A method for preparing a fermentation liquid of Streptomyces hygroscopicus and its application

Abstract

The present invention relates to a method for preparing a fermentation liquid of Streptomyces hygroscopicus and its application. The method for preparing a fermentation liquid of Streptomyces hygroscopicus comprises the following steps: inoculating a fermentation medium with a strain named Streptomyces hygroscopicus JN3-4, and fermenting and culturing, wherein the deposit number of Streptomyces hygroscopicus JN3-4 is CGMCC No. 27649. The fermentation liquid prepared by the above method can be used for biological control of pathogenic bacteria such as Aggregative Small Hole Shell Fungus, Red Bean Anthracnose Bacteria, Red Pine Blight Bacteria, Corn Big Spot Bacteria, Tobacco Target Spot Bacteria, Rice Bakanae Bacteria, Melon Vine Cut Bacteria, Pepper Anthracnose Bacteria, Pear Boy's Disk Fruit Rot Bacteria, Cucumber Sclerotinia Bacteria, Blueberry Canker Bacteria, Larch Blight Bacteria, etc., and has the advantages of good biological control effect, wide antibacterial spectrum, wide application range, etc.

Description

[0001] This invention is a divisional application. The original Chinese invention patent application number is: 202410127429.0, the application date is: January 30, 2024, and the patent name at the time of application is: A strain of Streptomyces hygroscopicus and its application. Technical Field

[0002] The invention belongs to the technical field of microorganisms, and in particular relates to a method for preparing a fermentation liquid of Streptomyces hygroscopicus and an application thereof. Background Art

[0003] Larix gmelinii belongs to the genus Larix of the Pinaceae family. It is light-loving, cold-resistant, highly adaptable, fast-growing, and has high economic value for its wood. It is a good tree species for artificially creating fast-growing and high-yield timber forests. The larch dieback pathogen Neofusicoccum laricinum can cause larch dieback. In recent years, larch dieback has become the main disease of larch plantations. The disease was first reported in Jilin Province in 1973. Since the disease's pathogenesis was not known at the time, and there were no effective prevention and quarantine methods, the disease spread rapidly. At present, the disease has spread to Shandong, Hebei, Shaanxi, and Shanxi. The area of ​​occurrence has been increasing year by year, and the damage is becoming more and more serious. Larch dieback mainly harms the new shoots of 1-35-year-old larch plantations in the current year. The disease is more severe and widespread in 6-15-year-old young forests. The disease begins in July, with peaks in August and September. Generally, the disease starts from the main shoots and gradually spreads downward from the branches and shoots in the upper part of the crown. The new shoots that become infected gradually fade from yellow-green to brown-gray, and the top bends and droops into a hook shape, gradually losing leaves downward, drying up, and shrinking and thinning. A cluster of leaves remains at the top of the shoot, and withers and turns purple-gray. The disease occurs year after year, and most of the shoots become dead, and the crown becomes broom-shaped. In severe cases, the growth of trees stops, forming small old trees or the whole plant dies. The leaf clusters withered at the top will have dense black spots on the back of the leaves 15-20 days after the disease occurs, which are the conidiophores of the pathogen and a small number of immature ascocarps.

[0004] In recent years, reports on the occurrence and damage of leaf blight in China have been common. Currently, the following measures are taken to prevent and control the disease: chemical control, breeding of disease-resistant varieties, cultivation of disease-free seedlings, optimization of cultivation sites and cultivation methods, etc. (Fu Xiaoxia, Wang Zhiming, Wang Xuanyao, et al. Research on the combined prevention and control of larch leaf blight and blight [J]. Jilin Forestry Science and Technology, 2016, 45(02): 30-34+40. DOI: 10.16115 / j.cnki.issn.1005-7129.2016.02.010.). Among the many disease prevention methods, chemical control and breeding of disease-resistant varieties are the most easily accepted by the public. However, both of the above control methods have obvious disadvantages. On the one hand, the breeding of disease-resistant varieties takes a long time and there are problems such as resistance degradation, which cannot achieve the expected disease prevention effect; on the other hand, the large-scale use of chemical pesticides is likely to cause environmental pollution and damage the ecological balance. Therefore, we study sustainable control technologies based on ecosystem regulation, use antagonistic microorganisms that have inhibitory effects on pathogens to prevent and control the disease, and use bacteria to treat bacteria to achieve green, environmentally friendly, and efficient comprehensive prevention and control goals.

