Preparation method of supernate for biological control

Abstract

The invention relates to a preparation method of a supernatant for biological control, which comprises the following steps: fermenting and centrifuging streptomyces fusiformis, and taking the supernatant; the strain name of the streptomyces fusiformis is streptomyces fusiformis JN4-2, and the preservation number of the streptomyces fusiformis JN4-2 is CGMCC (China General Microbiological Culture Collection Center) No. 28157. The supernate can be used for treating Korean pine rhizoctonia solani, red adzuki bean colletotrichum gloeosporioides, symbiotic microporus, apple shell monochromatic alternaria solani, exserohilum turcicum, tobacco target leaf spot, fusarium moniliforme, muskmelon vine cutting pathogen, pepper colletotrichum gloeosporioides, dahurian larch shoot pathogen, blueberry canker pathogen, poplar skin rot pathogen and valsa mali. The biological control agent can be used for biologically controlling pathogenic bacteria such as poplar canker pathogen, eggplant brown rot pathogen, pythium aphanidermatum, alternaria alternata, blueberry branch blight pathogen, apple fruit rot pathogen, cucumber sclerotinia sclerotiorum and the like, and has the advantages of good biocontrol effect, wide antibacterial spectrum, wide application range and the like.

Description

[0001] This invention is a divisional application. The original Chinese patent application number was: 202410127548.6, the application date was: January 30, 2024, and the patent name at the time of application was: A strain of Streptomyces fusiformis and its application. Technical Field

[0002] This invention belongs to the field of microbial technology, and particularly relates to a method for preparing a supernatant for biological control. Background Art

[0003] Pinus koraiensis belongs to the genus Pinus of the family Pinaceae. It is native to the northern regions of Eurasia and is the main constructive and dominant tree species in the forest areas of Northeast China. Pinus koraiensis is a light-loving plant with strong cold tolerance and good corrosion resistance. It is suitable for planting in moist slightly acidic or neutral soils. An atmospheric humidity above 0.7 promotes the growth of Pinus koraiensis.

[0004] Red pine damping-off is an infectious disease of seedlings caused by fungi (mainly Rhizoctonia and Fusarium). It occurs widely and causes serious damage. After the seedlings are infected, the mortality rate is extremely high. The incidence rate of 1-2-year-old seedlings is extremely high, and in severe cases, it reaches more than 60%, or even results in a complete crop failure. In production, chemical control and soil disinfectant treatment are still mainly used to prevent the occurrence and harm of this disease. The excessive use of chemical pesticides not only causes the pathogens to develop drug resistance but also causes irreversible damage to the ecological environment. The frequent use of soil disinfectants leads to the degradation of the nursery soil environment and the destruction of the microbial community, which further results in poor nutritional conditions and weak growth potential of the seedlings, and problems such as low survival rate and poor adaptability in subsequent afforestation and ecological restoration. In the past 40 years, China's agricultural production has mainly relied on chemical inputs, and the use of pesticides has increased significantly. According to the statistics of the State Environmental Protection Administration, in 2007, China produced approximately 2.5 million tons of pesticide raw materials, processed into more than 8 million tons of preparations, and applied them to the environment in various ways. 10%-20% adheres to the plants, and 80%-90% scatters in the soil, water, and atmosphere (Yang Yonghua, Yao Jian, Hua Xiaomei. The impact of pesticide pollution on the functional diversity of soil microbial communities [J]. Journal of Microbiology, 2000, (02): 23-25+47.). Facing the above problems, biological control is becoming more and more popular.

[0005] In the 1960s in China, Wu Yousan et al. first reported this disease. Since then, there have been numerous studies on the occurrence and control of this disease. The occurrence of damping-off of pine seedlings in Northeast China is affected by soil temperature. Rhizoctonia solani occurs more frequently at 18-22°C, Pythium at 17-22°C, and Fusarium at 23-28°C. The prevalence of damping-off of pine seedlings in Northeast China is mainly affected by soil moisture content and rainfall. High rainfall and high soil moisture content are conducive to the occurrence of the disease. In addition, the longer the onset period, the higher the overall incidence rate (Wu Yousan, Gao Ya, Gu Sifang, etc. Research on damping-off of pine seedlings II. Disease prevalence [J]. Acta Phytophylacica Sinica, 1963, (04): 399-408.). Tong Ying et al. (1979) found through experiments that the main pathogens causing damping-off of pine seedlings are Rhizoctonia solani, Fusarium, and Pythium, and Rhizoctonia solani has the strongest pathogenicity ([4] Tong Ying. Damping-off of pine seedlings and its control [J]. Journal of Jilin Forestry Science and Technology, 1979, (02): 117-120.). Zhang Lijun et al. (2012) found that the damping-off pathogen of Korean pine overwinters as mycelium and spores in the residues of various hosts and soil, and spreads among rows through mycelium. Adverse environmental conditions such as low temperature, rainy weather, and insufficient light will promote the infection of the pathogen, especially low temperature has the greatest impact on the disease (Zhang Lijun, Wang Baiping, Ni Tianbo, Li Chuncheng, Jia Junying. Occurrence regularity and control technology of damping-off of Korean pine [J]. Agriculture and Technology, 2012, 32(09): 116.).

