A method for preparing streptomyces fermentation broth for biological control

Abstract

The present application relates to a kind of preparation method of Streptomyces fermentation liquor for biological control, comprising the following steps: Streptomyces spindulus fermentation, centrifugation, take supernatant;The strain name of Streptomyces spindulus is Streptomyces netropsis JN4-2, and the preservation number is CGMCC No.28157.The Streptomyces fermentation liquor can be used for the biological control of red pine pythium, red bean anthracnose, polygenetic small hole shellfish, apple shell monochrome septum bacteria, corn big spot bacteria, tobacco target spot bacteria, rice malignant exanthema bacteria, melon vine cutting bacteria, pepper anthracnose bacteria, larch wilt bacteria, blueberry ulcer disease bacteria, poplar skin disease bacteria, apple rot bacteria, poplar ulcer disease bacteria, eggplant brown mottle bacteria, melon fruit pythium bacteria, tobacco brown spot bacteria, blueberry branch dry disease bacteria, apple fruit rot bacteria, cucumber sclerotinia rot bacteria and other pathogenic bacteria biological control, with the advantages of good biocontrol effect, broad-spectrum bacteriostatic, wide application range and the like.

Description

[0001] The application is a divisional application, the original Chinese patent application number is: 202410127548.6, the application date is: January 30, 2024, and the original patent application name is: A strain of Streptomyces spindalis and its application. TECHNICAL FIELD

[0002] The application belongs to the field of microbial technology, and particularly relates to a preparation method of Streptomyces fermentation liquor for biological control. BACKGROUND

[0003] Pinus koraiensis belongs to Pinaceae and Pinus, and is originally from the northern region of Eurasia. It is the main constructive species and dominant species in the northeast forest region of China. Pinus koraiensis is a light-loving plant with strong cold tolerance and good corrosion resistance, and is suitable for planting in humid slightly acidic or neutral soil. Air humidity above 0.7 promotes the growth of Pinus koraiensis.

[0004] Pinus koraiensis damping-off is a common and serious seedling infectious disease caused by fungi (mainly Rhizoctonia and Fusarium). The mortality rate of seedlings is extremely high after the disease occurs. The incidence rate of 1-2 year old seedlings is extremely high, and the incidence rate of severe seedlings is more than 60%, or even no production. In production, chemical control and soil disinfectant treatment are still mainly used to prevent the occurrence and harm of the disease. Excessive use of chemical pesticides causes pathogenic bacteria to produce drug resistance, and causes irreversible damage to the ecological environment. Frequent use of soil disinfectants leads to degradation of nursery soil environment and destruction of microbial community, thereby further leading to poor seedling nutrition, weak growth, and low survival rate and poor adaptability in subsequent afforestation and ecological restoration. In the past 40 years, China's agricultural production has relied on chemical input, and the amount of pesticides has increased significantly. According to statistics of the State Environmental Protection Bureau, about 250,000 tons of pesticide raw materials were produced in China in 2007, and more than 800,000 tons of preparations were processed, which were applied to the environment through various means. 10%-20% adheres to the plant body, and 80%-90% scatters in the soil, water and air (Yang Yonghua, Yao Jian, and Hu Xiaomei. Effects of pesticide pollution on the functional diversity of soil microbial communities [J]. Microbiology Journal, 2000, (02): 23-25+47.). In the face of the above problems, biological control is more and more favored by people.

[0005] The disease was first reported in the 1960s by Wu You San, and since then, research on the occurrence and control of the disease has been frequently reported. The occurrence of pine seedling damping-off in Northeast China is affected by soil temperature. Rhizoctonia solani occurs more at 18-22℃, Pythium at 17-22℃, and Fusarium at 23-28℃. The prevalence of pine seedling damping-off in Northeast China is mainly affected by soil moisture content and rainfall. High rainfall and soil moisture content are conducive to the occurrence of the disease. In addition, the longer the disease is delayed, the higher the overall incidence rate (Wu Y, Gao Y, Gu S, et al. Study on pine seedling damping-off Ⅱ. Disease prevalence [J]. Acta Phytopathologica Sinica, 1963, (04): 399-408.). Tong Ying et al. (1979) found that the main pathogenic fungi causing pine seedling damping-off were Rhizoctonia, Fusarium, and Pythium, and the pathogenicity of Rhizoctonia was the strongest (Tong Y. Pine seedling damping-off and its control [J]. Jilin Forestry Science and Technology, 1979, (02): 117-120.). Zhang Lijun et al. (2012) found that the red pine damping-off fungus overwinters in the form of mycelium and spores in the residues of various hosts and soil, and is transmitted between rows through mycelium. Unfavorable environmental conditions such as low temperature, rain, and insufficient light can promote the infection of the pathogen, especially low temperature has the greatest impact on the disease (Zhang L, Wang B, Ni T, Li C, Jia J. Occurrence pattern and control techniques of red pine damping-off [J]. Agriculture and Technology, 2012, 32(09): 116.).