[0005] Actinomycetes are the earliest discovered type of microorganisms with biological control effects and have been widely used in plant disease control in recent years. Streptomyces occupies an important position among actinomycetes. The actinomycetes isolated by Pandey et al. have antagonistic effects on dry root rot of chickpea (Nath RP, Gandhi KB, Manjunatha L, et al. In vitro evaluation of actinobacteria for biocontrol of dry root rot and Fusarium wilt diseases inchickpea (Cicer arietnum L.) [J]. Journal of Food Legumes, 2021, 34 (4): 245-247.), and Samac et al. found that most antibiotic-producing Streptomyces strains inhibited the growth of three different strains of Phytophthora alfalfa (LATORRE B, TORRES R, et al. Evaluation of the use of wound protectant fungicides and biological control agents against stem canker of blueberry [J]. Cien Inv Agr, 2013, 40 (3): 537-545.). In 2020, Northeast Agricultural University found that the active secondary metabolites of actinomycetes have an inhibitory effect on tomato bacterial wilt (Ling Ling. Study on the prevention effect of Streptomyces sp. NEAU-HV9 on tomato bacterial wilt and its active metabolites [D]. Northeast Agricultural University, 2021. DOI: 10.27010 / d.cnki.gdbnu.2020.000800); in 2023, Huaqiao University isolated the monomer compounds produced by Streptomyces Foc TR4 from banana orchard soil samples and had a strong inhibitory effect on banana wilt (Zou Niexia. Identification of active ingredients of Streptomyces H3-2 against banana wilt and study of its antibacterial mechanism [D]. Huaqiao University, 2022. DOI: 10.27155 / d.cnki.ghqiu.2022.000789.).

[0006] Different strains have different antibacterial spectra and antibacterial activities. In biological control, it is necessary to select appropriate strains to achieve better control effects. Therefore, providing appropriate strains is crucial for effective 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 fermentation broth of Streptomyces hygroscopicus and its application. Streptomyces hygroscopicus JN3-4 is discovered for the first time by the present invention. After further verification, it is found that the strain has the advantages of good biocontrol effect, broad 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 hygroscopicus, the strain name is Streptomyces hygroscopicus JN3-4 (Streptomyces hygroscopicus JN3-4), the preservation number is CGMCC No. 27649, and the strain was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on June 16, 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] The present invention isolated and obtained multiple actinomycetes from soil collected from different regions of Jilin Province. After multiple screenings, an antagonistic strain capable of effectively inhibiting the leaf dieback pathogen was finally obtained, which was marked as JN3-4. The present invention achieved a breakthrough in the application of actinomycetes in the field of leaf dieback disease and provided new strains and products for the biological control of larch dieback disease.

[0011] The present invention provides a bacterial agent, comprising the above-mentioned Streptomyces hygroscopicus and / or a fermentation product of the above-mentioned Streptomyces hygroscopicus.

[0012] The present invention has no particular 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 art for preparing biocontrol agents can also be added to facilitate their application.

[0013] The Streptomyces hygroscopicus and the bacterial agent thereof provided by the invention have the advantages of good biological control effect, broad antibacterial spectrum, wide application range and the like.

[0014] The present invention provides a fermentation method of the hygroscopic streptomyces, comprising the following steps: inoculating the hygroscopic streptomyces into a culture medium for fermentation and culture.

[0015] The formula of the fermentation medium may include: 2.5% peanut powder, 5.0% starch, 0.08% yeast powder, 0.02% glucose, 0.08% (NH4)2SO4, 0.32% CaCO3, 0.2% NaCl, pH 7.0-7.2.

[0016] Fermentation culture conditions may include: 28° C., culture for 6 days.