[0006] Plant diseases are causing increasingly serious harm to agriculture and forestry production, and the economic losses are incalculable. With the gradual aggravation of the disadvantages of chemical agent control, biological control has attracted widespread attention from the whole society. It has multiple advantages such as high efficiency, non-toxicity, and environmental protection, and will become the mainstream means of plant disease control. Among the more than 16,500 kinds of antibiotics reported in the world at present, actinomycetes account for more than half of the proportion. Actinomycetes are the first discovered class of microorganisms with biological control effects. The genus Streptomyces in actinomycetes is the genus that produces the most antibacterial substances. Gao Yuhong et al. (2009) from Northeast Agricultural University reported the novel agricultural antibiotic doramectin, an avermectin antibiotic produced by fermentation of a new species of recombinant Streptomyces avermitilis. Doramectin is one of the best anti-parasitic drugs in the avermectin family at present and is an internal and external killer, having good killing effects on nematodes and arthropods (Jiang Wei. Mutant 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 the low-concentration metabolites of S. philanthi RM-1-138, S. philanthi RL-1-178, and S. mycarofaciens SS-2-243 had an inhibition rate of 100% on the spore germination of the pathogen (Botrytis cinerea) of tomato gray mold (

[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 effects. Therefore, it is crucial to provide strains that can be effectively used for biological control. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides a method for preparing a supernatant for biological control. The supernatant is prepared using Streptomyces netropsis JN4-2, which is first discovered in the present invention. After further verification, it is found that the supernatant prepared using this strain has advantages such as good biocontrol effect, wide antibacterial spectrum, and wide application range.

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

[0009] The present invention provides a strain of Streptomyces, named Streptomyces netropsis JN4-2, with a preservation number of CGMCC No. 28157. This strain was preserved at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (CGMCC) on August 11, 2023, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0010] In the present invention, multiple strains of actinomycetes were isolated from soils collected from different regions of Jilin Province. Through multiple screening and determination of the control effect, finally, 1 strain of antagonistic strain with the best effect against the damping-off pathogen of Pinus koraiensis was obtained, labeled as JN4-2, achieving a breakthrough in the field of actinomycetes for controlling the damping-off of Pinus koraiensis and providing new strains and products for the biological control of the damping-off of Pinus koraiensis.

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

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

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

[0014] The present invention provides a preparation method for the above-mentioned microbial 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: 40 g of wheat bran, 20 g of corn flour, 60 g of sucrose, 4 g of potassium nitrate, 0.4 g of calcium carbonate, 0.2 g of dipotassium hydrogen phosphate, per 1000 mL of distilled water, pH 7.0. The fermentation culture conditions can include: culturing at 28 °C for 6 days.

[0015] The present invention provides a preparation method for a supernatant 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, with a preservation number of CGMCC No. 28157.

[0016] The fermentation temperature can be 28°C.

[0017] The fermentation time can be 6 days.

[0018] The present invention provides a supernatant for biological control. The preparation method of the supernatant may include the following steps: inoculating the seed liquid of the above-mentioned Streptomyces fusiformis into a fermentation medium, culturing at 28°C and 150 r / min under constant temperature oscillation for 6 days, then centrifuging and taking the supernatant.

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

[0020] The preparation method of the seed liquid may include the following steps: streaking and culturing the above-mentioned Streptomyces fusiformis on a plate, scraping the spores into sterile water to make a spore suspension, which is the seed liquid.

[0021] The present invention provides a supernatant for biological control, and the supernatant is prepared by the above preparation method.

[0022] The present invention provides the application of the above-mentioned Streptomyces fusiformis in the prevention and control of plant pathogenic bacteria.