[0006] Plant diseases cause harm to agricultural and forestry production, and the economic losses are immeasurable. With the increasing disadvantages of chemical pesticides, biological control has received widespread attention from the whole society. It has multiple advantages such as high efficiency, non-toxicity, environmental protection, and will become the mainstream means of plant disease control. Among more than 16500 antibiotics reported in the world, actinomycetes account for more than half of the proportion. Actinomycetes are the first discovered microorganisms with biological control effect. Streptomyces in actinomycetes is the genus that produces the most antibacterial substances. Gao Yuhong et al. (2009) reported a new type of agricultural antibiotic doramectin, which is produced by fermentation of a new species of recombinant avermectin-producing Streptomyces avermitilis. Doramectin is one of the best anti-parasitic drugs in the avermectin family, and it has good killing effect on nematodes and arthropods (Jiang Wei. Mutation biosynthesis and fermentation condition optimization of doramectin[D]. Northeast Agricultural University, 2009. Gao Yuhong, Guo Zhaocheng, Liu Fangyue, et al. Research progress and application of doramectin[J]. 2009, 40(4): 141-144). Boukaew et al. (2017) reported that low-concentration metabolites of S. philanthi RM-1-138, S. philanthi RL-1-178 and S. mycarofaciens SS-2-243 had 100% inhibition rate on spore germination of tomato gray mold pathogen (Botrytis cinerea) (

[16] Sawai Boukaew, Poonsuk Prasertsan, Claire Troulet, Marc Bardin. Biological control of tomato gray mold caused by Botrytis cinerea by using Streptomyces spp. [J]. BioControl, 2017, 62(6).). In biological control, it is necessary to select appropriate strains to achieve better control effect. Therefore, it is crucial to provide strains that can be effectively used for biological control. SUMMARY

[0007] In view of the problems existing in the prior art, the present application provides a preparation method of Streptomyces fermentation broth for biological control. The Streptomyces fermentation broth is prepared by using Streptomyces netropsis JN4-2. The strain is first discovered by the present application, and further verification shows that the Streptomyces fermentation broth prepared by using the strain has the advantages of good biocontrol effect, wide inhibition spectrum, and wide application range.

[0008] The technical solution of the present application to solve the above technical problems is as follows:

[0009] The present application provides a strain of Streptomyces, the strain name is Streptomyces netropsis JN4-2 (Streptomyces netropsis JN4-2), the preservation number is CGMCC No. 28157, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 11, 2023, and the preservation address is No. 1, Beichen West Road, Yard 3, Beijing, China.

[0010] The present application separates and obtains a plurality of strains of actinomycetes from soils collected from different regions of Jilin Province, adopts multiple screening and prevention and control effect determination, finally obtains a best strain of antagonistic bacteria for inhibiting red pine vascular wilt fungus, marked as JN4-2, realizes a breakthrough of actinomycetes in the field of preventing and treating red pine vascular wilt, and provides a new strain and product for biological control of red pine vascular wilt.

[0011] The present application provides a kind of bacterial agent, including above-mentioned Streptomyces netropsis and / or fermentation product of above-mentioned Streptomyces netropsis.The present application has no special restriction to the dosage form of bacterial agent, for example, the bacterial agent can be liquid preparation or solid preparation, can also be other types of preparation.In addition to the Streptomyces or fermentation product of Streptomyces provided by the present application, components commonly used in the art for preparing biocontrol preparation can also be added to facilitate its application.

[0012] The Streptomyces netropsis and its bacterial agent provided by the present application have the advantages of good biocontrol effect, wide antibacterial spectrum and wide application range.

[0013] The present application provides a fermentation method of the above-mentioned Streptomyces netropsis, including the following steps: inoculating the above-mentioned Streptomyces netropsis into a fermentation medium for fermentation culture.The formula of the fermentation medium can include: wheat bran 40g, corn flour 20g, sucrose 60g, potassium nitrate 4g, calcium carbonate 0.4g, dipotassium hydrogen phosphate 0.2g, per 1000mL distilled water, pH 7.0.The fermentation culture conditions can include: 28℃ culture for 6d.

[0014] The present application provides a preparation method of the above-mentioned bacterial agent, including the following steps: inoculating the above-mentioned Streptomyces netropsis into a fermentation medium for fermentation culture.The formula of the fermentation medium can include: wheat bran 40g, corn flour 20g, sucrose 60g, potassium nitrate 4g, calcium carbonate 0.4g, dipotassium hydrogen phosphate 0.2g, per 1000mL distilled water, pH 7.0.The fermentation culture conditions can include: 28℃ culture for 6d.

