroid color system, the growth, aerial mycelium color, intrabasal mycelium color and soluble pigment production of strain OPF-9 on nine culture media were studied (Table 1).

[0073] Table 1 Cultivation characteristics of strain OPF-9 on 9 culture media (14 days)

[0074]

[0075] 1 Growth conditions: +, poor growth; ++, good growth; +++, vigorous growth.

[0076] 2 Soluble pigments: –, no soluble pigments.

[0077] 2.3 Physiological and biochemical characteristics

[0078] (1) Hydrolase activity

[0079] The physiological characteristics of strain OPF-9 are that it can hydrolyze starch, peptone milk, and liquefy gelatin, but it does not produce urease and H 2 S, cannot hydrolyze cellulose.

[0080] (2) Carbon source utilization

[0081] The results of carbon source utilization are shown in Table 2. Strain OPF-9 can grow under carbon-free conditions and under 9 carbon source conditions. In addition, the mycelium grows vigorously and produces soluble pigments on the culture medium with D-glucose and sucrose as carbon sources. However, it grows poorly on the culture medium with other carbon sources and no soluble pigments are produced.

[0082] Table 2 Carbon source utilization characteristics of strain OPF-9

[0083]

[0084] 1 Growth conditions: +, poor growth; ++, good growth; +++, vigorous growth.

[0085] 2 Pigment: +, produces soluble pigment; -, does not produce soluble pigment.

[0086] (3) Salt tolerance

[0087] OPF-9 was able to grow at salt concentrations of 0–3%, grew vigorously in the range of 0.5%–1%, and was inhibited when the salt concentration was greater than 3% (Table 3).

[0088] Table 3 Salt tolerance of strain OPF-9

[0089]

[0090]

[0091] 1 Growth conditions: +, poor growth; ++, good growth; +++, vigorous growth.

[0092] 2 Spore production: +, spore production; –, no spore production.

[0093] 3 Production of soluble pigments: +, produces soluble pigments; –, does not produce soluble pigments.

[0094] 2.416S rDNA sequence phylogeny

[0095] The sequenced sequences were submitted to the NCBI database for Blast alignment, and the sequences with the highest homology were downloaded. The phylogenetic tree was constructed using the maximum likelihood method of MEGA11 software with Nocardiafarcinica IFM 11285 as the outgroup. Figure 2As shown, strain OPF-9 was found to be clustered with S.globosus LZH-48 and S.globosusVITLG011, with a self-sustaining rate of 98%. Based on macromorphology, micromorphology, culture, physiological and biochemical characteristics and molecular biological identification, strain OPF-9 was identified as Streptomyces globosus.

[0096] Optimization of liquid fermentation conditions and stability determination of antagonistic bacteria

[0097] 3.1 Methods and Materials

[0098] Table 4 Culture medium used in the experiment

[0099]

[0100]

[0101] The above culture medium was mixed with 1000 mL of deionized water according to the formula, and the pH was adjusted with 1 mol / L HCl and 1 mol / L NaOH solution. The culture medium was boiled and dispensed into conical flasks, and sterilized in an autoclave at 121°C for 20 min before use.

[0102] 3.2 Optimization of fermentation conditions of Streptomyces strain OPF-9

[0103] (1) Fermentation medium

[0104] An appropriate amount of strain OPF-9 was selected and placed in 50 mL of Gao's No. 1 liquid culture medium, and cultured at 28°C and 180 rpm for 48 h to prepare seed solution.

[0105] Use the 14 liquid culture media in Table 4, add seed liquid at an inoculation rate of 1%, and ferment at 28°C and 180rpm for 5 days. The test was conducted using the method of inhibiting mycelial growth rate. Centrifuge the fermentation broth at 4000rpm for 30min to obtain the supernatant, which was then filtered through a 0.22μm microporous membrane for later use. Prepare a PDA medium containing 10% sterile fermentation broth and pour it into a plate, and inoculate a 5mm A. longipes cake in the center of the medium.