[0023] The strain provided by the present invention can be used to prepare a biological control agent for preventing and controlling one or more of Rhizoctonia solani, Colletotrichum truncatum, Dothiorella gregaria, Diaporthe mali, Exserohilum turcicum, Corynespora cassiicola, Fusarium moniliforme, Fusarium oxysporum f. sp. niveum, Colletotrichum capsici, Botryosphaeria laricina, Botryosphaeria dothidea, Valsa mali, Dothiorella gregaria, Phomopsis vexans, Pythium aphanidermatum, Alternaria alternata, Botryosphaeria dothidea, Sclerotinia sclerotiorum, Botryosphaeria dothidea; for preventing and controlling the diseases caused by the above-mentioned pathogenic bacteria.

[0024] The present invention provides the application of the above-mentioned bacterial agent in the prevention and control of plant pathogenic bacteria. The plant pathogenic bacteria are selected from one or more of Rhizoctonia solani, Colletotrichum truncatum, Dothiorella gregaria, Diaporthe mali, Exserohilum turcicum, Corynespora cassiicola, Fusarium moniliforme, Fusarium oxysporum f. sp. niveum, Colletotrichum capsici, Botryosphaeria laricina, Botryosphaeria dothidea, Valsa mali, Dothiorella gregaria, Phomopsis vexans, Pythium aphanidermatum, Alternaria alternata, Botryosphaeria dothidea, Sclerotinia sclerotiorum, Botryosphaeria dothidea. The above-mentioned bacterial agent provided by the present invention can be used to prevent and control the diseases caused by the above-mentioned pathogenic bacteria.

[0025] The present invention provides the application of supernatant in the prevention and control of plant pathogenic bacteria.

[0026] The plant pathogenic bacteria are selected from one or more of Rhizoctonia solani, Colletotrichum truncatum, Dothiorella gregaria, Diaporthe eres, Exserohilum turcicum, Corynespora cassiicola, Fusarium moniliforme, Fusarium oxysporum f. sp. niveum, Colletotrichum capsici, Botryosphaeria laricina, Botryosphaeria dothidea, Valsa mali, Dothiorella gregaria, Phomopsis asparagi, Pythium aphanidermatum, Alternaria alternata, Botryosphaeria dothidea, Sclerotinia sclerotiorum, Botryosphaeria dothidea.

[0027] The supernatant can be prepared by the following method: inoculating the seed liquid of the above streptomyces into the fermentation medium, culturing at 28 °C and 150 r / min under constant temperature and shaking for 6 d, then centrifuging and taking the supernatant.

[0028] The formula of the fermentation medium may include: 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, per 1000 mL of distilled water, pH 7.0.

[0029] The preparation method of the seed liquid includes the following steps: streaking and culturing the above streptomyces on a plate, scraping the spores into sterile water to make a spore suspension, which is the seed liquid.

[0030] The above supernatant provided by the present invention can be used to prevent and control the diseases caused by the above plant pathogenic bacteria.

[0031] The plant pathogenic bacteria are selected from one or more of Rhizoctonia solani, Colletotrichum truncatum, Dothiorella gregaria, Diaporthe eres, Exserohilum turcicum, Corynespora cassiicola, Fusarium moniliforme, Fusarium oxysporum f. sp. niveum, Colletotrichum capsici, Botryosphaeria laricina, Botryosphaeria dothidea, Valsa mali, Dothiorella gregaria, Phomopsis asparagi, Pythium aphanidermatum, Alternaria alternata, Botryosphaeria dothidea, Sclerotinia sclerotiorum, Botryosphaeria dothidea.

[0032] The present invention provides the application of the above supernatant in the preparation of biological control agents.

[0033] The biocontrol agent is used to prevent and control diseases caused by plant pathogenic bacteria; the plant pathogenic bacteria are selected from one or more of Rhizoctonia solani, Colletotrichum truncatum, Dothiorella gregaria, Diaporthe eres, Exserohilum turcicum, Corynespora cassiicola, Fusarium moniliforme, Fusarium oxysporum f. sp. melonis, Colletotrichum capsici, Botryosphaeria laricina, Botryosphaeria dothidea, Valsa sordida, Valsa mali, Dothiorella gregaria, Pythium aphanidermatum, Alternaria alternata, Botryosphaeria dothidea, Sclerotinia sclerotiorum, Botryosphaeria dothidea, and Macrophoma kawatsukai.