[0017] The present invention provides a method for preparing the above-mentioned bacterial agent, comprising the following steps: inoculating the above-mentioned Streptomyces hygroscopicus into a fermentation medium for fermentation culture.

[0018] The formula of the fermentation medium may include: 2.5% peanut powder, 5.0% starch, 0.08% yeast powder, 0.02% glucose, 0.08% (NH4)2SO4, 0.32% CaCO3, 0.2% NaCl, pH 7.0-7.2.

[0019] Fermentation culture conditions may include: 28° C., culture for 6 days.

[0020] The invention provides a method for preparing a hygroscopic streptomyces fermentation liquid, comprising the following steps: inoculating a strain named Streptomyces hygroscopicus JN3-4 into a fermentation medium, and fermenting and culturing the strain, wherein the preservation number of the hygroscopic streptomyces JN3-4 is CGMCC No.27649.

[0021] Furthermore, after the fermentation culture, centrifugation and filtration are performed to collect the supernatant.

[0022] The formula of the fermentation medium may include: 2.5% peanut powder, 5.0% starch, 0.08% yeast powder, 0.02% glucose, 0.08% (NH4)2SO4, 0.32% CaCO3, 0.2% NaCl, pH 7.0-7.2.

[0023] The fermentation culture conditions may include: 28° C., culturing for 6 days. The fermentation culture may be carried out by shaking culture.

[0024] The present invention provides the use of the above-mentioned Streptomyces hygroscopicus in the prevention and treatment of plant pathogenic bacteria. The strain provided by the present invention can be used to prepare a biocontrol agent, used to prevent and treat one or more of the following: Aspergillus niger, red bean anthracnose, red pine damping-off, corn leaf blight, tobacco target spot, rice seedling blight, melon vine cutting, pepper anthracnose, pear fruit rot, cucumber sclerotinia, blueberry canker, larch dieback; used to prevent and treat diseases caused by the above-mentioned pathogenic bacteria.

[0025] The present invention provides an application of the above-mentioned microbial agent in the prevention and treatment of plant pathogenic bacteria. The plant pathogenic bacteria are selected from one or more of the following: Aspergillus niger, red bean anthracnose, red pine damping-off, corn leaf blight, tobacco target spot, rice seedling blight, melon vine slash, pepper anthracnose, pear fruit rot, cucumber sclerotinia, blueberry canker, and larch dieback. The above-mentioned microbial agent provided by the present invention can be used to prevent and treat diseases caused by the above-mentioned pathogenic bacteria.

[0026] The invention provides application of fermentation liquid of Streptomyces hygroscopicus in preventing and controlling plant pathogenic bacteria. The plant pathogenic bacteria are selected from one or more of the group consisting of Aspergillus niger, Colletotrichum spp., Pine blight pathogen, Corn leaf blight pathogen, Tobacco target spot pathogen, Rice seedling blight pathogen, Melon vine blight pathogen, Pepper anthracnose pathogen, Pear fruit rot pathogen, Cucumber sclerotinia pathogen, Blueberry canker pathogen and Larch dieback pathogen. The fermentation liquid of Streptomyces hygroscopicus is prepared by the preparation method.

[0027] The invention provides application of a fermentation liquid of Streptomyces hygroscopicus in preventing and treating symptoms caused by plant pathogenic bacteria. The plant pathogenic bacteria are selected from one or more of Aspergillus niger, Colletotrichum spp., Pine blight pathogen, Corn leaf blight pathogen, Tobacco target spot pathogen, Rice seedling blight pathogen, Melon vine blight pathogen, Pepper anthracnose pathogen, Pear fruit rot pathogen, Cucumber sclerotinia pathogen, Blueberry canker pathogen and Larch dieback pathogen. The fermentation liquid of Streptomyces hygroscopicus is prepared by the above-mentioned preparation method.

[0028] The invention provides application of fermentation liquid of Streptomyces hygroscopicus in preparing a biocontrol agent, wherein 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 Aspergillus niger, Colletotrichum spp., Rhizoctonia solani, Corn leaf blight, Tobacco target spot, Rice seedling blight, Melon vine spur, Pepper anthracnose, Pear fruit rot, Cucumber sclerotinia, Blueberry canker and Larch dieback, and the fermentation liquid of Streptomyces hygroscopicus is prepared by the preparation method.