[0106] PDA medium without sterile fermentation liquid was used as control and cultured in the dark in a constant temperature incubator at 25°C for 5 days. After the culture, the colony diameter (mm) was measured by the cross method and the inhibition rate was calculated using the following formula. Each treatment was repeated 3 times.

[0107]

[0108] (2) Fermentation temperature

[0109] The seed liquid was inoculated at 1% to No. 8 soybean cake powder soluble starch liquid culture medium and fermented at 24, 26, 28, 30 and 32°C, 180 rpm for 5 days. A. longipes was used as the target bacteria to test the antibacterial effect of the fermentation liquid. The method was the same as in part (1) above, and each treatment was repeated 3 times.

[0110] (3) Fermentation speed

[0111] The seed solution was added to medium No. 8 at a 1% inoculum rate and fermented at 140, 160, 180, 200 and 220 rpm and 28°C for 5 days. A. longipes was used as the target bacteria to test the antibacterial effect of the fermentation solution. The method was the same as in part (1) above, and each treatment was repeated 3 times.

[0112] (4) Fermentation inoculum volume

[0113] The seed solution was added to medium No. 8 at an inoculum rate of 1%, 3%, 5%, 10%, 15% and 20%, respectively, and fermented at 28°C and 200 rpm for 5 days. A. longipes was used as the target bacteria to test the antibacterial effect of the fermentation solution. The method was the same as in part (1) above, and each treatment was repeated 3 times.

[0114] (5) Fermentation days

[0115] The seed solution was added to medium No. 8 at a 1% inoculum rate and fermented at 200 rpm and 28°C for 1, 2, 3, 4, 5, 6 and 7 days respectively. A. longipes was used as the target bacteria to test the antibacterial effect of the fermentation solution. The method was the same as in part (1) above, and each treatment was repeated 3 times.

[0116] 3.3 Sterile fermentation broth stability

[0117] (1) Thermal stability of sterile fermentation broth

[0118] The sterile fermentation broth obtained by fermenting strain OPF-9 under optimal conditions was heated at 40, 60, 80, 100 and 121°C for 30 min, respectively. After cooling, it was mixed with PDA medium at a volume ratio of 10% and poured into plates. A 5mm bacterial cake of A. longipes was inoculated in the center of the medium. The untreated sterile fermentation broth was used as the control, and the PDA medium without sterile fermentation broth was used as the blank control. The culture was carried out in the dark at 25°C incubator for 5 days. After the culture was completed, the colony diameter (mm) was measured by the cross method, and the relative inhibition rate was calculated. The formula is as follows, and each treatment was repeated 3 times.

[0119]

[0120] (2) UV stability of sterile fermentation broth

[0121] The sterile fermentation broth obtained by fermenting strain OPF-9 under optimal conditions was placed at 30 cm from a 40 W ultraviolet lamp. The irradiation time was 0.5 h, 1 h, 2 h, 3 h and 4 h. The method was the same as in part (1) above. Each treatment was repeated 3 times and the relative inhibition rate was calculated.

[0122] (3) Acid-base stability of sterile fermentation broth

[0123] The sterile fermentation broth obtained by fermenting strain OPF-9 under optimal conditions was adjusted to pH 3, 5, 7, 9 and 11 with 1 mol / L HCl and 1 mol / L NaOH solutions and treated at room temperature for 2 h. The pH was then adjusted to 7 using the same method as in part (1) above. Each treatment was repeated 3 times and the relative inhibition rate was calculated.

[0124] (4) Sterile fermentation broth enzyme stability

[0125] Trypsin, alkaline protease and proteinase K at a final concentration of 1 mg / mL were added to the sterile fermentation broth obtained by fermenting strain OPF-9 under optimal conditions. The mixture was incubated in a water bath at 37°C for 2 h and then at 80°C for 30 min to inactivate the enzymes. The method was the same as in part (1) above. Each treatment was repeated 3 times and the relative inhibition rate was calculated.