[0034] The present invention provides the application of the above supernatant in the prevention and control of damping-off of Pinus koraiensis.

[0035] When applied, the present invention has the advantages of good control effect, broad antibacterial spectrum, wide application range, etc.

[0036] The present invention provides a biological control method, which includes the following steps: using the above streptomyces and / or the above bacterial agent for biological control. The above supernatant can also be used for biological control.

[0037] The method provided by the present invention has the advantages of good control effect, broad antibacterial spectrum, wide application range, etc. Description of the Drawings

[0038] Figure 1 It is the experimental result of Example 1 of the present invention. Among them, A is the inhibitory effect of JN4-2 viable bacteria on Rhizoctonia solani, and B is Rhizoctonia solani.

[0039] Figure 2 It is the culture characteristics of JN4-2 on Gause'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 viable bacteria against Dothiorella gregaria. Among them, A is JN4-2 viable bacteria against Dothiorella gregaria, and B is the control (Dothiorella gregaria).

[0043] Figure 6 It is the detection result of the inhibition zone of JN4-2 viable bacteria against Botryosphaeria dothidea. Among them, A is JN4-2 viable bacteria against Botryosphaeria dothidea, and B is the control (Botryosphaeria dothidea).

[0044] Figure 7Detection results of the antibacterial zone of JN4-2 live bacteria against tobacco target spot pathogen. Among them, A is JN4-2 live bacteria against tobacco target spot pathogen, and B is the control (tobacco target spot pathogen).

[0045] Figure 8 Detection results of the antibacterial zone of JN4-2 live bacteria against Pythium aphanidermatum. Among them, A is JN4-2 live bacteria against Pythium aphanidermatum, and B is the control melon (control of Pythium aphanidermatum).

[0046] Figure 9 Detection results of the antibacterial circle of JN4-2 fermentation broth against Rhizoctonia solani. Among them, A is JN4-2 fermentation broth against Rhizoctonia solani, and B is the control (Rhizoctonia solani).

[0047] Figure 10 Detection results of the antibacterial circle of JN4-2 fermentation broth against Colletotrichum truncatum. Among them, A is JN4-2 fermentation broth against Colletotrichum truncatum, and B is the control (Colletotrichum truncatum).

[0048] Figure 11 Detection results of the antibacterial circle of JN4-2 fermentation broth against Fusarium oxysporum f. sp. melonis. Among them, A is JN4-2 fermentation broth against Fusarium oxysporum f. sp. melonis, and B is the control (Fusarium oxysporum f. sp. melonis).

[0049] Figure 12 Detection results of the antibacterial circle of JN4-2 fermentation broth against Botryosphaeria laricina. Among them, A is JN4-2 fermentation broth against Botryosphaeria laricina, and B is the control (Botryosphaeria laricina). Detailed implementation manners

[0050] The principles and characteristics of the present invention will be described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0051] In the present invention, 87 kinds of actinomycetes were isolated from the understory soil in various forest areas of Jilin Province, and screened using Rhizoctonia solani as the target pathogen. The strain JN4-2 with the best inhibitory effect on Rhizoctonia solani was screened out. Through morphological observation and molecular biological analysis and identification methods such as 16S rDNA analysis, the strain JN4-2 was identified as Streptomyces netropsis. Therefore, it was named Streptomyces netropsis JN4-2, and on August 11, 2023, it was deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No. 28157.

[0052] In order to better understand the biological control effect and classification status of antagonistic actinomycetes that can effectively inhibit the pathogen of Korean pine damping-off, the present invention measured the antibacterial activity and antibacterial spectrum of JN4-2 live bacteria and fermentation liquid by plate confrontation method and cup-disc method respectively. The research results show that the strain has an inhibitory effect on 21 tested plant pathogens and a wide antibacterial spectrum. It is particularly worth mentioning that the strain has a strong inhibitory effect on the pathogen of Korean pine damping-off, the antibacterial bandwidth of the live bacteria reaches 22.54mm, and the diameter of the antibacterial zone of the fermentation liquid reaches 34.60mm. The present invention reports the fusiformis streptomyces for the first time, as well as the application of the fusiformis streptomyces in the biological control of Korean pine damping-off, which has a good application development prospect.