[0015] The present application provides a preparation method of Streptomyces fermentation liquid for biological control, including the following steps: fermenting Streptomyces netropsis, centrifuging, and taking the supernatant;the strain name of Streptomyces netropsis is Streptomyces netropsis JN4-2, and the preservation number is CGMCC No. 28157.

[0016] The fermentation temperature can be 28℃.

[0017] The fermentation time can be 6d.

[0018] The present application provides a Streptomyces fermentation liquid for biological control, and a preparation method of the Streptomyces fermentation liquid can comprise the following steps: inoculating seed liquid of the above-mentioned Streptomyces sp. into a fermentation medium, and culturing at 28℃ and 150r / min for 6d, and then centrifuging and taking supernatant.

[0019] The formula of the fermentation medium can comprise: wheat bran 40g, corn flour 20g, sucrose 60g, potassium nitrate 4g, calcium carbonate 0.4g, dipotassium hydrogen phosphate 0.2g, per 1000mL distilled water, and pH 7.0.

[0020] The preparation method of the seed liquid can comprise the following steps: streaking culture of the above-mentioned Streptomyces sp. on a plate, scraping spores into sterile water to prepare a spore suspension, which is the seed liquid.

[0021] The present application provides a Streptomyces fermentation liquid for biological control, which is prepared by the above-mentioned preparation method.

[0022] The present application provides application of the above-mentioned Streptomyces sp. in plant pathogen control.

[0023] The strain provided by the present application can be used for preparing a biocontrol preparation for preventing and treating one or more of the following plant pathogenic bacteria: red pine Pythium sylvaticum, red bean anthracnose fungus, Phialophora fastigiata, Venturia inaequalis, Alternaria longipes, Alternaria solani, Magnaporthe grisea, Pseudomonas syringae, Botrytis cinerea, Sclerotinia sclerotiorum, Monilinia fructicola, Pythium aphanidermatum, Pythium ultimum, Pythium catenatum, Phytophthora capsici, Phytophthora ramorum, Phytophthora cactorum, Phytophthora nicotianae, Phytophthora citri, Phytophthora capsici, and Pythium aphanidermatum.

[0024] The present application provides application of the above-mentioned biocontrol preparation in plant pathogen control. The plant pathogenic bacteria are selected from one or more of the following: red pine Pythium sylvaticum, red bean anthracnose fungus, Phialophora fastigiata, Venturia inaequalis, Alternaria longipes, Alternaria solani, Magnaporthe grisea, Pseudomonas syringae, Botrytis cinerea, Sclerotinia sclerotiorum, Monilinia fructicola, Pythium aphanidermatum, Pythium ultimum, Pythium catenatum, Phytophthora capsici, Phytophthora ramorum, Phytophthora cactorum, Phytophthora nicotianae, Phytophthora citri, Phytophthora capsici, and Pythium aphanidermatum. The above-mentioned biocontrol preparation provided by the present application can be used for preventing and treating diseases caused by the above-mentioned pathogenic bacteria.

[0025] The application provides application of a streptomyces fermentation liquor in prevention and treatment of plant pathogenic bacteria.

[0026] The plant pathogenic bacteria are selected from one or more of the following: red pine seedling blight, red bean anthracnose, polygenetic small hole shell fungus, apple shell single color septum ulcer disease bacterium, corn large spot disease bacterium, tobacco target spot disease bacterium, rice malignant exanthema bacterium, melon vine cutting disease bacterium, pepper anthracnose bacterium, larch dieback bacterium, blueberry ulcer bacterium, poplar bark rot bacterium, apple rot bacterium, poplar ulcer bacterium, eggplant brown mottle bacterium, melon fruit rot bacterium, tobacco brown spot bacterium, blueberry branch rot disease bacterium, apple fruit rot bacterium, cucumber sclerotinia disease bacterium and pear wave disc fruit rot bacterium.

[0027] The streptomyces fermentation liquor can be prepared by inoculating the seed liquid of the above-mentioned streptomyces into a fermentation medium, and then culturing at 28 DEG C and 150 r / min constant temperature oscillation for 6 days, and then centrifuging and taking the supernatant.

[0028] The formula of the fermentation medium can include the following components: wheat bran 40 g, corn flour 20 g, sucrose 60 g, potassium nitrate 4 g, calcium carbonate 0.4 g, dipotassium hydrogen phosphate 0.2 g, and 1000 mL of distilled water, and the pH is 7.0.

[0029] The preparation method of the seed liquid comprises the following steps: streaking culture of the above-mentioned streptomyces on a plate, scraping spores into sterile water to prepare a spore suspension, and then obtaining the seed liquid.

[0030] The above-mentioned streptomyces fermentation liquor provided by the application can be used for preventing and treating diseases caused by the above-mentioned plant pathogenic bacteria.