[0029] The invention provides application of fermentation liquid of Streptomyces hygroscopicus in preventing and treating larch dieback disease. The fermentation liquid of Streptomyces hygroscopicus is prepared by adopting the preparation method.

[0030] The invention has the advantages of good prevention and treatment effect, broad antibacterial spectrum, wide application range, etc. when used.

[0031] The present invention provides a biological control method, comprising the following steps: using the above-mentioned Streptomyces and / or the above-mentioned bacterial agent for biological control. The above-mentioned supernatant can also be used for biological control.

[0032] The method provided by the invention has the advantages of good prevention and treatment effect, broad antibacterial spectrum, wide application range, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the inhibitory effect of live bacteria JN3-4 on larch dieback pathogen.

[0034] Figure 2 These are the observation results of strain JN3-4, where A is the culture characteristics of JN3-4 on Gao's No. 1 agar medium, and B is the morphology of JN3-4 under an electron microscope.

[0035] Figure 3 Phylogenetic analysis of strain JN3-4 and related strains. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0037] The present invention targets larch dieback pathogen and obtains a biocontrol strain JN3-4 with good resistance through preliminary screening. Morphological and 16S rDNA sequence analysis and identification show that the strain belongs to Streptomyces hygroscopicus. In order to clarify the biocontrol effect of the antagonistic strain JN3-4, the antibacterial activity of its live bacteria and fermentation liquid was determined by plate confrontation method and cup-disc method respectively. The results show that the strain has an inhibitory effect on 12 tested plant pathogens, with a broad antibacterial spectrum, and has an inhibitory effect on larch dieback pathogen, polyphytic burrows, and pear fruit rot pathogen, among which the strongest inhibitory effect is on larch dieback pathogen, with an inhibition zone of 19.50 mm and an inhibition zone diameter of 31.44 mm in the fermentation liquid. The strain has a significant inhibitory effect on a variety of tested pathogens, and has a good prospect for application and development.

[0038] Plant pathogens tested:

[0039] Dothiorella gregari, Fusarium oxysporum, Neofusicoccum laricinum and Botryosphaeria dothidea were isolated and preserved by our laboratory; Exserohilum turcicum, Rhizoctonia solani, Fusarium moniliforme, Colletotrichum truncatum, Fusarium oxysporum, Colletortrichum capsici and Sclerotinia scleroriorum were donated by the Plant Virus Research Laboratory of Shenyang Agricultural University; Potebnia myces pyri 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.

[0040] The test soil samples were 18 soil samples collected from Dunhua, Jiaohe, Jilin Province, Jiangnan Forest Farm, Panshi City, Jilin City, Jilin Province from June to July 2022.

[0041] Test medium:

[0042] Gause's No.1 agar medium was prepared according to the following proportions: KNO31 g, K2HPO4 0.5 g, MgSO4 0.5 g, NaCl 0.5 g, FeSO4 0.01 g, soluble starch 20 g, agar 20 g, water 1000 mL, pH 7.2-7.4.

[0043] Potato dextrose agar (PDA) was prepared according to the following proportions: 200 g potato, 20 g glucose, 20 g agar, and 1000 mL distilled water.

[0044] Fermentation medium: peanut powder 2.5%, starch 5.0%, yeast powder 0.08%, glucose 0.02%, (NH4)2SO4 0.08%, CaCO3 0.32%, NaCl 0.2%, pH 7.0-7.2, all are mass percentages.

[0045] Test reagents: Resin-type TM genomic DNA extraction kit was purchased from Shanghai Saibaisheng Gene Technology Co., Ltd.; 16S rDNA Bacterial Identification PCR Kit, Agarose Gel DNA Purification KitVer 2.0 and DNA Marker DL2000 were all purchased from Bao Biotechnology (Dalian) Co., Ltd.