[0126] 3.4 Fermentation condition optimization results

[0127] 3.4.1 Optimal fermentation liquid medium

[0128] The results show that if Figure 3 As shown in the figure, the fermentation product produced by No. 8 culture medium (soybean cake powder soluble starch culture medium) has the highest antibacterial activity, with an inhibition rate of 75.45% against A. longipes, followed by No. 1 and No. 5, with inhibition rates of 71.97% and 70.86%, respectively. Therefore, No. 8 culture medium was used in subsequent experiments.

[0129] 3.4.2 Optimal fermentation temperature

[0130] Temperature is one of the important factors affecting the growth, development and metabolism of Streptomyces. The antibacterial activity of the aseptic fermentation broth of strain OPF-9 at different temperatures was determined. The strongest antibacterial activity was found at 28°C, with an inhibition rate of 71.69%. Figure 4 Therefore, the subsequent experiments were cultured at 28°C.

[0131] 3.4.3 Optimal fermentation speed

[0132] The fermentation speed is an important factor affecting the contact between the strain and the air. If the speed is too slow, the strain will have less contact with the air, and if the speed is too fast, the spores of the strain will break, which will affect the growth and development of the strain and the production of antibacterial substances. Figure 5As shown in the figure, different speed conditions have significant differences in the production of active substances by strain OPF-9. When the speed is 200 rpm, the fermentation liquid has the best antibacterial effect, with an inhibition rate of 79.05%. Therefore, the speed of 200 rpm was selected for cultivation in subsequent experiments.

[0133] 3.4.4 Optimal inoculum size

[0134] The inoculation amount has a certain effect on the production of antibacterial substances. The strain OPF-9 produced the most antibacterial substances when inoculated with an inoculation amount of 1%, and the inhibition rate against A. longipes was 73.99%. Therefore, the inoculation amount in subsequent experiments was 1%. Figure 6 shown.

[0135] 3.4.5 Optimal fermentation days

[0136] The experimental results show that Figure 7 As shown in the figure, after 4 days of fermentation, the inhibition rate of the fermentation broth on A. longipes reached 87.17%. As the fermentation time increased, the antibacterial effect of the fermentation broth decreased, so the fermentation time was determined to be 4 days.

[0137] Based on the above experimental results, the optimal fermentation conditions for strain OPF-9 were determined as follows: adding 1% seed solution to the No. 8 soybean cake powder soluble starch liquid culture medium, fermenting at 28°C and 200rpm for 4 days.

[0138] 3.5 Sterile fermentation broth stability

[0139] 3.5.1 Thermal stability of sterile fermentation broth

[0140] After the aseptic fermentation broth of strain OPF-9 was treated at 40℃, 60℃, 80℃, 100℃ and 121℃ for 30min respectively, the relative protection efficiency of the aseptic fermentation broth remained above 80% under the treatment of 40-100℃, but the relative protection efficiency of the aseptic fermentation broth decreased significantly after being treated at 121℃. Figure 8 As shown, it can be seen that the sterile fermentation broth has good stability at 40-100°C, but cannot withstand the high temperature of 121°C.

[0141] 3.5.2 UV stability of sterile fermentation broth

[0142] After 0.5-4h of ultraviolet irradiation, the relative inhibitory effect can be maintained above 90%. The ultraviolet stability of the sterile fermentation broth of strain OPF-9 is good, indicating that the antibacterial substances in the sterile fermentation broth are not easily decomposed and denatured under ultraviolet irradiation. Fig. 9 shown.