[0031] The plant pathogenic bacteria are selected from one or more of the following: red pine seedling blight, red bean anthracnose, polygenetic small hole shell fungus, apple shell single color septum ulcer disease bacterium, corn large spot disease bacterium, tobacco target spot disease bacterium, rice malignant exanthema bacterium, melon vine cutting disease bacterium, pepper anthracnose bacterium, larch dieback bacterium, blueberry ulcer bacterium, poplar bark rot bacterium, apple rot bacterium, poplar ulcer bacterium, eggplant brown mottle bacterium, melon fruit rot bacterium, tobacco brown spot bacterium, blueberry branch rot disease bacterium, apple fruit rot bacterium, cucumber sclerotinia disease bacterium and pear wave disc fruit rot bacterium.

[0032] The application provides application of the above-mentioned streptomyces fermentation liquor in preparation of a biocontrol preparation.

[0033] The biocontrol agent is used for preventing and treating diseases caused by plant pathogenic bacteria, and the plant pathogenic bacteria are selected from one or more of the following: red pine stand rot bacteria, red bean anthracnose bacteria, polygenetic small hole shell bacteria, apple shell single color septate ulcer bacteria, corn large spot bacteria, tobacco target spot bacteria, rice malignant exanthema bacteria, melon vine cutting bacteria, pepper anthracnose bacteria, larch dieback bacteria, blueberry ulcer bacteria, poplar bark rot bacteria, apple rot bacteria, poplar ulcer bacteria, eggplant brown spot bacteria, melon fruit rot bacteria, tobacco brown spot bacteria, blueberry branch rot bacteria, apple fruit rot bacteria, cucumber sclerotinia rot bacteria, and pear wave disc fruit rot bacteria.

[0034] The application provides application of the above-mentioned Streptomyces fermentation liquor in prevention and treatment of red pine stand rot.

[0035] The application has the advantages of good prevention and treatment effect, wide bacteriostatic spectrum, wide application range and the like.

[0036] The application provides a biological control method, which comprises the following steps: adopting the above-mentioned Streptomyces and / or the above-mentioned microbial agent for biological control.

[0037] The application has the advantages of good prevention and treatment effect, wide bacteriostatic spectrum, wide application range and the like. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is experimental results of Example 1 of the application, wherein A is the inhibition effect of JN4-2 live bacteria on red pine stand rot bacteria, and B is the red pine stand rot bacteria.

[0039] Figure 2 It is the culture trait of JN4-2 in Gao's No. 1 agar medium.

[0040] Figure 3 It is the morphology of JN4-2 under an optical microscope.

[0041] Figure 4 It is the phylogenetic analysis of strain JN4-2 and related strains.

[0042] Figure 5 It is the detection result of the inhibition zone of JN4-2 live bacteria on polygenetic small hole shell bacteria, wherein A is the JN4-2 live bacteria on polygenetic small hole shell bacteria, and B is the control (polygenetic small hole shell bacteria).

[0043] Figure 6 It is the detection result of the inhibition zone of JN4-2 live bacteria on apple fruit rot bacteria, wherein A is the JN4-2 live bacteria on apple fruit rot bacteria, and B is the control (apple fruit rot bacteria).

[0044] Figure 7It is the detection result of JN4-2 live bacteria on tobacco target alternaria alternata inhibition zone, wherein A is JN4-2 live bacteria on tobacco target alternaria alternata, B is control (tobacco target alternaria alternata).

[0045] Figure 8 It is the detection result of JN4-2 live bacteria on tobacco target alternaria alternata inhibition zone, wherein A is JN4-2 live bacteria on tobacco target alternaria alternata, B is control (tobacco target alternaria alternata).

[0046] Figure 9 It is the detection result of JN4-2 live bacteria on tobacco target alternaria alternata inhibition zone, wherein A is JN4-2 live bacteria on tobacco target alternaria alternata, B is control (tobacco target alternaria alternata).

[0047] Figure 10 It is the detection result of JN4-2 live bacteria on tobacco target alternaria alternata inhibition zone, wherein A is JN4-2 live bacteria on tobacco target alternaria alternata, B is control (tobacco target alternaria alternata).

[0048] Figure 11 It is the detection result of JN4-2 live bacteria on tobacco target alternaria alternata inhibition zone, wherein A is JN4-2 live bacteria on tobacco target alternaria alternata, B is control (tobacco target alternaria alternata).

[0049] Figure 12 It is the detection result of JN4-2 live bacteria on tobacco target alternaria alternata inhibition zone, wherein A is JN4-2 live bacteria on tobacco target alternaria alternata, B is control (tobacco target alternaria alternata). DETAILED DESCRIPTION

[0050] The principles and characteristics of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application and are not used to limit the scope of the present application.