[0046] Instruments: BX53 Olympus optical microscope was purchased from Olympus Corporation; Hitachi S-3400N scanning electron microscope was purchased from Hitachi, Ltd.

[0047] 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, which can be obtained through commercial channels or prepared by conventional methods.

[0048] In the embodiments, if data processing is involved, SPSS 23.0 software is used for statistical analysis, and Duncan's new multiple range method is used for difference significance test.

[0049] The following is an introduction through specific embodiments.

[0050] Example 1

[0051] The strain screening was carried out by the stepwise dilution and spreading separation method: 10 g of the test soil sample was baked at 120 °C for 1 h, then added to 100 mL of sterile water, shaken for 30 min, 1 mL of the suspension was drawn, and gradiently diluted with sterile water from 10 to 10. -3, 10 -4 and 10 -5 Pipette 0.1 mL of the dilution tube onto a plate of Gao's No. 1 agar medium, spread it evenly, then invert the culture dish in a 28°C incubator and culture it for 3-10 days. Observe and select strains with different colony morphology every day, and transfer them to the slant of Gao's No. 1 agar medium for culture in time. Re-purify them 3-5 times using the dilution separation method, and store them in a 4°C refrigerator with numbers for later use.

[0052] After purification, 87 purified strains with consistent morphology, size and color were obtained.

[0053] The activated larch dieback pathogen was inoculated into the PDA plate and cultured at 28°C for 7 days before use. All isolated strains were transferred to the plate of Gao's No. 1 agar medium for culture. After each strain produced a sufficient amount of spores, the larch dieback pathogen was used as the target bacteria, and the antibacterial activity of the isolated actinomycetes was determined by the plate confrontation culture method. The determination method includes the following steps: a 7mm bacterial cake was made on the Korean pine blight pathogen plate with a puncher, and the pathogenic bacteria cake was placed on one side 2 cm above and below the center of the PDA plate (diameter 90 mm), and the cultured strain to be tested was picked up and streaked with an inoculation loop on the other side. After constant temperature culture at 28°C for 72 hours, the width of the inhibition zone between the actinomycetes and the pathogenic fungi was measured, and each treatment was repeated 3 times. According to the size of the inhibition zone, the strain with the strongest antagonistic activity was selected as the next research object.

[0054] The results of the plate confrontation test showed that there were 11 strains with an inhibition zone width greater than 13 mm, and 3 strains with an inhibition zone width greater than 17 mm. Among them, strain JN3-4 had the best antibacterial effect, with an inhibition zone width of 19.50 mm ( Figure 1 ), so JN3-4 (isolated from the test soil sample collected from Jiangnan Forest Farm) was selected as the strain for the next test (Table 1).

[0055] Table 1 Antibacterial activity of living Streptomyces against larch dieback pathogen

[0056]

[0057] The data in the table are mean ± standard deviation.

[0058] Example 2

[0059] Observation of JN3-4 strain: The JN3-4 strain was inoculated onto a plate of Gao's No. 1 agar medium, and a sterilized cover glass was inserted into the medium at an angle of 45°. After culturing at 28°C for 5-7 days, the cover glass was removed and observed using an Olympus BX53 optical microscope and a Hitachi S-3400N scanning electron microscope.

[0060] like Figure 2As shown, strain JN3-4 grew vigorously on Gao's No. 1 agar medium; when cultured at 28°C for 1-3 days, the colonies were smooth and no spores were produced. Under a microscope, spores mostly occurred in single colonies; and the spore threads were autolyzed and absorbed water; obvious spiral spore chains were visible under an electron microscope, which had typical Streptomyces characteristics.

[0061] Example 3 16S rDNA Identification of Strain JN3-4

[0062] The DNA of strain JN3-4 was extracted according to the instructions of the resin-based TM genomic DNA extraction kit. The 16S rDNA of strain JN3-4 was amplified by PCR using the 16S rDNA Bacterial Identification PCR Kit. The upstream and downstream primers used to amplify 16S rDNA were purchased from Takara Biotech (Dalian) Engineering Co., Ltd.