[0053] The tested plant pathogens: Fusariumoxysporum, Valsasordida, Dothiorella gregari, Botryosphaeriastevensii, Botryosphaeria dothidea, Neofusicoccumlaricinum, Monilinia polystroma, and Valsa mali were isolated and preserved by Jilin Academy of Forestry Sciences; Colletotrichum sp., Exserohilumturcicum, Rhizoctonia solani, F.moniliforme, Fusariumoxysporum, C.gloeosporioides, Phomopsisvexans, and Pythium were isolated and preserved by Jilin Academy of Forestry Sciences. aphanidermatum, tobacco brown spot pathogen Alternaria alternata, cucumber sclerotinia pathogen Sclerotinia scleroriorum were donated by the Plant Virus Research Laboratory of Shenyang Agricultural University; blueberry canker pathogen Botryosphaeria dothidea and blueberry branch blight pathogen Neofusicoccum parvum were donated by Associate Professor Xu Chengnan of the School of Life Sciences of Yan'an University; pear fruit rot pathogen Phytophthora cactorum was provided by Researcher Li Xin of Dalian Customs Technology Center; the public can obtain the embodiments recorded in the present invention for non-commercial purposes only.

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

[0055] Test medium:

[0056] Gause's No. 1 agar medium: KNO 3 1 g, K 2 HPO 4 0.5 g, MgSO 4 0.5 g, NaCl 0.5 g, FeSO 4 0.01 g, soluble starch 20 g, agar 20 g, per 1000 mL of water, pH 7.2 - 7.4.

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

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

[0059] Test agents:

[0060] Ezup Column Bacterial Genomic DNA Extraction Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. (abbreviation: Sangon Biotech Co., Ltd.); 16S rDNA Bacterial Identification PCR Kit, Agarose Gel DNA Purification Kit Ver 2.0 and DNA Marker DL2000 were purchased from Takara Bio Inc. (Dalian); others are all domestic analytical pure.

[0061] Universal primers 27F and 1429R were both purchased from Changchun Kumei Biotechnology Co., Ltd.

[0062] Instrument: BX53 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; the phylogenetic tree is established using MEGAX version.

[0064] In the present invention, unless otherwise specified, the experimental methods used are all conventional experimental methods in the art; the materials, reagents, and instruments used are all conventional materials, reagents, and instruments in the art, and can be obtained through commercial channels or prepared by conventional methods.

[0065] The following is introduced through specific examples.

[0066] Example 1 Screening of biocontrol Streptomyces strains

[0067] From June to July 2022, 26 soil samples were collected from places such as Hunchun, Dunhua, Helong in Jilin Province, and Jiangnan Forest Farm in Panshi City, Jilin City, Jilin Province. After fully mixing 100 ml of sterile water with 10 g of soil sample and standing still, it was diluted into 10 -3 、10 -4 、10 -5 Different concentrations were spread on Gao's No. 1 agar medium to isolate and screen the strains. After separate colonies grew on the medium, they were transferred to Gao's No. 1 agar medium for purification culture. The purification culture was repeated 3 times. The selected strains were numbered and transferred to a slant and stored in a 4℃ refrigerator for standby.

[0068] After isolation and purification by the dilution culture method, 87 purified strains with different morphologies, sizes and colors were obtained. The antibacterial activities of the above strains were detected using the red pine damping-off pathogen as the target bacterium by the plate confrontation test.

[0069] The test method of the plate confrontation test includes the following steps: Cultivate the obtained strains until they grow vigorously, use the red pine damping-off pathogen as the target strain, and determine the inhibitory effect of the isolated strains on the red pine damping-off pathogen. The specific method is as follows: Make 7-mm-diameter bacterial cakes on the red pine damping-off pathogen plate with a puncher. At 2 cm above and below the center of the PDA plate (90 mm in diameter), place the pathogen bacterial cake on one side and streak the cultivated strain to be tested with an inoculation loop on the other side. Use the plate without inoculating actinomycetes as the control group and culture it at a constant temperature of 28℃. When the colonies in the control group cover the plate, use a vernier caliper to measure the average distance between the pathogen bacterial cake and the strain to be tested. Each treatment has 3 replicates. Select the strain with strong growth and the best antibacterial effect as the object for the next step of research.

[0070] The results of the plate confrontation test showed that among the 87 selected strains, 14 strains showed obvious antibacterial effects (Table 1). Among them, JN4-2 collected from Jiangnan Forest Farm had the best antibacterial effect on the red pine damping-off pathogen, and the antibacterial zone of JN4-2 could reach 22.54 mm( Figure 1 ). Therefore, JN4-2 was selected as the object for further research.