[0051] The present application separates 87 kinds of actinomycetes from the understory soil of each forest area in Jilin Province, and selects the strain JN4-2 with the best inhibitory effect on the target pathogen of red pine pythium aphanidermatum. The strain JN4-2 is determined as Streptomyces netropsis by morphological observation and molecular biology analysis identification method such as 16S rDNA analysis. Therefore, it is named as Streptomyces netropsis JN4-2 (Streptomyces netropsis JN4-2), and it is preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 11, 2023, the address is No. 1, Beichen West Road, Haidian District, Beijing, China, and the preservation number is CGMCC No. 28157.

[0052] In order to better understand the biological control effect and classification position of the antagonistic actinomycete which can effectively inhibit red pine fusarium wilt, the present application determines the bacteriostatic activity and bacteriostasis spectrum of the JN4-2 live bacteria and fermentation liquor by means of plate confrontation method and cup and dish method. The research results show that the strain has inhibitory effect on 21 kinds of tested plant pathogenic bacteria, and has wide bacteriostasis spectrum. Especially worth mentioning is that the strain has very strong inhibitory effect on the red pine fusarium wilt pathogen, the bacteriostatic band width of the live bacteria is 22.54mm, and the bacteriostatic circle diameter of the fermentation liquor is 34.60mm. The present application first reports the fusiform streptomyces, and the fusiform streptomyces is applied to the biological control of the red pine fusarium wilt, and has good application and development prospect.

[0053] Tested plant pathogenic bacteria: red pine fusarium wilt Fusarium oxysporum, poplar bark disease Valsa sordida, Dothiorella gregari, apple shell single color Botryosphaeria stevensii, poplar ulcer disease Botryosphaeria dothidea, larch dieback disease Neofusicoccum laricinum, apple fruit rot disease Monilinia polystroma, apple rot disease Valsamali isolated and preserved by Jilin Forest Science Research Institute; red bean anthracnose Colletotrichum sp, corn large spot disease Exserohilum turcicum, tobacco target spot disease Rhizoctonia solani, rice evil disease F.moniliforme, melon vine cutting disease Fusarium oxysporum, pepper anthracnose C.gloeosporioides, eggplant brown spot disease Phomopsis vexans, melon fruit rot disease Pythium aphanidermatum, tobacco alternaria disease Alternaria alternata, cucumber sclerotinia disease Sclerotinia scleroriorum, presented by Shenyang Agricultural University Plant Virus Research Room; blueberry ulcer disease Botryosphaeria dothidea and blueberry branch rot disease Neofusicoccum parvum presented by Professor Xu Chengnan of Yan'an University School of Life Sciences; pear Phytophthora cactorum provided by Dalian Customs Technical Center Li Xin Researcher; the public can obtain only a copy of the present application for non-commercial purposes.

[0054] Tested soil samples are collected from 26 soil samples in Hunchun, Dunhua, Helong and Jiangnan Forest Farm in Jilin Province.

[0055] Tested culture medium:

[0056] Gause's No. 1 agar: KNO3 1 g, K2HPO4 0.5 g, MgSO4 0.5 g, NaCl 0.5 g, FeSO4 0.01 g, soluble starch 20 g, agar 20 g, per 1000 mL water, pH 7.2-7.4.

[0057] Potato dextrose agar (PDA): potato 200 g, glucose 20 g, agar 20 g, per 1000 mL distilled water.

[0058] Fermentation medium: wheat bran 40 g, corn flour 20 g, sucrose 60 g, potassium nitrate 4 g, calcium carbonate 0.4 g, potassium phosphate dibasic 0.2 g, per 1000 mL distilled water, pH 7.0.

[0059] Test agent:

[0060] Ezup column bacterial genomic DNA extraction kit was purchased from Shengong Bioengineering (Shanghai) Co., Ltd. (hereinafter referred to as Shengong Bioengineering Co., Ltd.); 16S rDNA Bacterial Identification PCR Kit, Agarose Gel DNA Purification Kit Ver 2.0 and DNA Marker DL2000 were purchased from Baobioengineering (Dalian) Co., Ltd.; and the others were all domestic analytical pure.

[0061] Universal primers 27F and 1429R were purchased from Changchun Kumi Biological Technology Co., Ltd.

[0062] Instrument: BX53 type Olympus optical microscope was purchased from Olympus Corporation.

[0063] In the examples, if data processing is involved, SPSS 23.0 is used for data statistics; and MEGA X version is used to establish the phylogenetic tree.

[0064] In the present application, if not specifically stated, the experimental methods used are conventional experimental methods in the art; the materials, reagents and instruments used are conventional materials, reagents and instruments in the art, which can be obtained through commercial channels or prepared by conventional methods.

[0065] The following will be described through specific examples.