[0063] The PCR reaction system (50 μL) includes: PCR Mixture 25 μL, Template 2 μL, upstream and downstream primers 1 μL each, and ddH2O 21 μL.

[0064] PCR reaction conditions: denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 2 min, 35 cycles; extension at 72°C for 5 min, storage at 4°C.

[0065] The PCR product of sterile water was used as a negative control, and the PCR product of a known 16S rDNA strain was used as a positive control. The sample volume was 2 μL, and 2% agarose gel electrophoresis was performed. The DNA Marker used for electrophoresis was DNA Marker DL2000. The product was recovered using Agarose Gel DNA Purification Kit Ver 2.0 and sent to Takara Biotech (Dalian) Engineering Co., Ltd. for bidirectional sequencing. After the sequence was analyzed and manually corrected by software such as BioEdit7.0.1, the BLAST program of NCBI was used to compare the measured sequence with the sequence of known similar model species downloaded from GenBank for homology analysis, and the neighbor-joining method (NJ) of MEGA6.0 software was used to align the sequence and draw a phylogenetic tree.

[0066] After sequencing, the full length of the 16S rDNA gene amplified fragment of strain JN3-4 was 1217 bp. Nine sequences with high homology to strain JN3-4 were selected to establish a phylogenetic tree, such as Figure 3 As shown, strain JN3-4 was preliminarily determined to be Streptomyces hygroscopicus of the genus Streptomyces.

[0067] The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on June 16, 2023, with the deposited name as Streptomyces hygroscopicus JN3-4, the deposited number as CGMCC No.27649, and the deposited address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0068] Example 4 Determination of the antibacterial spectrum of live bacteria of Streptomyces JN3-4

[0069] The antibacterial spectrum of live Streptomyces JN3-4 was determined by plate confrontation culture method using Aspergillus niger, Colletotrichum spp., Psoralea corylifolia ... as test strains. The plate confrontation culture method is referred to Example 1.

[0070] The results showed that the antagonistic bacteria JN3-4 had an inhibitory effect on all the tested strains. JN3-4 had the strongest inhibitory effect on larch blight pathogen, with an obvious inhibition zone and a width of 19.50 mm, which was significantly different from other tested pathogens. It also had a strong inhibitory effect on Aspergillus niger, Pear Bordetella rot and Rice Bakanae spp., with inhibition zone widths of 19.31 mm, 17.06 mm and 15.05 mm, respectively. The inhibitory effect on cucumber sclerotinia and red bean anthracnose was relatively weak, but the inhibition zone width was also above 13.24 mm (Table 2).

[0071] Table 2 Antibacterial spectrum of antagonistic bacteria JN3-4

[0072]

[0073] The data in the table are mean ± standard deviation. The letters after the data in the same column indicate significant differences at the P < 0.01 level tested by Duncan's new multiple range method.

[0074] Example 5 Preparation of fermentation broth and determination of its antibacterial spectrum

[0075] Prepare 200 mL of fermentation medium and put it into a 500 mL Erlenmeyer flask, sterilize and set aside. Transfer strain JN3-4 to the fermentation medium, culture at 28°C and 150 r / min for 6 days, then centrifuge at 8000 r / min for 15 min, filter the supernatant through a 0.22 μm bacterial filter, remove the bacteria and store in a -20°C refrigerator for later use.

[0076] The test strains were Aspergillus niger, Colletotrichum oxysporum, Pine blight, Corn leaf blight, Tobacco target spot, Rice seedling blight, Melon vine disease, Pepper anthracnose, Pear fruit rot, Cucumber sclerotinia, Blueberry canker and Larix gmelinii dieback. All the tested plant pathogens were activated by the cup-and-disc method to prepare a spore suspension with 30-40 spores / field of view under a 10×15 magnification microscope. 300 μL of the spore suspension was added to 70 mL of PDA culture medium to prepare a mixed bacterial plate. An Oxford cup was placed in the center of the culture dish. 100 μL of fermentation liquid (i.e., the supernatant prepared by the above method) was added to the Oxford cup. After constant temperature culture at 28°C for 72 h, the diameter of the inhibition zone was measured by the cross method.