[0143] 3.5.3 pH stability of sterile fermentation broth

[0144] The pH value of the aseptic fermentation broth of strain OPF-9 was adjusted to 3, 5, 7, 9, and 11 with 1 mol / L HCl and 1 mol / L NaOH, and then the pH value was adjusted to 7 after standing at room temperature for 2 h. Fig.10 As shown in the figure, after treatment at pH 3-5, the relative inhibition rate of the aseptic fermentation broth of strain OPF-9 was greater than 90%, but at pH 11, the antibacterial activity decreased to 69.97%. The results show that the antibacterial active substances are not resistant to strong alkali and the suitable pH value is 3-9.

[0145] 3.5.4 Sterile fermentation broth enzyme stability

[0146] The sterile fermentation broth of strain OPF-9 was treated with three proteases, trypsin, alkaline protease and proteinase K, and the relative protection efficiency was greater than 80%. Fig.11 As shown, it indicates that the antibacterial active substances in the sterile fermentation broth are not sensitive to proteases.

[0147] Determination of antifungal spectrum of 4 strains OPF-9

[0148] The pathogenic bacteria strains tested were: Botrytis cinerea, Alternaria alternata, Phomopsis spp., Fusarium solani, Fusarium solani, Alternaria longifolia, Sclerotinia spp., and Fusarium solani.

[0149] OPF-9 was inoculated into liquid culture medium No. 8 at an amount of 1%, and the culture was shaken in a shaker at 28°C and 200 rpm / min for 4 days to obtain the fermentation liquid. The fermentation liquid was centrifuged in a centrifuge at 24°C and 4000 rpm / min for 30 minutes. The supernatant was filtered three times with a 0.22 μm microporous filter membrane to obtain a sterile fermentation liquid free of bacteria.

[0150] The sterile fermentation filtrate was thoroughly mixed with PDA in a volume ratio of 1:9 and poured into the plate. A pathogen cake with a diameter of 6 mm was inoculated in the center of the PDA plate containing the sterile fermentation liquid. The PDA plate with an equal volume of sterile water was used as the control. Each group was repeated 3 times and cultured at a constant temperature of 28°C until the control mycelium grew to the entire plate. The colony diameters of the control group and the treatment group were measured by the cross method, and the growth inhibition rate of the sterile fermentation liquid on Botrytis cinerea was calculated.

[0151]

[0152] The results are as follows:

[0153] OPF-9 sterile fermentation liquid has the best antibacterial effect on Botrytis cinerea, with an inhibition rate of 95.9%, followed by long-handled chain spores, chain spores, and phomopsis, with inhibition rates of 78.64%, 77.36%, and 70.70%, respectively. The 10% sterile fermentation liquid of Streptomyces can inhibit a variety of pathogenic fungi with a wide antibacterial spectrum, such as Fig.12 shown.

[0154] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0155] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A Streptomyces globosus strain, which is classified and named Streptomyces globosus OPF-9, with a deposit number of CCTCC NO: M 2024905, a deposit date of May 11, 2024, and a depository unit of China Center for Type Culture Collection.

2. A composition comprising the Streptomyces spheroides OPF-9 or a fermentation product thereof according to claim 1; the fermentation product comprises a sterile fermentation broth; the sterile fermentation broth is a product obtained by fermenting the Streptomyces spheroides OPF-9 in a soybean cake powder soluble starch culture broth.

3. The composition according to claim 2, characterized in that The soybean cake powder soluble starch culture solution is composed of the following components in parts by weight: 20 parts of glucose, 15 parts of soybean cake powder, 10 parts of soluble starch, 4 parts of sodium chloride, 2.5 parts of ammonium sulfate, 1 part of calcium carbonate, and 0.2 parts of potassium dihydrogen phosphate.

4. Use of the composition according to claim 2 or 3 as an antibacterial active substance in the prevention and control of plant diseases; the plant disease is kiwifruit soft rot; the composition has an antagonistic effect on Botrytis cinerea, Alternaria pedunculata, Alternaria alternata, and Phomopsis spp.

5. The use according to claim 4, characterized in that The invention comprises using 10% of the aseptic fermentation liquid of Streptomyces sphericalensis OPF-9 for biological control of kiwifruit soft rot.

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