[0066] Example 1: Screening of biocontrol streptomyces strains

[0067] In June-July 2022, 26 soil samples were collected from Hunchun, Dunhua, and Longjiang in Jilin Province, and Jiangnan Forest Farm in Jilin City, Jilin Province. 100 ml of sterile water was mixed with 10 g of soil sample, and then diluted to 10 -3 、10 -4 、10 -5 Different concentrations were coated on the high's agar medium, and the strains were isolated and screened. After the medium grew single colonies, they were transferred to high's agar medium for purification culture, and the purified culture was repeated 3 times. The screened strains were numbered, transferred to the slope, and stored in a 4°C refrigerator for standby.

[0068] After isolation, purification and dilution culture, 87 purified strains with different morphologies, sizes and colors were obtained. The antibacterial activity of the above strains was detected by plate confrontation test using red pine stand disease bacteria as target bacteria.

[0069] The test method of plate confrontation test includes the following steps: the obtained strain is cultured to grow vigorously, and the inhibition effect of the isolated strain on red pine stand disease bacteria is determined. The specific method is as follows: a 7mm fungus cake is made on the red pine stand disease bacteria plate with a puncher, and at a distance of 2cm from the center of the PDA plate(diameter 90mm), one side is placed with the pathogenic fungus cake, and the other side is streaked with the inoculation ring to pick the cultured test strain. The plate without actinomycetes inoculation is used as the control group, and the culture is incubated at 28°C. When the control group colony grows on the plate, the average distance between the pathogenic fungus cake and the test strain is measured with a vernier caliper, and each group is repeated 3 times. The strain with vigorous growth and best antibacterial effect is selected as the next research object.

[0070] The plate confrontation test results show that among the 87 screened strains, 14 strains show obvious antibacterial effect(Table 1), among which JN4-2 from Jiangnan Forest Farm has the best antibacterial effect on red pine stand disease bacteria, and the antibacterial band of JN4-2 can reach 22.54mm( Figure 1 ). Therefore, JN4-2 is selected as the further research object.

[0071] Table 1 Antimicrobial activity of Streptomyces strains against red pine stand disease bacteria

[0072] The data in the table are mean ± standard deviation.

[0073] Example 2 Morphological observation of JN4-2 strain

[0074] Strain JN4-2 was inoculated on Gause's No.1 agar medium, a sterilized cover glass was inserted in the medium at 45°, and the culture was incubated at 28°C for about 5 days. The cover glass was taken out and placed under a BX53 Olympus optical microscope to observe the morphology of the strain. According to the research methods of the International Streptomyces Project and the Actinomycete Classification Group of the Chinese Academy of Sciences Microbiology Institute, the strain was cultured on an actinomycete identification medium, and its growth and appearance characteristics were observed.

[0075] Morphological observation results of aerial hyphae and spore filaments: strain JN4-2 grew radially on Gause's No.1 agar medium, and the aerial hyphae were very lush. When incubated at 28°C for 1-3 days, the colonies were round and smooth, and no spores were generated. On the 5th day, white spores grew from the edge of the colonies, and gradually changed to meat shell white as the number of growth days increased. The color of the substrate mycelium was yellow, and no soluble pigment was produced. After continuous culture for 30 days, the colonies were cheese yellow. Figure 2 ) Microscopically, the hyphae were long and branched, straight, not broken, and had no transverse membranes. A large number of spores were dispersed around the hyphae Figure 3 ).

[0076] Example 3 Identification of JN4-2 strain

[0077] Molecular biology identification of JN4-2 by 16S rDNA sequencing included the following steps: genomic DNA of JN4-2 was extracted using Ezup column bacterial genomic DNA extraction kit (Sheng Wu Biotechnology Co., Ltd.).

[0078] PCR amplification was performed using 16S rDNA Bacterial Identification PCR Kit. The PCR reaction system (50 μL) included: PCR Mixture 25 μL, Template 2 μL, Universal Primer 27F, 1429R each 2 μL, ddH2O 19 μL. The PCR reaction conditions were: denaturation at 94℃ for 4 min; denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1 min, 35 cycles; extension at 72℃ for 10 min, 4℃ storage. The PCR product was used as a negative control with sterile water, and the PCR product of a strain with a measured 16S rRNA result in the laboratory was used as a positive control. The loading amount was 2 μL, and 2% agarose gel electrophoresis (DNA Marker was DL2000) was used. The PCR product was recovered using Agarose Gel DNA Purification Kit Ver 2.0. The recovered product was sent to Jilin Province Kumai Biotechnology Co., Ltd. for sequencing analysis. The obtained strain 16s sequence was spliced, and the measured sequence was compared with the known similar mode strain sequence downloaded in GenBank for homology analysis using the BLAST program of NCBI, and the neighbor-joining (NJ) method of MEGA X software was used for sequence alignment and phylogenetic tree drawing.