[0071] Table 1 Antibacterial activities of Streptomyces viable bacteria against the red pine damping-off pathogen

[0072]

[0073] The data in the table are mean ± standard deviation.

[0074] Example 2 Morphological observation of strain JN4-2

[0075] The strain JN4-2 was inoculated on Gause's No.1 agar medium. A sterilized cover glass was inserted obliquely into the medium at an angle of 45°. It was cultured at 28°C for about 5 days. Then the cover glass was taken out and placed under a BX53 Olympus optical microscope to observe the morphology of the strain. It was cultured using a medium for actinomycetes identification according to the research methods of the International Streptomyces Project and the Actinomycetes Taxonomy Group of the Institute of Microbiology, Chinese Academy of Sciences, and its growth status and appearance characteristics were observed.

[0076] Observation results of the morphology of aerial hyphae and spore chains: On Gause's No.1 agar medium, the strain JN4-2 grew radially, and the aerial hyphae were very lush. When cultured at a constant temperature of 28°C for 1-3 days, the colonies were round, smooth, and no spores were formed. On the 5th day, white spores grew from the edge of the colony and gradually changed to the color of oyster white with the increase of the growth days. The color of the substrate mycelium was yellow, and no soluble pigment was produced. After continuous culture for 30 days, the colonies were butyric yellow ( Figure 2 ). Under the microscope, it could be seen that the hyphae had a large number of branches, were slender, straight, did not break, had no cross membranes, and a large number of spores were scattered around the hyphae ( Figure 3 ).

[0077] Example 3 Identification of Strain JN4-2

[0078] Molecular biological identification of JN4-2 was carried out by 16S rDNA sequencing, including the following steps: The Ezup column bacterial genomic DNA extraction kit (Sangon Biotech Co., Ltd.) was used to extract the genomic DNA of JN4-2.

[0079] PCR amplification was carried out using the 16S rDNA Bacterial Identification PCR Kit. The PCR reaction system (50 μL) included: 25 μL of PCR Mixture, 2 μL of Template, 2 μL each of the universal primers 27F and 1429R, ddH 219 μL. PCR reaction conditions: denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min, stored at 4°C. The PCR product of sterile water was used as the negative control, and the PCR product of the strain with the determined 16S rRNA result in the laboratory was used as the 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 Kumei Biotechnology Co., Ltd. for sequencing and analysis. The obtained 16S sequences of the strain were spliced, and the sequenced sequences were compared and analyzed for homology with the sequences of known similar model strains downloaded from GenBank using the BLAST program of NCBI, and the neighbor-joining (NJ) method of MEGAX software was used for sequence alignment and phylogenetic tree diagram drawing.

[0080] After PCR amplification, purification, and sequencing of strain JN4-2, the length of its 16S rDNA sequence was measured to be 1409 bp. Twelve sequences with high homology to strain JN4-2 were selected to construct a phylogenetic tree, as Figure 4 shown. Compared with the 16S rDNA sequences in the 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) clustered in one branch and had the closest genetic relationship. Strain JN4-2 (OR758449) was identified as Streptomyces netropsis.

[0081] Therefore, it was named Streptomyces netropsis JN4-2 and was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC) on August 11, 2023. The deposit address was the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number was CGMCC No. 28157.

[0082] Example 4 Determination of the antibacterial activity of the antagonistic strain JN4-2

[0083] The plate confrontation test was used to detect the antibacterial activity of the antagonistic strain JN4-2 against the tested strains. The test method of the plate confrontation test was referred to Example 1.

[0084] The test strains were respectively: Fusarium oxysporum f. sp. pini, Colletotrichum truncatum, Dothiorella gregaria, Diaporthe eres, Exserohilum turcicum, Corynespora cassiicola, Gibberella fujikuroi, Fusarium oxysporum f. sp. niveum, Colletotrichum capsici, Botryosphaeria laricina, Botryosphaeria dothidea, Valsa sordida, Valsa mali, Dothiorella gregaria, Phytophthora aphanidermata, Alternaria alternata, Botryosphaeria dothidea, Botrytis cinerea, Sclerotinia sclerotiorum, Botryosphaeria dothidea.