[0077] The antibacterial activity of the fermentation liquid of the antagonistic strain JN3-4 was determined by the cup-disc method. The results showed that the fermentation liquid still maintained good antibacterial activity. The fermentation liquid of this strain had the strongest antibacterial effect on larch dieback pathogen, reaching 31.44 mm, which was significantly different from other tested strains. It had a strong antagonistic effect on cucumber sclerotinia and corn leaf blight pathogen, with an inhibition diameter of more than 24.69 mm. It also had a good inhibitory effect on pepper anthracnose, polymorphic burr hole fungus, and rice seedling pathogen, with inhibition diameters of 24.34 mm, 23.36 mm, and 22.68 mm, respectively. The fermentation liquid of this antagonistic bacterium had a significant inhibitory effect on all tested pathogens (Table 3), and the minimum inhibition diameter also reached 10.77 mm.

[0078] Table 3 Antibacterial spectrum of the fermentation broth of antagonistic bacteria JN3-4

[0079]

[0080] The data in the table are mean ± standard deviation. The letters after the data in the same column indicate significant differences at the P < 0.01 level tested by Duncan's new multiple range method.

[0081] In summary, the JN3-4 strain provided by the present invention has fungicidal activity, and its live bacteria and fermentation liquid both show good inhibitory effects on larch dieback pathogens, which may be due to the fact that the strain produces antifungal substances during its physiological metabolism.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a fermentation broth of Streptomyces hygroscopicus, characterized in that: The following steps are involved: Streptomyces hygroscopicus JN3-4 ( Streptomyces hygroscopicus The strain JN3-4) is inoculated into the fermentation medium for fermentation culture. The preservation number of Streptomyces hygroscopicus JN3-4 is CGMCC No. 27649.

2. The method for preparing a hygroscopic Streptomyces fermentation broth according to claim 1, characterized in that: After fermentation, centrifugation and filtration were performed to collect the supernatant.

3. The method for preparing a hygroscopic Streptomyces fermentation broth according to claim 1 or 2, characterized in that: The formula of the fermentation medium includes: 2.5% peanut cake powder, 5.0% starch, 0.08% yeast powder, 0.02% glucose, 0.08% (NH4)2SO4, 0.32% CaCO3, 0.2% NaCl, and pH 7.0-7.

2.

4. The method for preparing a hygroscopic Streptomyces fermentation broth according to claim 1 or 2, characterized in that: The fermentation time was 6 days.

5. The method for preparing a hygroscopic Streptomyces fermentation broth according to claim 1 or 2, characterized in that: The fermentation temperature was 28°C.

6. The method for preparing a hygroscopic Streptomyces fermentation broth according to claim 1 or 2, characterized in that: The fermentation culture was carried out by shaking culture.

7. Application of the fermentation liquid of Streptomyces hygroscopicus in the prevention and treatment of plant pathogenic bacteria, characterized in that: The plant pathogenic bacteria are selected from one or more of the group consisting of Aspergillus niger, Colletotrichum sphaeroides, Pine blight, Corn leaf blight, Tobacco target spot, Rice seedling blight, Melon vine blight, Pepper anthracnose, Pear fruit rot, Cucumber sclerotinia, Blueberry canker, and Larch dieback, and the fermentation broth of Streptomyces hygroscopicus is prepared by the preparation method described in any one of claims 1 to 6.

8. Use of the fermentation liquid of Streptomyces hygroscopicus in the preparation of 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 group consisting of Aspergillus niger, Colletotrichum sphaeroides, Pine blight, Corn leaf blight, Tobacco target spot, Rice seedling blight, Melon vine blight, Pepper anthracnose, Pear fruit rot, Cucumber sclerotinia, Blueberry canker, and Larch dieback, and the fermentation liquid of Streptomyces hygroscopicus is prepared by the preparation method described in any one of claims 1 to 6.

9. The use of the fermentation liquid of Streptomyces hygroscopicus in preventing and treating larch dieback disease, characterized in that: The fermentation broth of Streptomyces hygroscopicus is prepared by the preparation method according to any one of claims 1 to 6.

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