[0079] After PCR amplification, purification and sequencing of strain JN4-2, the length of its 16S rDNA sequence was measured to be 1409 bp. Twelve strains with higher homology to strain JN4-2 were selected to establish a phylogenetic tree, as shown in Figure 4 compared with the 16S rDNA sequence of GenBank database, the homology with Streptomyces netropsis was the highest, with a similarity value of 100%. The results showed that strain JN4-2 and Streptomyces netropsis (GenBank: EU119185) were clustered in one branch, with the closest genetic relationship. Strain JN4-2 (OR758449) was identified as Streptomyces netropsis.

[0080] Therefore, it was named Streptomyces netropsis JN4-2, and was preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 11, 2023, with an address of No. 1 Yard, West Beichen Road, Chaoyang District, Beijing, China, and a preservation number of CGMCC No. 28157.

[0081] Example 4 Determination of antagonistic activity of strain JN4-2

[0082] The antibacterial activity of the antagonistic strain JN4-2 against the tested strain was detected using a plate confrontation test. The test method for the plate confrontation test is the same as in Example 1.

[0083] The tested strains were: *Pseudomonas aeruginosa*, *Pseudomonas erythrinae*, *Sclerotium chrysogenum*, *Pseudomonas monochromaticis*, *Pseudomonas maize*, *Pseudomonas truncatum*, *Pseudomonas taeniformis*, *Pseudomonas jaundice*, *Pseudomonas citrinum ...

[0084] Experimental results showed that the antagonistic bacterium JN4-2 had an inhibitory effect on all tested strains. Among them, JN4-2 had the strongest inhibitory effect on *Pseudomonas aeruginosa*, the pathogen of *Pinus koraiensis*, with a clear inhibition band of 22.54 mm in width, showing a significant difference in effect compared to other tested pathogens (Table 2); it also showed an inhibitory effect on *Cryptococcus faecium* (…). Figure 5 ), blueberry canker fungus and apple fruit rot fungus ( Figure 6 It also has a strong inhibitory effect, with inhibition bands of 17.19 mm, 16.23 mm, and 16.33 mm, respectively, against tobacco target spot pathogens (…). Figure 7 ), corn leaf blight fungus, and fruit rot fungus ( Figure 8 The inhibitory effect of bacteria such as 10.13 mm is relatively weak, but the width of the antibacterial band still reaches more than 10.13 mm.

[0085] Table 2. Antibacterial spectrum of antagonistic bacteria JN4-2 (live bacteria)

[0086]

[0087]

[0088] The data in the table are mean ± standard deviation.

[0089] Example 5: Preparation of fermentation broth for strain JN4-2 and determination of its antibacterial spectrum.

[0090] Because strain JN4-2 exhibits the strongest antibacterial activity, it was selected for cultivation using fermentation medium. Strain JN4-2 was streaked onto Gao's No. 1 agar plates. After vigorous growth, spores were scraped into sterile water to prepare a spore suspension as seed culture. 2 ml of the seed culture was added to 50 mL of fermentation medium and incubated at 28°C with constant shaking at 150 rpm for 6 days. The resulting fermentation broth was centrifuged at 6000 rpm / min at 4°C for 10 min. The supernatant was transferred to a 50 mL centrifuge tube, sealed, and stored at -20°C for later use.

[0091] The cup and dish method is used to detect the bacteriostatic spectrum of the fermentation liquor of the strain JN4-2, including the following steps: 7mm pathogenic bacteria are prepared, the pathogenic bacteria are symmetrically inverted on the four sides of the PDA culture medium, the Oxford cup is placed in the center of the plate, the fermentation liquor (i.e. the supernatant prepared by the above method) is inoculated in the Oxford cup, the inoculation amount is 200 μL, distilled water is used as a control, and constant temperature culture is carried out at 28 DEG C; after the control group of bacteria grows on the culture plate, the diameter of the bacteriostatic circle of the treatment group is measured by the cross method.

[0092] The test pathogenic bacteria are as follows: red pine blight fungus, red bean anthracnose fungus, aggregate poria fungus, apple shell single-color septate ulcer disease fungus, corn large spot fungus, tobacco target spot fungus, rice malignant edema fungus, melon vine cutting fungus, pepper anthracnose fungus, larch dieback fungus, blueberry ulcer disease fungus, poplar bark rot fungus, apple rot fungus, poplar ulcer disease fungus, eggplant brown mottle fungus, melon fruit rot fungus, tobacco brown spot fungus, blueberry branch rot disease fungus, apple fruit rot fungus, cucumber sclerotinia disease fungus, pear boshuan fruit rot fungus.