[0085] The experimental results showed that the antagonistic bacterium JN4-2 had inhibitory effects on all the test strains. Among them, JN4-2 had the strongest inhibitory effect on Fusarium oxysporum f. sp. pini, with an obvious inhibition zone, and the width of the inhibition zone reached 22.54 mm, showing a significant difference from other test pathogens (Table 2); it also had a strong inhibitory effect on Dothiorella gregaria ( Figure 5 ), Botryosphaeria dothidea and Botrytis cinerea ( Figure 6 ), and the widths of the inhibition zones were 17.19 mm, 16.23 mm, and 16.33 mm respectively. It had relatively weaker inhibitory effects on Corynespora cassiicola ( Figure 7 ), Exserohilum turcicum, Phytophthora aphanidermata ( Figure 8 ), etc., but the width of the inhibition zone still reached more than 10.13 mm.

[0086] Table 2 Antibacterial spectrum of the viable cells of antagonistic bacterium JN4-2

[0087]

[0088]

[0089] The data in the table are the mean ± standard deviation.

[0090] Example 5 Preparation of the fermentation broth of strain JN4-2 and determination of its antibacterial spectrum

[0091] Since the strain JN4-2 had the strongest antibacterial activity, the strain JN4-2 was selected for culture using a fermentation medium. The strain JN4-2 was streaked and cultured on a plate of Czapek-Dox agar medium. After it grew vigorously, the spores were scraped into sterile water to make a spore suspension as the seed liquid; 2 ml of the seed liquid was pipetted into 50 mL of the fermentation medium, and cultured at 28 °C with constant shaking at 150 r / min for 6 d. The obtained fermentation broth was centrifuged at 6000 rpm / min at 4 °C for 10 min, and the supernatant was taken and placed in a 50 mL centrifuge tube, sealed and stored in a -20 °C refrigerator for standby.

[0092] The antibacterial spectrum of the fermentation broth of strain JN4-2 was detected by the cup-plate method, including the following steps: Prepare 7-mm pathogen disks, place the disks symmetrically around the periphery of the PDA medium, place Oxford cups in the center of the plate, inoculate the fermentation broth (i.e., the supernatant prepared by the above method) into the Oxford cups, with an inoculation volume of 200 μL, use distilled water inoculation as a control, incubate at a constant temperature of 28°C, and measure the diameter of the antibacterial circle of the treatment group by the cross method after the control group's bacteria cover the culture dish.

[0093] The test pathogens were respectively: Rhizoctonia solani of Pinus koraiensis, Colletotrichum truncatum of Vigna angularis, Dothiorella gregaria, Diaporthe eres of Malus domestica, Setosphaeria turcica of Zea mays, Corynespora cassiicola of Nicotiana tabacum, Fusarium moniliforme of Oryza sativa, Fusarium oxysporum f. sp. niveum of Cucumis melo, Colletotrichum capsici of Capsicum annuum, Botryosphaeria laricina of Larix principis-rupprechtii, Botryosphaeria dothidea of Vaccinium uliginosum, Valsa sordida of Populus, Valsa mali of Malus domestica, Dothiorella gregaria of Populus, Phyllosticta solani of Solanum melongena, Pythium aphanidermatum of Cucumis sativus, Alternaria alternata of Nicotiana tabacum, Botryosphaeria dothidea of Vaccinium uliginosum, Physalospora piricola of Pyrus pyrifolia, Sclerotinia sclerotiorum of Cucumis sativus, Botryosphaeria dothidea of Pyrus pyrifolia.

[0094] The results showed that the antibacterial activity of the fermentation broth of the antagonistic strain JN4-2 was determined by the cup-plate method, and its fermentation broth still maintained good antibacterial activity. The fermentation broth of this strain had the strongest antibacterial effect on Rhizoctonia solani of Pinus koraiensis ( Figure 9 ), reaching 34.60 mm, with a significant difference from other test strains; it had a strong antagonistic effect on Colletotrichum truncatum of Vigna angularis ( Figure 10 ) and Valsa mali of Malus domestica, with antibacterial diameters of 31.92 mm and 29.87 mm respectively. It also had a good inhibitory effect on Phyllosticta solani of Solanum melongena, Fusarium oxysporum f. sp. niveum of Cucumis melo ( Figure 11 ), Botryosphaeria laricina of Larix principis-rupprechtii ( Figure 12 ), with antibacterial diameters of 27.60 mm, 27.07 mm, and 28.46 mm respectively. The fermentation broth of this antagonistic bacterium had an obvious inhibitory effect on all test pathogens (Table 3). Among them, the antibacterial diameter of Corynespora cassiicola of Nicotiana tabacum was the smallest, but still reached 18.25 mm.

[0095] Table 3 Antibacterial spectrum of the fermentation broth of the antagonistic strain JN4-2

[0096]

[0097]

[0098] The data in the table are mean ± standard deviation.

[0099] In summary, the JN4-2 strain provided by the present invention has antifungal activity, and its live bacteria and fermentation broth both show strong inhibitory effects on Rhizoctonia solani of Pinus koraiensis. It is speculated that this may be due to the production of antifungal substances during the physiological metabolism process of this strain.

[0100] The antagonistic strain JN4-2 obtained in the present invention has broad development prospects. It has a wide antibacterial spectrum and great potential for biological control. The active antibacterial components in its fermentation broth can be developed as biological pesticides for the prevention and control of various plant diseases after processes such as separation and purification. At the same time, compared with chemical pesticides, the fermentation broth of strain JN4-2 has multiple advantages such as low toxicity, no residue, and environmental friendliness, and is more in line with the environmental protection concept of sustainable control.

[0101] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a supernatant for biological control, characterized in that: The following steps are involved: The Streptomyces netropsis was fermented, centrifuged and the supernatant was collected; the strain name of the Streptomyces netropsis was Streptomyces netropsis JN4-2 and the deposit number was CGMCC No.28157.

2. The preparation method according to claim 1, characterized in that: The fermentation temperature was 28°C.

3. The preparation method according to claim 1 or 2, characterized in that: The fermentation time is 6 days.

4. The preparation method according to claim 1 or 2, characterized in that: The medium formula for fermentation includes: 40 g wheat bran, 20 g corn flour, 60 g sucrose, 4 g potassium nitrate, 0.4 g calcium carbonate, 0.2 g dipotassium hydrogen phosphate, per 1000 mL distilled water, pH 7.

0.

5. A supernatant for biological control, characterized in that: The supernatant is prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the supernatant according to claim 5 in the prevention and treatment of plant pathogenic bacteria, characterized in that: The plant pathogenic bacteria are selected from one or more of the following: red pine damping-off pathogen, red bean anthracnose pathogen, aggregated burr shell fungus, apple shell monochromatic spore canker pathogen, corn leaf blight pathogen, tobacco target spot pathogen, rice seedling blight pathogen, melon vine cut pathogen, pepper anthracnose pathogen, larch tip dieback pathogen, blueberry canker pathogen, poplar bark rot pathogen, apple rot pathogen, poplar canker pathogen, eggplant brown streak pathogen, melon fruit rot pathogen, tobacco brown star pathogen, blueberry branch blight pathogen, apple fruit rot pathogen, cucumber sclerotinia pathogen, and pear fruit rot pathogen.

7. Use of the supernatant according to claim 5 in preventing and treating symptoms caused by plant pathogenic bacteria, characterized in that: The plant pathogenic bacteria are selected from one or more of the following: red pine damping-off pathogen, red bean anthracnose pathogen, aggregated burr shell fungus, apple shell monochromatic spore canker pathogen, corn leaf blight pathogen, tobacco target spot pathogen, rice seedling blight pathogen, melon vine cut pathogen, pepper anthracnose pathogen, larch tip dieback pathogen, blueberry canker pathogen, poplar bark rot pathogen, apple rot pathogen, poplar canker pathogen, eggplant brown streak pathogen, melon fruit rot pathogen, tobacco brown star pathogen, blueberry branch blight pathogen, apple fruit rot pathogen, cucumber sclerotinia pathogen, and pear fruit rot pathogen.

8. Use of the supernatant according to claim 5 in preparing a biocontrol agent, characterized in that: The biocontrol agent is used for preventing and treating diseases caused by plant pathogenic bacteria; the plant pathogenic bacteria are selected from one or more of the following: red pine damping-off pathogen, red bean anthracnose pathogen, aggregated burr shell fungus, apple shell monochromatic spore canker pathogen, corn leaf blight pathogen, tobacco target spot pathogen, rice seedling blight pathogen, melon vine cut pathogen, pepper anthracnose pathogen, larch dieback pathogen, blueberry canker pathogen, poplar bark rot pathogen, apple rot pathogen, poplar canker pathogen, eggplant brown streak pathogen, melon fruit rot pathogen, tobacco brown star pathogen, blueberry branch blight pathogen, apple fruit rot pathogen, cucumber sclerotinia pathogen, and pear fruit rot pathogen.

9. Use of the supernatant according to claim 5 in preventing and treating Korean pine damping-off disease.

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