[0093] The results show that the cup and dish method is used to determine the bacteriostatic activity of the fermentation liquor of the antagonistic strain JN4-2, and the fermentation liquor still has good bacteriostatic activity; the fermentation liquor of the strain has the strongest bacteriostatic effect on the red pine blight fungus ( Figure 9 ), and the difference is significant compared with other test strains; the fermentation liquor has very strong antagonistic effect on the red bean anthracnose fungus ( Figure 10 ) and apple rot fungus, and the bacteriostatic diameters are 31.92 mm and 29.87 mm respectively; the fermentation liquor also has good inhibition effect on the eggplant brown mottle fungus, melon vine cutting fungus ( Figure 11 ), larch dieback fungus ( Figure 12 ), and the bacteriostatic diameters are 27.60 mm, 27.07 mm and 28.46 mm respectively; the fermentation liquor of the antagonistic strain has obvious inhibition effect on all test pathogenic bacteria (Table 3), and the bacteriostatic diameter of the tobacco target spot fungus is the smallest, but still reaches 18.25 mm.

[0094] Table 3 Bacteriostatic spectrum of the fermentation liquor of the antagonistic strain JN4-2

[0095]

[0096]

[0097] The data in the table are average value ± standard deviation.

[0098] In summary, the JN4-2 strain provided in the application has fungicidal activity, and the viable bacteria and fermentation liquor have strong inhibition effect on the red pine blight fungus, and it is analyzed that the strain may produce antifungal substances in the physiological metabolism process.

[0099] The obtained antagonistic strain JN4-2 has a broad development prospect, has a wide bacteriostatic spectrum, and has great biological control potential; the active bacteriostatic component in the fermentation liquor of the strain JN4-2 can be developed and applied as a biological pesticide in the prevention and treatment of various plant diseases after separation and purification and other processes, and compared with chemical pesticides, the fermentation liquor of the strain JN4-2 has the advantages of low toxicity, no residue, environmental friendliness and the like, and is more in line with the sustainable control and environmental protection concept.

[0100] The above merely describes preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing Streptomyces fermentation broth for biological control, characterized in that, Includes the following steps: *Streptomyces fusiforme* was fermented at 28℃ for 6 days. The fermentation medium consisted of: 40g wheat bran, 20g corn flour, 60g sucrose, 4g potassium nitrate, 0.4g calcium carbonate, 0.2g dipotassium hydrogen phosphate, and 1000mL distilled water at pH 7.

0. The mixture was centrifuged, and the supernatant was collected. The strain of *Streptomyces fusiforme* was named *Streptomyces fusiforme* (…). Streptomyces netropsis JN4-2, with accession number CGMCC No.28157.

2. A Streptomyces fermentation broth for biological control, characterized in that, Prepared using the preparation method described in claim 1.

3. The application of the Streptomyces fermentation broth according to claim 2 in the control of plant pathogens, characterized in that, The plant pathogens mentioned are selected from one or more of the following: *Pseudomonas aeruginosa*, *Pseudomonas erythropus*, *Sclerotium chrysogenum*, *Pseudomonas monochromaticis*, *Pseudomonas maize*, *Pseudomonas tobacco*, *Pseudomonas bakanae*, *Pseudomonas melonensis*, *Pseudomonas chili*, *Pseudomonas larchensis*, *Pseudomonas blueberry*, *Pseudomonas poplarensis*, *Pseudomonas appleensis*, *Pseudomonas pustulosa*, *Pseudomonas auricula-judae*, *Pseudomonas auricula-judae*, *Pseudomonas auricula-judae*, *Pseudomonas blueberryensis ... appleensis*, *Pseudomonas cucumberensis*, and *Pseudomonas pearensis*.

4. The application of the Streptomyces fermentation broth according to claim 2 in the prevention and control of symptoms caused by plant pathogens, characterized in that, The plant pathogens mentioned are selected from one or more of the following: *Pseudomonas aeruginosa*, *Pseudomonas erythropus*, *Sclerotium chrysogenum*, *Pseudomonas monochromaticis*, *Pseudomonas maize*, *Pseudomonas tobacco*, *Pseudomonas bakanae*, *Pseudomonas melonensis*, *Pseudomonas chili*, *Pseudomonas larchensis*, *Pseudomonas blueberry*, *Pseudomonas poplarensis*, *Pseudomonas appleensis*, *Pseudomonas pustulosa*, *Pseudomonas auricula-judae*, *Pseudomonas auricula-judae*, *Pseudomonas auricula-judae*, *Pseudomonas blueberryensis ... appleensis*, *Pseudomonas cucumberensis*, and *Pseudomonas pearensis*.

5. The application of the Streptomyces fermentation broth according to claim 2 in the preparation of biocontrol agents, characterized in that, Biocontrol agents are used to control diseases caused by plant pathogens; the plant pathogens are selected from one or more of the following: *Pseudomonas aeruginosa*, *Pseudomonas aeruginosa*, *Pseudomonas aeruginosa*, *Pseudomonas monochromaticis*, *Pseudomonas aeruginosa ...

6. The application of the Streptomyces fermentation broth according to claim 2 in the prevention and control of damping-off disease in Korean pine.

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